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Heat shock protein 27 inhibits the Hippo tumor suppressor pathway by facilitating MST1 proteasomal degradation Vahid, Sepideh 2016

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HEAT SHOCK PROTEIN 27 INHIBITS THE HIPPO TUMOR SUPPRESSOR PATHWAY BY FACILITATING MST1 PROTEASOMAL DEGRADATION  by  Sepideh Vahid Pharm.D., Tehran University of Medical Sciences, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  January 2016 © Sepideh Vahid, 2016 ii  Abstract  Heat shock protein 27 (Hsp27) is a molecular chaperone highly and ubiquitously expressed in aggressive cancers where it controls a variety of pro-tumorigenic signaling pathways. Using gene expression profiling in prostate cancer cells with loss of Hsp27 function, we identified for the first time that Hsp27 regulates target genes in signaling pathways dependent on YAP and TAZ. Suppression of these transcriptional co-activators occurs via their phosphorylation and cytoplasmic retention by the Hippo tumor suppressor pathway. Mechanistic studies revealed that Hsp27 expression is associated with reduced YAP phosphorylation and enhanced transcription of YAP/TAZ target genes. Examination of the core components of the Hippo kinase cascade revealed that Hsp27 facilitates the proteasomal degradation of the core Hippo kinase, MST1, leading to reduced phosphorylation/activity of other main kinases responsible for YAP phosphorylation/inactivation, LATS1 and MOB1.  Importantly, our data from cell lines was supported by data from human tumors; clinically, high expression of Hsp27 correlates with increased expression of YAP target genes in prostate cancer as well as reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. Together, our data reveal a novel mechanism by which Hsp27 regulates the Hippo tumor suppressor pathway, providing further rationale to target Hsp27 in multiple cancers.     iii  Preface The data presented in this thesis are based on the work that I carried out during the completion of my MSc program.  A version of this thesis has been submitted to be published and is currently under review as noted below:  Hsp27-dependent degradation of MST1 leads to inactivation of the Hippo tumor suppressor pathway in cancer. Sepideh Vahid, Daksh Thaper, Kate Gibson, Martin E. Gleave, Jennifer L. Bishop, Amina Zoubeidi Dr. Zoubeidi was the principal investigator in this study. The study concept and design as well as data interpretation was conducted by myself, Daksh Thaper, Dr. Bishop and Dr. Zoubeidi. I performed all of the experiments presented in this thesis, while Daksh Thaper and Kate Gibson provided technical support. I drafted the original manuscript, which was reviewed by Daksh Thaper, Dr. Bishop and Dr. Zoubeidi.  Dr. Gleave provided Hsp27 antisense oligonucleotide (OGX-427) for one of the experiments in this thesis. He is listed as one of the inventors on the patent for OGX-427 submitted by the University of British Columbia and licensed to OncoGenex Technologies, a Vancouver-based company that Dr.Gleave has founding shares in.  I and others involved in the following thesis project declare no conflict of interest.    iv  Table of Contents  Abstract ......................................................................................................................................................... ii Preface ......................................................................................................................................................... iii Table of Contents ......................................................................................................................................... iv List of Tables ............................................................................................................................................... vii List of Figures ............................................................................................................................................. viii List of Abbreviations ..................................................................................................................................... x Acknowledgments ....................................................................................................................................... xii Dedication ................................................................................................................................................... xiv 1. Introduction .......................................................................................................................................... 1 1.1. Heat Shock Proteins ...................................................................................................................... 1 1.1.1. Biology, function and transcription....................................................................................... 1 1.1.2. Small Heat Shock Proteins .................................................................................................... 2 1.1.3. Heat Shock Protein 27 ........................................................................................................... 2 1.1.4. Hsp27 in human cancer......................................................................................................... 3 1.1.5. Hsp27 and oncogenic signaling pathways ............................................................................ 4 1.1.6. Targeting Hsp27 .................................................................................................................... 4 1.2. Hippo Tumor Suppressor Pathway ............................................................................................... 5 1.2.1. Core components .................................................................................................................. 5 1.2.2. Biology and function in mammals ......................................................................................... 6 1.2.3. Upstream regulators of the Hippo pathway ......................................................................... 7 1.2.4. Hippo tumor suppressor pathway in cancer ......................................................................... 8 1.3. Hypothesis Formation ................................................................................................................... 9 1.3.1. Gene expression profiling as a method to paint a global picture of cellular function.......... 9 1.3.2. Ingenuity Pathway Analysis ................................................................................................ 10 1.3.3. Hypothesis ........................................................................................................................... 12 2. Materials and Methods ....................................................................................................................... 13 v  2.1. Cell culture .................................................................................................................................. 13 2.2. Transfections ............................................................................................................................... 13 2.2.1. Small interfering RNA transfection ..................................................................................... 13 2.2.2. Plasmid transfection for overexpression ............................................................................ 13 2.3. Gene Expression profiling ........................................................................................................... 13 2.4. Western blotting ......................................................................................................................... 14 2.5. Immunoprecipitation .................................................................................................................. 14 2.6. Reagents and antibodies ............................................................................................................. 14 2.7. Immunofluorescence .................................................................................................................. 15 2.8. Quantitative real time PCR (qRT-PCR) ........................................................................................ 15 2.9. Luciferase assay .......................................................................................................................... 15 2.10. Public data mining ................................................................................................................... 16 2.10.1. CBioportal for Cancer Genomics ......................................................................................... 16 2.10.2. The UCSC Xena Genome Browser (http://genome.ucsc.edu/) ........................................... 16 2.11. Statistical analyses .................................................................................................................. 16 3. Results ................................................................................................................................................. 17 3.1. Identification of Hsp27 as a possible regulator of YAP/TAZ activity ........................................... 17 3.1.1. The effect of Hsp27 