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Characterization of verteporfin as an inhibitor of autophagosome formation and its therapeutic potential… Donohue, Elizabeth 2013

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CHARACTERIZATION OF VERTEPORFIN AS AN INHIBITOR OF AUTOPHAGOSOME FORMATION AND ITS THERAPEUTIC POTENTIAL IN CANCER  by Elizabeth Donohue  B.Sc.H., McGill University, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Biochemistry & Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2013  © Elizabeth Donohue, 2013  ABSTRACT  Autophagy is a cellular “self-eating” process that enables cells to degrade and recycle cytoplasmic materials both as a housekeeping mechanism and in response to extracellular stress. Based on preclinical studies, autophagy promotes cell survival in the nutrient-deprived tumour environment and in response to several cancer therapy agents, making it a prospective therapeutic target. Due to a lack of pharmacologically suitable and selective autophagy inhibitors, a phenotypic automated fluorescence microscopy assay was designed and used to screen > 3,500 drugs and pharmacological agents for novel inhibitors of autophagosome accumulation. Verteporfin, a benzoporphyrin derivative used in photodynamic therapy, was the only active compound identified. As a photosensitizer, verteporfin generates large amounts of singlet oxygen upon light irradiation, which elicits a cell-death response. In the absence of light activation, verteporfin is nontoxic and inhibits starvation- and drug-induced autophagy. Biochemical and microscopy assays revealed that verteporfin prevents autophagic sequestration and degradation downstream of LC3 lipidation and membrane association. p62 is a scaffold protein that oligomerizes and links poly-ubiquitinated proteins to the autophagosome membrane by binding LC3, thus delivering both its cargo and itself for lysosomal degradation. Western blot analysis revealed that verteporfin produces SDS-stable high-MW p62, which is highly oxidized, and is likely a product of p62 crosslinking. The mechanism of high-MW p62 generation by verteporfin was discovered to be low-level singlet oxygen production, and the appearance of high-MW p62 correlated with autophagy inhibition. p62 co-immunoprecipitation experiments revealed that its association with EGFP-  i  LC3 was not affected by verteporfin, but binding to poly-ubiquitinated cargo was disrupted. Therefore, non-photoactivated verteporfin generates low-level singlet oxygen that induces p62 oxidation and high-MW products that may interfere with autophagosome formation. Verteporfin was used to evaluate the therapeutic potential of autophagy inhibition using two different tumour xenograft models. Verteporfin did not show anti-tumour activity in a JIMT-1 breast cancer model, but it did enhance the anti-tumour and survival effects conferred by gemcitabine in a BxPC-3 pancreatic cancer model. The characterization of an early autophagy inhibitor among FDA-approved drugs that shows in vivo potential has significant implications for understanding autophagy modulation as a therapeutic strategy.  ii  PREFACE  This thesis is presented in six chapters. A general review of the research area is presented in Chapter 1. Chapter 2 describes the Materials and Methods used in the experiments presented in this study. A version of Chapter 3 was published with co-authors Andrew Tovey, A. Wayne Vogl, Steve Arns, Ethan Sternberg, Robert N. Young, and Michel Roberge (Donohue E, Tovey A, Vogl AW, Arns S, Young RN, and Roberge M. Inhibition of autophagosome formation by the benzoporphyrin derivative verteporfin. J Biol Chem 2011; 286: 7290-7300). The verteporfin regioisomers tested in Figure 3.3 were separated from a mixture of commercial verteporfin by Dr. Arns. The electron microscopy presented in Figure 3.5 was done in collaboration with Dr. Vogl. After I treated the cells, Dr. Vogl carried out the sample preparation and imaging; analysis was done in collaboration. The benzoporphyrin derivatives presented in Figure 3.11 were synthesized by Andrew Tovey and other members of Dr. David Dolphin’s laboratory at UBC. Andrew Tovey and Ethan Sternberg also assisted Dr. Roberge and myself in the structure-activity analysis. All other work presented in Chapter 3 is my own, and I wrote the manuscript. The in vivo experiments presented in Chapter 5 were carried out in collaboration with the Centre for Drug Research and Development (CDRD). The methodologies used in these animal studies were reviewed and approved by the Institutional Animal Care Committee (IACC) prior to conducting the studies, as per protocol # A10-0171. Dr. Norbert Maurer and Dr. Anitha Thomas formulated verteporfin in DSPE-PEG micelles for both the JIMT-1 and BxPC-3 tumour xenograft studies. The technical aspects of the in vivo studies including animal care, drug administration, monitoring of animal morbidity and tumour volume, and  iii  tumour excision were all carried out at the BC Cancer Research Agency by members of Dr. Marcel Bally’s laboratory and in accordance with the Canadian Council on Animal Care Guidelines. The experimental design was determined collaboratively by Dr. Roberge, Dr. Bally, Dr. Murray Webb (CDRD), Dr. Maurer, Dr. Thomas, and myself. The pharmacokinetic analysis of tumour tissue presented in Figures 5.3 and 5.10 was carried out by Dr. Thomas and other members of the Analytical Chemistry Department at the CDRD. All other experiments, including those presented in Chapter 4, were designed and analyzed by Dr. Roberge and myself, and carried out by me.  iv  TABLE OF CONTENTS  ABSTRACT .............................................................................................................................. i PREFACE ............................................................................................................................... iii TABLE OF CONTENTS ....................................................................................................... v LIST OF TABLES ............................................................................................................... viii LIST OF FIGURES ............................................................................................................... ix LIST OF ABBREVIATIONS ............................................................................................. xiii ACKNOWLEDGEMENTS ................................................................................................ xvi CHAPTER 1: INTRODUCTION......................................................................................... 1 1.1 Autophagy ................................................................................................................. 1 1.1.1 Molecular mechanisms of autophagy ................................................................... 3 1.1.1.1 Isolation membrane nucleation ..................................................................... 4 1.1.1.2 Isolation membrane elongation and autophagosome formation ................... 8 1.1.1.3 Autophagome-lysosome fusion and degradation ........................................ 10 1.1.2 Physiological roles for autophagy....................................................................... 11 1.1.3 Selective autophagy ............................................................................................ 13 1.2 p62........................................................................................................................... 15 1.2.1 p62 in autophagy ................................................................................................. 15 1.2.2 Other physiological roles of p62 ......................................................................... 20 1.3 Autophagy and cancer............................................................................................. 22 1.4 Autophagy inhibitors .............................................................................................. 25 1.5 Chemical biology as an approach to study biological processes and to discover novel drugs .......................................................................................................................... 26 1.6 Cell-based high throughput screening .................................................................... 29 1.7 Objectives ............................................................................................................... 35 1.7.1 Development of a high content screen to identify novel small molecule inhibitors of autophagy ................................................................................................................... 35 1.7.2 Investigate the mechanism of action of novel autophagy inhibitors................... 35 1.7.3 Determine if autophagy inhibition is a promising therapeutic strategy for cancer therapy ............................................................................................................................ 36 CHAPTER 2: MATERIALS AND METHODS ............................................................... 37 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8  Cell lines ................................................................................................................. 37 Chemicals................................................................................................................ 37 Screening assay for inhibitors of autophagosome formation .................................. 38 Starvation procedures.............................................................................................. 39 Immunofluorescence microscopy ........................................................................... 39 SDS-PAGE and immunoblotting ............................................................................ 40 Subcellular fractionation ......................................................................................... 42 Long-lived protein degradation assay ..................................................................... 42 v  2.9 FITC-dextran sequestration assay ........................................................................... 43 2.10 Benzoporphyrin derivatives .................................................................................... 43 2.11 Electron microscopy ............................................................................................... 43 2.12 Cell viability and proliferation assays..................................................................... 44 2.13 Recombinant p62 assays ......................................................................................... 45 2.14 Immunoprecipitation ............................................................................................... 45 2.15 Formulation of verteporfin for animal studies – mPEG2000-DSPE micelles ........ 45 2.16 Animal husbandry ................................................................................................... 46 2.17 Efficacy studies of verteporfin ................................................................................ 47 2.18 Pharmacokinetic studies of verteporfin mPEG2000-DSPE micelles in BxPC-3 tumour-bearing mice ........................................................................................................... 48 2.19 Measurement of verteporfin concentration in plasma and tumour tissue ............... 48 CHAPTER 3: IDENTIFICATION AND CHARACTERIZATION OF VERTEPORFIN AS AN INHIBITOR OF AUTOPHAGOSOME FORMATION........ 50 3.1 Synopsis .................................................................................................................. 50 3.2 Screen for chemical inhibitors of autophagosome accumulation ........................... 50 3.3 Verteporfin inhibits drug- and starvation-induced autophagosome accumulation . 57 3.4 Verteporfin inhibits autophagic degradation .......................................................... 60 3.5 Verteporfin inhibits non-specific autophagic sequestration ................................... 63 3.6 Verteporfin does not inhibit LC3 processing or LC3II membrane association ...... 65 3.7 Verteporfin sensitizes cells to starvation ................................................................ 69 3.8 Structural requirements for inhibition of autophagy by benzoporphyrin derivatives ........................................................................................................................... 69 3.9 Discussion ............................................................................................................... 73 CHAPTER 4: INDUCTION OF HIGH-MOLECULAR WEIGHT P62 BY VERTEPORFIN AND ITS POTENTIAL ROLE IN AUTOPHAGY INHIBITION .... 79 4.1 Synopsis .................................................................................................................. 79 4.2 Verteporfin induces the appearance of a high-molecular weight form of p62 ....... 80 4.3 Verteporfin-induced high-MW p62 does not represent p62 aggregates ................. 86 4.4 Verteporfin can induce high-MW p62 in vitro ....................................................... 89 4.4.1 Experiments with cell lysates.............................................................................. 89 4.4.2 Experiments with p62 immunoprecipitates ........................................................ 91 4.4.3 Experiments with pure p62 protein ..................................................................... 93 4.5 Verteporfin-mediated high-MW p62 forms are due to protein oxidation by singlet oxygen ..................................................................................................................... 95 4.6 p62 is oxidized by singlet oxygen generators ....................................................... 101 4.7 Rose bengal inhibits starvation-induced autophagosome accumulation .............. 103 4.8 Rose bengal induces the appearance of high MW-p62 forms in vivo................... 105 4.9 High-MW p62 shows reduced ability to bind to ubiquitinated proteins but not to LC3 ......................................................................................................................... 108 4.10 Discussion ............................................................................................................. 116  vi  CHAPTER 5: EFFICACY OF VERTEPORFIN IN ANIMAL MODELS OF CANCER ....................................................................................................................... 125 5.1 Synopsis ................................................................................................................ 125 5.2 Efficacy of verteporfin in a JIMT-1 breast cancer xenograft model .................... 126 5.2.1 In vitro effects of verteporfin on JIMT-1 cells ................................................. 127 5.2.2 Verteporfin tumour accumulation following single administration.................. 131 5.2.3 Verteporfin induces high-MW p62 forms in vivo in tumour tissue .................. 133 5.2.4 Verteporfin does not inhibit JIMT-1 tumour growth in vivo alone or in combination with gefitinib ............................................................................................ 133 5.3 Efficacy of verteporfin in a BxPC-3 pancreatic adenocarcinoma xenograft model ................................................................................................................ 136 5.3.1 In vitro effects of verteporfin on pancreatic adenocarcinoma cells .................. 136 5.3.2 In vivo effects of gemcitabine on BxPC-3 and SU86.86 tumour growth ......... 142 5.3.3 Verteporfin accumulates in BxPC-3 tumour tissue following single administration ............................................................................................................... 147 5.3.4 Verteporfin treatment induces high-MW p62 forms in BxPC-3 tumour tissue in vivo.................................................................................................................. 150 5.3.5 Verteporfin moderately enhances the anti-tumour activity of gemcitabine in BxPC-3 xenografts ........................................................................................................ 151 5.3.6 Discussion ......................................................................................................... 156 CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS .................................. 163 6.1 High-throughput screen for early autophagy inhibitors ........................................ 163 6.2 Identification and characterization of verteporfin as an inhibitor of autophagosome formation via singlet oxygen ............................................................................................ 164 6.2.1 Verteporfin inhibits autophagosome formation ................................................ 164 6.2.2 Verteporfin produces oxidized high-MW p62 products via singlet oxygen production ..................................................................................................................... 165 6.2.3 p62 crosslinking disrupts binding to poly-ubiquitinated cargo......................... 168 6.2.4 Singlet oxygen is the mechanism underlying autophagy inhibition by verteporfin ................................................................................................................ 169 6.3 Characterization of the anti-tumour effects of verteporfin in vivo ....................... 170 6.3.1 Verteporfin does not show anti-tumour efficacy on its own in vivo ................. 170 6.4 Verteporfin enhances the anti-tumour effect of gemcitabine in a BxPC-3 tumour xenograft model ................................................................................................................ 171 BIBLIOGRAPHY ............................................................................................................... 173  vii  LIST OF TABLES  Table 5.1 Summary of PDAC cell line origin, mutational status of common PDAC markers, and verteporfin sensitivity............................................................................................. 141  viii  LIST OF FIGURES  Figure 1.1 The process of (macro)autophagy ........................................................................... 2 Figure 1.2 Regulation of the ULK1/2-Atg13 complex upon autophagy induction by mTORC1 ........................................................................................................................... 5 Figure 1.3 Ubiquitin-like conjugation reactions involved in isolation membrane elongation . 9 Figure 1.4 The functional domains of p62 .............................................................................. 16 Figure 1.5 Selective autophagy of ubiquitinated proteins by p62 .......................................... 18 Figure 3.1 Cell-based assay for the identification of inhibitors of autophagosome accumulation. .................................................................................................................. 53 Figure 3.3 Identification of verteporfin as an inhibitor of autophagosome accumulation...... 55 Figure 3.4 Effect of verteporfin regioisomers on chloroquine-induced autophagosome accumulation. .................................................................................................................. 56 Figure 3.5 Verteporfin inhibits autophagosome accumulation stimulated by rapamycin or serum starvation. ............................................................................................................. 58 Figure 3.6 Ultrastructural examination of inhibition of chloroquine-induced autophagosome accumulation by verteporfin. .......................................................................................... 59 Figure 3.7 Inhibition of EGFP-LC3 degradation by verteporfin. ........................................... 62 Figure 3.8 Inhibition of protein degradation by verteporfin. .................................................. 64 Figure 3.9 Inhibition of the sequestration of cytosolic FITC-dextran into vesicles by verteporfin. ...................................................................................................................... 66 Figure 3.10 Verteporfin does not inhibit LC3 processing or membrane association. ............ 67 Figure 3.11 Effect of verteporfin on cell survival in different starvation conditions. ............ 70  ix  Figure 3.12 Effect of selected verteporfin analogues on chloroquine-induced autophagosome accumulation. .................................................................................................................. 72 Figure 3.13 Hypothetical model for the general scheme of inhibition of autophagosome formation by verteporfin. ................................................................................................ 76 Figure 4.1 Verteporfin induces high-MW p62 in cells. .......................................................... 81 Figure 4.2 Verteporfin-mediated p62 modification is amplified by light exposure during lysate preparation. ........................................................................................................... 82 Figure 4.3 High-MW and 60-kDa p62 immunoprecipitate from untreated and verteporfintreated cells. .................................................................................................................... 85 Figure 4.4 High-MW p62 generated by verteporfin is not indicative of p62 aggregates. ...... 88 Figure 4.5 In vitro effect of verteporfin on p62 in cell lysates. .............................................. 90 Figure 4.6 In vitro effect of verteporfin on immunoprecipitated p62. .................................... 92 Figure 4.7 Effect of verteporfin on purified GST-p62. ........................................................... 94 Figure 4.8 Rose bengal induces high-MW p62 in vitro. ......................................................... 97 Figure 4.9 Quenching singlet oxygen reduces the generation of high-MW p62 by verteporfin. .................................................................................................................... 100 Figure 4.10 Singlet oxygen increases the carbonyl content in p62 and high-MW p62. ....... 102 Figure 4.11 Rose bengal does not inhibit autophagosome accumulation by chloroquine in the presence of serum. ........................................................................................................ 104 Figure 4.12 Rose bengal inhibits starvation-induced autophagosome accumulation in the absence of serum ........................................................................................................... 106 Figure 4.13 Rose bengal induces high-MW p62 in cells in the absence of serum. .............. 107  x  Figure 4.14 High-MW p62 shows impaired association with poly-ubiquitinated proteins, but not with LC3 ................................................................................................................. 115 Figure 4.15 Proposed model for singlet oxygen-mediated inhibition of autophagosome formation involving p62 crosslink products. ................................................................ 122 Figure 5.1 Verteporfin inhibits gefitinib-induced EGFP-LC3 degradation. ......................... 128 Figure 5.2 Long-term verteporfin treatment inhibits JIMT-1 cell survival on its own and enhances the effect of gefitinib in vitro. ....................................................................... 129 Figure 5.3 Verteporfin tumour concentration peaks 8 h after administration and remains above its IC 50 for 24 h in JIMT-1 tumour-bearing mice. ............................................. 132 Figure 5.4 Verteporfin induces high-MW p62 in JIMT-1 tumour tissue in vivo.................. 134 Figure 5.5 Verteporfin does not exhibit anti-tumour efficacy on its own or in combination with gefitinib in a JIMT-1 tumour mouse model. ......................................................... 135 Figure 5.6 PDAC cell lines show variable sensitivity to long-term verteporfin tretment in vitro. .............................................................................................................................. 139 Figure 5.7 Verteporfin inhibits gemcitabine-induced autophagosome accumulation. ......... 143 Figure 5.8 Gemcitabine is anti-proliferative and toxic to BxPC-3 and SU86.86 PDAC cell lines in vitro. ................................................................................................................. 145 Figure 5.9 Gemcitabine has varying efficacy between an SU86.86 tumour xenograft model and a BxPC-3 tumour xenograft model. ....................................................................... 146 Figure 5.10 Verteporfin tumour concentration peaks 8 h after administration and remains above its IC 50 for 24 h in BxPC-3 tumour-bearing mice. ............................................. 148 Figure 5.11 Verteporfin induces high-MW p62 forms in BxPC-3 tumour tissue in vivo. .... 149  xi  Figure 5.12 Combining verteporfin treatment with gemcitabine in a BxPC-3 tumour model inhibits tumour growth and increases survival. ............................................................ 155  xii  LIST OF ABBREVIATIONS  3-MA  3-methyladenine  AMBRA1  activated molecule in beclin-1 regulated autophagy  ATG  autophagy-related  ATP  adenosine triphosphate  BPD  benzoporphyrin derivative  CDRD  Centre for Drug Research and Development  CQ  chloroquine  DFCP1  double FYVE-containing protein 1  DMEM  Dulbecco’s modified Eagle medium  DMSO  dimethyl sulfoxide  DNPH  2,4-dinitrophenylhydrazine  DPBS  Dulbecco’s phosphate buffered saline  DSPE  1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine  DTT  dithiothreitol  EBSS  Earl’s balanced salt solution  EGFP  enhanced green fluorescent protein  EGFR  epidermal growth factor receptor  ER  endoplasmic reticulum  ESCRT  endosomal sorting complex required for transport  FBS  fetal bovine serum  FITC  fluorescein isothiocyanate  xiii  HCQ  hydroxychloroquine  HCS  high content screening  HER2  human epidermal growth factor receptor 2  HRP  horseradish peroxidase  HTS  high-throughput screening  IC 50  half maximal inhibitory concentration  i.p.  intraperitoneally  Keap1  kelch like-ECH-associated protein 1  LAMP-2A  lysosomal-associated membrane protein 2A  LC3  microtubule-associated protein 1 light chain 3  LIR  LC3 interacting region  LRS  LC3 recognition sequence  MS  mass spectrometry  mTORC1  mammalian target of rapamycin complex 1  MTT  3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide  MW  molecular weight  NAC  N-acetylcysteine  NBR1  neighbor of BRCA1  NDP52  nuclear dot protein 52  NF-κB  nuclear factor kappa B  Nrf2  nuclear factor (erythroid-derived-2)-like 2  PBS  phosphate buffered saline  PDAC  pancreatic ductal adenocarcinoma  xiv  PDT  photodynamic therapy  PE  phosphatidylethanolamine  PEG  polyethylene glycol  PI3K  phosphatidylinositol 3-kinase  PI3P  phosphatidylinositol 3-phosphate  PVDF  polyvinylidene fluoride  RNAi  RNA interference  ROS  reactive oxygen species  RPMI  Roswell Park Memorial Institute medium  SDS  sodium dodecyl sulfate  SNARE  soluble N-ethylmaleimide-sensitive factor attachment protein receptor  siRNA  small interfering RNA  TBS  Tris-buffered saline  Tris  tris(hydroxylmethyl)aminomethane  ULK1/2  uncoordinated-51-like kinase  UPS  ubiquitin proteasome system  V-ATPase  vacuolar-type H+-ATPase  WIPI1/2  WD repeat domain phosphoinositide-interacting 1/2  UBA  ubiquitin-associated  xv  ACKNOWLEDGEMENTS  First of all, I would like to thank my supervisor, Dr. Michel Roberge, for his mentorship and encouragement throughout my graduate studies. Your dedication and passion for science inspired me immensely. I really appreciate your collaborative approach to science and willingness to try new and interesting avenues of research. Thank you for helping me to develop a number of skills, particularly writing and presenting, and for providing such a pleasant research environment. Past and present members of the Roberge laboratory have provided experimental support and helpful discussion: Hilary Anderson, Aruna Balgi, Pamela Dean, Bruno Fonseca, Danielle Kemmer, Karen Lam, Lianne McHardy, Alym Moosa, Jenna Riffell, and Carla Zimmerman. I would like to thank my supervisory committee, Dr. Roger Brownsey and Dr. Eric Jan, for their insights and mentorship. Thanks to our collaborators, particularly CDRD members Dr. Marcel Bally, Dr. Norbert Maurer and Dr. Anitha Thomas whose resources and expertise were invaluable for the animal studies presented. I want to thank my fellow graduate students through the years for their support, suggestions, and commiseration, particularly Kristina McBurney and Jenna Riffell. I would like to acknowledge my family for their endless emotional support and belief in me. To my parents, John and Lorraine Donohue, your unrelenting hard work and dedication have been an inspiration for as long as I can remember. Thank you Lorraine and Suzanne, my sisters, for always looking out for me and making me laugh. Thank you to my friends who have been incredibly encouraging over the years and understanding of my limited free time. Finally, thank you Richard for your love and support.  xvi  CHAPTER 1: INTRODUCTION  1.1  Autophagy Autophagy is a general term for the lysosomal degradation of cytoplasmic components.  Macromolecules, such as proteins, glycogen, lipids, and nucelotides, and organelles, including mitochondria, peroxisomes, and lipid droplets can all be degraded by autophagy (1,2). In eukaryotes, there are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy (3,4). Macroautophagy (hereafter referred to as autophagy) is a conserved pathway characterized by the formation of an isolation membrane called the phagophore, which expands and sequesters cytoplasmic constituents in a doublemembrane-bound (double-bilayer) vesicle called the autophagosome. The autophagosomes eventually fuse with lysosomes, and the engulfed material is degraded by acidic hydrolases, and released to the cytoplasm for recycling (Figure 1.1). Microautophagy is poorly characterized, and involves the direct sequestering of cytoplasmic material by invagination of the lysosomal membrane itself (5). In microautophagy, lysosomal invagination extends to form autophagic tubes that constrict at the neck near the lumen of the lysosomal membrane in a dynamic process mediated by ubiquitin-like systems that are also involved in macroautophagy (6). This microautophagic tube then pinches off the lumen off the lysosome to form a small vesicle that contains soluble intracellular substrates. It is believed that microautophagy cooperates with macroautophagy and chaperone-mediated autophagy to consume superabundant materials and to maintain the lipid composition and size of the lysosome (6). Chaperone-mediated autophagy describes the  1  Figure 1.1 The process of (macro)autophagy An isolation membrane forms as a vesicle in the cytoplasm and expands to engulf and sequester cytoplasmic constituents in a double-membrane bound organelle called the autophagosome (blue). The completed autophagosome then fuses with the lysosome (yellow), delivering its contents for degradation by lysosomal hydrolases. The degradation products are then released into the cytoplasm for macromolecular synthesis.  2  selective translocation of cytosolic proteins with a specific pentapeptide sequence called the “KFERQ” motif across the lysosomal membrane (7,8). Chaperone-mediated autophagy is characterized by individual recognition of single proteins, chaperone-mediated protein unfolding, and translocation of those proteins into the lysosome. The pentapeptide motif is recognized by the multifunctional chaperone hsc70, followed by hsc70/substrate binding to LAMP2A, which then multimerizes and mediates translocation into the lysosome (9). In addition to its role maintaining homeostasis, chaperone-mediated autophagy is believed to regulate tissue- and cell-specific roles through the selective degradation of transcription factors and co-regulators. Constitutive autophagy functions as a housekeeping mechanism by controlling the turnover of long-lived proteins and organelles (4). Autophagy can also be stimulated as a cytoprotective response to intracellular and extracellular stresses including starvation, hypoxia, low cellular ATP levels, oxidative stress, accumulation of damaged organelles or misfolded proteins, and pathogen invasion (10–13). Considering its role maintaining cellular homeostasis, it is unsurprising that autophagy has been implicated in a number of pathophysiological states including, but not limited to cancer (reviewed in Section 1.3), neurodegeneration (including Parkinson’s, Alzheimer’s, and Huntington’s diseases), myopathies, and Crohn’s disease (14–16). 1.1.1  Molecular mechanisms of autophagy  Christian de Duve was the first to describe autophagosomes as vesicles for cargo delivery to the lysosome on the basis of electron microscopy in 1963 (17). During the following twenty years, microscopy was used to characterize autophagy as a catabolic response controlled by hormones and amino acids to sustain metabolism (18,19). In 1993 and  3  1994, two seminal yeast genetic studies were published that identified 15 ‘core’ autophagyrelated (Atg) genes essential for autophagosome formation (20,21), many of which are conserved in humans (22). The identification of the core Atg proteins provided the tools and markers necessary for deciphering many of the underlying mechanisms of canonical nonselective autophagy in mammals (15,23). In mammals, the Atg proteins primarily mediate the membrane dynamics for autophagosome biogenesis and membrane fusion. 1.1.1.1 Isolation membrane nucleation Upon autophagy induction, a cup-shaped protrusion forms from the ER membrane, called the omegasome that acts as the platform for isolation membrane formation. Previous studies have also implicated the plasma membrane and the mitochondrial membrane as isolation membrane sources (24,25), while a recent report out of the Yoshimori laboratory (26) localized the omegasome to ER-mitochondrial contact points, suggesting multiple sites and membrane sources during autophagosome biogenesis. Autophagy was initially characterized as a cellular response to starvation, particularly in conditions lacking amino acids and/or growth factors (18,19). These nutrient-sensing signaling pathways converge at the master regulatory complex, mTORC1 (27). Rapamycin and other mTORC1 inhibitors have been widely used to induce autophagy, suggesting that autophagy is negatively regulated by mTORC1 (28,29). Autophagy induction and isolation membrane formation are mediated by the autophagy regulatory complexes, the Atg1/ULK protein kinase complex and the hVps34 class III PI3K complex. 1.1.1.1.1 ULK1/2-Atg13-FIP200-Atg101 kinase complex The autophagy effector immediately downstream of mTORC1 is the ULK1/2-Atg13FIP200-Atg101 complex (30–32). ULK1/2 are mammalian orthologs of Atg1, the only  4  Figure 1.2 Regulation of the ULK1/2-Atg13 complex upon autophagy induction by mTORC1 ULK1/2 forms a stable complex with Atg13, FIP200, and Atg101 irrespective of mTORC1 activation. Under nutrient-rich conditions, the active mTORC1 associates with the ULK complex, phosphorylates ULK1/2 and hyperphosphorylates Atg13, thus inhibiting the kinase activity of ULK1/2 and blocking autophagy induction. In the absence of nutrients, mTORC1 inactivation leads to dissociation from the ULK complex, preventing phosphorylation of Atg13 and ULK1/2 by mTORC1. This leads to increased ULK1/2 kinase activity, which phosphorylates Atg13, itself, and FIP200, leading to autophagy induction. Abbreviations: P, phosphorylation; mTOR, mammalian target of rapamycin.  5  protein kinase among Atg proteins (Figure 1.2). In nutrient-replete conditions where mTORC1 is active, mTORC1 constitutively interacts with the stable ULK1/2-Atg13-FIP200Atg101 complex, and phosphorylates ULK1/2 and Atg13 (32–34), thereby inhibiting ULK1/2 kinase activity. Following amino acid withdrawal, ULK1/2 dissociates from mTORC1, resulting in dephosphorylation (activation) of ULK1/2. Activated ULK1/2 phosphorylates Atg13, FIP200, and itself, causing translocation of the entire complex to the isolation membrane (30,31) (Figure 1.2). The role of the ULK1/2 complex at the isolation membrane is unknown, but it is believed to recruit other Atg proteins and regulatory complexes (1). In addition to mTORC1, ULK1/2 is directly phosphorylated by AMPK during glucose deprivation, to induce autophagy (35). The rapid loss of phosphorylation of ULK1/2 after mTORC1 dissociation is mediated by unidentified phosphatases (36,37). The fact that multiple dephosphorylation and kinase reactions mediate ULK1/2-Atg13-FIP200-Atg101 activation and translocation is indicative of complex and multifaceted regulation of starvation-induced autophagy. Notably, unlike knockout mice of essential autophagy genes, Atg5 (38) and Atg7 (39), Ulk1 -/- Ulk2 -/- mice do not display early neonatal lethality, evidence that developmental starvation-induced autophagy does not require ULK1/2. Moreover, cells from these mice were unable to induce autophagy in response to amino acid withdrawal, but did show autophagy in the presence of increasing ammonia concentrations (40). It is therefore likely that starvation-induced autophagy has different molecular requirements and regulation depending on the cellular and physiological context (41).  6  1.1.1.1.2 Class III PI3K complex Phosphatidylinositol 3-phosphate (PI3P) is a phospholipid that recruits proteins to intracellular membranes (42), and is essential for recruitment of key regulatory factors to the autophagosome formation site (1). hVps34 is the class III PI3-kinase (PI3K) responsible for this phosphorylation, and is found in distinct complexes that either mediate autophagy induction, autophagosome maturation, or endocytosis (43–45). During autophagosome induction, hVps34 is complexed with hVps15, Beclin-1, and Atg14L. Atg14L recruits the autophagy-specific hVps34 complex to the ER membrane, where it produces a PI3P-rich ER region (46) to recruit the PI3P effectors, WIPI1/2 and DFCP1 (47–50). The ER subdomain where DFCP1 binds PI3P is termed the omegasome due to its Ω shape. Concurrently WIPI1/2 localizes adjacently to the omegasome, at the presumed isolation membrane (29,49,50). The kinase activity of hVps34 is enhanced by beclin-1, whose availability is highly regulated by transient interactions with a number of cofactors, including AMBRA1, Bif-1, and anti-apoptotic proteins, Bcl-2 or Bcl-X L (1,51). In fact, AMBRA1 anchors the hVps34 complex to the microtubule under nutrient rich conditions, but in starvation-induced autophagy, ULK1 phosphorylates AMBRA1, releasing hVps34, perhaps enabling its translocation to the autophagosome initiation site (52). Additionally, regulation of local PI3P by two phosphatases, MTMR3 and MTMR14, was shown to mediate autophagy induction and autophagosome size, revealing another level of control over autophagy induction (53,54). hVps34 also exists in two other stable complexes with distinct functions in autophagosome maturation (44,55), further highlighting its critical role in autophagy.  7  1.1.1.2 Isolation membrane elongation and autophagosome formation The elongation stage of autophagosome formation is believed to involve membrane incorporation from other organelles, mediated by the protein Atg9; however, this has not been formally proven in mammalian cells. Atg9 is a transmembrane protein that cycles between the expanding autophagosome, the Golgi network, and endosomes. Found primarily in the Golgi network and endosomes under nutrient-rich conditions, Atg9 translocates to the ER during amino acid withdrawal, partially colocalizing with the DFCP1 enriched omegasome (56). It was recently demonstrated that despite dynamically interacting with the expanding autophagosome, Atg9 is not incorporated into the autophagosome membrane, and its association is transient (56). It is currently speculated that Atg9 removes and/or delivers membrane or protein components that mediate autophagosome elongation and maturation but further experimentation is required to clarify its role (1,57,58). 1.1.1.2.1 Ubiquitin-like conjugation systems Two ubiquitin-like conjugation systems directly contribute to membrane elongation and autophagosome maturation: the Atg12 and Atg8/LC3 (lipidation) conjugation systems (Figure 1.3) (59,60). Atg12 is activated by the E1-like enzyme Atg7, transferred to the E2like enzyme Atg10, and conjugated to Atg5 (61,62). Atg12-Atg5 noncovalently interacts with Atg16L to form the Atg12-Atg5-Atg16L multimeric complex, which is recruited to the isolation membrane in a PI3P-dependent manner, but dissociates prior to autophagosome formation (63–66). The second ubiquitin-like conjugation reaction involves Atg8/LC3 conjugation to the phospholipid phosphatidylethanolamine (PE), and is commonly referred to as LC3 lipidation (Figure 1.3). This conjugation reaction is common to all known mammalian Atg8 homologs  8  Figure 1.3 Ubiquitin-like conjugation reactions involved in isolation membrane elongation Two ubiquitin-like conjugation reactions involving core autophagy machinery mediate membrane expansion during autophagosome biogenesis. The first system involves conjugation of Atg12 to Atg5. Atg7, and E1-like enzyme, activates Atg12 and passes it to an E2-like enzyme Atg10. Atg12 then forms a stable conjugate with Atg5, which then binds Atg16L noncovalently. The second conjugation reaction mediates lipidation of LC3. pro-LC3 is cleaved at its c-terminus by Atg4B to LC3-I. LC3-I is activated by Atg7, and transferred to a distinct E2-like enzyme, Atg3. Finally, LC3 is conjugated to phosphatidylethanolamine (PE) to form LC3-II. It is believed that Atg12-Atg5 acts as an E3-ligase in the lipidation of LC3.  9  (67), but LC3 is the best characterized of these proteins, and the only protein to stably associate with autophagosomes (60). LC3 is synthesized as ProLC3, which is cleaved by Atg4B to form LC3-1 with an exposed C-terminal glycine (68). LC3-1 is activated by Atg7, and transferred to the E2 ligase, Atg3, leading to a covalent bond between the carboxylterminal glycine and the amino group of PE (60,69). Lipidated LC3, called LC3-II, is targeted to the elongating isolation membrane, and remains associated with the complete autophagosome until lysosomal fusion, after which Atg4B can delipidate LC3-II facing the cytosol, releasing LC3-1 for recycling (60). The actions of these two ubiquitin-like reactions are tightly coordinated, such that the site of LC3 lipidation is directed by Atg16L, and the Atg12-Atg5 conjugate functions as an E3-like ligase for LC3 lipidation (12,64). The final step of autophagosome biogenesis requires fusion of the tips of the phagophore to generate a complete double-membrane bound vesicle. This step is poorly understood, but requires the GABARAP family of Atg8 homologues. Therefore the ubiquitin-conjugation system for lipidation is required for both the elongation and the completion of autophagosomes. 1.1.1.3 Autophagome-lysosome fusion and degradation Autophagosomes undergo stepwise maturation and lumen acidification through fusion with endocytic vesicles including endosomes, multivesicular bodies, and finally lysosomes (47). These fusion events are mediated in part by the hVps34 complex such that UVRAGhVps34-hVps15-beclin1 positively regulates autophagosome-lysosome fusion, but additional binding of Rubicon to the UVRAG-hVps34 complex antagonizes fusion (44). Other proteins involved in autophagosome fusion events are SNAREs, the ESCRT III complex, LAMP-2, and rab7 (70–75). In the final steps of autophagy lysosomal acidic hydrolases, including cathepsins B, D, and L, degrade the inner autophagosomal membrane and its contents, and  10  the monomeric units are released to the cytoplasm through lysosomal efflux permeases (76– 78). 1.1.2  Physiological roles for autophagy  As a dynamic cellular response to environmental changes and hormones, autophagy plays a critical role in cell differentiation and development. Autophagy-deficient embryos derived from oocyte-specific Atg5-/- mice do not survive embryogenesis because autophagy is upregulated shortly after fertilization to degrade proteins and maternal mRNA, in order to ‘reprogram’ the highly-differentiated oocyte into a highly undifferentiated zygote (79,80). Conventional knockout mice of many autophagy genes including Atg7, Atg16L, and Atg9 survive embryogenesis, but die within one day of birth (81–83). Autophagy is required to withstand this neonatal period of nutrient deprivation caused by the sudden termination of the placental blood supply in order to maintain the amino acid pool, needed to meet high energy demands in neonatal tissue (38). It is also hypothesized that autophagy has tissue-specific roles during this period to mediate cellular remodeling for tissue differentiation, which requires both the elimination and generation cytoplasmic constituents (84). For example, autophagy is required for the removal of mitochondria during erythrocyte and adipocyte maturation (85,86). Protein degradation and organelle turnover are required for the survival of cells, and impairment of these processes can result in cellular abnormalities or cell death, leading to various disease states (87). The ubiquitin-proteasome system (UPS) and autophagy work cooperatively to regulate cytoplasmic quality control where the UPS primarily degrades short-lived proteins and autophagy mediates degradation of long-lived proteins and organelles (88). Neural-specific Atg5-/- or Atg7-/- mice show a loss of neurons and  11  accumulation of ubiquitin-positive protein aggregates despite having a functional proteasome (15,89). Additionally, intracytoplasmic aggregate-prone proteins that cause neurodegenerative diseases, such as mutant forms of huntingtin, are autophagy substrates that accumulate in toxic amounts when autophagy is compromised (90,91). Similarly, dysregulation of autophagy in other tissues has been shown to cause accumulation of unwanted aggregates that contribute to hepatic diseases and muscular myopathies (92). Just as autophagy functions to remove abnormal protein aggregates, it also protects cells against various pathogens including certain bacteria, parasites, and viruses. Xenophagy describes the autophagic removal of extracellular bacteria that invade intracellularly, bacteria and parasites that reside in the cytosol, phagosomes, or other pathogen-containing vacuoles, as well as newly synthesized virions released from the nucleus into the cytoplasm (93,94). One of the most studied forms of xenophagy is the targeted removal of Salmonella enterica through ubiquitination of surface proteins, resulting in recognition and degradation by the autophagic machinery (92). Some pathogens have evolved to evade or exploit the autophagic machinery for their benefit. This has been shown with Mycobacterium tuberculosis (Mtb) and Herpes Simplex Virus-1 (93,95). Mtb, which resides in single-membraned phagosomes within macrophages, manipulates the host cell intracellular membrane trafficking events by secreting a protein phosphatase that prevents phagosome acidification, thus protecting the pathogen from lysosomal degradation (96). Herpes Simplex Virus-1 encodes a protein ICP34.5, which inhibits autophagy both by binding Beclin-1 and by promoting dephosphorylation of eukaryotic initiation factor 2-α, a positive regulator of viral-induced autophagy (95).  12  Autophagy is overwhelmingly considered to be a cytoprotective process by the scientific community, but there is also evidence that autophagy can cause a type of cell death that is distinct from apoptosis and necrosis. This phenomenon was termed Type II Programmed Cell Death and was originally characterized by morphological features of excessive autophagy (97), making it unclear whether death by autophagy was due to a failed cytoprotective response or whether it was a distinct function of autophagy (15,98). Most evidence showing autophagic cell death in mammalian cells was conducted under conditions of deficient apoptosis or pharmacological manipulation, further complicating the matter. However, it was recently shown that hippocampal neural stem cells undergo autophagic cell death in response to insulin withdrawal despite having an intact apoptotic machinery, and Atg7 knockdown in these cells blocked this response (99,100). Therefore, it is likely that autophagy does in fact mediate cell death in circumstances specific to species, tissue, and stimuli (92,98). This phenomenon and its physiological significance are currently the subject of scrutiny and require further experimentation. 1.1.3  Selective autophagy  Although autophagy has long been described as nonselective, recent findings and an increasing body of evidence suggest that substrate recognition is a critical factor for proper autophagic degradation of cellular constituents including mitochondria (mitophagy), microorganisms (xenophagy), and both protein aggregates and cytosolic proteins (1,101). Selective autophagy of the 60S ribosomal subunit (ribophagy) has been described in yeast, but there is currently no evidence of this process occurring in mammals (1). Selective autophagy typically involves ubiquitination of substrates and recruitment of adaptor proteins, which bind ubiquitin and deliver substrates to the core autophagic machinery (12,102,103).  13  The list of adaptor proteins involved in selective autophagy is rapidly expanding, and includes SQSTM1/p62 (reviewed in detail in Section 1.2), NBR1, and NDP52 as well as others (104–106). Mitophagy is the selective degradation of damaged mitochondria mediated by the proteins, Parkin and PINK1. PINK1 recruits and translocates Parkin, an E3-ubiqutin ligase, into the membrane of damaged mitochondria (107,108). Parkin then mediates the ubiquitination of the outer mitochondrial membrane proteins, voltage-dependent anion channel 1 and mitofusins, which recruits p62, causing mitochondrial clustering and incorporation into the autophagosome by binding LC3 and/or GABARAP family members (1,109,110). In p62 knockout mice, Parkin-mediated autophagy is delayed, but still occurs, suggesting there is some functional redundancy between p62 and other adaptor proteins (101,108,111). Ubiquitin-independent mitophagy has also been described, which requires Nix/BNIP3L. This type of mitophagy was first characterized in developing erythrocytes, which require mitochondrial degradation for proper differentiation (112,113). As mentioned previously, xenophagy is another form of selective autophagy. Both p62 and NDP52 have been shown to be recruited to ubiquitinated bacteria, resulting in their delivery to the autophagosome (106,114). p62 was the first described autophagy adaptor protein involved in selective autophagy, and it is the best characterized. In addition to its functions in mitophagy and xenophagy, it is also involved in the degradation of ubiquitinated protein aggregates and ubiquitinated cytosolic proteins. p62 sequesters cytoplasmic long-lived and misfolded proteins by binding poly-ubiquitin and, via oligomerization, forming cytoplasmic aggregates. LC3 binds p62, enabling the sequestration of p62 and its cargo into the autophagosome for lysosomal  14  degradation (104,115). p62 and its role in selective protein degradation are reviewed in detail in Section 1.2. 1.2  p62 SQSTM1/p62 is a multifunctional scaffold protein with diverse cellular functions arising  from its ability to interact with a number of proteins involved in various signaling and regulatory pathways. In addition to its role as a regulator and substrate of autophagy, p62 is involved in nutrient sensing, regulation of oxidative stress, and mitosis, and has been implicated in a number of disease states including protein aggregation diseases of the brain and liver, bone disease, and cancer (101,116). Human p62 is 440 amino acids long with multiple domains (Figure 1.4). In this thesis work, I identified p62 as a target of verteporfin, and explored its potential role in verteporfin-mediated autophagy inhibition. 1.2.1  p62 in autophagy  p62 has an N-terminal Phox/Bem 1p (PB1) domain that governs its dimerization and oligomerization with other PB1 containing proteins including itself, aPKCs, and NBR1 (Figure 1.4) (117–119). As an adaptor protein for selective autophagy, p62 binds ubiquitinated cargo through its C-terminal UBA domain (120) and it binds LC3 via its LC3interacting region (LIR), which is also called the LC3-recognition sequence (LRS). Pankiv et al. (104) first mapped the LIR to a 22-amino acid region spanning aa321-342; a subsequent report by Ichimura et al. (121) identified an 11-amino acid region within the LIR, the LRS, which spans aa334-344, and is necessary for LC3 binding by p62. The LIR/LRS contains a conserved motif of an acidic cluster followed by hydrophobic residues (DDD or DEE and WXXL or WXXV) that is necessary for LC3 binding by p62 (121,122). The crystal structure  15  Figure 1.4 The functional domains of p62 This figure depicts several p62 protein-protein interaction motifs and their location in the p62 protein. The PB1 domain mediates oligomerization and binding to other PB1-containing proteins. RIP1 binds the ZZ domain and Traf6 binds the TB domain, both of which cause NF-κB activation. p62 interacts with raptor between the ZZ domain and the TB domain. The LRS domain mediates interaction with LC3 and GABARAP family members for degradation by autophagy. The KIR domain is involved in stress reduction by binding Keap1, leading to Nrf2 activation, and transcription of antioxidant genes. The UBA domain binds polyubiquitinated proteins targeted for degradation by the proteasome or autophagy.  16  of the LC3-LRS interaction and NMR analysis revealed that the LRS acidic cluster (aa337339) interacts with N-terminal basic residues of LC3, and the LRS Trp-340 and Leu343 insert into two hydrophobic pockets on the ubiquitin-like domain of LC3 (104,122,123). The significance of the LRS was verified by experiments substituting the acidic cluster or the hydrophobic residues to alanine, which disrupted p62-LC3 binding (101,124). Moreover, p62 proteins carrying mutations in the LRS still bind ubiquitin and form p62-positive inclusions, but they are no longer degraded by autophagy (115,121). The C-terminal UBA domain mediates the noncovalent interaction between p62 and ubiquitinated proteins, which it targets for degradation by autophagy or the proteasome. The PB1 domain of p62 can bind the S5A/Rpn10 subunit of the 26S proteasome, and tau protein in neurons is one of many p62-substrates shown to be degraded by the proteasome as well as autophagy (125,126). The UBA domain can bind mono- and poly-ubiquitin, and it has been shown to readily form dimers (127,128). It was proposed by Long et al. (127) that UBA dimerization increases its binding affinity for poly-ubiquitinated over mono-ubiquitinated proteins. However, a recent report by Isogai et al. (128) concluded that UBA dimerization and ubiquitin binding are mutually exclusive. It is therefore likely that UBA dimerization is a regulatory process for its function, and further studies are needed to elucidate this relationship. As previously mentioned, the role of p62 in mediating degradation of ubiquitinated proteins was discovered upon the identification of ubiquitin- and p62-positive cytoplasmic protein inclusion bodies in autophagy-deficient mice (39,129). Interestingly, knocking down p62 in addition to autophagy prevented the formation of inclusion bodies in the same model, suggesting that p62 is required to form ubiquitinated protein inclusions (39). Additionally,  17  Figure 1.5 Selective autophagy of ubiquitinated proteins by p62 Misfolded proteins destined for autophagic degradation are tagged with poly-ubiquitin, which binds p62. p62 self-oligomerization causes p62- and ubiquitin-positive protein aggregates to form in the cytoplasm. Binding of LC3 to p62 via its LRS/LIR targets the aggregates for autophagic degradation by sequestration into the autophagosome.  18  disruption of p62 self-oligomerization through PB1 domain mutation or deletion of the UBA domain prevents the formation of p62- and ubiquitin-positive aggregates, suggesting that ubiquitin binding and p62 self-oligomerization are both required for the formation of cytosolic misfolded protein inclusions (115,121). Therefore, it is likely that ubiquitinated proteins interact with p62, which sequesters p62 into cytoplasmic aggregates that are targeted for degradation by autophagy (Figure 1.5). In this manner, p62 reduces the toxicity associated with dispersed accumulation of soluble ubiquitinated misfolded proteins like mutant huntingtin (101,130,131). Studies showing accumulation of p62- and ubiquitinpositive protein aggregates after knockdown of autophagy in neurons (89,132), hepatocytes (39), pancreatic β-cells (133,134), and both skeletal (135) and cardiac muscle (136) suggest that constitutive autophagy is particularly important for quality control in differentiated, nonproliferating cells. p62 is commonly found in cytoplasmic and nuclear aggregates characteristic of a number of diseases. In diseases where particular mutant proteins cause protein aggregates, like polyglutamine expansion in Huntington’s disease, it is unclear whether p62 is recruited to the aggregates, whether it is required for their formation, or both (115,137). Such protein aggregates often contain additional adaptor proteins, such as NBR1 and ALFY, that appear to function similarly to p62 (71,105,138). Matsumoto et al. (139) recently demonstrated that phosphorylation of p62 at serine 403 (S403) by casein kinase 2 increases its affinity for poly-ubiquitinated chains, thus enhancing their aggregation and autophagy-mediated degradation. In their proposed model, there is a mixed population of S403-phosphorylated and unphosphorylated p62 under normal conditions. Binding of poly-ubiquitinated substrates prevents dephosphorylation, thus  19  increasing the relative amount of S403-phosphorylated-p62, which continues to bind and sequester poly-ubiquitinated into cytoplasmic inclusions, called sequestosomes. The isolation membrane is then recruited to the sequestosome through LC3-binding, and the autophagosome forms around its cargo (139). Whether other kinases can mediate S403 phosphorylation remains unknown. 1.2.2  Other physiological roles of p62  Through its interactions with other proteins, p62 is involved in a number of signaling pathways seemingly unrelated to autophagy. However, since p62 is itself degraded in selective autophagy, its turnover may influence the abundance and lifetime of its other functional complexes (101,116). Furthermore, in addition to the S403 phosphorylation site, two other inducible serine phosphorylation sites were identified in functional domains of p62, but their significance remains to be determined (139). Binding of p62 to ERK1 via its PB1 domain negatively regulates adipogenesis while its PB1-mediated interaction with PKCζ plays a role in NF-κB signaling. In response to cytokine activation, p62 binds either aPKCs (PB1 domain), RIP1 (ZZ domain), or TRAF6 (TB domain), which activates IKK and induces nuclear translocation of the transcription factor NF-κB, necessary for proper osteoclastogenesis (Figure 1.4) (140–142). Pathological K-Ras activation or pathological levels of p62 can lead to conditions of high NF-κB activity and chronic inflammation, which can promote tumourigenesis (140,143,144). This situation is amplified by the fact that NF-κB activates p62 transcription, causing a feed-forward response (143,144). p62 also plays an important role in mediating cellular oxidative stress. p62 binds Keap1 through its KIR domain (Figure 1.4). In the absence of p62, Keap1 binds Nrf2, targeting Nrf2 for degradation via the ubiquitin-proteasome pathway (145). In the presence of oxidative and  20  electrophilic stresses, Keap1 dissociates from Nrf2, likely though oxidation of key cysteine residues in Keap1, stabilizing Nrf2, thus allowing it to translocate into the nucleus where it activates transcription of a number of cytoprotective genes that contain antioxidant response elements, including NQO1 and p62 (145,146). p62 binding to Keap1 sequesters it from Nrf2, thus participating in a feed-forward loop that amplifies and sustains the Keap1-Nrf2 oxidative stress response (147,148). Recent findings implicate p62 as a sensor of amino acid availability in the mTORC1 pathway. p62 specifically associates with mTORC1 through the protein raptor (149). It is believed that p62 binds raptor and Rag GTPases, promoting activation of mTORC1 by mediating Rag dimerization (149). Therefore, in the absence of nutrients, autophagy is stimulated, causing p62 degradation, thus making it unavailable for mTORC1 activation. This provides another level of autophagy regulation in response to nutrient availability. p62 has been shown to shuttle between the cytoplasm and nucleus using its two nuclear localization sequence domains (NLS1 and NLS2) and one nuclear export motif (NES) (Figure 1.4) (150). Notably, inhibition of nuclear export causes significant accumulation of endogenous p62 in the nucleus, demonstrating that p62 subcellular localization is quite dynamic (150). It is believed that p62 mediates proteasomal degradation of nuclear proteins via nuclear shuttling, but this role needs further exploration. p62 participates in a variety of distinct cellular roles, but its primary function appears to be as a mediator of cellular stress through its roles in selective autophagy, NF-KB signalling, and in response to oxidative stress. As a scaffolding protein, p62 is regulated by its oligomerization with itself and other proteins, as well as its degradation by autophagy.  21  Understanding its role in pathological states is only just beginning, and appears to be quite complex considering its multifunctional nature. 1.3  Autophagy and cancer As both a quality control mechanism and an adaptive response to stress, autophagy plays  a critical role in cancer development and disease progression. The prevailing view in the field is that autophagy inhibits tumourigenesis through the elimination of sources of genotoxic stress; yet, conversely, autophagy enables tumour cells to survive under metabolic stress and in response to chemotherapeutic agents. The first study implicating autophagy in cancer demonstrated that beclin-1, an autophagy-promoting gene, was monoallelically deleted in most human sporadic breast, ovarian, and prostate cancers, and that its forced expression in breast carcinoma cells reduced their tumourigenicity in nude mice (151). beclin-1 has since been characterized as a haploinsufficient tumour suppressor gene such that aging beclin-1+/- mice show a high incidence of malignancies including lymphomas, lung cancers, and liver cancers (152,153). Several other tumour suppressor genes have been shown to positively regulate autophagy, including PTEN and LKB1, both negative regulators of mTORC1 (154). Genetic alterations of other autophagy-related genes have been association with tumourigenesis: Atg4C knockout mice show increased susceptibility to carcinogen-induced fibrosarcomas (155) and Atg5 or Atg7 deficient mice develop benign liver tumours (156). The suppression of oncogenesis by autophagy is most likely due to limiting cellular ROS production by removing aberrant organelles and protein aggregates. Mice with autophagy defects accumulate abnormal mitochondria, and tumours formed by autophagy-deficient cells are susceptible to enhanced gene amplification and chromosomal instability, which is due in  22  part to excess cellular ROS (157,158). High levels of p62 in autophagy-deficient tissues also contribute to tumourigenesis by activating NF-κB signalling, which leads to chronic inflammation and tissue damage (159). Three recent publications have proposed that the basal autophagy machinery is required for oncogenic Ras-induced malignant transformation (160–162). Specifically, Guo et al. (160) showed that ectopic expression of mutant H-Ras or K-Ras upregulates autophagy, and that inhibiting autophagic flux decreased the survival of a subset of Ras-activated human cancer cell lines. It has been proposed that Ras activation alters metabolism in a way that requires autophagy for proliferation (161); however, not all Ras-transformed cell types show elevated autophagy levels and not all cancer cells with elevated autophagy show mutated Ras, suggesting there are other factors that contribute to autophagy-dependent Rastransformation (161,163). Finally, Lock et al. (162) observed that Ras-mediated adhesionindependent transformation was attenuated in Atg5-/- MEF cells as was their ability to upregulate glycolysis in glucose-deficient media. In established tumours, autophagy appears to have a protective effect by allowing tumour cells to survive and proliferate in a nutrient-deprived environment. Autophagy is often enhanced in tumour tissue compared to adjacent corresponding noncancerous tissue (164). It has been shown that stimulated autophagy localizes to hypoxic and poorly vascularized regions within tumours, and genetic ablation of autophagy selectively kills these metabolically stressed cells (165). In addition to its role maintaining cellular energy, autophagy may promote tumour cell survival by limiting ROS-induced cell damage through the clearance of damaged organelles and proteins (57,157,166). The dependence on autophagy for tolerating nutrient deprivation is variable among cancer cell types, and is likely  23  influenced by a variety of factors including tissue type, tumour microenvironment, and oncogenic factors (167); however, these studies provide a rationale for inhibiting autophagy as a strategy to selectively target cancer cells (164). The most compelling evidence for targeting autophagy as an anticancer strategy came from studies investigating autophagy in response to chemotherapeutic agents and other currently used anticancer strategies. Multiple cancer therapy agents, such as temozolamide (168), tamoxifen (169), imatinib (170), and ionizing radiation (171) induce autophagy in human cancer cell lines. Ultrastructural analysis of tumour tissues from patients and animals treated with many of the aforementioned agents shows a substantial increase in the number of autophagosomes, suggesting that excessive autophagy induced by these agents elicits autophagic cell death (172,173). An alternative hypothesis for the increased autophagic activity observed in response to cancer therapy is that it constitutes a protective response against drug-induced cellular stress (157,173). This hypothesis was investigated in a number of studies using RNA interference (RNAi) technology to suppress autophagy while exposing cells to autophagy-stimulating cancer agents. RNAi-mediated knockdown of the autophagy gene Atg5 resulted in decreased cell survival in tamoxifen-treated MCF-7 cells (174) and in glioma cells treated with a DNA damaging agent (175) or the tyrosine kinase inhibitor imatinib (170). The first in vivo study confirming this concept used the lysosomotropic inhibitor chloroquine (CQ) to inhibit autophagic degradation, and it enhanced the ability of p53 activation or alkylating agents to induce tumour regression in a myc-induced lymphoma mouse model (176). Despite these encouraging results, there is a hurdle in pursuing autophagy inhibition as an anticancer strategy due to the limitations of available pharmacological inhibitors of autophagy. Selective autophagy inhibition has been achieved  24  using shRNA or siRNA to Atg3, Atg4B, Atg4C, Atg5, Atg7, Atg12, or beclin-1 (174,176– 178), but this approach is not currently suitable for human therapy. Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) have been used extensively to inhibit autophagic degradation by inhibiting lysosomal function, but they do not specifically target autophagy. Interestingly, a recent study by Maycotte et al. (179) showed that CQ sensitized breast cancer cells to death induced by a DNA-damaging agent or an mTOR inhibitor, but sensitization was not mimicked by Atg12 or beclin-1 knockdown. Moreover, sensitization was still achieved using CQ in the absence of Atg12, implying that CQ sensitization of these cells is independent of its modulation of autophagy. By contrast, the same group observed that 4T1 breast cancer cells could not be sensitized to radiation by either CQ or Atg12 knockdown (180). Therefore, the identification of specific autophagy inhibitors has the potential to elucidate the interplay between autophagy and cancer, and to improve anti-cancer therapies. 1.4  Autophagy inhibitors There is a lack of autophagy inhibitors that are both selective and pharmacologically  suitable (157,181–183). Most of the compounds currently employed to inhibit autophagy act at a late stage of the process, after sequestration of material into autophagosomes. Lysosomotropic agents (weak bases that accumulate within lysosomes, CQ and HCQ), VATPase inhibitors (bafilomycins, RTA-203), lysosomal protease inhibitors (pepstatin A, E64d, and zVAD), clomiporamine (184) and lucanthone (185), all interfere with lysosomal function. These agents prevent the degradation of autophagosomes, thus leading to cytoplasmic accumulation of abnormal autophagosomes, which can be toxic to cells (182). To my knowledge, the only previously described early stage inhibitors of autophagy are compounds that inhibit class III PI3-kinases, such as 3-methyladenine (3-MA), wortmannin,  25  and LY294002; however, they do not act selectively on the autophagy process and have pharmacologically undesirable properties (183). 3-MA, the most widely used inhibitor, is known to inhibit a number of other cellular processes, including class I PI3-kinases, and must be used at high millimolar concentrations that are not achievable in vivo. It is also toxic to cells upon prolonged exposure (181,186). Wortmannin is more potent than 3-MA, but it also inhibits many lipid and protein kinases and is toxic in vivo (187). Likewise, LY294002 inhibits a number of other kinases (188). The late stage inhibitors CQ and HCQ are the only autophagy inhibitors being tested in clinical trials because they are already used in humans as anti-malarial agents. One trial for glioblastoma patients treated with irradiation or temozolomide in the presence of HCQ showed enhanced patient survival (189); however, whether this effect was due to autophagy inhibition, autophagosome accumulation, or inhibition of the lysosome is unknown. Another cause for concern when interpreting the outcome of clinical trials using CQ and HCQ is that it is difficult to deliver the amounts of drug to the tumour site in humans that are required to inhibit autophagy in preclinical studies (190). Finally, recent evidence suggests that prolonged inhibition of lysosomal degradation may stimulate the production of more autophagosomes (191). Thus there is a need for selective early autophagy inhibitors that instead prevent autophagosome formation and are suitable for human use to assess the potential contribution of autophagy inhibition to cancer therapy. 1.5  Chemical biology as an approach to study biological processes and to discover novel drugs Chemical biology broadly describes a field of research that utilizes chemical tools to  answer biological questions. Specifically, small molecules can be used both in vitro and in  26  vivo to perturb a particular protein or pathway of interest in order to better understand its function and assess its role in physiological and pathophysiological processes. When the target of interest is associated with a disease phenotype, chemical probes can validate potential therapeutic targets and act as lead candidates for drug discovery (192,193). Chemical biology and molecular biology are complementary techniques that both involve developing and using tools to investigate complex biological processes. While molecular biology uses DNA and RNA technology (e.g. DNA expression, RNA interference) to manipulate gene expression, chemical biology uses small molecules to interfere with target proteins and pathways. Two major advantages to using chemical modulators are experimental control and versatility. Chemicals act rapidly and allow conditional control of the target through both timing and dosage (193,194). In contrast to RNA interference (RNAi), chemicals can be studied in multiple cell types and large-scale experiments with relative ease. Furthermore, a specific function, such as enzymatic activity, of multifunctional proteins can be modulated with chemicals whereas knockdown through RNAi causes complete ablation of the protein and all of its interactions (195). RNAi also has advantages, the most notable being its specificity and relative lack of off-target effects, thus providing concrete evidence of the functional role of a particular protein of interest. Together both approaches are important for a comprehensive understanding of biological processes and the identification and characterization of druggable targets (192,195,196). Chemical biology has been particularly useful for studying cancer and developing more targeted treatments (197). An illustrative example of the power of chemical biology is the small molecule inhibitor rapamycin, which has been used successfully to elucidate the role and regulation of the serine-threonine kinase, mTOR, and has been developed into several  27  analogs and prodrugs for clinical use (198). Rapamycin is a macrocyclic lactone produced by bacteria that was discovered in a screen for antifungal agents in the 1970s (199–201). Over time, rapamycin was observed to have both immunosuppressive and anti-proliferative properties (202), but its mechanism of action was unknown until 1991 when its cellular target was identified in yeast as TOR (target of rapamycin) (203) and then in mammals as mTOR (204–206). Using rapamycin to probe the mTOR pathway, it became clear that mTOR exists in two distinct complexes: mTORC1 (mTOR complex 1), which is rapamycin sensitive and mTORC2 (mTOR complex 2), which is largely rapamycin insensitive (198,201,207). Rapamycin binds FKBP12, and FKBP12-rapamycin only binds to and inhibits the kinase activity of mTORC1. Further research in this area has revealed that mTORC1 and mTORC2 contain several different regulatory components and have distinct cellular targets. Facilitated by work with rapamycin, the biological processes mediated by mTORC1 are well characterized and include mRNA translation, ribosome biogenesis, and autophagy (10,208,209). There is less known about mTORC2, but it has been shown to affect the actin cytoskeleton and cell survival (210–212). Notably, RNAi and genetic techniques would not have revealed these intricacies since knockdown of mTOR would affect all its biological functions and genetic ablation of mTOR or its distinct interactors is embryonically lethal in mice (211,213,214). Finally, to overcome poor bioavailability and low solubility, rapamycin analogs have been developed for clinical use as immunosuppressive (215) and anticancer agents (216–218), further highlighting the potential impact of identifying small molecule modulators of biological processes.  28  1.6  Cell-based high throughput screening Discovering novel small molecule modulators has been of great interest to scientists  throughout history, but progress was limited due to its laborious nature and limited resources. Rapid technological advancements made in the late-1980s to mid-1990s revolutionized the field of chemical biology with the introduction of high-throughput screening (HTS) (219). HTS is an efficient method of experimentation in which large collections of chemicals or natural products are tested for biological activity, ultimately to generate drug discovery candidates in a pharmaceutical setting and/or biological probes for basic research. The emergence of HTS was due in large part to concurrent developments in the fields of genomics and combinatorial chemistry. Progress in genetic sequencing and manipulation revealed a number of potential novel drug targets (220,221) while advancements in combinatorial chemistry led to the production of large collections of purified chemicals available for testing (219,222). The potential of combining these resources drove first pharmaceutical companies and then academic researchers to rapidly develop, automate, and miniaturize biological assays (223). Successful implementation of robotics and automated liquid handling over the years has enabled the reduction of individual reaction volumes while simultaneously scaling up the chemicals screened in each experiment through the use of 96well, 384-well, and 1536-well assay formats. Despite great improvements in the efficiency and speed of primary small molecule screens, the identification of high-quality leads largely depends on the nature and robustness of the biological assay being used (222,224). The two general categories of assays developed for HTS are biochemical and cell-based. Biochemical assays are target-based in vitro assays and have been the dominant methodology in industrial HTS. Due to the uniform nature of in  29  vitro reactions, biochemical assays are relatively easy to scale-up, miniaturize, and automate. Biochemical-based screens also benefit from being direct and target-based, so target validation is not a challenge (219). Successful biochemical-based in vitro screens have been developed to identify modulators of a number of targets including protein kinases (225), ion channels (226), and protein-protein interactions (227). However, despite its success, biochemical-based HTS has significant limitations as well. Since biochemical assays rely on purified proteins and substrates, they are limited by the ability to produce and purify large amounts of the assay reagents. Additionally, many hits from primary biochemical-based screens ultimately fail due to cytotoxicity, off-target effects, solubility issues, or a general inability to show activity in a cellular environment. Cell-based assays overcome some of the drawbacks of biochemical assays by providing a more physiologically relevant experimental system. Screening in a cellular context can reduce the number of hits with pharmacologically undesirable properties including poor solubility and membrane impermeability, cytotoxic agents, and chemicals that are readily metabolized or degraded by the cell. Additionally, it eliminates small molecules that cannot reach their intracellular target, perhaps due to the presence of binding partners. Another value associated with cell-based assays is that instead of targeting a specific protein, the goal of is to discover modulators of a specific biological pathway or cellular response. Therefore, modulators of physiological and pathophysiological phenotypes can be identified, even when the causative molecular targets are unknown (228). Since this approach is not focused on one particular protein, it expands the potential for discovering modulators of different arms of targeted pathways. Therefore, compared to biochemical assays, utilizing cell-based assays in  30  HTS provides the potential for discovering a more diverse collection of chemical modulators with greater biological relevance. Two types of cell-based assays exist: those that produce a single readout, such as overall fluorescence or luminescence, typically measured with a plate reader, or those that produce cell images using fluorescence microscopy. In the early days of HTS, image-based analysis was unsuitable for screening because it was time-intensive and highly subjective. Two significant technologies in the last twenty years enabled the implementation of highthroughput image-based screens: automated microscopes and image analysis software. Automated microscopes capable of a) mechanically positioning and focusing each sample and b) high-quality and rapid detection of a variety of fluorophores using multiple excitation and emission filters, dramatically improved a previously labour-intensive process. The development of image-analysis software enabled the extraction of quantified data from acquired images, making interpretation less subjective and, of course, faster. Once this technology was introduced, rapid progress in software development generated a diverse collection of sophisticated algorithms for analyzing various types of image-based assays. The first system to integrate these technologies with chemical screening platforms was introduced in 1996, and the new methodology was termed high content screening (HCS) (229,230). HCS describes “multifunctional screening based on imaging multiple targets in the physiologic context of intact cells by extraction of multicolor fluorescence information” (229). The ultimate goal of HCS is to identify more specific and less toxic compounds in a primary screen in the hopes of identifying higher quality leads for future validation and development. Where traditional plate-reader assays give one value per well that represents thousands of cells, HCS enables quantified analysis of a number of individual cell features  31  per well. This aspect allows users to primarily monitor and analyze relevant subpopulations of cells, which is particularly useful for assays involving transient transfections or highly variable cell populations (228). Moreover, by imaging multiple cellular parameters, a single experiment can provide quantitated information regarding the effect of a compound on the specific phenotype(s) of interest and correlate it to other cellular properties (222,231). A distinguishing element of HCS compared to traditional biochemical-based HTS is that images from primary screens are stored and remain available for further quantitative and qualitative analysis, such as number, size, and spatial distribution of objects. Therefore, HCS provides more flexibility in the type of assay it supports and the interpretable data it generates. In fact, this strength of HCS is also its greatest limitation since it requires massive amounts of virtual storage, and for the vast majority of chemicals screened, far more data is generated than will realistically be used. Since the development of HCS platforms, several general strategies for small molecule image-based screening have emerged that highlight its capabilities. The most versatile assay utilized in HCS is the translocation assay, and it is the basis for most published HCS screens (229). In general, translocation assays measure changes in cellular distribution and accumulation of fluorescent biological markers. This assay was successfully used to identify PI3-kinase inhibitors by screening for compounds that prevent insulin-induced translocation of AKT-EGFP from the cytoplasm to the cell membrane (232). Moreover, translocation assays are particularly relevant for disease-related phenotypic screens monitoring the improper localization or aggregation of biological components. Other common HCS approaches include, but are not limited to, cell cycle analysis (233), neurite outgrowth (234), and cell motility (235).  32  In order to conduct a successful cell-based screen, there are four necessary components: experimental design, high-throughput assay development, screen execution, and target validation. Regardless of the inherent advantages of HCS, the quality of the screen’s data is critically dependent on the development of a robust biological assay to probe the target or phenotype of interest. This process typically involves developing and optimizing a standard microscopy assay where the localization or amount of a fluorescent protein or marker responds to a biologically relevant stimulus. It is therefore important to choose an appropriate biological system. Most high-content screens use either primary cells or a native or engineered cell line, but more recently model organisms have also been used including yeast (236) and nematodes (237). This selection depends on whether the system is representative of the biological process of interest in addition to whether it is compatible with the assay approach (reporter, functional, or phenotypic). Biological assay development can take some time as it typically entails optimization of immunostaining, proper tagging and expression of the protein(s) of interest, or engineering a cell line to produce a representative phenotypic response (222). Once a robust assay has been developed, it must be optimized for high-throughput screening. Considerations for optimization include, but are not limited to titration of cell densities and other reagents, stability of the reagents, incubation times, and concentration of screening compounds (219). These factors determine the degree to which a particular assay can be scaled up and miniaturized without compromising quality. A widely-accepted measurement to assess the robustness of a screen is the Z’-factor (219,228). By comparing the means and standard deviations of values of positive and negative controls, Z’-factor indicates whether there is a sufficient screening window to confidently identify hits (238).  33  Selecting an appropriate chemical library for screening is another determinant for a successful screen. Since HCS is particularly useful for studying processes with unknown targets, diverse chemical libraries with good bioavailability are needed (239). As mentioned previously, the capabilities for multivariate analysis in HCS can be overwhelming; therefore, in primary high-content screens, it is important to base analysis on a manageable number of parameters. In a robust assay, it will be relatively obvious which screened compounds are of interest, and the raw data from the primary screen can be reanalyzed with secondary criteria to determine exclude chemicals with unintended cellular effects (240,241). When screening for small molecule probes of a particular pathway, the final and most challenging step is target validation. There is no broadly-applicable method for finding the target of a bioactive small molecule. If the compound retains biological activity after chemical modification, affinity-based approaches, such as immobilizing the compound to pull-down its target are commonly used (219). More recently, chemically-based approaches, namely click chemistry, have been successfully used to isolate the active compound bound to its target, but this also requires successful chemical modification of the bioactive compound (242), which is often not possible. Proteomic approaches have also been successful, but require a lot of material, and likely only yield a defined target if the binding is very strong and selective (243). Finally, as mentioned in Section 1.1, knowledge from chemical screens can be combined with that from RNAi screens for inference and systemic validation of phenotype-function relationships. The identification of bioactive small molecules as biological probes and potential drugs is an exciting discovery-based approach for understanding complex biological and diseaserelated processes. Like forward genetics, where systemic genetic manipulation identifies  34  genes responsible for certain phenotypes, forward chemical genetics uses high-content screening to identify small molecules inducers of a desired phenotype. Follow-up comprehensive target-driven characterization studies can uncover previously unknown biological functions, and in some cases, reveal novel targets for therapeutic intervention. 1.7  Objectives  1.7.1  Development of a high content screen to identify novel small molecule inhibitors of autophagy  At the time of beginning the work described in this thesis, the only pharmacological autophagy inhibitors known were lysosomotropic agents and class III PI3-kinase inhibitors, neither of which selectively inhibit autophagy (173,183). Identification of LC3-II as a protein stably associated with autophagosomes provided a tool for tracking autophagosome formation by tagging it to EGFP (68). Under conditions of low autophagy, EGFP-LC3 is localized diffusely throughout the cytoplasm; autophagy induction promotes EGFP-LC3 lipidation and the insertion of EGFP-LC3-II into autophagosomal membranes generates punctate EGFP-LC3-II fluorescence. Since autophagy is regulated by a number of signal transduction pathways linked to a variety of intracellular and extracellular environmental cues rather than one particular protein, and it can be monitored phenotypically, it was suitable for high-content screening. Therefore, I aimed to identify inhibitors of autophagosome formation by designing and carrying out a high-content screen. 1.7.2  Investigate the mechanism of action of novel autophagy inhibitors  Using biochemical and cell biology assays, I aimed to characterize how inhibitors identified in Objective 1 prevent autophagosome formation at a molecular level. Insight into  35  mechanism of action may reveal novel factors involved in autophagosome formation or clarify the role of known autophagy components. 1.7.3  Determine if autophagy inhibition is a promising therapeutic strategy for cancer therapy  The identification of an inhibitor of autophagosome formation may help elucidate the role of autophagy in cancer progression. I aimed to evaluate the potential impact of using an inhibitor of autophagosome formation both on its own and in combination with current anticancer drugs both in vitro and in vivo using tumour xenograft mouse models.  36  CHAPTER 2: MATERIALS AND METHODS  2.1  Cell lines The breast cancer cell lines MCF-7 and MCF-7 stably expressing EGFP-LC3 were  maintained in RPMI 1640 (Sigma-Aldrich, #R8758) supplemented with 1 mM Hepes, pH 7.4 (Gibco, #15630), as were the pancreatic cancer cell lines BxPC-3 and SU86.86 (gifts from Dr. Sylvia Ng, BC Cancer Agency). EGFP-LC3-expressing cells (28) were supplemented with 400 μg/ml G418 (Sigma-Aldrich, Gibco). Capan 2, HS766T, and Panc-1 cells (gifts from Dr. Sylvia Ng) were maintained in DMEM (Sigma-Aldrich, #D6429) as were MIA PaCa-2 cells purchased from the American Type Culture Collection (ATCC). Capan-1 and CFPAC-1 cells (gifts from Dr. Sylvia Ng) were maintained in IMDM (Gibco, #12440-053). All cell culture media were supplemented with 10% (v/v) fetal bovine serum (Gibco, SigmaAldrich, PAA Laboratories), 100 units/ml penicillin and 100 µg/mL streptomycin (Gibco), and cells were maintained 37oC in 5% CO 2 . BxPC-3 and SU86.86 cells used for the animal studies were purchased from ATCC and tested negative for mycoplasma contamination. Cells were grown in RPMI 1640 supplemented with 2 mM L-glutamine (Gibco) and 10% FBS without antibiotics. Cells were sub-cultured once a week and cell cultures with passage number 3-10 were used for in vivo studies. 2.2  Chemicals Verteporfin was purchased from Prestwick Chemical or from US Pharmacopeia.  Chloroquine, 3-MA, wortmannin, and MG132 were from Sigma-Aldrich. Bafilomycin A1  37  was purchased from LC Laboratories and rapamycin from Calbiochem. Valine and histidine were purchased from BioShop Canada Inc. All screening chemicals came from a collection of >30,000 drugs and pharmacologically active compounds that comprise the Canadian Chemical Biology Network (CCBN) collection in the Roberge Laboratory. A searchable database is available in the laboratory. 2.3  Screening assay for inhibitors of autophagosome formation MCF-7 cells stably expressing EGFP-LC3 were seeded in PerkinElmer View 96-well  plates at 20,000 per well. Eighteen hours after seeding, chemicals from the Prestwick, Sigma LOPAC, Microsource Spectrum and Biomol natural products collections were added to plates at ~10 μM using a Biorobotics Biogrid II robot equipped with a 0.4 mm diameter 96pin tool. Chloroquine (30 μM) was added immediately after to all but negative control wells. Plates were incubated for 4 h at 37°C and then cells were fixed with 3.7% paraformaldehyde (Invitrogen) in PBS for 15 min at room temperature. Nuclei were simultaneously stained with 500 ng/ml Hoechst 33342 (Invitrogen). Fixative and stain were removed and replaced with PBS for storage and imaging. Punctate EGFP-LC3 was determined quantitatively using a Cellomics Arrayscan VTI automated fluorescence imager and the Compartmental Analysis Bioapplication as described in detail previously (28). Briefly, cells were photographed using a 20x objective in the Hoechst and GFP channels (XF-100 filter) and the compartmental analysis algorithm was used to identify the nuclei, apply a cytoplasmic mask, and to quantitate GFP spots above a fixed threshold. Cells were gated such that only those with an average GFP fluorescence intensity of 10 and above were analyzed. Spots were distinguished from the background using a spot kernel radius parameter of 6 and a fixed threshold set at 500 pixel intensity units. The punctate fluorescence indicates the total intensity of all pixels  38  inside the spot within the cytoplasmic mask. Analysis of 1,000 cells per well resulted in a Z’ factor of 0.54. Images were examined to exclude toxic compounds. 2.4  Starvation procedures For starvation experiments, cells were washed twice with DPBS (Invitrogen, #14040)  before incubation in the denoted starvation media. Serum starvation was carried out by incubating cells in the previously described growth medium lacking FBS. Glucose starvation was carried out by incubating cells in DPBS supplemented with amino acids from a 10X mixture (244) and 10% FBS. Amino acid starvation was carried out in DPBS containing 11 mM glucose (Fisher Scientific), and DPBS was used for serum, glucose, and amino acid starvation. Earl’s Balanced Salt Solution (EBSS) was purchased from Sigma-Aldrich. 2.5  Immunofluorescence microscopy In experiments showing EGFP-LC3 fluorescence microscopy, MCF-7 cells stably  expressing EGFP-LC3 were cultured on 7x detergent-treated glass coverslips in 6-well plates at 600,000 cells/ml. Eighteen hours after plating, drugs were added for 4 h. The cells were fixed with 3% paraformaldehyde in PBS and stained with 500 ng/ml Hoechst 33342. The coverslips were mounted in DABCO or CelVol mounting medium and viewed using the 60X objective of an Olympus Fluoview FV1000 laser scanning microscope equipped with Olympus-selected Hamamatsu photomultiplier tubes. Images were analyzed using Olympus FV10-ASW1.7 software. In p62 immunofluorescence experiments, MCF-7 EGFP-LC3 cells were seeded on 7x detergent-treated glass coverslips in 12-well plates at 300,000 cells/well in normal culture medium. The next day, the cells were treated as indicated. At the end of the incubation period, cells were washed once with PBS and fixed and permeabilized in 100% methanol  39  (Sigma-Aldrich) at -20°C for 6 min. After multiple PBS washes to remove methanol, cells were blocked in 1% bovine serum albumin (BSA) in PBS for 10 min. The cells were incubated with mouse α-p62 antibody (1:250, Santa Cruz Biotechnology sc-28359) in 3% BSA in PBS for 45 min and then washed twice with PBS. The cells were then incubated in goat α-mouse AlexaFluor 568 secondary antibody (1:1000, Molecular Probes) in 3% BSA in PBS for 45 min and washed twice with PBS. DNA was stained with Hoechst 33342 at 500 ng/ml for 5 min, and washed twice more with PBS. Coverslips were mounted on glass slides using CelVol, and images were acquired by confocal microscopy. 2.6  SDS-PAGE and immunoblotting Cultured cells were washed in PBS and lysed on ice in lysis buffer containing 20 mM  Tris-HCl (Fisher Scientific), pH 7.5, 150 mM NaCl (Fisher Scientific), 1% Triton X-100 (LabChem, Inc.), 1 mM EDTA (Sigma-Aldrich), 1 mM EGTA (Sigma-Aldrich), 2.5 mM sodium pyrophosphate (Fisher Scientific), 1 mM β-glycerophosphate (Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), and 1x cOmplete protease inhibitor cocktail (Roche Molecular Biochemicals). Lysates were centrifuged at 18,000 x g at 4°C, and the supernatant was collected and quantified by Bradford assay (Bio-Rad). To prepare tumour tissue, sections were washed in PBS and chopped finely with a scalpel on ice. Tumour tissue was then homogenized in a 10X volume of cold lysis buffer and centrifuged 18,000 x g at 4°C, and the supernatant was collected and quantified. Lysates were normalized for protein content in SDS-PAGE sample buffer containing 50 mM Tris-HCl, pH 6.8, 2% SDS (Fisher Scientific), 0.1% bromophenol blue (Sigma-Aldrich), 10% glycerol (Sigma-Aldrich), and 25 mM DTT (Sigma-Aldrich). Samples were resolved by SDS-PAGE and electroblotted onto a nitrocellulose (Bio-Rad) or polyvinylidene fluoride membrane (Millipore Immobilon-P) and  40  blocked in 5% (w/v) non-fat milk (Nestle) in TBS containing 0.1% Tween-20 (MP Biomedical) (TBS-T) for 30 min. Membranes were incubated overnight at 4°C in the appropriate antibody diluted in 5% low-fat milk powder (Nestle Carnation) in TBS-T unless otherwise noted. Membranes were then washed 3 x 10 min with TBS-T and incubated for 1 h at room temperature in the secondary antibody diluted in 5% milk in TBS-T. Following another 3 x 10 min washes in TBS-T, membranes were imaged by chemiluminescence (Millipore Immobilon Western). Deviations from this general procedure follow. LC3 lipidation was assessed by resolving 70-100 μg of protein on a 16% polyacrylamide gel. Electroblotting to PVDF was immediately followed by cross-linking using 0.2% (v/v) glutaraldehyde (Sigma-Aldrich) in PBS+0.02% (v/v) Tween-20 for 30 min and then blocking in 5% milk. Primary LC3 antibody was diluted in 5% BSA in TBS-T. For immunodetection of polyubiquitin, nitrocellulose membranes were boiled for 10 min following protein transfer and before the blocking step. For immunodetection of oxidized proteins, the Oxyblot Immunodetection Kit (Millipore) was used according to the manufacturer’s instructions. Briefly, proteins samples were reduced with 50 mM DTT, denatured with SDS, and derivatized with DNPH for 15 min at RT when neutralization buffer was added to stop the reaction. The proteins were resolved on a 10% polyacrylamide gel and electroblotted to nitrocellulose membrane. Blocking was carried out in 1% BSA in PBS-T for 1 h. Provided primary α-DNP and secondary antibodies were diluted in 1% BSA in PBS-T at 1:150 and 1:300, respectively. Membranes were imaged as described previously. Primary antibodies used were mouse α-GFP (1:7000, Roche Molecular Biochemical 11814460001), mouse α-SQSTM1/p62 (1:250, Santa Cruz Biotechnology, Inc sc-28359),  41  mouse α-LC3 (1:1000, NanoTools 0260), mouse α-polyubiquitin (1:1000, Santa Cruz Biotechnology, Inc sc-53509), rabbit α-β-tubulin (1:20000, Santa Cruz Biotechnology, Inc sc-9104), and mouse α-transferrin receptor (1:1000, Zymed Laboratories, Inc 13-6800). The following secondary antibodies used were HRP conjugated goat α-mouse IgG, Light Chain Specific (1:10000, Jackson ImmunoResearch 115-035-174), HRP conjugated goat α-mouse IgG (1:10000, ThermoScientific 31430), and HRP conjugated goat α-rabbit IgG (1:10000, KPL 074-1506). 2.7  Subcellular fractionation MCF-7 cells were treated as described for 4 h at 37°C. After treatment, cells were  washed once with cold PBS, and collected in subcellular fractionation buffer (250 mM sucrose (Fisher Scientific), 20 mM hepes (pH 7.4), 10 mM KCl (Sigma-Aldrich), 1.5 mM MgCl 2 , 1 mM EDTA, 1mM EGTA). The lysate was passed through a 26G1/2 needle 10 times and incubated on ice for 20 min. The nuclear pellet was collected by centrifugation at 500 x g for 10 min. The supernatant was ultracentrifuged at 100,000 x g to obtain a cytosolic supernatant and a pellet containing total membrane (68). The pellet was resuspended in subcellular fractionation buffer containing 1% Triton X-100 and 10% glycerol. Identical amounts of the supernatant and the membrane fractions (~30-50 μg of protein), respectively, were resolved on a 16% polyacrylamide gel and immunoblotted as described above. 2.8  Long-lived protein degradation assay MCF-7 EGFP-LC3 cells were seeded in a 6-well plate at 1,000,000 cells per well and  grown overnight. Cells were incubated for 24 h at 37°C with 0.2 µCi/mL of L-[14C]valine (Perkin Elmer, #NEC291) in RPMI 1640 supplemented as described above. After radiolabelling, cells were washed three times with PBS, and incubated in complete RPMI  42  supplemented with 10 mM cold valine for 2 h. After this cold chase period during which short-lived proteins were degraded, the cells were treated for 6 h as described in either complete RPMI or EBSS, both supplemented with 10mM cold valine. Protein degradation was measured as described previously (245). 2.9  FITC-dextran sequestration assay FITC-dextran was scrape-loaded into MCF-7 cells as described (246), except cells were  suspended in medium containing 0.1% DMSO, 10 mM 3-methyladenine, or 10 μM verteporfin after scrape loading. The suspended cells were plated on coverslips for 2 h or 24 h before fixation with 3% paraformaldehyde in PBS and DNA staining with Hoechst 33342. Slides were then analyzed using confocal microscopy. 2.10  Benzoporphyrin derivatives The ring A and B benzoporphyrin derivatives were synthesized using standard  techniques (247–249). All derivatives were characterized using 1H NMR and elemental analysis. The verteporfin regioisomers were separated by reversed phase HPLC. Using a 250 × 10 mm Phenomenex Synergi 4µ MAX-RP 80A C-12 column and a mobile phase of 95 % MeOH/0.01 M ammonium formate, verteporfin isomer A eluted at 11.3 min and verteporfin isomer B eluted at 11.9 min. 2.11  Electron microscopy MCF-7 EGFP-LC3 cells were seeded onto BD 24-well cell culture inserts with PET  track-etched membrane at 30,000 cells per well. Thirty-six hours after seeding, cells were treated as described. Medium in transwells was replaced with fixative (1.5% paraformaldehyde, 1.5% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.3) at room temperature. After 30 min, membranes were cut from the transwells and immersed for an  43  additional 2.5-3 h in fixative. The fixative was replaced with buffer (0.1 M sodium cacodylate, pH 7.3) and left overnight at room temperature. The next morning, the membranes were washed twice (10 min each wash) with buffer and then post-fixed for 1 h on ice in 1% OsO 4 in 0.1 M sodium cacodylate (pH 7.3). Membranes were washed three times for 10 min each at room temperature with dH 2 O, stained en bloc with 1% aqueous uranyl acetate for 1 h, washed three times with dH 2 O, and then dehydrated through an ascending concentration series of ETOH. The membranes were treated three times for 10 min in 100% ETOH, twice with 100% propylene oxide (15 min each), and then infiltrated overnight in 1:1 propylene oxide:EMbed-812 (Electron Microscope Sciences, Hatfield, PA). After two changes of EMbed–812 (2 h each), the membranes were embedded and the resin polymerized for 24 h at 60oC. Thin sections were stained with uranyl acetate and lead citrate and photographed on a Philips 300 electron microscope operated at 60 kV. 2.12  Cell viability and proliferation assays To assess cell viability and proliferation in transient starvation assays, MCF-7 cells were  seeded at 8,000 per well in 96-well plates and grown overnight. Cells were washed twice with DPBS and treated as described. The drugs were then removed, and cells were grown in complete medium for 48 h. Cell viability and proliferation were measured by 3-[4,5Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay (250). To monitor cell proliferation of various PDAC cell lines during long-term verteporfin treatment, cells were seeded at 500 - 1000 cells per well in 96-well PerkinElmer View plates and grown overnight. The following day (t=0), four plates were identically treated as indicated for 1-7 days. At t = 1 d, 3 d, 5 d, and 7 d, the appropriate plate was fixed and stained as described previously (see Section 2.3), and analyzed using the Cellomics  44  Arrayscan VTI. Cells were identified and quantified by their nuclear stain using the Target Activation bioapplication, and fifteen fields were analyzed per well. 2.13  Recombinant p62 assays Purified recombinant GST-p62 (H00008878-P01) was purchased from Abnova and  purified recombinant His-p62 (NBP-44490) from Novus Biologicals. The buffer used for all recombinant p62 reactions was 50 mM Tris-HCl, 150 mM NaCl, and 10 mM glutathione, pH 8.0. For immunoblot analysis, 33 nM protein was treated as described for 1-4 h at 37°C in the dark. Reactions were stopped by the addition of 4X SDS-PAGE sample buffer and reactions were analyzed (Section 2.6). 2.14  Immunoprecipitation Cells were grown to 80% confluency in 10-cm tissue culture dishes and treated as  indicated. After treatment, lysates were prepared and quantified exactly as described (see Section 2.6 immunoblotting section). Cell lysates were incubated with mouse αSQSTM1/p62 or mouse α-GFP for 4 h at 4°C, followed by overnight incubation with Protein-G-agarose beads (Roche Molecular Biochemicals). Immunoprecipitates were washed 3 x 20 min in lysis buffer at 4°C. Samples were eluted by boiling beads in 2X SDS-PAGE sample buffer containing 50mM dithioreitol (Sigma-Aldrich) for 10 min, and present proteins were detected by western blotting (Section 2.6). 2.15  Formulation of verteporfin for animal studies – mPEG2000-DSPE micelles 200 mg verteporfin was dissolved in 2 ml DMSO. 1500 mg mPEG2000-DSPE was  dissolved in 50 ml PBS at pH 7.4 using bath sonication. The DMSO solution of verteporfin was slowly added to mPEG2000-DSPE solution while stirring. Stirring was continued for 1 h at 23°C followed by dialysis overnight against PBS using Spectra/Por dialysis membrane  45  (15,000 MWCO, Spectrum Laboratories Inc). The concentration of verteporfin was measured in triplicates and quantified against an external standard curve using UPLC (Waters® ACQUITY®) equipped with PDA detector. Separations were done using a C18 column (Waters® BEH; column size 50 x 2.1 mm, particle size 1.7 µm) and a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile:methanol (1:1; B) [10-80% B over 2 min at 0.5 ml/min flow rate]. The concentration of verteporfinPEG2000-DSPE micelles was adjusted to 2.25 mg/ml followed by filter-sterilization and sterile vialing. The concentration was reconfirmed before proceeding with the animal studies. Stability of the formulation was assessed using UPLC for both regioisomers and also by optical microscopy (Olympus) for any micro particles or crystals. 2.16  Animal husbandry Animal studies were approved by the University of British Columbia Animal Care  Committee and were performed in accordance with the Canadian Council on Animal Care guidelines (protocol #A10-0171). Mice were housed under standard conditions and had access to sterile food and water ad libitum. Mice were handled aseptically and cages were changed every 10-14 days. Animals were closely monitored post administration of test articles and were monitored twice daily for morbidity. Weights were recorded 3 times per week or more if deemed necessary during the pre-treatment and treatment periods. These data were recorded on a comprehensive health monitoring standard operating procedure which included assessments of body weight loss, change in appetite, changes in stool consistency as well as behavioural changes such as altered gait, lethargy and gross manifestations of stress. Animals were euthanized by CO 2 asphyxiation if signs of severe toxicity or tumour-related illness were observed or when the tumour reached a maximum  46  weight of 800 mg , an approved humane end point of the study. Macroscopic necropsy was performed to assess signs of toxicity. Liver, gall bladder, spleen, lung, kidney, heart, intestine, lymph nodes and bladder were examined during necropsy and any unusual findings were noted. 2.17  Efficacy studies of verteporfin Female, Rag2M mice (20-25g; n=8) were inoculated subcutaneously into the centre of  the lower back with 5 x 106 BxPC-3 cells (1:1 RPMI:matrigel; 100 µL volume; expressed as day 0). Tumours appeared within three weeks of implantation. Tumour growth was monitored by measuring tumour dimensions with digital calipers once the tumours were palpable. When the tumours reached a size of 100-150 mg, mice were randomized in groups of eight animals based on tumour weight such that average tumour weights of the animals remain the same between various groups and the treatment was initiated. Tumour volumes were calculated according to the equation (length X width2)/2 with the length (mm) being the longer axis of the tumour. Measured dimensions were converted to tumour weight in milligrams (1 mm3 = 1 mg). Animals were dosed i.p. Monday, Wednesday, Friday for 4 weeks with 45 mg/kg verteporfin under low light conditions (injection volume 400 µl/20 g mouse). Gemcitabine was administered i.p. once weekly for 4 weeks at dosages of 120 mg/kg or 240 mg/kg (injection volume 200 µl/20 g mouse). Groups treated with both verteporfin and gemcitabine received gemcitabine every Monday 6 h after verteporfin administration in order to have maximum verteporfin levels in the tumor (reached 8 h postadministration) coincide with maximum gemcitabine tumor levels (reached 2 h postadministration). Mice were injected with the required volume to administer the prescribed dose (mg/kg) to the animals based on individual mouse weights obtained the day of injection.  47  Following verteporfin administration, animals were housed in dark conditions until the next day morning and then exposed to normal light conditions. Animals in the control group were treated with the delivery vehicle DSPE-mPEG2000 at the same concentration and the same dosing schedule as the verteporfin DSPE-mPEG2000. Appropriate care was taken not to expose the animals to bright light conditions after verteporfin administration as verteporfin is a photosensitizer and exposing to bright light could be harmful for the animals. A One-Way ANOVA with Tukey’s multiple comparison test was used to compare differences in tumor growth (GraphPad Prism Version 6.00). 2.18  Pharmacokinetic studies of verteporfin DSPE-mPEG micelles in BxPC-3 tumour-bearing mice  Rag2-M mice (20-25g; n=3) were inoculated subcutaneously with 5 x 106 BxPC-3 cells. Mice were injected with verteporfin 45 mg/kg i.p. when tumours were approximately 200250 mg. Mice were euthanized by CO 2 inhalation, and blood and tumour were collected at 2, 8, 16, and 24 h post administration of verteporfin. Tumours were excised, rinsed in PBS, and were placed into separate pre-labeled, pre-weighed cryovials. Cryovials were snap-frozen in liquid nitrogen and kept frozen at -80°C until analysis. Tumours were sectioned while frozen. One half of each tumour was used for determining verteporfin concentration by ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) and the other half was used for immunoblot analysis. 2.19  Measurement of verteporfin concentration in plasma and tumour tissue Tumour samples were analyzed for verteporfin concentration by UPLC-MS/MS.  Samples were thawed for approximately 15 min at room temperature, homogenized and then extracted with acetonitrile containing 0.1% formic acid. Protein precipitation and filtration  48  was carried out using ISOLUTE® PPT+ protein precipitation plates (Biotage). Samples were analyzed using a Waters® ACQUITY® UPLC system with mass spectrometry detection. Separations were performed using an isocratic method where mobile phase A was 0.1% formic acid in water and B was 0.1% formic acid in acetonitrile (70% B for 2.5 min followed by 95% B for the wash). Verteporfin regioisomer A was eluted at 1.77 min and regioisomer B was eluted at 2.16 min with a total run time of 4.5 min per sample. The MS/MS system was operated with an ESI interface in a positive ionization mode. Quantification was performed using multiple reactions monitoring (MRM) mode with a precursor mass m/z of 719.27 and product mass m/z of 645.36. The levels of verteporfin in the samples were measured against external calibration standards prepared using the same process as samples.  49  CHAPTER 3: IDENTIFICATION AND CHARACTERIZATION OF VERTEPORFIN AS AN INHIBITOR OF AUTOPHAGOSOME FORMATION  3.1  Synopsis The goal of this work was to discover novel small molecule inhibitors of autophagy. The  first step towards this goal was to design and carry out a high content screen to identify inhibitors of autophagosome accumulation. 3800 pure chemicals were screened in a cellbased automated fluorescence microscopy assay using MCF-7 breast cancer cells. Verteporfin, an approved drug, was found to prevent autophagosome accumulation and was chosen for further study. In this chapter, I describe the effects of verteporfin on both drug- and starvation-induced autophagy. Specifically, verteporfin inhibited autophagic degradation and sequestration of cytoplasmic material into autophagosomes, but it did not prevent LC3 lipidation or membrane association. Transient exposure to verteporfin in starvation conditions reduced cell viability whereas cells in nutrient-rich medium were unaffected by drug treatment. Additionally, analysis of 16 structural analogs indicated that the activity of verteporfin requires the presence of a substituted cyclohexadiene at ring A of the porphyrin core but that it can tolerate a number of large substituents at rings C and D. 3.2  Screen for chemical inhibitors of autophagosome accumulation To identify chemical inhibitors of autophagy, I used MCF-7 breast cancer cells stably  expressing LC3 tagged to EGFP at its N-terminus (MCF-7 EGFP-LC3). LC3 is a cytosolic protein that is recruited to autophagosomes via cleavage of its C-terminus to expose a glycine 50  residue which is then conjugated to phosphatidylethanolamine, enabling its insertion into autophagosomal membranes (68). In nutrient-rich conditions associated with low levels of autophagy, EGFP-LC3 is primarily distributed diffusely throughout the cytoplasm with a few EGFP-LC3 spots, indicative of basal autophagy. Stimulation of autophagy by exposure to medium lacking serum or to the mTORC1 inhibitor rapamycin increases autophagosome formation and punctate EGFP-LC3 fluorescence intensity accordingly. However, the rate of autophagic degradation also increases and autophagosome-associated EGFL-LC3 is degraded (Figure 1.1), resulting in a modest increase in EGFP-LC3 punctate fluorescence that is not sufficiently high to identify inhibitors in a screening assay. I overcame this problem by using chloroquine (CQ), a lysosomotropic agent that neutralizes the acidic pH of lysosomes (251), rendering acidic hydrolases inactive, thereby preventing autophagic protein degradation and causing autophagosome accumulation (Figure 3.1) (252–254). Since CQ prevents autophagic degradation, it causes significant autophagosome accumulation and a corresponding redistribution of EGFP-LC3 from the cytoplasm to autophagosome membranes. Incubation of MCF-7 EGFP-LC3 cells with 30 µM CQ caused a large increase in punctate EGFP-LC3 compared to control cells where EGFPLC3 was predominantly localized diffusely throughout the cytoplasm (Figure 3.2A). 3methyladenine (3-MA), a PI3-kinase inhibitor, is known to prevent autophagosome formation efficaciously but with low potency and selectivity (181). As expected, 10 mM 3MA considerably reduced the ability of CQ to induce autophagosome accumulation (Figure 3.2A). I wished to search for chemicals that, like 3-MA, inhibit autophagosome formation. Cellular autophagosomal content was detected quantitatively by automated fluorescence  51  52  Figure 3.1 Cell-based assay for the identification of inhibitors of autophagosome accumulation and EGFP-LC3 processing confirmation assay. (A) Chloroquine (CQ) causes autophagosome accumulation by raising the pH of the lysosome, thus inhibiting acidic hydrolases responsible for autophagic degradation. (B) MCF-7 EGFP-LC3 cells were treated with CQ and screening chemicals for 4 h. Any chemical that inhibited punctate EGFP-LC3 accumulation was considered active and tested further. (C) Upon autophagy induction, EGFP-LC3I is lipidated to EGFP-LC3II, which inserts into the autophagosome membrane. Upon lysosomal fusion, acidic hydrolases degrade the autophagosome and its contents including EGFP-LC3II. The EGFP moiety is resistant to proteolysis and transiently accumulates as a marker of autophagic degradation.  53  microscopy in 96-well plates (28). Chloroquine caused a 5-10-fold increase in punctate EGFP-LC3 fluorescence over that of untreated control cells. Cells were exposed for 4 h to CQ and chemicals from a collection of 3,584 drugs and pharmacological agents at a concentration of 10 μM (Figure 3.1). Compounds were designated as active if they decreased CQ-induced punctate fluorescence by ≥50% and induced less than 20% cell death during a 4 h incubation period. Verteporfin was the only active compound identified in this screen. It showed concentration-dependent inhibition of CQ-induced punctate EGFP-LC3 with an IC 50 of 1 μM, and reducing punctate fluorescence below that of DMSO controls at ≥ 15 μM (Figure 3.2B). Verteporfin is a benzoporphyrin derivative used clinically for photodynamic therapy (PDT) of age-related macular degeneration. Red light irradiation, in the presence of oxygen, causes the rapid generation of large amounts of singlet oxygen, eliciting oxidative damage that nonselectively kills cells exposed to verteporfin (255). Verteporfin shows little or no cellular toxicity in the absence of light activation (256,257). Verteporfin is composed of an equal mixture of two regioisomers (Figure 3.3A), each of which consists of a pair of enantiomers. The regioisomers were separated by HPLC, and both were active in the automated microscopy assay, reducing CQ-induced punctate fluorescence >60% at 10 µM (Figure 3.3B). Importantly, the ability of verteporfin to inhibit CQ-induced autophagosome accumulation occurred in the absence of light, suggesting its activity was independent of photoactivation. All experiments described in this chapter were conducted without direct light.  54  Figure 3.2 Identification of verteporfin as an inhibitor of autophagosome accumulation. (A) MCF-7 EGFP-LC3 cells were exposed to 0.1 % DMSO, 10 mM 3-MA, or 10 µM verteporfin without or with 30 µM chloroquine (CQ) for 4 h in complete cell culture medium. The images were acquired by confocal microscopy (Section 2.5). Scale bar, 10 µm. (B) Cells were exposed to 30 µM CQ and different concentrations of verteporfin for 4 h in complete medium. The cells were fixed and stained and punctate EGFP-LC3 fluorescence was quantitated using a Cellomics Arrayscan VTI automated imager (Section 2.3). The level of punctate EGFP-LC3 fluorescence observed in control DMSO-treated cells is indicated by the dotted line. (mean ± S.D., n=3). 55  Figure 3.3 Effect of verteporfin regioisomers on chloroquine-induced autophagosome accumulation. (A) Chemical structures of verteporfin regioisomers. (B) MCF-7 EGFP-LC3 cells were exposed for 4 h to 75 µM chloroquine (CQ) without or with the indicated concentration of each verteporfin (VP) regioisomer or commercial verteporfin. Punctate EGFP-LC3 was quantitated using the automated microscopy assay (Section 2.3) (mean ± S.D., n=3).  56  3.3  Verteporfin inhibits drug- and starvation-induced autophagosome accumulation Having identified verteporfin as an inhibitor of autophagosome accumulation in the  presence of CQ, I next tested whether this drug can inhibit autophagosome formation in response to well characterized autophagic stimuli such as rapamycin or serum deprivation. Cellular exposure to 30 nM rapamycin or to serum-free medium caused a ~3-fold increase in punctate EGFP-LC3 fluorescence (Figure 3.4). Simultaneous treatment with verteporfin considerably reduced both rapamycin-induced and starvation-induced punctate EGFP-LC3 fluorescence and increased diffuse cytoplasmic fluorescence (Figure 3.4). Electron microscopy was used to examine this effect at the ultrastructural level. Sections of control cells treated with DMSO rarely if ever contained any autophagosomes (Figure 3.5) while exposure to 75 μM CQ caused a significant perinuclear accumulation of autophagic vacuoles containing lamellar structures and undigested cytoplasmic material (Figure 3.5c, arrowheads), as expected. Noticeably, incubation with 10 µM verteporfin alone caused the distinct appearance of small empty rounded single-membraned vesicles; however, it did not alter the integrity of most distinguishable organelles, such as mitochondria and multivesicular bodies (Figure 3.5b). Cells exposed to both 75 μM CQ and 10 μM verteporfin contained significantly fewer autophagosomes (Figure 3.5d, arrowheads) than those exposed to CQ alone. Interestingly, these cells also contained numerous empty single-membraned vesicles that were larger in size than in cells treated with verteporfin alone (Figure 3.5d). There was no clear accumulation of structures resembling elongation membranes in any of the samples; however, these are difficult to identify (258).  57  Figure 3.4 Verteporfin inhibits autophagosome accumulation stimulated by rapamycin or serum starvation. (A) MCF-7 EGFP-LC3 cells were exposed for 4 h to 30 nM rapamcycin without or with 10 µM verteporfin in complete cell culture medium or to medium lacking serum without or with 10 µM verteporfin. Images were acquired by confocal microscopy (Section 2.5). Scale bar, 10 µm. (B) Quantification of verteporfin inhibition of rapamycin- and serum starvationinduced autophagosome accumulation. Punctate EGFP-LC3 was quantified (left) using the automated microscopy assay (Section 2.3). (mean ± S.D., n=6) along with (right) quantification of the number of autophagosome in confocal images by visual inspection. (mean ± S.D., n=3). 58  Figure 3.5 Ultrastructural examination of inhibition of chloroquine-induced autophagosome accumulation by verteporfin. MCF-7 EGFP-LC3 cells were exposed for 4 h to 0.1% DMSO (a,b) or 75 µM CQ (c,d) without (a,c) or with 10 µM verteporfin (b,d). Images were acquired by transmission electron microscopy (Section 2.11). Arrowheads point to autophagic vesicles. Scale bar, 0.5 µm.  59  3.4  Verteporfin inhibits autophagic degradation After demonstrating that verteporfin inhibits autophagosome accumulation, it was  important to determine whether it also modulated autophagic flux by monitoring EGFP-LC3 processing and degradation. Autophagic flux describes the entire process of autophagy from autophagosome synthesis to fusion with the lysosome and degradation of autophagic substrates within the lysosome (259). EGFP-LC3 recruited to the membranes of autophagosomes is degraded upon fusion with lysosomes, but EGFP is less sensitive to lysosomal proteases than the LC3 moiety, leading to transient EGFP accumulation (Figure 3.1C). Therefore, the relative levels of EGFP-LC3 and free EGFP reflect autophagic flux (28). MCF-7 cells treated with 30 nM rapamycin for 4 h showed a significant increase in free EGFP levels compared to controls, consistent with its stimulation of autophagy (Figure 3.6A). As expected, co-treatment with 3-MA prevented LC3 degradation and thus the appearance of the free EGFP band. Bafilomycin A1, a V-ATPase inhibitor that prevents lysosomal acidification and lysosomal protein degradation (253), also prevented the appearance of the free EGFP band in cells treated with rapamycin (Figure 3.6A). Having established these conditions with well-characterized compounds, I then tested the effects of different combinations of verteporfin and rapamycin on EGFP-LC3 processing. Exposure of cells to verteporfin alone led to a decrease in the intensity of the free EGFP band in a concentration-dependent manner, indicating that verteporfin inhibits basal autophagy (Figure 3.6B). Verteporfin also caused a concentration-dependent decrease in the intensity of the free EGFP band when co-incubated with rapamycin (Figure 3.6B), further demonstrating its effect on autophagic degradation. Notably, verteporfin also inhibited autophagic degradation by all other chemicals tested, including autophagy modulators previously  60  61  Figure 3.6 Inhibition of EGFP-LC3 degradation by verteporfin. MCF-7 EGFP-LC3 cells were treated for 4 h with (A) 30 nM rapamycin without or with 10 mM 3-MA or 100 nM bafilomycin A1 in complete medium; (B) different concentrations of verteporfin without or with 30 nM rapamycin in complete medium; (C) 3 μM rottlerin, 1 μM rapamycin, 10 μM perhexiline, or 50 μM amiodarone without or with 10 μM verteporfin in complete medium. (D) 10 μM verteporfin in complete medium or in serum-free medium; (A-D) Cells were exposed to 0.1% DMSO as a vehicle control and EGFP-LC3 processing and degradation was monitored by western blotting with anti-GFP antibody (Section 2.6).  62  characterized in the Roberge lab (28), rottlerin, amiodarone, and perhexiline (Figure 3.6C) as well as many others (data not shown). The effects of verteporfin on autophagic flux were also tested in serum starvation conditions. Cells exposed to serum-free medium for 4 h showed a significant increase in the free EGFP band compared to controls, demonstrating that starvation induces autophagic degradation of EGFP-LC3 (Figure 3.6D). When cells were exposed to 10 μM verteporfin in serum-free conditions, the intensity of the free EGFP band decreased, showing that verteporfin inhibits starvation-induced autophagic degradation. The effect of verteporfin on autophagic flux was verified and quantified by monitoring the degradation of long-lived proteins. Cells were incubated with 14C-valine for 24 h followed by an additional 2 h cold chase to allow degradation of short-lived proteins before treatment. Over 6 h, the basal long-lived protein degradation in complete medium was 1.4%/h while cells exposed to EBSS, which lacks serum and amino acids, showed more proteolysis at 2.6%/h, consistent with previous studies (e.g.(253)). Exposure to verteporfin significantly reduced starvation-induced proteolysis (Figure 3.7). In this cell line, exposure to the PI3-kinase inhibitor wortmannin only showed minimal inhibition of long-lived protein degradation. These results further confirm that verteporfin inhibits degradation by starvationinduced autophagy. 3.5  Verteporfin inhibits non-specific autophagic sequestration Having demonstrated that verteporfin inhibits autophagosome accumulation and  autophagic protein degradation, I next investigated whether it affected the sequestration of cytoplasmic material. An early step in autophagy involves the expansion of phagophores into bowl-shaped structures that surround cytoplasmic material and capture it when the edges of  63  Figure 3.7 Inhibition of protein degradation by verteporfin. The amount of [14C]valine-labeled long-lived protein degradation was measured in MCF-7 EGFP-LC3 cells treated with 0.1% DMSO, 10 μM verteporfin (VP), or 100 nM wortmannin (Wort) in EBSS and cells exposed to complete cell culture medium (CM) for 6 h (Section 2.8). (mean± S.D., n=3). *, p<0.05 versus corresponding DMSO treatment.  64  the phagophores fuse to form double-membraned autophagosomes (260,261). This process may be monitored experimentally by examining the transfer of fluorescently labelled dextran from the cytoplasm into autophagic vesicles (246). FITC-dextran was introduced into the cytoplasm of MCF-7 cells by scrape-loading at a temperature of 4°C to prevent uptake by fluid-phase endocytosis. Two hours after loading, FITC-dextran was localized diffusely throughout the cytoplasm but it redistributed to punctate structures within 24 h, reflecting autophagosomal sequestration (Figure 3.8). When cells were treated with 3-MA, FITCdextran remained diffuse in the cytoplasm 24 h after loading (Figure 3.8), consistent with its demonstrated ability to inhibit autophagic sequestration (262). In cells treated with verteporfin, FITC-dextran remained completely diffuse in the cytosol (Figure 3.8), showing that it too inhibits sequestration of cytoplasmic material into autophagosomes. 3.6  Verteporfin does not inhibit LC3 processing or LC3II membrane association Having established that verteporfin inhibits autophagic vacuole formation, sequestration  and degradation, I tested whether verteporfin inhibits the processing, lipidation and membrane association of endogenous LC3 in MCF-7 cells. Lipidated LC3 (LC3II) associates with the isolation membrane of nascent autophagosomes and is believed to participate in phagophore expansion (263). LC3II was not detected in either vehicle- or rapamycin-treated MCF-7 cells, consistent with previous observations in MEF and HeLa cell lines (28,264). While rapamycin caused an increase in autophagosome accumulation (Figure 3.4) and stimulation of autophagic degradation (Figure 3.6), 4 h treatment did not cause detectable accumulation of LC3II, perhaps due to the transient nature of LC3II as a kinetic intermediate.  65  Figure 3.8 Inhibition of the sequestration of cytosolic FITC-dextran into vesicles by verteporfin. FITC-dextran was scrape-loaded into MCF-7 cells and its distribution was analyzed by confocal microscopy. DMSO-treated MCF-7 cells were fixed 2 h or 24 h after FITC-dextran loading. MCF-7 cells loaded with FITC-dextran were incubated in 10 mM 3-MA or 10 µM verteporfin (VP) for 24 h in complete medium (Section 2.9). Cells were fixed, DNA was stained and images were acquired by confocal microscopy (Section 2.5). Scale bar, 10 µm.  66  Figure 3.9 Verteporfin does not inhibit LC3 processing or membrane association. (A) MCF-7 cells were exposed to 0.1% DMSO, 30 nM rapamycin, or 75 μM chloroquine without or with 10 μM verteporfin for 4 h in complete medium. Endogenous LC3 processing was examined by western blotting with anti-LC3 antibody and protein loading was monitored with anti-β tubulin antibody (Section 2.6). (B) MCF-7 cells were exposed to 0.1% DMSO or 75 μM chloroquine without and with 10 μM verteporfin for 4 h in complete medium. Lysates were subjected to ultracentrifugation and the resulting supernatant (S) and pellet (P) fractions were immunoblotted with anti-LC3 antibody. Subcellular fractionation and protein loading was monitored by immunoblotting for the membrane marker, Transferrin Receptor (TfR), and the cytosolic marker, β tubulin (Section 2.7).  67  However, treatment with CQ inhibited autophagic degradation, causing significant LC3II accumulation (Figure 3.9A). Interestingly, cells treated with both CQ and verteporfin showed a similar increase in LC3II despite inhibition of autophagosome accumulation, indicating that LC3 becomes lipidated. These results suggest that verteporfin acts downstream of LC3 processing. Since verteporfin prevents autophagosome accumulation without inhibiting LC3 lipidation, I sought to establish if verteporfin affects the intracellular distribution of LC3II. LC3II has been shown to specifically associate with autophagosome membranes while unprocessed LC3 is cytosolic (68). Lysates of treated cells were subjected to ultracentrifugation to separate membrane and cytosolic fractions. In both DMSO and verteporfin-treated cells, LC3 was predominantly in the supernatant cytosolic fraction, and no LC3II was detected in either cytosolic or pellet membrane fractions (Figure 3.9B). In CQ-treated cells, there was a large increase in LC3II, all of which was found in the membrane fraction as expected (Figure 3.9B). Interestingly, in cells co-treated with verteporfin and CQ, LC3II was also only detected in the membrane fraction (Figure 3.9B), demonstrating that verteporfin does not prevent LC3II membrane association. In a majority of experiments, the amount of LC3II detected in the membrane pellet was similar between cells treated with CQ and those co-treated with verteporfin and CQ; however, in some experiments, there was a noticeable LC3II decrease in the membrane fraction of co-treated cells. This variability was most likely caused by washing the membrane pellet and may indicate that LC3II is not bound as tightly in the co-treated cells, resulting in a loss of material during the wash.  68  3.7  Verteporfin sensitizes cells to starvation Having identified verteporfin as an inhibitor of autophagy, it was important to investigate  its effect on cell survival and proliferation. MCF-7 cells were exposed to increasing concentrations of verteporfin in complete medium for 8 h. Verteporfin was then washed away and the cells were incubated in complete medium for 48 h to monitor their ability to recover and proliferate. Verteporfin had no effect on viability suggesting that transient inhibition of autophagy under nutrient-rich conditions does not affect MCF-7 proliferation and viability (Figure 3.10). Similar experiments were then carried out using different starvation conditions. Exposure of cells to medium lacking glucose, amino acids or serum (DPBS) for 8 h resulted in only a small decrease in cell numbers after 48 h, showing that transient exposure to starvation did not drastically affect cell survival. However, when cells were exposed to DPBS and verteporfin, cell survival at 48 h decreased substantially and in a concentration-dependent manner (Figure 3.10). Supplementing DPBS with amino acids did not rescue cells. However, supplementation of DPBS with glucose considerably increased cell survival and further addition of serum resulted in complete cell survival. Therefore, transiently inhibiting autophagy with verteporfin in nutrient-rich conditions does not affect MCF-7 cell proliferation and viability, but verteporfin sensitizes cells to glucose and serum deprivation. 3.8  Structural requirements for inhibition of autophagy by benzoporphyrin derivatives It has already been established that both regioisomers of verteporfin efficiently inhibit  autophagosome accumulation (Figure 3.3). This observation indicates that the propionic acid and propionic acid methyl ester on rings C and D could be interchanged without affecting  69  Figure 3.10 Effect of verteporfin on cell survival in different starvation conditions. MCF-7 cells were incubated for 8 h with different concentrations of verteporfin in complete medium or in the different starvation conditions shown (Section 2). Chemicals and medium were washed away and the cells were incubated for 40 h in complete medium without chemicals before measuring cell viability using the MTT assay (Section 2). (mean ± S.D., n=4).  70  activity. Interestingly, verteporfin may be described as a derivative of protoporphyrin IX (apo-heme), bearing modifications to rings A, C and D (Figure 3.11A). Protoporphyrin IX itself showed no inhibition of autophagy (not shown), demonstrating the dependence on one or more of these modifications for activity. To examine this question, a number of analogues modified at these positions (Figure 3.11A) were tested at different concentrations for inhibition of autophagy (Figure 3.11B). In protoporphyrin IX, two propionic acid groups are attached to rings C and D while verteporfin has a propionic acid methyl ester at one of these positions. Verteporfin analogue 1 with two propionic acid methyl esters at rings C and D was fully active, but analogue 2, with two propionic acid groups was essentially inactive, showing that the presence of one carboxylic acid is tolerated, but not two. Verteporfin analogues 5 and 6 with propanol functionalities attached to rings C and D remained active. To determine whether additional C and D ring substitutions would affect activity, a number of different groups were also incorporated at these positions. Inhibition of autophagy by verteporfin derivatives was not affected by the presence of various large substituents on rings C and D, including some bearing distal OH groups (not shown). However, the presence of two carboxylic acid groups close to the C and D rings prevented activity. Verteporfin also differs from protoporphyrin IX by the presence, fused to ring A, of a cyclohexadiene bearing two methanoic acid methyl esters. Analogue 1 with one methanoic acid methyl ester and one methanoic acid group retained full activity suggesting that a variety of substitutions are likely tolerated at this position. Thirteen analogues of verteporfin were generated with a disubstituted cyclohexadiene fused to ring B instead of ring A. Interestingly, 11 derivatives showed no activity and the  71  Figure 3.11 Effect of selected verteporfin analogues on chloroquine-induced autophagosome accumulation. (A) Structures of protoporphyrin IX, verteporfin, and benzopophyrin derivatives (BPD). (B) MCF-7 EGFP-LC3 cells were incubated for 4 h in complete medium with different concentrations of each verteporfin analogue in the presence of 75 μM chloroquine. Punctate EGFP-LC3 was quantitated using the automated microscopy assay (Section 2.3). (mean ± S.D., n=3). 72  remaining two were only weakly active: analogue 13 (one methanoic acid on ring B) was much less potent than its corresponding ring A analogue 1 while analogue 14 (two methyl esters on ring B and propionic acids attached to C and D) was comparable in activity to its corresponding ring A derivative 2. These data show that fusing the substituted cyclohexadiene to ring B instead of ring A almost completely abolishes activity. 3.9  Discussion This chapter describes the first screening assay for inhibitors of autophagosome  formation and the identification and characterization of verteporfin as an early-stage inhibitor of autophagy. The chemical collections screened comprised mainly approved drugs and pharmacological agents, and yielded verteporfin as the only active compound. The same collection yielded over 30 chemical stimulators of autophagosome formation ((28) and unpublished), suggesting that, unlike stimulators of autophagy, few inhibitors of autophagosome formation exist among known drugs. I showed that verteporfin inhibits the basal level of autophagy in fed cells, as demonstrated by reduced numbers of EGFP-LC3 puncta, reduced EGFP-LC3 degradation by western blot analysis, and reduced long-lived protein degradation. Verteporfin also inhibited autophagy stimulated by serum starvation, a physiological stimulus, and by rapamycin, the chemical inhibitor of mTORC1. Moreover, verteporfin inhibited autophagy induced by all other chemicals tested to date including rottlerin, amiodarone, perhexiline, and niclosamide. The observation that verteporfin inhibits autophagy triggered by a wide variety of stimuli suggests that it targets the autophagic process itself rather than an upstream control mechanism.  73  Autophagy may be arbitrarily divided into four steps: phagophore nucleation, phagophore elongation, sequestration of cytoplasmic constituents, and lysosomal degradation. Electron microscopy analysis did not reveal the presence of partially formed phagophores or phagophores with unfused tips. Rather, the cytoplasm of cells treated with verteporfin alone contained small empty vesicles with a single membrane. Verteporfin also inhibited the sequestration of fluorescent dextran loaded into the cytoplasm. Based on this evidence, I speculate that verteporfin binds to the membrane of expanding phagophores or to a factor involved in phagophore expansion and prevents expanding phagophores from adopting their characteristic cup shape, so they are unable to capture cytoplasmic cargo and only form empty single-membrane vesicles, as depicted in the model presented in Figure 3.12. The number and size of these vesicles were increased in cells treated with both verteporfin and CQ. While CQ is a known lysosomal inhibitor, it is believed to cause autophagosome accumulation not only by inhibiting lysosomal degradation but also by stimulating the expression of certain autophagy-associated genes including LC3 (265). Furthermore, under starvation-induced autophagy, mTORC1 activity is rapidly inhibited, which induces autophagy. mTORC1 localizes near the lysosomal membrane, and it was recently shown that increased amino acid levels in lysosomes reactivate mTORC1 signaling, resulting in feedback inhibition of autophagosome formation (266). Since CQ inhibits lysosomal degradation, intralysosomal amino acids do not accumulate, resulting in mTORC1 inhibition and increased autophagosome formation. Therefore, in addition to inhibiting autophagic flux through lysosome inactivation, it is likely that autophagosome accumulation by CQ is also due to stimulation of autophagosome biogenesis through mTORC1 inactivation (191). In my proposed model, verteporfin would also affect the formation of CQ-induced  74  phagophores, resulting in an increase in the size and number of these empty vesicular structures compared to verteporfin treatment alone. In yeast, the processing and lipidation of LC3/Atg8 is required for phagophore elongation (267,268). Localization of lipidated LC3 to the phagophore appears necessary for expansion into bowl-shaped structures that engulf cytoplasmic materials and generate the characteristic double membrane of the autophagosome (68). Verteporfin did not inhibit the generation of the LC3II band nor did it inhibit membrane association of LC3II. However, the subcellular fractionation data suggest that LC3II may be less tightly bound to membranes in the presence of verteporfin than in its absence. Whether lipidated LC3 is associated with the membranes of the abnormal cytoplasmic vesicles seen by electron microscopy remains to be determined. What is clear at present is that verteporfin inhibits autophagosome formation via a mechanism not previously described that is downstream of the widely used PI3-kinase inhibitors, which prevent LC3 lipidation, and upstream of lysosomal inhibitors, which cause the accumulation of autophagosomes containing undigested materials, making verteporfin a useful pharmacological tool to study autophagy in cells and animals. Further mechanistic characterization of verteporfin-mediated autophagy inhibition will be discussed in Chapter 4. Verteporfin was used to examine the effect of inhibiting autophagy on cell survival under starvation. In the presence of glucose, amino acids and serum, transient exposure of cells to verteporfin for 8h had essentially no effect on proliferation and viability. In such nutrient-rich conditions, autophagy occurs at a basal level and downregulation of autophagy is not expected to be deleterious, as demonstrated in previous studies using siRNA knockdown to target components of autophagy machinery including Atg5, Atg7, and Beclin-1(174,253). However, in starvation conditions that stimulate autophagy, verteporfin significantly, and  75  Figure 3.12 Hypothetical model for the general scheme of inhibition of autophagosome formation by verteporfin. Verteporfin prevents phaphore elongation and closure, which causes the double membrane structure to lose its shape, thus forming an empty single-membrane vesicle.  76  dose-dependently, reduced cell proliferation and viability after transient exposure. These results are consistent with a considerable body of evidence that autophagy is a mechanism of cellular protection under nutrient-limiting conditions (165,253). Cell death was not rescued by the addition of amino acids, was partially rescued by the addition of glucose, and was completely rescued by the addition of glucose and serum, suggesting that cell death depends on the interaction between the energy state of the cell, the nutrients available, and the degree of autophagy inhibition. Verteporfin is a benzoporphyrin derivative used clinically for photodynamic therapy of age-related macular degeneration. Macular degeneration is characterized by the formation of abnormal blood vessels in the vascular layer of the eye, adjacent to the retina. These new blood vessels are leaky, causing fluid buildup and hemorrhaging in the subretinal space, which damages photoreceptors, the retina, and leads to severe vision loss. Verteporfin is administered systemically and irradiation of the retina with red laser light causes the generation of singlet oxygen and secondary reactive oxygen species (ROS) that damage the local neovascular endothelium leading to occlusion of abnormal blood vessels (269). By contrast, verteporfin inhibits autophagy in the absence of light activation, the mechanism of which is explored in detail in Chapter 4. Our examination of verteporfin-related compounds in the EGFP-LC3 screening assay provided a clearer picture of the structural elements important for activity and the possible mode of binding of verteporfin to its putative target. The data showed that the “tails” attached to rings C and D are unlikely to interact with the putative binding site and might simply jut out into the solvent. Acid moieties positioned close to the planar porphyrin ring hindered activity but moving them to more distal positions did not. The presence of propanol groups did not abolish activity. This data suggests verteporfin  77  inserts into a region that does not tolerate the presence of charged groups. One of the methyl ester groups attached to the cyclohexadiene moiety fused to the A ring is attached to an sp3 hybridized carbon and is oriented substantially out of plane from the otherwise flat verteporfin molecule. This group can be replaced with other substituents without affecting activity, indicating that it does not make important contacts with the binding site and it could project outside the binding pocket. Finally, the observation that none of the ring B benzoporphyrin derivatives show significant activity indicates that the overall shape of the molecule is important for binding to its target. Even though analogous ring A and B derivatives have the same elemental composition, the "bulk volume" and orientation of that volume is substantially different. Whether verteporfin inhibits autophagy by binding to a protein or a membrane target cannot be predicted from these data. Evidence suggesting that autophagy promotes tumour growth in a nutrient-deprived environment and in response to several cancer therapy agents makes inhibition of autophagy an attractive strategy for cancer treatment. The facts that verteporfin is not toxic to cells under nutrient-rich conditions and in the absence of light irradiation, that it selectively kills cells under metabolic stress, that it is well tolerated provided that patients are not exposed to intense light (255,269), and that it is already approved for clinical use, make it a prime candidate for testing of its therapeutic potential, and is explored extensively in Chapter 5.  78  CHAPTER 4: INDUCTION OF HIGH-MOLECULAR WEIGHT P62 BY VERTEPORFIN AND ITS POTENTIAL ROLE IN AUTOPHAGY INHIBITION  4.1  Synopsis Having determined that verteporfin inhibits autophagosome formation, this chapter  explores its mechanism of action, and describes p62 as a molecular target of verteporfin. As a substrate of autophagy, the 60-kDa protein p62 is expected to accumulate during autophagy inhibition, but I observed that verteporfin treatment induced the appearance of SDS-stable high-MW forms of p62 ranging in size from ~120 kDa and exceeding 170 kDa. In vitro exposure of cell lysate, p62 immunoprecipitate, and purified p62 to verteporfin also produced high-MW p62, thus revealing p62 as a direct target verteporfin. Moreover, the presence of overhead laboratory light amplified the effect of verteporfin on p62 in vitro, suggesting a photochemical mechanism may contribute to non-photoactivated generation of high-MW p62. Since verteporfin is known to produce large amounts of singlet oxygen upon photoactivation, the effect of verteporfin on p62 in the absence of light was compared with rose bengal, a structurally unrelated singlet oxygen producer. Like verteporfin, in vitro exposure of purified p62 or p62 immunoprecipitates to rose bengal induced high-MW p62. Co-treatment with excess histidine quenched the appearance of high-MW by both verteporfin and rose bengal, implicating singlet oxygen production as the mechanism of p62 modification due to thermal activation. In biological systems, singlet oxygen reacts with proteins, causing amino acid oxidation, and often leading to protein-protein crosslinking;  79  therefore, it is likely that high-MW p62 induced by verteporfin is the product of oxidative crosslinking via low-level singlet oxygen generation. Furthermore, like verteporfin, cell treatment with rose bengal inhibited starvationinduced autophagy in a concentration-dependent manner that correlated with the appearance of high-MW p62, and suggests that singlet oxygen mediates autophagy inhibition by verteporfin and rose bengal. Co-immunoprecipitation experiments demonstrated that the generation of high-MW p62 decreased binding of p62 to poly-ubiquitinated cargo, reflecting impaired p62 function due to oxidation by verteporfin. I propose a model for verteporfinmediated inhibition of autophagosome formation due to the presence of abnormal high-MW p62 crosslink products. 4.2  Verteporfin induces the appearance of a high-molecular weight form of p62 SQSTM1/p62 is a multifunctional protein that is widely used as a marker for autophagic  degradation (270). As an adapter protein, p62 binds to both poly-ubiquitin and LC3, thus delivering ubiquitinated cargo for autophagic degradation. Consequently, p62 is itself degraded in the process, along with cargo protein (124). Therefore, in autophagy-stimulating conditions, p62 levels decrease, while when autophagy is inhibited, p62 accumulates (115). When MCF-7 cells were treated with 100 nM bafilomycin A1, an inhibitor of autophagic degradation (Section 3.4) in complete medium, no difference in p62 levels was observed as expected after short-term inhibition in low basal autophagy conditions (Figure 4.1). As anticipated, cells exposed to serum-free medium for 8 h, where autophagic flux is stimulated, contained noticeably less p62 while treatment with 100 nM bafilomycin A1 in serum-free medium showed similar levels to the nutrient-rich control (Figure 4.1). As an inhibitor of autophagy, verteporfin was similarly expected to cause p62 accumulation under autophagy-  80  Figure 4.1 Verteporfin induces high-MW p62 in cells. MCF-7 EGFP-LC3 cells were treated for 8 h with (A) 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin in the presence or absence of serum or to (B) 0.1% DMSO, 10 µM verteporfin (VP), or 10 µM B benzoporphyrin derivative diol (BBPD diol) in complete medium. p62 levels were examined by western blotting (Section 2.6).  81  Figure 4.2 Verteporfin-mediated p62 modification is amplified by light exposure during lysate preparation. MCF-7 EGFP-LC3 cells were treated for 4 h with 0.1% DMSO, 100 nM bafilomycin A1 (Baf A1), or 10 µM verteporfin (VP) in complete medium. Cells were kept in the dark during the course of treatment. Cell lysates from DMSO, Baf A1, and VP + light were collected, quantified, and normalized in the presence of overhead laboratory light. The lysate from VP-light was collected, quantified, and normalized in the dark. p62 levels were examined by western blotting (Section 2.6).  82  stimulating conditions. However, in both nutrient-rich and starvation media, verteporfin treatment caused the appearance of an altered form of p62 that showed reduced SDS-PAGE electrophoretic mobility under reducing and denaturing conditions (Figure 4.1A, High-MW p62). Compared to the expected p62 form at ~60 kDa, the high MW-form is not a distinct band, but migrates as a smear starting from ~120 kDa and surpassing the highest MW band of the protein ladder at 170 kDa. Bafilomycin A1 did not induce high-MW p62, showing that it was not merely a consequence of autophagy inhibition (Figures 4.1, 4.2). Cells were also exposed to a verteporfin structural analogue lacking autophagy-inhibiting activity, ring B benzoporphyrin derivative, BBPD diol (Compound 7, Section 3.8), which did not produce high-MW p62 forms (Figure 4.1B). It is important to note that initial investigations into the effects of verteporfin on p62 gave variable results. In some experiments no 60-kDa p62 was detected in verteporfin-treated cells (Figure 4.1B) while other experiments showed a faint to moderate 60-kDa form (Figure 4.2). The high-MW form was always present after verteporfin treatment, but its intensity and range of sizes also appeared to vary in initial experiments. Specifically, when no 60-kDa p62 was detected, the high-MW p62 signal was more intense and broader in size than when 60-kDa p62 was present (Figures 4.1-2). The explanation for these differences is technical. One variable in these initial experiments was the amount of ambient light during and after cell lysis. Due to the photoactivatable nature of verteporfin, exposure to light was always avoided during exposure of cells to verteporfin, but light exposure was not initially as tightly controlled during cell lysis and subsequent steps. I observed that more high-MW p62 and less 60-kDa p62 was detected when cell lysis, protein quantification, and normalization were done in the presence of ambient light. To address the contribution of light, two dishes of MCF-7 EGFP-LC3 cells  83  were treated identically with 10 µM verteporfin for 4 h in the dark, but one was lysed in the dark while the other was lysed in the presence of overhead fluorescent light. Both verteporfin-treated samples contained high-MW p62 that was not present in DMSO- or bafilomycin A1- treated cells; however, the lysate from verteporfin-treated cells exposed to ambient light showed a stronger and broader high-MW p62 band as well as noticeably less of the 60-kDa form compared to the verteporfin sample not exposed to light (Figure 4.2). Other autophagy markers were investigated in the same samples, but none were affected by light (data not shown). These results suggest that the effect of verteporfin on p62 occurs in the absence of illumination but is amplified in vitro, after cell lysis, in the presence of light. This phenomenon will be described further in Section 4.4, but it should be noted that exposure to overhead laboratory light was carefully avoided in subsequent experiments unless otherwise noted. Initial p62 immunoprecipitation experiments showed that both the 60-kDa and high-MW p62 forms were effectively immunoprecipitated from cells treated for 4 h with verteporfin. When p62 was immunoprecipitated from vehicle-treated control cells, a small amount of high-MW p62 was detected when sufficient material was loaded (Figure 4.3, third lane) implying that this form is present under physiological conditions, but in much lower amounts than in verteporfin-treated cells. Interestingly, immunoprecipitates from verteporfin-treated cells contained more of the 60-kDa form of p62 than controls, as well as larger amounts of high-MW p62. The fact that both 60-kDa and high-MW p62 were present in verteporfintreated lysates at significantly higher amounts than in the equivalent DMSO- treated samples demonstrates that verteporfin causes p62 accumulation as well as a protein modification that generates a high-MW form (Figure 4.3).  84  Figure 4.3 High-MW and 60-kDa p62 immunoprecipitate from untreated and verteporfin-treated cells. MCF-7 EGFP-LC3 cells were treated for 4 h with 0.1% DMSO or 10 µM verteporfin (VP). Indicated amounts of each lysate were then immunoprecipitated with anti-p62 antibody and analyzed by western blot (Section 2.6).  85  4.3  Verteporfin-induced high-MW p62 does not represent p62 aggregates Several studies have shown p62 is a common component of protein aggregates including  Lewy bodies, huntingtin aggregates, Mallory bodies, and hyaline bodies (137,271,272). Many of these p62 aggregates have been shown to be degraded by autophagy (104,115,273,274) via binding to poly-ubiquitinated proteins and tethering to LC3 for delivery to autophagosomes. It was previously demonstrated that ubiquitin-positive inclusion bodies accumulate in autophagy-deficient cells (39,89,132), so I wanted to determine whether the high-MW p62 form accumulates as aggregates in cells as a consequence of autophagy inhibition. p62 abundance and localization were analyzed in MCF-7 EGFP-LC3 cells by immunofluorescence microscopy after 4 h treatment with different compounds. As expected, 100 nM bafilomycin A1, which inhibits autophagic degradation, caused a large increase in punctate p62 structures (Figure 4.4A), which could represent cytoplasmic aggregates or autophagosomes. The p62 punctate staining largely colocalized with punctate EGFP-LC3, suggesting these structures were indeed undigested autophagosomes containing p62-tethered cargo. Another compound that has been shown to induce accumulation of p62-containing protein aggregates is the proteasome inhibitor MG132. Treatment with MG132 perturbs protein homeostasis by interfering with the ubiquitin-proteasome system (UPS), and causes dispersed protein aggregate formation (275,276). In previous studies, MG132 induced protein inclusion bodies containing p62 and often colocalizing with LC3 (276–278), demonstrating overlap between UPS and autophagy substrates. It is believed that inclusion bodies form, in part, when the protein degradation capacity of the proteasome is overwhelmed, particularly in times of cellular stress, and these aggregates are instead  86  degraded by autophagy (276,278). MCF-7 EGFP-LC3 cells treated with 5 µM MG132 contained noticeably more punctate p62 staining compared to the DMSO-treated control (Figure 4.4A). Interestingly, EGFP-LC3 localization in MG132-treated cells was mostly diffuse, showing little co-localization with punctate p62 (Figure 4.4A). A possible explanation is that short-term inhibition of the proteasome was enough to induce accumulation of poly-ubiquitinated proteins into p62-positive inclusion bodies, but not long enough to elicit a strong stimulation of autophagy. Analysis of p62 by western blotting showed that despite induction of punctate p62 staining, no high-MW p62 was detected after MG132 treatment (Figure 4.4B). Therefore, two drugs that cause p62 aggregates through different mechanisms, do not generate high-MW p62, implying that the decreased electrophoretic mobility of p62 in verteporfin-treated cells is not due to protein aggregation. Additionally, 4 h treatment with 10 µM verteporfin did not cause any significant increase in punctate p62 staining, implying that verteporfin does not induce p62 aggregates (Figure 4.4). Therefore, the high-MW p62 form is distinct from p62 aggregates since punctate p62 structures were not present after 4 h verteporfin treatment. As expected, verteporfin did not affect EGFP-LC3 localization since the cells were treated in complete medium where basal autophagy is low (Figure 4.4). The punctate p62 observed after bafilomycin A1 is a result of accumulation of p62 trapped in autophagosomes. Since verteporfin prevents autophagosome formation, the vehicle for p62 accumulation and delivery for autophagic degradation is no longer present in the cell, thus its localization is largely unchanged after verteporfin treatment (Figure 4.4). Unlike previous studies showing, in the absence of autophagy, accumulation of ubiquitin- and p62-positive inclusion bodies appearing as punctate aggregates by immunofluorescence (89,132,278), no evidence of  87  Figure 4.4 High-MW p62 generated by verteporfin is not indicative of p62 aggregates. (A) MCF-7 EGFP-LC3 cells were treated for 4 h with 0.1% DMSO, 10 µM verteporfin (VP), 100 nM bafilomycin A1 (Baf A1), or 5 µM MG132 in complete medium. The cells were fixed and stained with p62 antibody, and images were acquired by confocal microscopy (Section 2.5). (B) MCF-7 EGFP-LC3 cells were treated for 4 h with 0.1% DMSO, 10 µM verteporfin, or 5 µM MG132 for 4 h. Cells were lysed and normalized as describe in Chapter 2. Five µg of cell lysate were loaded for p62 immunoblot analysis (Section 2.6).  88  punctate p62 inclusion bodies was present after short-term verteporfin treatment. The aforementioned cited observations were all made in primary cells of autophagy-deficient mice or during long-term autophagy inhibition in cultured neuronal cells, compared to my experiments focusing on short-term autophagy inhibition in cultured cancer cells. 4.4  Verteporfin can induce high-MW p62 in vitro  4.4.1  Experiments with cell lysates  After observing that the formation of high-MW p62 was amplified by light in vitro (Section 4.2), I wanted to determine whether the formation of high-MW p62 was a direct effect of verteporfin on p62 or resulted from a cellular response to verteporfin treatment. Lysates were prepared from untreated BxPC-3 (used in the next chapter) cells and incubated without or with 10 µM verteporfin for 30 min at 4°C or 37°C in the absence (dark) or in the presence of overhead fluorescent and ambient light (light) in the laboratory. Immunoblotting for p62 showed that, in the absence of verteporfin, only the 60-kDa form was present after incubation at 4°C or 37°C in light or dark conditions. Interestingly, exposure to verteporfin in the presence of light at 4°C caused the generation of a range of high-MW p62 forms appearing above the 130 kDa protein marker and spanning beyond 170 kDa (Figure 4.5). Exposure to verteporfin at 37°C in the absence of light also induced high-MW p62 forms spanning the same range, but the smear was much less intense. Exposure of cell lysate to verteporfin at 37°C in the presence of light produced the most intense and broadest range of high-MW p62 forms starting from ~110 kDa and spanning beyond 170 kDa (Figure 4.5). I concluded that, in vitro, verteporfin is capable of generating high-MW p62 in the presence of thermal or light energy, and that light stimulation has a stronger effect. These observations offer further evidence that in vitro light exposure amplifies the effect of verteporfin on p62.  89  Figure 4.5 In vitro effect of verteporfin on p62 in cell lysates. Untreated BxPC-3 cell lysate was treated with 10 µM verteporfin in the presence or absence of light at 4°C or 37°C for 30 min. p62 was immunoprecipitated and analyzed by western blot using anti-p62 antibody (Section 2.6).  90  The fact that verteporfin could generate high-MW p62 even in the presence of light at 4°C implies that enzymatic mechanisms do not confer the modification(s) to p62 by verteporfin. Moreover, considering that light or heat was required to induce high-MW p62 by verteporfin implies that high-MW p62 is the product of a chemical reaction rather than a conformational change or an oligomerization product. 4.4.2  Experiments with p62 immunoprecipitates  To further investigate the effect of verteporfin on p62, p62 was immunoprecipitated from untreated cells, washed extensively and then treated with 10 µM verteporfin in the presence or absence of light at 4°C or 37°C. Untreated p62 immunoprecipitates incubated at 4°C in the dark or light contained only 60-kDa p62, but exposure to verteporfin and light at 4°C generated a range of high-MW p62 forms (Figure 4.6). Interestingly, incubating untreated immunoprecipitates at 37°C in the dark or light generated a small amount of highMW p62, detected as a faint smear near 170 kDa. However, exposing p62 immunoprecipitates to verteporfin under the same conditions generated noticeably more high-MW p62 forms than observed in the respective untreated samples (Figure 4.6). Immunoprecipitated p62 was also modified in vitro by verteporfin in the presence of both mild and stringent detergents (data not shown), implying that verteporfin modifies p62 directly or through a very strongly associated protein(s). The fact that a small amount of high-MW p62 was detected in the untreated lysates incubated at 37°C raises the possibility that p62 is susceptible to modification by heat or light, even in the absence of verteporfin. These in vitro experiments clearly demonstrate verteporfin can generate high-MW p62 when exposed to light or heat; however, it remains unclear whether this effect by verteporfin is direct or if it occurs through p62-associated proteins. As a scaffolding protein involved in a  91  Figure 4.6 In vitro effect of verteporfin on immunoprecipitated p62. p62 was immunoprecipitated from untreated BxPC-3 cells. The immunoprecipitate was then treated for 30 min in lysis buffer with 10 µM verteporfin in the presence or absence of light at 4°C or 37°C and analyzed by western blot (Section 2.6).  92  variety of cellular functions, p62 associates with a number of proteins including PKCξ, TRAF6, LC3, Keap1, raptor, Hsp70, and several mitochondrial proteins (104,118,141,149,279,280). Theoretically, any protein(s) bound directly or indirectly to p62 in the immunoprecipitates might be responsible for generating high-MW p62 in the presence of verteporfin and heat or light. Even using the most stringent immunoprecipitation conditions, we cannot exclude the presence of p62-interacting proteins; therefore, the most obvious way to investigate whether it is a direct effect is to treat purified p62 protein with verteporfin. 4.4.3  Experiments with pure p62 protein  To determine whether the effect of verteporfin on p62 is direct, recombinant GST-p62 was purchased and its purity was assessed by SDS-PAGE and Coomassie blue staining, which showed a single protein band at 80 kDa (data not shown), the expected size for GSTp62. GST-p62 was incubated at a concentration of 66 nM in the presence or absence of 10 µM verteporfin for 4 h at 37°C in the dark or light. Immunoblotting for p62 revealed a banding pattern (Figure 4.7) very similar to that seen after verteporfin treatment on cells (Figures 4.1, 4.3), and in vitro treatments on cell lysate (Figure 4.5) and immunoprecipitated p62 (Figure 4.6). In the absence of light, untreated GST-p62 was detected as a single 80 kDa band, but exposure to verteporfin decreased the intensity of the 80 kDa band and generated an intense high-MW band, detected above the largest MW marker at 170 kDa (Figure 4.7). Similar to the previous experiment, a small amount of high-MW p62 was observed in the untreated GST-p62 sample after exposure to light and heat, but the majority of p62 was detected at 80 kDa. These observations demonstrate that verteporfin treatment in the absence of light at 37°C directly affects p62 molecules and confers a modification or crosslinking  93  Figure 4.7 Effect of verteporfin on purified GST-p62. 100 ng (66 nM) purified GST-p62 was incubated with 10 µM verteporfin (VP) for 4 h at 37°C in the absence or presence of light (Section 2.13). Samples were immunoblotted for p62 as (Section 2.6).  94  event that increases the molecular weight significantly. When GST-p62 was exposed to verteporfin in the presence of both light and heat, its signal almost completely disappeared from the blots (Figure 4.7). I speculate that the combination of verteporfin, heat, and light, each of which alone can modify p62, either caused p62 degradation or prevented it from entering the gel. In summary, both heat and light cause verteporfin to form high-MW p62 and the banding pattern is reproducible regardless of the energy source. Since the immunoprecipitates were washed extensively and the purified GST-p62 was prepared only with buffered salts, it must generate high-MW p62 directly, without any need for protein cofactors, enzymatic activity, or energy from ATP. 4.5  Verteporfin-mediated high-MW p62 forms are due to protein oxidation by singlet oxygen Verteporfin is known to produce large amounts of singlet oxygen (1O 2 ) upon red laser  light irradiation at 690 nm (281–283). Singlet oxygen (1O 2 ) is a type of reactive oxygen species (ROS) generated by an input of energy, classically photodynamic activation, that results in a rearrangement of electrons in the oxygen atom (284). Singlet oxygen has the ability to oxidize a number of biological molecules including DNA, lipids, and proteins (270,285,286). Proteins are the primary target of singlet oxygen because they are more abundant than other potential targets and singlet oxygen rapidly reacts with the residues tryptophan, tyrosine, histidine, cysteine, and methionine at physiological pH (286–288). These protein oxidation products are then capable of crosslinking with other oxidized residues to produce interprotein crosslinks, with His-His, His-Lys, His-Arg, His-Cys, and Tyr-Tyr interactions the most commonly reported (286,289–293), thus producing high-  95  molecular mass products (294,295). In most studies, protein crosslinking has been only observed upon intense light irradiation of singlet oxygen producers (292,296), but Bae et al. (297) showed that in the presence of some drugs, protein crosslinking occurred after brief exposure to laboratory light, just as I observed with p62 and verteporfin. I have previously stated that verteporfin inhibits autophagy by a mechanism independent of photoactivation (298). The results above now lead me to hypothesize that thermal energy is sufficient to activate verteporfin to generate a low level of singlet oxygen, and that this is increased by even low levels of light. During photodynamic therapy (PDT), verteporfin is activated by laser light such that a total energy dose of 25-50 J/cm2 is administered (255,297,299). In contrast, 5 min exposure to laboratory lights has been reported to achieve 0.039 J/cm2, 800X less (297). Therefore, I speculate that p62 is particularly susceptible to modification by singlet oxygen and this modification results in inhibition of autophagy. To test this hypothesis I examined the effects on p62 of rose bengal, a widely used singlet oxygen producer that is structurally unrelated to verteporfin (Figure 4.8A) (291,292,300). Rose bengal is a chemical dye that can be photoactivated to produce singlet oxygen (300,301). To determine whether singlet oxygen produced by rose bengal also generates high-MW p62 forms, recombinant GST-p62 was exposed to 5 µM rose bengal or 5 µM verteporfin for 1 h at 4°C or 37°C in the dark. An equal amount of untreated GST-p62 was also analyzed, which contained no high-MW p62 (Figure 4.8B, untreated). A small amount of high-MW GST-p62 was observed in the DMSO control after exposure to heat, demonstrating again that heat alone can generate some high-MW p62 in vitro. Exposure of recombinant p62 to rose bengal at both 4°C and 37°C induced high-MW p62 forms similar to those generated by verteporfin under the same conditions (Figure 4.8B). Combining rose  96  Figure 4.8 Rose bengal induces high-MW p62 in vitro. (A) Chemical structure of rose bengal (B) 50 ng (33 nM) purified GST-p62 was incubated with 0.5% DMSO (D), 5 µM verteporfin (VP), or 5 µM rose bengal (RB) for 1 h at 4°C or 37°C in the dark. The untreated control contained only GST-p62 (Section 2.13). (C) p62 was immunoprecipitated from untreated BxPC-3 cells. The immunoprecipitated material was treated for 30 min in lysis buffer with 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal in the dark at 4°C or 37°C. p62 was analyzed by western blot (Section 2.6).  97  bengal treatment with heat strongly stimulated the formation of high-MW GST-p62, further implicating singlet oxygen generation as the source of verteporfin-induced high-MW p62. The fact that significant amounts of high-MW GST-p62 forms were generated in reactions containing only recombinant protein, buffered salt, a photosensitizer, and heat strongly suggests that singlet oxygen generation causes p62 oxidation and subsequent crosslinked GST-p62 products. The in vitro effect of rose bengal was also demonstrated on cellular p62 using p62 immunoprecipitates. p62 was immunoprecipitated from untreated cells and then treated with 10 µM rose bengal or 10 µM verteporfin for 30 min at 4°C or 37°C in the dark. Neither had any effect at 4°C, but at 37°C immunoprecipitated p62 was modified by rose bengal to generate a range of high-MW forms even in the dark, as seen with verteporfin treatment (Figure 4.8C). The effects of singlet oxygen can be quenched in vitro using excess histidine (285,297,302,303). The imidazole side chain of histidine has been shown to be one of the most vulnerable to modification by photooxidation via singlet oxygen (288,304), and its reactive product is implicated in the generation of His-His, His-Lys, and His-Arg adducts (286,289,291). The half-life of singlet oxygen in aqueous solutions is extremely short, in the range of µs; therefore, singlet oxygen will react rapidly with the first available substrate (255,303). Excess histidine added in the presence of singlet-oxygen acts as an abundantly available substrate for oxidation, thus reducing cellular protein oxidation and subsequent reactions (286). p62 was immunoprecipitated as previously described from untreated BxPC-3 cells. The immunoprecipitates were then treated with 10 µM verteporfin or 10 µM rose bengal in the presence or absence of 20mM histidine for 30 min at 37°C. High-MW p62 was detected in all treatments lacking histidine, even the DMSO-treated control although at a very  98  low level compared with the rose bengal and verteporfin treatments (Figure 4.9A). The intensity of the high-MW p62 signal was decreased in all samples containing histidine, supporting the hypothesis that high-MW p62 generated by verteporfin is mediated by low levels of singlet oxygen generation (Figure 4.9A). The presence of a small amount of highMW p62 in control samples reaffirms that p62 is extremely sensitive to oxidation. In another experiment, cells were treated with N-acetylcysteine (NAC), a precursor to the ROS scavenger glutathione, that is often used as a cellular antioxidant in ROS studies (163,305), and has been shown to inhibit the effects of singlet oxygen with conflicting efficacy (306,307). BxPC-3 cells were treated for 4 h with 10 µM verteporfin in the presence or absence of 3mM NAC. Exposure of cells to verteporfin in the absence of light led to the appearance of high-MW p62 forms that were not present in the untreated control (Figure 4.9B). Co-treatment with NAC did not decrease the intensity of high-MW bands generated by verteporfin, demonstrating that, despite its antioxidant properties, NAC cannot inhibit the generation of high-MW p62 forms by verteporfin. A closer review of the literature reveals that despite its ability to chemically scavenge singlet oxygen through its thiol group (308), NAC is actually a poor quencher of singlet oxygen compared to other scavengers including histidine and sodium azide (303,307). In fact, in a cell-free screen for singlet oxygen scavengers, the IC 50 for NAC was found to exceed 100mM (306), 30-fold higher than the concentration used in my experiment. When singlet oxygen is generated by photodynamic activation in the presence of laser light, cellular ROS including superoxide, hydrogen peroxide, and the hydroxyl radical are produced by the mitochondria as a secondary response, leading to cellular apoptosis that can be attenuated by NAC treatment (309–311). An elegant study by Matroule et al (309) showed that NAC-  99  Figure 4.9 Quenching singlet oxygen reduces the generation of high-MW p62 by verteporfin. (A) p62 was immunoprecipitated from untreated BxPC-3 cells. The immunoprecipitated material was treated for 30 min in lysis buffer with 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal in the dark at 37°C with or without 20mM histidine (His). (B) BxPC-3 cells were exposed for 4 h to 0.1% DMSO or 10 µM verteporfin without or with 3mM Nacetylcysteine (NAC) in complete medium. Samples were analyzed by immunoblotting for p62 and protein loading was monitored using anti-β-tubulin antibody (Section 2.6)  100  mediated quenching of cellular ROS during photosensitization prevented apoptosis but had no effect on singlet oxygen generation, suggesting that the inability of NAC to quench the effect of verteporfin on p62 actually supports my hypothesis that high-MW p62 forms are a consequence of low-level singlet oxygen generation rather than other ROS. 4.6  p62 is oxidized by singlet oxygen generators It has been shown that singlet oxygen-mediated protein oxidation is characterized, in  part, by conversion of some amino acid residues into carbonyl derivatives (312–314). Carbonyl content can be measured by derivatization with 2,4-dinitrophenylhydrazine (DNPH), which reacts with ketones and aldehydes to produce stable DNP-hydrazone amino acid products that are specifically detected with anti-DNP antibodies (314–316). Using this “Oxyblot” procedure, purified recombinant His-tagged p62 was exposed to 5 µM rose bengal or 5 µM verteporfin for 1 h at 4°C or 37°C in the dark. An equal amount of untreated Hisp62 was also analyzed (Figure 4.10, untreated). At 4°C very little signal was detected after exposure to DMSO, exactly like the untreated control. Exposure to either rose bengal or verteporfin at 4°C showed a significant increase in carbonyl content at ~40 kDa, the size of His-p62, and at the high MW-region ≥ 90 kDa, (Figure 4.10). When the same treatments were carried out at 37°C the carbonyl signal was entirely localized to the high-MW region ≥ 90 kDa, and exposure to rose bengal or verteporfin increased the intensity. At 37°C, no oxidized His-p62 was detected at 40 kDa, even in the DMSO-treated sample. The presence of oxidized high-MW His-p62 after incubation at 37°C in the DMSO control confirmed previous findings that heat alone can generate high-MW p62 (Figure 4.7, Figure 4.8B) and demonstrated a correlation with its production and an increase in protein oxidation. Oxidized His-p62 at 40 kDa was observed only when His-p62 was exposed to singlet oxygen at 4°C,  101  Figure 4.10 Singlet oxygen increases the carbonyl content in p62 and high-MW p62. 25 ng (33nM) nM purified His-p62 was incubated with 0.5% DMSO (D), 5 µM verteporfin (VP), or 5 µM rose bengal (RB) for 1 h at 4°C or 37°C in the dark. The untreated control contained only 25 ng His-p62. After incubation samples were derivatized with DNPH, and subjected to SDS-polyacrylamide gel electrophoresis for immunoblotting with anti-DNP antibody solution according to the Oxyblot kit manufacturer’s instructions (Section 2.6).  102  suggesting that at 37°C oxidized His-p62 is highly reactive, and leads to the generation of high-MW His-p62, most likely through crosslinking. Of the amino acids with which singlet oxygen has a tendency to react, histidine and tryptophan are the most likely to be converted directly into carbonyl derivatives at physiological pH (286,288,304). Additionally, it has been shown that the formation of interprotein crosslinks via oxidized His residues can produce more carbonyl derivatives in the process (286,287,317,318). Therefore, the increased intensity of oxidized His-p62 migrating at ≥ 90 kDa observed after exposure to verteporfin or rose bengal at 37°C provides strong evidence of protein crosslinking. This experiment was carried out only once just prior to submission of my thesis, and needs to be confirmed and extended. However, the fact that conditions which have consistently produced high-MW p62 also increased its carbonyl content provides substantial evidence that verteporfin-induced high-MW p62 is a product of singlet oxygen-mediated crosslinking. 4.7  Rose bengal inhibits starvation-induced autophagosome accumulation Having established that rose bengal, a structurally unrelated photosensitizer, generates  high-MW p62 forms like those observed after verteporfin treatment, it was important to determine whether it also affected autophagy. MCF-7 EGFP-LC3 cells were exposed to 10 µM rose bengal for 4 h in complete medium in the presence and absence of 75 µM chloroquine and autophagosome accumulation was monitored by automated fluorescence microscopy (Section 2.3). Chloroquine treatment caused a large increase in punctate EGFPLC3 fluorescence that was completely prevented by verteporfin treatment, but was not affected by rose bengal (Figure 4.11). Rose bengal has been shown to readily bind serum albumin, with 90% of the drug bound when used at 5 µM (319,320), raising the possibility that only a fraction of rose bengal  103  Figure 4.11 Rose bengal does not inhibit autophagosome accumulation by chloroquine in the presence of serum. MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 10 µM verteporfin (VP), or 10 µM rose bengal (RB) without or with 75 µM chloroquine (CQ) for 4 h in complete cell culture medium. The cells were fixed and stained, and images were acquired using a Cellomics VTI automated imager (Section 2.3).  104  actually entered the cell during 4 h treatment. To account for potential interference by serum proteins, MCF-7 EGFP-LC3 cells were exposed to rose bengal in serum-free medium for 4 h. Serum starvation caused an increase in punctate EGFP-LC3 fluorescence compared to cells treated with DMSO in complete cell culture medium, demonstrating autophagosome accumulation in response to starvation (Figure 4.12). Treatment with rose bengal in serumfree medium considerably reduced starvation-induced punctate EGFP-LC3 fluorescence in a concentration-dependent fashion. Exposure of cells to either 10 µM rose bengal or 10 µM verteporfin in serum-free medium completely inhibited the appearance of punctate EGFPLC3 and increased diffuse cytoplasmic fluorescence (Figure 4.12). The fact that two photosensitizers known to generate singlet oxygen show similar in vitro protein modification and in vivo autophagy modulation in the absence of photoactivation strongly suggests a common mechanism of action. 4.8  Rose bengal induces the appearance of high MW-p62 forms in vivo Having established that, like verteporfin, rose bengal inhibits starvation-induced  autophagosome accumulation in vivo and induces high-MW forms of recombinant and cellular p62 in vitro, it was important to determine whether high-MW p62 forms were detected in cells after rose bengal treatment. MCF-7 EGFP-LC3 cells were exposed to two concentrations of rose bengal or to 10 µM verteporfin in the presence or absence of serum for 4 h, and p62 was monitored by immunoblotting. As a consequence of starvation-induced autophagy, less 60-kDa p62 was present after DMSO treatment in the absence of serum compared to complete medium (Figure 4.13). In the presence of serum, a small amount of high-MW p62 was detected in the DMSO-treated control and verteporfin treatment significantly increased high-MW p62. Only 60-kDa p62 was detected in cells treated with  105  Figure 4.12 Rose bengal inhibits starvation-induced autophagosome accumulation in the absence of serum MCF-7 EGFP-LC3 cells were exposed for 4 h to 0.1% DMSO, 10 µM verteporfin (VP), or different concentrations of rose bengal (RB) in the presence or absence of serum. The cells were fixed and stained, and images were acquired using a Cellomics VTI automated imager (Section 2.3).  106  Figure 4.13 Rose bengal induces high-MW p62 in cells in the absence of serum. MCF-7 EGFP-LC3 cells were exposed for 4 h to 0.1% DMSO, 10 µM verteporfin (VP), or different concentrations of rose bengal (RB) in the presence or absence of serum. Cell lysates were immunoblotted for p62 and β-tubulin (Section 2.6).  107  rose bengal in complete medium (Figure 4.13). By contrast, in the absence of serum, high MW-p62 was detected after exposure to 10 µM verteporfin or to 10 µM rose bengal. Furthermore, those treatments that generated high-MW p62 forms also showed accumulation of 60-kDa p62 compared to the serum-starved DMSO control. Exposure to 1 µM rose bengal in the absence of serum did not induce detectable amounts of high-MW p62 forms and there was less 60-kDa p62 present than in cells treated in complete medium (Figure 4.13). These experiments reveal a correlation between inhibition of autophagosome accumulation and generation of high-MW p62 by both rose bengal and verteporfin. In vivo rose bengal treatment only produced high-MW p62 forms in conditions where it also inhibited autophagosome accumulation (Figure 4.12-13). The observation that 10 µM rose bengal did not induce high-MW p62 forms in complete medium further implies that serum affected its ability to modulate autophagy and modify p62. Taken with earlier data showing the inability of a verteporfin analogue lacking autophagy inhibiting activity to generate highMW p62 forms in vivo (Figure 4.1), these observations infer a correlation between high-MW p62 forms and inhibition of autophagy by both verteporfin and rose bengal. The fact that high-MW p62 forms are generated by verteporfin and rose bengal in vitro demonstrate that it is a direct effect by drug treatment rather than a downstream consequence of autophagy inhibition; however, accumulation of 60-kDa p62 is most likely a consequence of autophagy inhibition. 4.9  High-MW p62 shows reduced ability to bind to ubiquitinated proteins but not to LC3 Verteporfin treatment generates high-MW forms of p62 via singlet oxygen, and we  hypothesize that this modification affects p62 function. p62 contains a Phox/Bem 1p (PB1)  108  protein-protein binding domain that governs its interaction with a number of binding partners (118,119), including itself. In fact, the ability to self-oligomerize via the PB1 domain has been implicated in targeting p62 to autophagosomes (321). In addition to the PB1 domain, p62 has a number of other protein interacting domains including the C-terminal ubiquitinassociated (UBA) domain that binds ubiquitin and the LC3 recognition sequence (LRS) (104). These two domains facilitate p62-mediated delivery of poly-ubiquitinated proteins to the autophagosome for degradation. Structural analysis of the UBA domain shows that it dimerizes and that this dimerization affects p62 affinity for mono- and poly-ubiquitin chains (127). To provide mechanistic insight into how verteporfin-induced singlet oxygen inhibits autophagosome formation, I next examined whether exposure to verteporfin affects the ability of p62 to interact with LC3 and ubiqutinated proteins. p62 was immunoprecipitated from cells treated or not with verteporfin and immunoblotted for known interacting proteins. To investigate whether verteporfin treatment affects interaction of proteins with the 60-kDa and high-MW p62, whose levels are affected themselves by drug treatment, I ensured 100% immunoprecipitation of p62 to account for all forms of p62 present in the cell. Treatments were carried out in both MCF-7 EGFP-LC3 and BxPC-3 cells. In each experiment, 2.5% of the input was collected before and after immunoprecipitation to confirm presence of the protein of interest in the lysate, and its depletion by immunoprecipitation. In both cell lines, verteporfin treatment caused the appearance of high-MW p62 that was completely immunoprecipitated along with 60-kDa p62 from the lysates (Figure 4.14 A-C, where A and B are independent experiments with MCF-7 EGFP-LC3 cells). p62 western blot analysis showed that nearly equal amounts of 60kDa p62 were immunoprecipitated from DMSO- and verteporfin-treated cells while high-  109  MW p62 was detected in immunoprecipitates only from verteporfin-treated cells (Figure 4.14 A-C). To determine whether verteporfin treatment affected p62 binding to ubiquitinated proteins, p62 immunoprecipitates from DMSO- and verteporfin-treated cells were immunoblotted for co-immunoprecipitated ubiquitinated proteins using an antibody that recognizes both mono- and poly-ubiquitinated protein chains (109). In both MCF-7 EGFPLC3 and BxPC-3 cells, exposure of cells to verteporfin led to an increase in polyubiquitinated proteins compared to control cells as detected by ubiquitin immunoblotting in the cell lysates prior to immunoprecipitation (Figure 4.14 A-C). Analysis of p62 immunoprecipitates for co-precipitated poly-ubiquitinated proteins clearly showed that a fraction of cellular poly-ubiquinated proteins bind to p62 in untreated cells (Figure 4.14 AC). However, despite the increase in polyubiquitinated proteins detected after verteporfin treatment, p62 immunoprecipitates from verteporfin-treated MCF-7 EGFP-LC3 cells contained less poly-ubiquitinated proteins compared to control cells (Figure 4.14 A, B). When the corresponding immunoprecipitate supernatants were probed for poly-ubiquitin, the supernatant from verteporfin-treated cells contained more poly-ubiquitinated proteins than found in the control supernatant (Figure 4.14 A, B), which most likely reflects both the increased cellular poly-ubiquitin content and its reduced binding to p62 caused by verteporfin. p62 immunoprecipitates from verteporfin-treated BxPC-3 cells appeared to contain amounts of co-immunoprecipitated poly-ubiquitinated proteins equal to DMSOtreated cells. However, keeping in mind that more p62 was present, and therefore immunoprecipitated, after exposure to verteporfin, equal amounts of co-immunoprecipitated poly-ubiquitin suggest there was relatively less ubiquitin bound to p62 after verteporfin  110  treatment than beforehand (Figure 4.14C). Due to unequal amounts of p62 between controland verteporfin-treated immunoprecipitates, densitometric analysis was performed to quantify the entire co-immunoprecipitated poly-ubiquitin signal relative to the entire immunoprecipitated p62 signal before and after drug treatment (Figure 4.14D). The ratio of poly-ubiquitin to p62 was markedly decreased after drug treatment in both MCF-7 EGFPLC3 and BxPC-3 cell lines despite there being an overall increase in cellular polyubiquitinated content. In all three experiments presented, the relative amount of polyubiquitin associated with p62 decreased by ~60% after exposure to verteporfin (Figure 4.14D). Therefore, generation of high-MW p62 by verteporfin interferes with its ability to associate with poly-ubiquitin, providing a possible mechanism for inhibition of autophagy by verteporfin. Since verteporfin decreased p62 binding to poly-ubiquitinated proteins, I wanted to determine whether it also interfered with LC3 binding. Because of low endogenous LC3 levels, this question was examined in MCF-7 EGFP-LC3 cells by immunoprecipitating EGFP-LC3 from cells treated with 10 µM verteporfin for 4 h and probing for p62. EGFPLC3 was immunoprecipitated from the same cell lysates used to generate p62 immunoprecipitates in Figure 4.14A. Analysis of the input after immunoprecipitation with GFP antibody showed complete isolation of EGFP-LC3 from the lysate (Figure 4.14E). A few striking observations were made after probing EGFP-LC3 immunoprecipitates for p62. Co-immunoprecipitated p62 was strongly detected in EGFP-LC3 immunoprecipitates from both DMSO- and verteporfin-treated cells at 60 kDa; however, high-MW p62 forms were also detected in the EGFP-LC3 immunoprecipitate from verteporfin-treated cells, implying that high-MW p62 forms can still associate with LC3 (Figure 4.14E). p62 western blot  111  analysis of the lysate after EGFP-LC3 immunoprecipitation did not show any 60-kDa or high-MW p62 forms remaining, indicating that all endogenous p62 was bound to EGFP-LC3. This observation was surprising since p62 has so many known binding partners, and it implies that LC3 and p62 are bound in the cytoplasm even in basal autophagy conditions. GFP immunoprecipitation was also carried out in BxPC-3 cells, which do not express EGFPLC3, to control for any nonspecific binding of p62 to the antibody or agarose protein G beads: no p62 was detected (Figure 4.14E). These results demonstrate that both 60-kDa and high-MW p62 forms present after verteporfin treatment efficiently bind EGFP-LC3 despite the decreased association with poly-ubiquitinated proteins observed. Our data suggest that p62 is constitutively bound to LC3, even after treatment with DMSO, an observation that has not been made previously. It is possible that in cells not overexpressing EGFP-LC3, not all p62 is associated with cytosolic LC3. Even if this interaction is enhanced in this EGFP-LC3 overexpressing system, the results demonstrate that p62 has a high affinity for EGFP-LC3 that is not affected by verteporfin treatment. It was previously shown that mutational disruption of p62 self-oligomerization via the PB1 domain prevents p62 localization to the autophagosomes (321). Furthermore, deletion of either the PB1 domain or UBA domain prevents the formation of p62-positive aggregates and p62-mediated degradation (115,321), implying that protein dimerization mediates its function in autophagic degradation. Dimerization of the UBA domain has been shown to differentially affect binding affinity of mono- and poly-ubiquitin to p62. It has been shown that UBA dimerization actually occludes a part of the ubiquitin binding surface, indicating that UBA dimerization and Ub-binding are mutually exclusive (127). While polyubiquitination targets proteins for degradation by the proteasome or autophagy, monomeric  112  113  114  Figure 4.14 High-MW p62 shows impaired association with poly-ubiquitinated proteins, but not with LC3 (A,B) MCF-7 EGFP-LC3 or (C) BxPC-3 cells were exposed for 4 h to 0.1% DMSO (D) or 10 µM verteporfin (VP) in complete medium. p62 was immunoprecipitated and bound polyubiquitinated proteins were detected in the immunoprecipitate (IP) fraction using an antibody that recognizes mono- and poly-ubiquitin (Ub) n . 2.5% of the lysate was run both prior to and post p62 immunoprecipitation. Immunoprecipitation was confirmed by western blot for p62. (D) Densitometry analysis was performed on the experiments presented using Quantity One software. (E) Using the same lysates prepared in (A), EGFP-LC3 was immunoprecipitated and bound p62 was detected in the IP fraction using an anti-GFP antibody. 2.5% of the lysate was run both prior to and post p62 immunoprecipitation. To control for non-specific binding, EGFP-LC3 was immunoprecipitated from BxPC-3 cell lysates from C and probed for coprecipitated p62. Immunoprecipitation was confirmed by western blot for GFP. A-C represent three separate experiments. A and C were carried out simultaneously using identical experimental conditions.  115  ubiquitin is believed to play an important role in signaling. For example, monomeric Ub binding to p62 regulates downstream NF-κB signaling (322,323). While UBA-mediated dimerization ablates mono-ubiquitin binding, it may actually favour binding of polyubiqutin-linked protein chains (127). Verteporfin is a hydrophobic molecule that we suspect localizes preferentially to membranes. Presuming p62 localizes to the autophagosome formation site, and singlet oxygen generation by verteporfin causes p62 crosslinking, it is possible that crosslinking events alter the UBA binding surface, thus reducing binding to poly-ubiquitinated proteins. By contrast, verteporfin treatment did not affect LC3 association with p62; this observation may offer insight into how verteporfin inhibits autophagosome formation. 4.10  Discussion Having characterized verteporfin as an inhibitor of autophagosome formation in Chapter  3, this chapter identifies SQSTM1/p62 as a cellular target of verteporfin and describes its modification via non-photoactivated singlet oxygen production by verteporfin. In autophagy, p62 delivers cargo for autophagic degradation, and is itself degraded in the process; therefore, its relative abundance at 60 kDa has been used to determine the autophagic state of the cell for years (115,124,270). However, due to its role in a number of different cellular processes including the UPS, there are limitations to interpreting autophagy through p62 levels alone (149,270). High-MW forms of p62, stable to SDS denaturation, starting from ~120 kDa and exceeding 170 kDa were detected in cell lysates and p62 immunoprecipitates collected after verteporfin treatment. The observation that in vitro exposure of cell lysate from verteporfin-treated cells to laboratory room lighting strongly amplified the effect of  116  verteporfin on p62 suggested a photochemical mechanism may contribute to the nonphotoactivated generation of high-MW p62 observed. To date, verteporfin has been essentially only studied in PDT; its effects in the absence of light activation are largely unknown (255,299,324,325). In in vitro experiments controlling for light exposure and heat, I established that verteporfin generated high-MW p62 from cell lysates either exposed to laboratory light at 4°C or incubated at 37°C in the dark, and that the pattern of p62 modification by verteporfin was similar irrespective of the energy source. Furthermore, the banding pattern matched that observed after cell treatment. Thus, a similar mechanism probably underlies p62 modification by verteporfin in cells and in vitro and in the presence of light. Verteporfin similarly produced high-MW p62 in vitro from both p62 immunoprecipitates and purified recombinant GST-p62, demonstrating that the generation of high-MW p62 by verteporfin is a direct effect of drug treatment rather than a downstream consequence of autophagy inhibition, and that it requires light or thermal energy only, without any cellular constituents other than p62 itself. The major photochemical reactions elicited by verteporfin in PDT are due to singlet oxygen production. Verteporfin is administered and subsequently activated by red light, usually using a laser. Verteporfin absorbs the light, exciting the molecule to a triplet state that initiates both primary and secondary photochemical reactions resulting in cytotoxic cellular damage. In biological environments, the light-activated triplet state of verteporfin transfers its energy to ground state oxygen, leading to the formation of singlet oxygen (255,285,286,297,326). Showing that rose bengal, another singlet oxygen producer, also generated high-MW p62, and that excess histidine, but not NAC, decreased p62 modification confirms that singlet oxygen formation is the mechanism behind p62 modification by  117  verteporfin, even in the absence of light. In biological systems singlet oxygen primarily reacts with and oxidizes proteins due to their high abundance and the presence of tryptophan, histidine, tyrosine, methionine, and cysteine, 5 amino acids that react readily with the electrophilic singlet oxygen (255,326). As these amino acids become oxidized by singlet oxygen, they react with other amino acid residues, and protein crosslinking occurs (286,291,292,326). Preliminary results showing increased carbonyl content, a marker for oxidative modification, in purified recombinant His-p62 after exposure to either rose bengal or verteporfin in the dark revealed that high-MW His-p62 is oxidized, implying it is indeed a product of crosslinking. In a study by Bae et al., (297) the protein PCNA was photocrosslinked by a number of different photosensitizers, and in the presence of some drugs crosslinking occurred after brief exposure to laboratory light, just as I observed with p62 and verteporfin. The facts that verteporfin generates high-MW p62 via singlet oxygen formation, that this effect was still observed after verteporfin treatment of purified recombinant p62, and that verteporfin increases the carbonyl content of p62, are all consistent with observations of singlet oxygen-mediated protein crosslinking made by others, and suggests that high-MW forms of p62 are actually covalently crosslinked p62 oligomers. Moreover, that singlet oxygen has an extremely short lifetime in the range of microseconds and a restricted diffusion range of 10-200 nm (327,328) makes crosslinking more favorable among proteins that exist as oligomers, like p62 (297,329). Assuming p62-p62 crosslinking occurs, since the high-MW forms were observed at sizes ≥ 120 kDa, we infer that they represent p62 dimers and oligomers. Observations of p62 crosslinking are supported by evidence in two publications showing accumulation of high-MW p62 in cells exposed to either cigarette smoke or UVA light  118  (330,331). Cigarette smoke contains high levels of free radicals of ill-defined structure (332,333); however, due to high levels of both nitric oxide and hydrogen peroxide, singlet oxygen is probably generated and contributes to cigarette smoke-induced oxidative damage (334–336). Both UVA and UVB radiation are known sources of singlet oxygen and have been implicated in the aging process (286,337,338). High-MW p62 was detected in alveolar macrophages from long-term smokers while only 60-kDa p62 was found in macrophages extracted from nonsmokers’ lungs (330). Likewise, Zhao et al. observed high-MW p62 in keratinocytes exposed to UVA or UVB radiation, but not in unexposed cells (331). In these two papers, p62 immunofluorescence showed aggregation under the same conditions, leading the authors to attribute the high-MW p62 observed by western blot to p62 aggregation. However, my p62 immunofluorescence and western blot data refute that assumption. In my hands, verteporfin generated high-MW p62 without causing p62 aggregation and induction of p62 aggregates did not generate SDS-stable high-MW p62. Based on my observations, I suspect p62 crosslinks were present in addition to p62-positive protein aggregates in the aforementioned studies. Interestingly, two additional publications describe high-MW p62 in aged cells in the absence of external singlet oxygen producers. Gamerdinger et al. (339) showed accumulation of high-MW p62, which they termed “SDS-stable SQSTM1 polymers” in protein extracted from the cerebellum of aged mice and not in young cerebellum tissue, but the authors did not discuss this further. Kang et al. (340) observed a decrease in the amount of 60-kDa p62 detected in aged fibroblasts, which they attributed to a corresponding increase in “SDSresistant high molecular weight polymers”. These observations were again interpreted as p62 aggregation, but no direct evidence of that was presented. It is well established that oxidized  119  and crosslinked proteins accumulate as a consequence of age (339,341). Therefore, high-MW p62 has also been observed under physiological conditions in cellular and animal models of aging that are associated with accumulation of biological oxidative stress. In addition to mediating selective autophagy of protein aggregates, p62 participates in cellular oxidative stress responses. p62 expression is induced during oxidative stress by the transcription factor NRF2. In unstressed conditions, NRF2 is bound and targeted for ubiquitin-mediated degradation by Keap1. Upon oxidative stress, p62 binds and chelates Keap1, allowing NRF2 to translocate into the nucleus, where it binds the Antioxidant Response Element (ARE) in the promoter region of a number of antioxidant genes, including p62, creating a feed-forward loop (120,145,342,343). Zinc finger motifs have been described as being particularly susceptible to singlet oxygen due to the presence of cysteine and histidine residues which act as available ligands for the zinc ion to stabilize protein-protein or DNA-protein interactions (344,345). Interestingly, p62 contains a ZZ-type zinc finger domain, which may contribute to its role in redox signaling (346). In my hands, high-MW p62 crosslinks were periodically observed even in the absence of verteporfin treatment in very low amounts, raising the question whether p62 is intrinsically susceptible to exogenous and endogenous sources of protein oxidation, perhaps acting as a physical sensor of oxidative stress as well. Such susceptibility would also account for the accumulation of high-MW p62 observed in models of aging (339,340). Oxidative stress produced by PDT and other ROS sources has widely been reported to stimulate autophagy (347–350). Those studies primarily focused on ROS species produced intracellularly, particularly mitochondrial ROS such as superoxide and hydrogen peroxide, but to my knowledge, no one has looked at the effects of low-level singlet oxygen on  120  autophagy. According to my studies singlet oxygen production is the underlying mechanism behind verteporfin-mediated autophagy inhibition. Like verteporfin, rose bengal, a structurally distinct singlet oxygen producer, inhibited starvation-induced autophagosome accumulation in a concentration-dependent manner that correlated with the appearance of p62 crosslink products. While the extent of singlet oxygen generation by verteporfin without light activation is unknown, it is considerably lower than in PDT conditions. In PDT, photoactivated verteporfin elicits a rapid apoptotic response due, in part, to mitochondrial photochemical damage (257,351,352); however, it is well characterized as being nontoxic in the absence of light (255,298,352). Furthermore, other than the appearance of singlemembrane vesicles, ultrastructural analysis in Chapter 3 revealed no abnormal cellular effects from verteporfin treatment; the mitochondria appeared normal and intact (Figure 3.5). Protein oxidation and crosslinking can lead to loss of function depending on the site(s) and extent of modification (302,312). p62 co-immunoprecipitates showed impaired binding to poly-ubiquitinated proteins after verteporfin treatment. Based on these findings, I propose a model for verteporfin-mediated inhibition of autophagosome formation via p62 crosslinking (Figure 4.15). Itakura & Mizushima (321) showed that p62 colocalizes with ULK1 and Atg14 at the autophagosome formation site, and that localization requires p62 self-oligomerization. p62 has been described as the major interacting protein of LC3 (104) and my data implies that p62 and LC3 are constitutively associated, even after verteporfin treatment. Because of limited diffusion of singlet oxygen from its site of production, sites of immediate PDT damage are indicative of its subcellular localization (353,354). Verteporfin-mediated PDT immediately causes mitochondrial swelling and  121  Figure 4.15 Proposed model for singlet oxygen-mediated inhibition of autophagosome formation involving p62 crosslink products. As p62 oligomers are recruited to the autophagosome membrane, they become oxidized and crosslinked to each other due to low-level singlet oxygen generation by verteporfin. This crosslinking event interferes with p62 binding to poly-ubiquitinated cargo, but does not affect LC3 binding. The generation of large p62 crosslink products with impaired function either physically disrupts proper autophagosome elongation and closure or it interferes with the function of other molecules necessary for complete autophagosome formation.  122  damages mitochondrial proteins (256), thus suggesting it localizes in or near the mitochondria (355), where structurally similar sensitizers, hematoporphyrin and protoporphyrin IX are also known to accumulate (354). Therefore, it is likely that both verteporfin and p62 oligomers are present at the membrane of the autophagosome formation site and within the range of singlet oxygen diffusion, generating p62 crosslinks. Verteporfin treatment does not affect LC3 lipidation, subsequent membrane association, or its association with p62. Therefore, lipidated LC3 which is bound to p62 is recruited to the expanding phagophore membrane, where p62 gets oxidized by verteporfin and forms more p62 crosslinks (Figure 4.15). In the presence of verteporin, p62 crosslinking disrupts its association with poly-ubiquitinated cargo, supporting observations that p62 binding to cargo is tightly regulated by both p62 oligomerization and dimerization of the UBA domain (83,128). Under normal autophagy conditions, LC3 participates in p62-mediated recruitment of ubiquitinated cargo during expansion (321). Regulation of expansion during autophagosome biogenesis is arguably the least understood subject in autophagy, but there is consensus in the field that it must be highly regulated to facilitate membrane curvature and eventual closure (58,261). I propose that irregular products of p62 crosslinking prevent autophagosome formation through physical and mechanical disruption of the expanding membrane or that the association of poly-ubiquitinated proteins with p62 facilitates proper membrane expansion structures targeted for degradation. The production of irregular p62 crosslinks as the proposed mechanism preventing autophagosome formation by verteporfin is consistent with my hypothesis in Chapter 3 (Figure 3.12), where disruption of membrane curvature resulted in small single-membrane vesicles, such as those observed by electron microscopy (Figure 3.5). However, it is possible that singlet oxygen-mediated inhibition of  123  autophagy inhibition occurs through a mechanism distinct from or in combination with p62 crosslinking.  124  CHAPTER 5: EFFICACY OF VERTEPORFIN IN ANIMAL MODELS OF CANCER  5.1  Synopsis In Chapter 3, I showed that transient verteporfin treatment sensitized MCF-7 breast  cancer cells to starvation. It is well established that autophagy promotes tumour cell survival under conditions of environmental and drug-induced stress; therefore, I wanted to explore its therapeutic potential in animal models of cancer. The anti-tumour efficacy of verteporfin was assessed in vivo in two human tumour xenograft models: JIMT-1 breast carcinoma and BxPC-3 pancreatic ductal adenocarcinoma. The effects of verteporfin on JIMT-1 cells were assessed in vitro prior to the animal study. Verteporfin was found both to inhibit gefitinib-induced autophagy and to enhance JIMT-1 cell death in response to gefitinib treatment. However, in the JIMT-1 xenograft study, verteporfin did not show anticancer effects in vivo either on its own or in combination with the EGFR inhibitor, gefitinib. In light of recent evidence suggesting that pancreatic cancers are dependent on autophagy for growth, verteporfin was tested on a panel of pancreatic cancer cell lines, and cell survival was monitored. Two cell lines, BxPC-3 and SU86.86, were found to be particularly sensitive to long-term verteporfin treatment at concentrations previously shown to inhibit autophagy. Furthermore, verteporfin was shown to inhibit autophagy induced by gemcitabine, the standard treatment for pancreatic cancer. In a BxPC-3 xenograft model, verteporfin did not show anti-tumour efficacy on its own, but it enhanced the in vivo effects of gemcitabine on tumour growth and mouse survival. Both the JIMT-1 and BxPC-3 in vivo 125  studies were accompanied by pharmacokinetic analysis, which showed that verteporfin reached the tumour site and accumulated at concentrations shown to inhibit autophagy in vitro. Additionally, analysis of tumour tissue showed that verteporfin induced the appearance of high-MW p62 in vivo, suggesting that its effects in tumour tissue reflect those described in Chapters 3 and 4. 5.2  Efficacy of verteporfin in a JIMT-1 breast cancer xenograft model Evidence suggesting that autophagy promotes tumour growth in a nutrient-deprived  environment and in response to several cancer therapy agents makes inhibition of autophagy an attractive strategy for cancer treatment (10,57,157). The identification of verteporfin, an FDA-approved drug already used clinically, as an inhibitor of starvation- and drug- induced autophagy makes it an excellent subject for evaluating the therapeutic potential of inhibiting autophagy in vivo. To date, the only pharmacological autophagy inhibitor for in vivo studies has been chloroquine (CQ) and its derivative hydroxychloroquine (HCQ); however, these lysosomotropic agents affect lysosomal processes that are distinct from autophagy (57). Moreover, recent studies indicate that CQ can sensitize breast cancer cells to chemotherapy independently of autophagy (179). Clearly there is a need for additional small molecule tools to study the therapeutic efficacy of autophagy inhibition in vivo. In collaboration with the Centre for Drug Research and Development (CDRD), I was able to explore the in vivo anti-tumour efficacy of verteporfin on its own and in combination with gefitinib, a chemotherapeutic epidermal growth factor receptor (EGFR) inhibitor that cytoprotectively induces autophagy (57,356) (Dragowska et al. submitted 2013) in a JIMT-1 breast carcinoma xenograft model. One characteristic of chemotherapy resistance in breast cancer is overexpression and co-expression of members of the HER receptor family, EGFR  126  (HER1), HER2, and HER3 (357,358), rendering patients resistant to monoclonal antibody therapy using trastuzumab (TZ). Currently there are limited therapeutic options available for combating HER2-positive breast cancers (359–361), and there is a need to develop novel drugs or combinations of drugs to improve therapeutic outcome. As an EGFR tyrosine kinase inhibitor, gefitinib has been studied to combat HER2-positive breast cancer, but showed only moderate effects in vivo (362) and did not appear to improve therapy in a phase II clinical trial (363). Blocking EGFR has been reported to stimulate autophagy in a number of cancer cell lines (356,364), and Han et al.(356) demonstrated that autophagy knockdown enhanced cytotoxicity by gefitinib in small cell lung carcinoma cells. The JIMT-1 cell line was originally isolated from a pleural metastasis of breast cancer in a patient resistant to the antibody therapy, trastuzumab (365), and is a good tumour model to investigate the combinatorial effects of inhibiting EGFR with gefitinib and inhibiting autophagy with verteporfin. 5.2.1  In vitro effects of verteporfin on JIMT-1 cells  Before starting the in vivo study, the effect of verteporfin on gefitinib-induced autophagy in JIMT-1 cells was determined. MCF-7 EGFP-LC3 cells were exposed to gefitinib for 24 h in the presence or absence of 10 µM verteporfin, and EGFP-LC3 processing was monitored. As expected of an autophagy stimulator, gefitinib treatment caused a decrease in EGFP-LC3 intensity and an increase in free EGFP due to increased autophagic degradation. Cotreatment with verteporfin decreased the intensity of the free EGFP band demonstrating its inhibition of gefitinib-induced EGFP-LC3 degradation (Figure 5.1).  127  Figure 5.1 Verteporfin inhibits gefitinib-induced EGFP-LC3 degradation. MCF-7 EGFP-LC3 cells were exposed for 24 h to different concentrations of gefitinib in the presence or absence of 10 µM verteporfin. Cells were treated with 0.5% DMSO as a vehicle control and 30 nM rapamycin as a positive control. EGFP-LC3 processing and degradation was monitored by western blot using anti-GFP antibody and protein loading was monitored using anti-tubulin antibody (Section 2.6).  128  Figure 5.2 Long-term verteporfin treatment inhibits JIMT-1 cell survival on its own and enhances the effect of gefitinib in vitro. JIMT-1 cells were seeded in 96-well plates and exposed (A) to 0-10 µM verteporfin for up to 7 days. Media and drugs were replenished every third day. Cells were stained with Hoechst 33342 and quantified using an automated fluorescence microscope (Section 2.3). (mean ± S.D. (error bars), n=4) (B) to 0-10 µM verteporfin and 10-20 µM gefitnib for 48 h. Cell survival was measured using the MTT assay (Section 2.12) (mean ± S.D. (error bars), n=3).  129  Having previously established that transient verteporfin treatment (4-24 h) in nutrientrich conditions shows no toxicity to various cancer cell lines (Figure 3.10 and data not shown), I explored the effects of long-term verteporfin treatment on JIMT-1 cell proliferation. Cells were seeded at a low density and treated with 0-10 µM verteporfin for 7 days in complete medium, and live cells were stained and quantified by automated fluorescence microscopy. Continuous treatment with 10 µM verteporfin strongly inhibited JIMT-1 cell proliferation, with cells doubling only ~1.5 times over 7 days. Verteporfin did not appear to kill cells as no reduction in cell numbers was observed. Exposure to < 10 µM verteporfin treatment had no deleterious effects on cell proliferation or viability, with all cells reaching ~90-100 % confluence by the end of the assay (Figure 5.2A). The effects of combining gefitinib with verteporfin treatment on JIMT-1 cell survival were determined using a 48 h MTT assay. Cells were exposed to combinations of 0-10 µM and 0-20 µM gefitinib. Compared to untreated cells, 10 µM verteporfin reduced cell survival by 60% and 5 µM verteporfin reduced cell survival by 45% (Figure 5.2B). Exposure to gefitinib moderately reduced cell survival in a concentration-dependent manner, and on its own was less efficacious than verteporfin (Figure 5.2B). Combining verteporfin with gefitinib reduced cell survival more than either drug alone, showing an additive effect. For instance, exposure to 10 µM verteporfin and 20 µM gefitinib allowed only ~30% cell survival compared to 40% and 60%, respectively, by either drug alone. Interestingly, in combination experiments, increasing the gefitinib dose from 10 to 20 µM did not significantly reduce cell survival; however, increasing the verteporfin dose from 5 to 10 µM reduced cell survival by ~ 15% (Figure 5.2B). The observation that sensitization of JIMT-1 cells to gefitinib by verteporfin is concentration-dependent implies that it is dependent on  130  autophagy inhibition, particularly since my earlier characterization work showed partial autophagy inhibition using 5 µM verteporfin and complete inhibition with 10 µM (Figures 3.1B, 3.6B). There is an obvious discrepancy between the effects of 5 µM verteporfin on cell survival in the two assays presented which may be due to differences in the duration of treatment, the cell density at the beginning of the experiment, or the different readouts for each assay. MTT assays measure cellular metabolic activity, so one possibility is that 5 µM verteporfin reduced metabolic output, as shown in the 48 h assay, without affecting cell proliferation. Further experimentation is needed to confidently decipher these inconsistent results. While the in vitro characterization of verteporfin treatment in JIMT-1 cells was limited, it established that on its own, 10 µM verteporfin significantly reduced cell numbers in both the 48 h MTT assay and the 7 day proliferation assay, suggesting long-term autophagy inhibition may have anti-tumour effects in vivo. We also established that verteporfin inhibits gefitinibinduced autophagy, and that JIMT-1 cell treatment with both verteporfin and gefitinib produces additive cytotoxic effects. Taken together, our data suggest that combining verteporfin with gefitinib in a JIMT-1 tumour model is appropriate for investigating whether inhibiting autophagy in response to anti-cancer therapy enhances anti-tumour efficacy. 5.2.2  Verteporfin tumour accumulation following single administration  A pharmacokinetic study was done to establish how much verteporfin reached the tumour site after one intraperitoneal (i.p) administration of 60 mg/kg of verteporfin in Rag2M mice bearing subcutaneous JIMT-1 tumour xenografts. Tumours were harvested 2, 8, 16, and 24 h after drug administration, and verteporfin concentration was measured using UPLC-MS/MS analysis. Verteporfin tumour concentrations peaked 8 h post administration  131  Figure 5.3 Verteporfin tumour concentration peaks 8 h after administration and remains above its IC 50 for 24 h in JIMT-1 tumour-bearing mice. JIMT-1 tumour-bearing Rag2M mice were treated with verteporfin intraperitoneally at a dose of 60 mg/kg and tumours were harvested 2, 8, 16, and 24 h after administration for pharmacokinetic analysis. The concentration of verteporfin in tumour tissue was determined by liquid chromatography combined with mass spectrometry analysis (Section 2.19). (mean ± S.D. (error bars), n=3).  132  at 9.1 µg/g (12.7 µM, assuming 1g tumour tissue = 1 ml), and were above its in vitro autophagy inhibition IC 50 of 1 µM for a period of 24 h (Figure 5.3). A similar analysis was carried out for gefitinib after oral dosing with 50 mg/kg. Gefitinib was found to accumulate in tumours well over its IC 50 (data not shown). These preliminary pharmacokinetic experiments demonstrated that dosing mice with 60 mg/kg verteporfin intraperitoneally and with 50 mg/kg gefitinib orally delivered each drug to the tumour tissue to levels comparable to those used in vitro. 5.2.3  Verteporfin induces high-MW p62 forms in vivo in tumour tissue  Having established that verteporfin reached the tumour at concentrations sufficiently high to inhibit autophagy, I wanted to determine whether it had similar cellular effects on p62. Tumours collected for pharmacokinetic analysis were cut in half prior to analysis, and one half was used for western blot analysis of p62. Immunoblotting for p62 showed that in vivo verteporfin treatment induced the appearance of high-MW p62. Tumours from control mice, treated with saline, contained only 60-kDa p62, but 2 h after verteporfin administration, high MW-forms were detected (Figure 5.4). Furthermore, the intensity of the high-MW p62 was stronger in tumours collected 8 h and 16 h after drug administration than at 2 h, which correlated with the pharmacokinetic analysis showing peak tumour drug levels 8 h post-administration (Figure 5.4). 5.2.4  Verteporfin does not inhibit JIMT-1 tumour growth in vivo alone or in combination with gefitinib  An anti-tumour efficacy study was carried out in Rag2M mice bearing subcutaneous JIMT-1 Herceptin-resistant human breast carcinoma xenografts. Verteporfin was formulated in DSPE-PEG micelles and administered intraperitoneally at 60 mg/kg once a day for five  133  Figure 5.4 Verteporfin induces high-MW p62 in JIMT-1 tumour tissue in vivo. JIMT-1 tumour-bearing Rag2M mice were treated with verteporfin intraperitoneally at a dose of 60 mg/kg and tumours were harvested 2, 8, 16, and 24 h after administration for western blot analysis. Tumour sections were homogenized and immunoblotted for p62 (Section 2.6). (Control: n=2; VP: n=3 per group).  134  Figure 5.5 Verteporfin does not exhibit anti-tumour efficacy on its own or in combination with gefitinib in a JIMT-1 tumour mouse model. JIMT-1 tumour-bearing Rag 2M mice were treated with gefitinib orally and verteporfin intraperitoneally over a period of 3 weeks. Tumour growth was monitored every Monday, Wednesday, and Friday using digital callipers (Section 2.17). Tumour growth is presented as both average tumour volume (left) and relative tumour size (right), where the average of each group was normalized to 1 on the first day of treatment. Treatment was initiated when tumours reached 100-150 mm3. Arrows indicate last day of treatment (mean ± S.E.M (error bars), n=6 for all groups at the start of the study).  135  consecutive days (q1dx5). Gefitinib was administered orally at 100 mg/kg when used alone and at 50 mg/kg and 100 mg/kg when used with verteporfin, at the same time as verteporfin administration. After the first week of dosing, toxicity was observed in some of the mice treated with 60 mg/day verteporfin; therefore, only gefitinib was administered the second week, and the third week verteporfin dosing was resumed (q1dx5), but was reduced to 45 mg/kg. Efficacy was evaluated by monitoring tumour volume 3 times a week throughout the study. A slight decrease in relative tumour volume was observed after treatment with 100 mg/kg gefitinib (Figure 5.5 left), but it was not statistically significant (Student t-test). Verteporfin showed no anti-tumour activity on its own or with gefitinib. Thus verteporfin and gefitinib alone as well as in combination were not efficacious in the JIMT-1 tumour xenograft model. JIMT-1 cells have been shown to express several markers of drug resistance mechanisms (366) in addition to EGFR overexpression, suggesting these cells elicit drug resistance through a number of pathways. Although this study produced negative anticancer results, it demonstrated that autophagy-modulating verteporfin concentrations can be achieved and measured in vivo and that verteporfin-induced p62 modification and inhibition of autophagy do not necessarily translate into anti-tumour activity. 5.3  Efficacy of verteporfin in a BxPC-3 pancreatic adenocarcinoma xenograft model  5.3.1  In vitro effects of verteporfin on pancreatic adenocarcinoma cells  The role of autophagy in pancreatic adenocarcinoma (PDAC) has been of particular interest in recent years due to a number of studies implicating activated autophagy in pancreatic malignancies (367–369). Recently, Yang et al. suggested that some pancreatic cancers are inherently addicted to autophagy for growth, even in nutrient-rich conditions, and  136  showed that inhibition of autophagic flux using CQ resulted in moderately reduced growth of cells in vitro and of tumours in mice (163). The encouraging preclinical studies showing inhibition of autophagy enhancing cytotoxic effects of anticancer therapies in a number of cancer types prompted the start of over 20 clinical trials exploring autophagy inhibition using CQ and HCQ (157,370). Unfortunately, the preclinical successes have not yet been reproduced in the clinic. A major issue is that the high micromolar concentrations of HCQ required to inhibit autophagy in vitro are inconsistently achieved in humans (190,369). Therefore, there is an urgent need for other autophagy inhibitors to use in the clinic. The previous JIMT-1 in vivo pharmacokinetic study demonstrated that p62-modifying and autophagy-modulating amounts of verteporfin do accumulate in the tumour; therefore, I wanted to investigate the therapeutic potential of verteporfin in a PDAC model that may be more sensitive to autophagy inhibition. To determine whether inhibiting autophagy with verteporfin affected the proliferation or survival of PDAC cells, a panel of 8 human PDAC cell lines was exposed to 0-10 µM verteporfin continuously for up to 7 days and live cells were quantified using automated fluorescence microscopy to count nuclei or by measuring absorbance with an MTT assay. Four of the PDAC cell lines, Capan 1, Capan 2, HS766T, and CFPAC-1 were essentially insensitive to long-term verteporfin treatment at all concentrations (Figure 5.6A). Panc-1 and MIA PaCa-2 cells exposed to ≤ 5 µM verteporfin grew normally, but cell proliferation was significantly inhibited when exposed to 10 µM verteporfin. Notably, BxPC-3 and SU86.86 cells were more sensitive to 10 µM verteporfin, which completely inhibited cell proliferation. Furthermore, unlike the other PDAC cell lines tested, 5 µM verteporfin inhibited BxPC-3 and  137  138  Figure 5.6 PDAC cell lines show variable sensitivity to long-term verteporfin treatment in vitro. Eight different PDAC cell lines were seeded in 96-well plates and treated with 0-10 µM verteporfin for up to 7 days and cell proliferation was monitored. Media and drugs were replenished every third day. To monitor cell viability and proliferation, Capan 1 and Capan 2 cells were assayed using MTT and measuring OD 570 nm while all other PDAC cell lines were stained with Hoechst 33342 and quantified using an automated fluorescence microscope (Section 2.12). (mean ± S.D. (error bars), n=4). (B) BxPC-3, SU86.86, and MIA PaCa-2 cells were exposed to vehicle control or 100 nM bafilomycin A1 in complete medium for 4 h. Lysates were collected and immunoblotted for LC3 or p62. β-tubulin was monitored as a loading control (Section 2.6).  139  SU86.86 cell proliferation by >50%, showing a clear dose-response (Figure 5.6A). The observed variability in verteporfin sensitivity suggests that PDAC cell lines have varying dependencies on autophagy that need to be considered when designing and analyzing therapeutic studies. Basal autophagy was monitored in highly verteporfin-sensitive BxPC-3 and SU86.86 cells and in relatively insensitive MIA PaCa-2 cells using two previously described autophagy markers, LC3-II and p62, in the presence and absence of the lysosomal inhibitor bafilomycin A1. In the presence of bafilomycin A1, LC3-II accumulated in all three cell lines compared to the respective control, but the relative accumulation between the three cell lines did not differ greatly (Figure 5.6B) and p62 levels were unchanged (Figure 5.6B). Therefore, the level of basal autophagy is comparable between the three cell lines, and does not show correlation with verteporfin sensitivity. Activating K-Ras mutations are a hallmark of pancreatic cancer (144,371) and recent studies suggest Ras activation is a key to autophagy addiction (160,162). However, no correlation between K-Ras mutation and verteporfin sensitivity was observed in these cell lines: BxPC-3 and SU86.86 cells, both of which are highly sensitive to verteporfin express wild type K-Ras and activated K-Ras, respectively (372–374), and Panc-1 cells express activated K-Ras due to the same genetic mutation as SU86.86 cells, but are relatively insensitive to verteporfin (374). Other frequent mutations found in PDAC were examined including tumour suppressor genes p53 and DPC4 (Table 5.1), but no obvious correlation could be inferred. Considering the genetic variability among PDAC cell lines, it is unlikely that there is a single determinant for verteporfin sensitivity. Regardless of the underlying  140  Table 5.1 Summary of PDAC cell line origin, mutational status of common PDAC markers, and verteporfin sensitivity Cell Line  Site of Derivitization  K-Ras  p53  P16Ink4A  Capan-1  Liver metastasis  12 Val  Capan-2  Primary Tumour  12 Val  Homozygous deletion 6 bp insertion 7 bp insertion  HS766T  Lymph node metastasis  61 His; WT  159 Val WT Intron 4 200 bp deletion WT Mut 225-282 Exons 2-4  CFPAC-1 Panc-1 MIA PaCa-2 BxPC-3 SU86.86  Liver metastasis Primary Tumour Primary Tumour Primary Tumour Primary Tumour  12 Val 12 Asp 12 Cys WT 12 Asp  242 Arg 273 His 248 Trp 220 Cys 245 Ser  WT Intron 2 splice site WT Promoter methylation HD HD WT/HD11 HD  SMAD4/DPC4 577 Leu 343 STOP  VP Sensitivity  -  WT  -  HD  -  HD WT WT 100 Thr WT  + + ++ ++  141  cause, the effects of verteporfin on BxPC-3 and SU86.86 cell lines were encouraging and these two cell lines were selected for in vivo studies. The standard of care for patients with unresectable pancreatic cancer is gemcitabine, a nucleoside analog that provides a modest survival benefit of merely 5 weeks (375,376), due largely to both innate and acquired chemoresistance (377). It has been established that gemcitabine treatment stimulates autophagy. However, whether the cellular autophagic response is protective or toxic remains unclear (378–380). Highlighting interest in the role of autophagy in pancreatic cancer is the fact that two phase I/II clinical trials are currently recruiting pancreatic cancer patients for combinatorial treatment with gemcitabine and HCQ. Therefore, combining verteporfin with gemcitabine may help elucidate the role of autophagy in response to gemcitabine treatment. I confirmed that verteporfin also inhibited gemcitabineinduced autophagy by measuring autophagosome accumulation. MCF-7 EGFP-LC3 cells were exposed to 500 nM gemcitabine for 24 h in the presence or absence of 10 µM verteporfin, they were fixed, and images were taken with an automated fluorescence microscope. Gemcitabine induced autophagosome accumulation and co-treatment with verteporfin clearly prevented punctate EGFP-LC3 accumulation in response to gemcitabine (Figure 5.7), confirming that verteporfin inhibits gemcitabine-induced autophagy. The high sensitivity of two PDAC cell lines to verteporfin treatment in vitro and the inhibition of gemcitabine-induced autophagy by verteporfin was justification for studying the anti-tumour effects of verteporfin both on its own and in combination with gemcitabine in animal models of PDAC.  142  Figure 5.7 Verteporfin inhibits gemcitabine-induced autophagosome accumulation. MCF-7 EGFP-LC3 cells were exposed to 500 nM gemcitabine (Gem) for 24 h in the presence or absence of 10 µM verteporfin (VP). Cells were exposed to 0.1% DMSO or 75 µM chloroquine (CQ) in the presence or absence of 10 µM verteporfin as controls. Cells were fixed and stained with Hoechst and images were acquired using an automated fluorescence microscope (Section 2.3).  143  5.3.2  In vivo effects of gemcitabine on BxPC-3 and SU86.86 tumour growth  Since we wanted to investigate the in vivo anti-tumour efficacy of verteporfin on its own or in combination with gemcitabine, we first examined the effects of gemcitabine treatment alone on BxPC-3 and SU86.86 PDAC cell lines. Exposing BxPC-3 and SU86.86 cells to 501000 nM gemcitabine for 7 days in vitro strongly and potently inhibited cell proliferation (Figure 5.8). Moreover, images of both cell lines taken throughout the assay showed very few intact cells remained on day 7, evidence of gemcitabine cytotoxicity in vitro. Unfortunately, due to the potent anti-proliferative and cytotoxic effects of gemcitabine, it was difficult to assess whether verteporfin enhanced its effects in vitro. Combining verteporfin with gemcitabine was tested in one preliminary experiment, but no obvious increased killing was observed since the effects of gemcitabine were so profound, even at the lowest concentration (data not shown). Since its introduction to the clinic in 1997, multiple studies have shown large discrepancies between in vitro and in vivo effects of gemcitabine, largely attributed to innate and acquired drug resistance, issues with drug delivery, and heterogeneity within the tumour microenvironment (377,381,382). Therefore, it was important to also characterize the in vivo response of BxPC-3 and SU86.86 xenografts tumours exposed to gemcitabine. Rag2M mice bearing subcutaneous BxPC-3 or SU86.86 xenograft tumours were treated intraperitoneally with gemcitabine at 120 mg/kg or 240 mg/kg once a week for 4 weeks (Q7Dx4) and tumour size was monitored. Gemcitabine was highly active at both 120 mg/kg and 240 mg/kg in SU86.86 tumour xenografts. During the first two weeks of dosing, all SU86.86 tumours, both control and gemcitabine-treated, nearly doubled in tumour volume, but in the third week, tumours in the gemcitabine-treated mice stopped growing, and then  144  Figure 5.8 Gemcitabine is anti-proliferative and toxic to BxPC-3 and SU86.86 PDAC cell lines in vitro. (A) BxPC-3 and (B) SU86.86 cells were seeded in 96-well plates and treated with 50-1000 nM gemcitabine for up to 7 days. Media and drugs were replenished every third day. To monitor cell viability and proliferation, live cells were stained with Hoechst 33342 and quantified using an automated fluorescence microscope (Section 2.3).  145  Figure 5.9 Gemcitabine has varying efficacy between an SU86.86 tumour xenograft model and a BxPC-3 tumour xenograft model. (A) SU86.86 and (B) BxPC-3 tumour bearing mice were treated with saline as a control or with gemcitabine at 120 mg/kg or 240 mg/kg intraperitoneally once per week over a period of four weeks (Section 2.17). Treatment was initiated when tumours reached 100-150 mm3, at day 24 for BxPC-3 tumours and day 25 for Su86.86 tumours. Tumour growth was monitored every Monday, Wednesday, and Friday using digital callipers (Section 2.17). Tumour growth is presented as both average tumour volume (left) and relative tumour size (right), where tumor volumes were normalized with respect to the tumor volume of each mouse on the initial day of treatment and the average of each group was plotted. Arrows indicate the last day of treatment for each tumour model. (mean ± S.E.M (error bars), n=6 for all groups at the start of the study). 146  regressed for the remainder of treatment (Figure 5.8A). After treatment cessation on day 50, SU86.86 tumour growth remained stagnant until the end of the study at day 62. Overall, in response to 120 mg/kg and 240 mg/kg gemcitabine dosing, SU86.86 tumours were unable to double in tumour volume compared to the saline-treated control group that grew more than 6fold in volume from the first day of treatment to the end of the study (Figure 5.8A). By contrast, BxPC-3 tumour xenografts were significantly less sensitive to gemcitabine treatment. By the end of the study, 61 days post tumour implantation, BxPC-3 tumours exposed to saline or 120 mg/kg gemcitabine grew > 9-fold and 7-fold, respectively, over the first day of treatment. BxPC-3 tumour growth responded more to 240 mg/kg gemcitabine treatment such that tumour size increased only 5-fold by the end of the study (Figure 5.8B). The moderate dose-dependent anti-tumour effects observed in BxPC-3 xenografts in response to gemcitabine treatment established it as a good model for investigating whether verteporfin can increase gemcitabine’s activity. By contrast, the dramatic anti-tumour response to gemcitabine treatment observed in SU86.86 xenografts indicates it is an inappropriate in vivo model for testing gemcitabine drug combinations. Since gemcitabine completely inhibited tumour growth in SU86.86 xenografts, enhanced anti-tumour effects in combination with verteporfin would be difficult to characterize. 5.3.3  Verteporfin accumulates in BxPC-3 tumour tissue following single administration  Due to the slight toxicity observed in JIMT-1 tumour bearing mice after daily verteporfin dosing at 60 mg/kg Rag2M, dosing was reduced to 45 mg/kg for the BxPC-3 in vivo study. Pharmacokinetic analysis was performed to determine the amount of verteporfin in the tumour tissue after dosing Rag2M mice bearing BxPC-3 tumours one time with  147  Figure 5.10 Verteporfin tumour concentration peaks 8 h after administration and remains above its IC 50 for 24 h in BxPC-3 tumour-bearing mice. BxPC-3 tumour-bearing Rag2M mice were treated with verteporfin at 45 mg/kg intraperitoneally and tumours were harvested 2, 8, 16, and 24 h after administration for pharmacokinetic analysis. The concentration of verteporfin in tumour tissue was determined by liquid chromatography combined with mass spectrometry analysis (Section 2.19). (mean ± S.D. (error bars), n=3).  148  Figure 5.11 Verteporfin induces high-MW p62 forms in BxPC-3 tumour tissue in vivo. BxPC-3 tumour-bearing Rag2M mice were treated with verteporfin intraperitonally at 45 mg/kg and tumours were harvested 2, 8, 16, and 24 h after administration for western blot analysis (Section 2.6). (n=2 per group)  149  verteporfin at 45 mg/kg. Verteporfin was administered intraperitoneally and tumours were harvested 2-24 h later for UPLC-MS/MS analysis to determine tumour drug levels. Verteporfin tumour concentration peaked 8 h post administration at 10.4 µg/g (14.5 µM), and remained > 2.6 µg/g (3.5 µM) for 24 h (Figure 5.10). This pharmacokinetic data illustrates that dosing with 45 mg/kg verteporfin achieves tumour drug levels comparable to those that elicited anti-proliferative and autophagy-inhibiting effects in vitro. 5.3.4  Verteporfin treatment induces high-MW p62 forms in BxPC-3 tumour tissue in vivo  Having established that verteporfin reached BxPC-3 tumour tissue at concentrations used in vitro, I wanted to determine whether it had similar effects on p62. Tumours collected for pharmacokinetic analysis were cut in half prior to analysis, and one half was used for western blot analysis of p62. Immunoblotting for p62 showed that in vivo verteporfin treatment induced the appearance of high-MW p62 (Figure 5.11). One of the tumours harvested from control mice (Ctl left), treated intraperitoneally with DSPE-PEG micelles, showed an intense dark smear in the p62 immunoblot and no tubulin, suggesting its protein content was largely degraded during preparation; therefore, only the second control (Ctl right) was considered for analysis. Only 60 kDa p62 was detected in this control tumour. 2 h after verteporfin administration, small amounts of high-MW p62 were detected along with 60-kDa p62 (Figure 5.11). Furthermore, the intensity of the high-MW p62 forms detected increased dramatically 8 h post administration, which correlated with the pharmacokinetic analysis. Interestingly, the amount of verteporfin detected in tumours 2 h and 24 h post administration was about equal (Figure 5.10), but significantly more high-MW p62 was detected after 24 h compared to 2 h, implying that high-MW p62 remains in the tumour for  150  some time after drug clearance. Since the final sample was taken 24 h post drug administration, it remains unknown how long high-MW p62 can be detected in tumours after a single drug administration. The fact that verteporfin administration induces high-MW p62 in PDAC tumour tissue demonstrates that it elicits similar cellular effects in vivo as seen in vitro. Additionally, since high-MW p62 forms have only been detected when verteporfin inhibits autophagy, it is an indirect indication that autophagy is being modulated in the tumour tissue. 5.3.5  Verteporfin moderately enhances the anti-tumour activity of gemcitabine in BxPC-3 xenografts  To determine whether inhibiting autophagy with verteporfin is a potential therapeutic approach against PDAC, Rag2M mice bearing subcutaneous BxPC-3 tumour xenografts were dosed with verteporfin on its own or in combination with gemcitabine. Gemcitabine was also administered as a single agent to compare verteporfin results with the current standard of care. When tumours reached 100-150 mm3, 25 days post tumour inoculation, the mice were separated into different cohorts, and treatment was commenced. Animals were dosed intraperitoneally Monday, Wednesday, and Friday for 4 weeks with 45 mg/kg verteporfin under low light conditions while gemcitabine was administered intraperitoneally once weekly for 4 weeks at 120 mg/kg or 240 mg/kg. Animals in the control group were treated with the delivery vehicle DSPE-PEG micelles at the same concentration and the same dosing schedule as the verteporfin-loaded DSPE-PEG. To monitor anti-tumour effects, tumour volumes were measured 3 times a week over the course of the study. After the last day of treatment, 50 days post tumour inoculation, animals continued to be monitored until their tumours reached a volume of 800 mm3, the humane  151  endpoint as dictated by the UBC animal protocol. No toxicity was observed in any of the treatment groups. Treatment with verteporfin alone had minimal effect on tumour volume and animal survival. At day 57, tumours exposed to verteporfin increased in relative size 7.1fold over the first day of treatment compared to the 8.4-fold increase observed in control mice (Figure 5.12 A,B). During the course of treatment, verteporfin initially appeared to slow tumour growth, delaying the time for tumours to increase 2-fold over its size at the beginning of treatment by an average of 5 days. However, by the time tumours doubled in size again, the delay was only 3 days, and by the end of the experiment very little difference in growth was observed. A significant anti-tumour effect was observed in mice treated with 120 mg/kg or 240 mg/kg gemcitabine as a single agent (Figure 5.12, top). One week after the conclusion of treatment, day 57, tumours exposed to 120 mg/kg and 240 mg/kg gemcitabine showed relative tumour volume increases of 5.5- and 6- fold, respectively, over the course of the study, both statistically significant (p<0.05, days 53 and 55) (Figure 5.12, top). Kaplan-Meier survival curves were generated by monitoring the number of surviving mice after treatment (no mice died naturally - mice were terminated when tumours reached 800 mm3). None of the groups treated with a single agent showed an obvious survival advantage over the control, but in all three treatment regimens at least one animal survived to 71 days, 4 days more than in the control group (Figure 5.12A,B bottom), showing a small effect. Based on both relative tumour growth and Kaplan-Meier survival curves, exposing BxPC-3 xenograft tumours to verteporfin alone resulted in no improved efficacy over the current standard of care, gemcitabine. The anti-tumour efficacy was also analyzed in response to combination treatments with verteporfin and gemcitabine. Comparing relative tumour growth on day 57, tumours exposed  152  to verteporfin and 120 mg/kg gemcitabine showed a relative tumour volume increase of 5.2fold over the first day of treatment, and those exposed to verteporfin and 240 mg/kg gemcitabine showed a 4.7-fold increase (Figure 5.12A-B, top), almost 50% less than that observed in the control group. Treatment with verteporfin and 120 mg/kg gemcitabine did not show an enhanced response compared to gemcitabine alone (Figure 5.12A top). However, tumours exposed to verteporfin and 240 mg/kg gemcitabine grew noticeably slower throughout the entire study, producing a highly significant effect on relative tumour growth compared to the control (p<0.01, days 53 to 60). Therefore, mice treated with verteporfin and 240 mg/kg gemcitabine responded better than any single-agent treatment group and showed the most promising anti-tumour responses compared to the control group. Comparing Kaplan-Meier survival curves, treatment with gemcitabine or verteporfin as single agents caused a modest survival benefit of 4 days, and combining verteporfin with 120 mg/kg gemcitabine extended that by about 5 days (Figure 5.12A, bottom). However, upon further scrutiny, it appears that until day 74, the survival curve for treatment with verteporfin and 120 mg/kg gemcitabine was practically identical to 120 mg/kg gemcitabine alone, and the apparent 5 day survival benefit is representative of only 2 out of 8 mice in the group (Figure 5.12A, bottom). In contrast, an obvious survival advantage of about 10 days was conferred by treatment with verteporfin and 240 mg/kg gemcitabine compared to 240 mg/kg gemcitabine alone, a 14 day advantage over the vehicle control (Figure 5.12B, bottom). The arm of the Kaplan-Meier curve representing the combined treatment regimen of verteporfin and 240 mg/kg gemcitabine shifted to the right and did not overlap with any other treatment arm, depicting a convincing survival advantage over the control and all other treatments tested (Figure 5.12A,B bottom).  153  154  Figure 5.12 Combining verteporfin treatment with gemcitabine in a BxPC-3 tumour model inhibits tumour growth and increases survival. BxPC-3 tumour-bearing Rag2M mice were given i.p. administrations of (A) a combination of 45 mg/kg verteporfin (VP) and 120 mg/kg gemcitabine (Gem) or each drug on its own or (B) a combination of 45 mg/kg verteporfin (VP) and 240 mg/kg gemcitabine (Gem) or each drug on its own for a period of four weeks. Treatment was initiated when tumours reached 100150 mm3, day 25 (Section 2.17). DSPE-PEG micelles without drug were delivered as the control. Tumour growth is presented as both average tumour volume (top) and relative tumour size (middle), where tumor volumes were normalized with respect to the tumor volume of each mouse on the initial day of treatment and the average of each group was plotted. Kaplan-Meier survival curves (bottom) illustrate the number of surviving mice in treatment group post drug administration. Arrows indicate the last day of treatment. (** p<0.01)(mean ± S.E.M (error bars), n=8 for all groups at the start of the study).  155  In summary, combining verteporfin with 240 mg/kg gemcitabine enhanced the anti-tumour effects of either drug on its own. The observed enhanced efficacy and survival advantage using verteporfin, an autophagy inhibitor, in the presence of gemcitabine reinforces the notion that gemcitabine-induced autophagy is a cytoprotective response, autophagy inhibition is a promising therapeutic target, and verteporfin has potential therapeutic applications in combinatorial therapy that should be explored further. It should be noted that none of the treatments showed tumour regression; however, in the context of pancreatic cancer, which has a 5-year survival rate of only 6% (383,384), any advancement in therapeutic efficacy should be considered valuable, and is worthy of further exploration. 5.3.6  Discussion  This chapter explores the anticancer therapeutic potential of verteporfin using two different mouse xenograft tumour models: a JIMT-1 breast cancer model and a BxPC-3 pancreatic cancer model. Neither verteporfin nor gefitinib was efficacious against JIMT-1 tumour growth as a single agent despite both drugs showing anti-proliferative effects in vitro. Considering that JIMT-1 cells have been characterized as showing multiple mechanisms for drug resistance in addition to HER2 gene amplification and EGFR overexpression, the negative response to either drug alone was not entirely surprising (366). A recent report by Dragowska et al. using the same gefitinib dose delivered on a similar schedule to our study confirmed its inability to inhibit JIMT-1 xenograft growth (385). In vitro exposure to 10 µM verteporfin strongly inhibited JIMT-1 cell proliferation, but < 10 µM showed very little effect. Despite confirmation that tumour drug levels reached > 10 µM for a 8-10 h period after a single verteporfin administration, no growth delay was observed. The amount of drug detected in the tumour by pharmacokinetic analysis is an average of the entire tumour tissue,  156  and due to the complex 3-dimensional tumour structure, the microenvironment is not uniform, and drug distribution is variable throughout the tissue (386). Another consideration is that verteporfin administration was completely stopped the second week of treatment and was resumed at a reduced 45 mg/kg dose the third week. Therefore, it is likely that throughout the course of treatment much of the tumour was exposed to < 10 µM verteporfin, which showed low-moderate in vitro effect, perhaps contributing to the lack of in vivo efficacy observed by verteporfin treatment alone. Combining gefitinib with verteporfin did not enhance the anti-tumour effects of either drug, indicating inhibiting autophagy in vivo does not sensitize JIMT-1 cells to gefitinib. The facts that PK data showed tumour accumulation of verteporfin exceeding its in vitro IC 50 for autophagy inhibition of 1 µM for the entire 24 h post administration period, and that significant amounts of high-MW p62 were detected during that period, suggest that autophagy inhibition was achieved in vivo by a single 60 mg/kg dose. To date, there are no reliable markers for autophagy modulation in vivo, so the appearance of high-MW p62 as a marker of verteporfin activity in tumour tissue was extremely useful. The lack of observed tumour growth delay after the first week with daily administration of both gefitinib and verteporfin indicates that autophagy inhibition did not sensitive JIMT-1 cells to gefitinib. However, the unanticipated changes in frequency and dose of verteporfin administration over the following weeks make it unclear whether autophagy-modulating levels of verteporfin were maintained longer-term. While our results imply that autophagy inhibition is not an effective strategy for sensitization of JIMT-1 tumours to HER2 targeted therapy, a recent report showing ATG12 silencing by shRNA robustly sensitized JIMT-1 tumour xenografts to TZ treatment appears  157  to contradict our results (387). One explanation for this apparent discrepancy is prolonged autophagy inhibition by RNA interference throughout the entire course of treatment, which was likely not achieved in our study due to dosing complications. However, a more likely explanation can be attributed to a key difference in the experimental design. In the study by Cufi et al., xenografts were inoculated with ATG12shRNA-JIMT-1 cells, and TZ treatment was initiated 7 days after inoculation, when tumour volume was much less than 100 mm3. By inoculating mice with autophagy deficient cells, this study actually assessed the contribution of autophagy to initial JIMT-1 tumour progression and to intrinsic resistance to HERtargeting drugs exhibited by JIMT-1 cells (387). In contrast, by evaluating whether sensitization to a HER-targeting drug could be achieved through autophagy modulation of an established tumour at the same time as gefitinib treatment, we evaluated the contribution of protective autophagy elicited by gefitinib treatment, and whether its modulation increased efficacy. However, to provide conclusive evidence on the latter, verteporfin dosage needs to be optimized such that autophagy inhibition can be maintained throughout the course of treatment. Although the JIMT-1 xenograft model gave negative anticancer results, it demonstrated that autophagy-modulating amounts of verteporfin can be achieved and monitored in vivo. Pancreatic cancer has recently been described as addicted to autophagy for growth (163), and elevated levels of autophagy have been linked to poor patient outcome (368). While encouraging data showing autophagy inhibition by CQ inhibited PDAC growth in vitro and in vivo have been reported, the results are variable among PDAC cell lines and xenograft models (163,388), demonstrating a need for further study with other clinically relevant autophagy inhibitors.  158  From a panel of 8 PDAC cell lines, only BxPC-3 and SU86.86 cells were determined to be highly sensitive to long-term verteporfin treatment, showing a clear dose response where 5 µM inhibited cell survival by ~50% and 10 µM completely inhibited cell proliferation. The observation that PDAC cell lines showed variable sensitivity to long-term in vitro verteporfin treatment implies that there is more complexity to the claim that PDAC is addicted to autophagy for proliferation. This notion is supported by the existing literature reporting variable PDAC response to CQ-mediated autophagy inhibition (163,388). In light of recent discoveries presented in Chapter 4 regarding p62 oxidation by verteporfin, it would be interesting to investigate the amount of oxidative stress in each of the cell lines. Perhaps sensitivity to autophagy inhibition by verteporfin is determined by the cellular balance between cellular dependence on autophagy, basal oxidative stress, and the amount of p62. Uncovering the mechanism(s) underlying verteporfin sensitivity was not explored further in this thesis, but is an interesting topic for further study. Characterization of BxPC-3 and SU86.86 cells revealed BxPC-3 cells to be the most suitable for testing verteporfin on PDAC xenograft tumour growth in vivo. Both cell lines were extremely sensitive to in vitro exposure to gemcitabine at all concentrations, but that response was recapitulated in vivo only in the SU86.86 tumour xenograft. BxPC-3 xenografts showed a moderate dose-dependent delay in tumour growth, but even the more effective dose at 240 mg/kg showed a remarkably small effect, particularly considering its potency in vitro. This result illustrates one of the hurdles in pancreatic cancer research, and cancer research in general, where promising in vitro results cannot be extended in vivo (381,388,389). For the purposes of our study, the less responsive BxPC-3 xenograft model was better-suited since it  159  provided a therapeutic window for evaluating whether autophagy inhibition by verteporfin could enhance gemcitabine efficacy. Verteporfin failed to show any anti-tumour effect on BxPC-3 xenografts in vivo, despite evidence the drug accumulated at the tumour site for 18 – 20 h at concentrations (5-10 µM) that affected cell proliferation in vitro. As mentioned previously, one caveat of pharmacokinetic data is that it measures the average drug accumulation across tumour tissue, but due to the 3-dimensional structure and complex tumour microenvironment, there is likely a heterogeneous accumulation of verteporfin. This is a challenge for drugs targeting autophagy because the dense, poorly vascularized, hypoxic tumour tissue that is highly dependent on autophagy (165) and susceptible to autophagy modulation is also less accessible to drugs. We hoped that using a cancer model previously described as addicted to autophagy for growth that also showed in vitro verteporfin sensitivity might be particularly susceptible to in vivo autophagy modulation, perhaps overcoming issues of drug targeting within the tumour site. The generation of high-MW p62 in BxPC-3 tumour tissue as a marker for in vivo verteporfin activity suggested autophagy was inhibited within 24 h of drug administration, but this effect alone was not enough to hinder tumour growth. In addition to our study, a recent report describes two cancer cell lines that gave encouraging in vitro results, but negative anti-tumour responses to autophagy inhibition in vivo, one being BxPC3 (388). Based on our findings and others, two issues have arisen with this strategy: 1) it is apparent that PDAC dependence on autophagy is highly variable among cell lines; 2) even extremely encouraging in vitro data is not necessarily indicative of in vivo success. Whether the latter is due to issues with drug delivery in vivo may be overcome by higher or more frequent doses or better drug delivery technology. However, due to the large number of  160  variables involved, targeting autophagy as a single strategy is an unlikely prospect for combating pancreatic cancer. The most encouraging in vivo anticancer response to verteporfin was observed in combination with gemcitabine where tumour growth was clearly inhibited and a significant survival advantage was conferred. This result supports a cytoprotective role for gemcitabineinduced autophagy, in contrast with other reports claiming autophagy contributes to tumoural cell death through VMP-1 – mediated selective autophagy (378,379). There are a number of experimental differences between the aforementioned studies and ours that may contribute to the conflicting results, particularly the use of 3-MA to inhibit autophagy, which has a number of other targets, and is unsuitable for in vivo use. Further studies using multiple autophagy inhibitors and PDAC xenograft models may clarify the role of autophagy in response to gemcitabine treatment. Modifying the dose and frequency of verteporfin administration in the BxPC-3 xenograft study successfully eliminated toxicity issues that were of concern in the JIMT-1 study, and allowed for 4 weeks of uninterrupted treatment. These modifications presumably enabled prolonged autophagy inhibition, which had been a concern in the JIMT-1 study. Since pancreatic cancer is such an aggressive disease, any improvement in therapy should be pursued further. Therefore, additional optimization of the frequency, amount, and timing of verteporfin administration should be explored. With regards to targeting autophagy as a therapeutic strategy, our results using JIMT-1 and BxPC-3 xenograft models suggest that the type of cancer is an important determinant of efficacy. A second variable in in vivo studies is optimization of the dosing concentration and frequency of drug administration. For combination studies, the relative timing of drug  161  administration is also very important and should be explored further. Finally, considering autophagy functions to alleviate cellular stress, its regulation is highly influenced by the extracellular environment. Therefore, assessing the role of autophagy in cancer progression and chemotherapeutic resistance likely depends heavily on the tumour microenvironment in the animal model used. While there are a number of advantages to using human tumour xenografts such as relative ease generating the tumour and monitoring its growth, the xenografts do not maintain the same structure and tumour microenvironment found clinically (390,391). For the purposes of studying autophagy modulation in vivo the tumour microenvironment should be carefully considered, and representative tumour models, such as orthotopic xenografts or genetically engineered mice, may provide more conclusive results.  162  CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS  6.1  High-throughput screen for early autophagy inhibitors Autophagy is a conserved intracellular catabolic process responsible for the bulk  degradation of organelles and long-lived proteins. Upon induction, cytoplasmic components are sequestered in double-membraned autophagic vesicles, digested in lysosomes, and recycled to the cytoplasm. Autophagy enables cells to degrade and recycle cytoplasmic materials both as a housekeeping mechanism and in response to extracellular stress such as nutrient deprivation. As a cytoprotective mechanism, autophagy protects tumour cells from both environmental and therapeutic stresses; therefore, inhibition of autophagy is considered as a promising anticancer strategy. Due to a lack of pharmacological autophagy inhibitors, my first objective was to develop a high-throughput cell-based assay for early inhibitors of autophagy. While several screens for autophagy inducers have been described (28,241), this was the first screening assay for compounds that prevent autophagosome formation. After screening the Prestwick collection, composed of > 3,500 off-patent drugs and pharmacological agents, verteporfin, an FDAapproved benzoporphyrin derivative used for photodynamic therapy, was the only active compound. It is noteworthy that no other inhibitors of autophagosome formation have been reported in the literature. Considering the low hit-rate observed in my screen of known pharmacological compounds, it may be advantageous to screen natural product collections instead of the more commonly used chemical libraries for novel small molecules with autophagy inhibiting activity. Our laboratory has screened ~1,500 marine extracts, which led to the identification  163  of four new potential inhibitors of autophagosome accumulation: aerothionine, homoaerothionine, cribrostatin 6, and mimosamycin (data not shown). All four compounds were confirmed to inhibit chloroquine-induced autophagosome accumulation at > 10-30 uM. At this time, the effects of these compounds have not been pursued further due to limited amounts of material. However, in the search for novel potent inhibitors of autophagy they may be revisited by future laboratory members. 6.2  Identification and characterization of verteporfin as an inhibitor of autophagosome formation via singlet oxygen  6.2.1  Verteporfin inhibits autophagosome formation  In Chapter 3, I have described results showing the identification and initial characterization of verteporfin as an inhibitor of autophagosome formation. Inhibition of autophagosome accumulation by chloroquine has an IC 50 of 1 µM, showing complete autophagy inhibition at 10 µM. I have shown verteporfin inhibits autophagy induced by all chemical stimulators tested thus far, implying that it targets the autophagic machinery rather than upstream effectors. This was confirmed through experiments showing that verteporfin does not affect LC3 lipidation or membrane association, both of which occur after induction of the isolation membrane and prior to autophagosome closure (12,29,67). Ultrastructural analysis confirmed that autophagosomes were not formed in the presence of verteporfin, either alone or in combination with CQ. The appearance of single membrane vesicles after verteporfin exposure led to the hypothesis that verteporfin disrupts the dynamics of proper autophagosome formation, perhaps causing the double membrane to ‘pop out’, generating an empty single membrane vesicle instead (298). This discovery is significant since all other chemical autophagy inhibitors traditionally used either affect lysosomal function or inhibit  164  the PI3K pathway; therefore, they are not selective and many have pharmacologically undesirable properties (181,183). Whether these single-membraned vesicles are remnants of failed autophagosome formation by verteporfin remains unknown. Since LC3II associates with membranes, even in the presence of verteporfin, we may be able to localize EGFP-LC3II to those singlemembrane vesicles, using immunogold anti-GFP labeling. Immunogold electron microscopy can be a technically laborious process, and autophagosome membranes have been difficult to study due to the dynamic nature of autophagy and the relatively low protein:lipid ratio (58,261). Furthermore, this approach is complicated by the fact that EGFP-LC3 is also cytosolic, making it difficult to discern between the two forms in the absence of mature autophagosomes. More sensitive and integrative microscopy techniques, such as Correlative Light and Electron Microscopy, which was recently used in to monitor the live-cell dynamics of LAMP-1 in endosomes (392) may provide more information, but these approaches require specialized equipment and technical expertise. As the only known inhibitor of autophagosome formation, verteporfin may be a useful tool for exploring the expansion of the autophagosome membrane using sensitive integrative microscopy. 6.2.2  Verteporfin produces oxidized high-MW p62 products via singlet oxygen production  Results reported in Chapter 4 demonstrate the generation of high-MW p62 by verteporfin, and explores its role in mediating autophagy inhibition. The observation that verteporfin causes the formation of altered high-MW forms of p62 that resist denaturing conditions led to the identification of p62 as a target of verteporfin. As a photodynamic agent, verteporfin is known to generate singlet oxygen upon light irradiation, which elicits a  165  number of secondary ROS, and causes cellular apoptosis or necrosis (255). In the absence of light, verteporfin has been characterized as non-toxic (257), but its non-photoactivated effects have not been described further. I determined that, even in the absence of light, verteporfin generates high-MW p62 through singlet oxygen generation and that this is a direct chemical reaction. Due to their cellular abundance, location, and the presence of chromophores in amino acid side chains, proteins are the major target of singlet oxygen, directly causing protein oxidation, which often results in protein crosslinking (286,287). Having established that singlet oxygen mediates the generation of high-MW p62 in the absence of other cellular constituents and that this effect correlates with increased carbonyl content strongly implies that observed high-MW p62 is a product of p62 crosslinking. While other studies have observed high-MW p62 (331,340), its presence was assumed to reflect p62-positive protein aggregates only. My study is the first to show that high-MW p62 is an oxidized product, and that high-MW p62 does not necessarily represent protein aggregation. I suspect that short-term verteporfin treatment does not produce obvious cytoplasmic aggregates detected by immunofluorescence, such as those observed when the proteasome or lysosome is inhibited, because its ability to sequester cytoplasmic misfolded proteins is compromised. I would like to characterize this further by using ultracentrifugation to fractionate the cell lysate to see if high-MW p62 is detected in both soluble and insoluble fractions. While characterizing the effect of singlet oxygen on p62, I observed that exposure of purified protein or p62 immunoprecipitate to light or heat alone produced high-MW p62, even in the absence of singlet oxygen generators. Likewise, exposure to thermal energy at 37°C oxidized purified His-p62 in the absence of drug, suggesting p62 is susceptible to  166  oxidation. The fact that I also observed high-MW p62 from untreated cells leads me to suspect that its oxidation is physiologically relevant. Since p62 also has an established role in the cellular antioxidant response, it is possible that, like Keap1, it is intrinsically sensitive to oxidation, and functions as a sensor of oxidative stress. To begin addressing this issue, I propose exposing cells to other sources of ROS such as hydrogen peroxide and superoxide and monitoring the state of p62. In this manner, we can determine whether p62 crosslinking is a consequence of multiple sources of oxidative stress or if it is specific to singlet oxygen. To characterize the potential role of high-MW p62 in redox signaling, I would like to explore its function under conditions producing high-MW p62 crosslink products, particularly in the Keap1-Nrf2 pathway (145,279). Zinc finger motifs present in transcription factors like Sp1 and the glucocorticoid receptor have been shown to be redox sensitive, which affects DNA-protein binding (393,394). Since p62 has a zinc finger motif, I suspect this region is oxidized by verteporfin. To identify the sites of p62 oxidation by verteporfin, I propose using biotin probes that contain a hydrazine-like moiety that will react with carbonyl groups (395). By exposing purified p62 to either heat alone or verteporfin and using tryptic digestion and streptavidin beads to capture carbonylated p62 peptides, we can use mass spectrometry to identify specific sites of oxidation (396). To fully characterize the mechanism of verteporfinmediated p62 oxidation, it will be interesting to determine whether verteporfin merely enhances naturally occurring oxidation by thermal energy or whether it oxidizes distinct residues.  167  6.2.3  High-MW p62 shows decreased binding to poly-ubiquitinated cargo  Protein oxidation and crosslinking have been shown to disrupt protein function through a number of mechanisms including the modification of an amino acid important for catalytic function, susceptibility to misfolding, and by preventing proper binding to cofactors and/or substrates (302,312,314). Co-immunoprecipitation experiments revealed that the generation of high-MW p62 by verteporfin decreased binding of p62 to poly-ubiquitinated cargo, reflecting impaired p62 function due to protein oxidation and/or crosslinking. This observation led to my hypothesized model of singlet oxygen-mediated autophagy inhibition due to the presence of bulky p62 crosslinks that disrupt proper autophagosome formation. To prove empirically that the presence of high-MW p62 disrupts autophagosome formation, it is important to test whether verteporfin inhibits autophagosome formation in cells lacking p62. Autophagy can still occur in the absence of p62, but all our experiments were done in cells expressing p62, so it is not known whether p62 is required for verteporfinmediated autophagy inhibition. In my upcoming work, I would like to knockdown p62 using siRNA and determine whether autophagy is inhibited. If autophagy is still inhibited in those cells, it would be worth blotting for other mediators of selective autophagy, such as NBR1 and NDP52, which contain similar domains to see if they produce high-MW crosslink products. Along these lines it would also be informative to determine whether p62 oligomerization, which is required for its function in autophagy facilitates crosslinking, and therefore, autophagy inhibition. To explore this, we can express a p62 K7A/D69A double mutant, which is unable to self-oligomerize, and monitor verteporfin-mediated autophagy inhibition. These experiments will expand our knowledge regarding the specificity and selectivity of both p62 crosslinking and inhibition of autophagy by verteporfin.  168  As a scaffolding protein, p62 functions to bring the right proteins together at the right time. Whether verteporfin-induced high-MW p62 prevents its association with other interactors needs to be explored. p62 immunoprecipitates should be collected from untreated and treated cells, and probed for the presence of p62 binding proteins including NBR1, TRAF 6, raptor, and Keap1. Since many of these proteins bind to different domains of p62, these results may offer insight into which domains are susceptible to oxidation. Moreover, considering p62 is inducible upon oxidative stress, potential disruption of binding to some associated proteins should be determined after short-term and long-term treatment. To further characterize the effects of singlet oxygen on p62 function, we can monitor the expression of Nrf2 and NQO1, two downstream proteins of the Keap1-Nrf2 signaling pathway. This should also be addressed under different types of cellular stress, particularly those involving ROS production and signaling, such as hypoxia and starvation. Any differences observed may provide a rationale for variable sensitivity to verteporfin observed in PDAC cell lines. 6.2.4  Singlet oxygen is the mechanism underlying autophagy inhibition by verteporfin  Characterization of high-MW p62 by verteporfin via singlet oxygen production using rose bengal led to the discovery that rose bengal also inhibits autophagy. Throughout my studies, I consistently observed a correlation between the generation of high-MW p62 and inhibition of autophagy. Notably, rose bengal does not enter cells in the presence of serum; therefore, it was unable to inhibit autophagy in complete medium, and there was no evidence of high-MW p62 generation in these conditions. In the absence of serum, cells treated with rose bengal contained high-MW p62 and autophagy was successfully inhibited. These results suggest that the mechanism of verteporfin-mediated autophagy inhibition is not specific to  169  the drug itself, but it is dependent on the production of singlet oxygen. Therefore, this represents a novel mechanism of autophagy inhibition. Characterization of other singlet oxygen producers, such as proflavine, acridine orange, hypericin, and photofrin may offer additional insight regarding the specificity of verteporfin. While I suspect that verteporfin induces the release of a small amount of singlet oxygen in the absence of light, determining the amount released may provide useful information for identification of other potential inhibitors and optimization of verteporfin as an autophagy inhibitor, particularly for clinical use. The most common analytical technique for measuring singlet oxygen is electron resonance spectroscopy, which measures the conversion of a spin-trapping agent TMPD to its stable nitroxide radical adduct TAN (326). These studies are out of the scope of our work, but may be addressed by other laboratories. 6.3  Characterization of the anti-tumour effects of verteporfin in vivo  6.3.1  Verteporfin does not show anti-tumour efficacy on its own in vivo  Exploration of the anti-tumour efficacy of verteporfin as a single agent gave negative results in two different xenograft tumour models: a JIMT-1 breast cancer model and a BxPC3 pancreatic cancer model. Supporting pharmacokinetic studies revealed that a dose of 45 mg/kg verteporfin accumulated in tumour tissue at concentrations that inhibit autophagy in vitro and it produced high-MW p62 in tumour tissue, implying that autophagy was inhibited. Therefore, it appears that autophagy inhibition as a single treatment is not efficacious against tumour growth. While previous studies have shown that dense tumour tissue is hypoxic and nutrient-starved, thus relying on autophagy for growth (165), that tissue is also presumably difficult for the drug to access, making it a difficult therapeutic avenue to pursue further.  170  6.4  Verteporfin enhances the anti-tumour effect of gemcitabine in a BxPC-3 tumour xenograft model Combining verteporfin treatment with gemcitabine showed encouraging results where  the anti-tumour growth of gemcitabine was enhanced by autophagy inhibition and a survival advantage was observed over any single treatment course. These results suggest that autophagy protects BxPC-3 cells from gemcitabine treatment. This is particularly encouraging in the context of pancreatic cancer, which has such a poor prognosis. A major advantage of verteporfin is that it is already approved for use in humans and could progress rapidly to clinical trials. Therefore, this effect on PDAC in combination with gemcitabine should be explored further and optimized, if possible. More representative in vivo tumour models, such as orthotopic pancreatic tumours that are more likely to reflect the tumour environment and structure should be pursued. Additionally, having overcome initial toxicity issues with verteporfin dosing, it may be optimized further to increase its accumulation in tumour tissue. This work may be done with the CDRD in the near future. Preliminary in vitro characterization of PDAC to long-term autophagy inhibition showed large variations in verteporfin sensitivity, and BxPC-3 was one of the most sensitive. It should be noted that most pancreatic cancers are characterized by oncogenic K-Ras activation, but BxPC-3 cells uncharacteristically express wild type Ras (371). In the presence of inflammatory stimuli, which are common in PDAC development, NF-κB participates in a feed-forward mechanism that amplifies Ras to pathological levels, and involves p62 (144). The treatment of activated K-Ras PDAC with verteporfin is attractive since it would target autophagy and p62 simultaneously. Despite the fact that many K-Ras activated PDAC cell lines were insensitive to verteporfin in vitro, no combinations with gemcitabine were tested.  171  To truly explore the therapeutic potential of verteporfin in PDAC, it is imperative to test it in combination with gemcitabine in a K-Ras activated PDAC model that is more relevant to the clinic. This should tell us whether inhibiting gemcitabine-induced autophagy should be broadly applied to PDAC or if it is specific to a subset of PDAC subtypes. To enhance our understanding of in vivo verteporfin treatment, tumour sections should be preserved for immunohistochemistry using p62 and carbonyl-specific probes for confocal microscopy. Monitoring the amount of oxidized p62 in representative tumour sections may provide information on drug perfusion and the amount of oxidative stress in the tumour, both of which presumably contribute to in vivo efficacy. The work presented in this thesis demonstrates how a high-content screen can lead to the identification of a powerful biological probe with therapeutic potential. Verteporfin is the only inhibitor of autophagosome formation identified thus far, and its characterization surprisingly revealed p62 as a target of singlet oxygen as the mechanism of inhibition. As a mediator and substrate of selective autophagy, p62 is not an obvious target for autophagy modulation since it is not part of the core machinery responsible for autophagosome biogenesis. 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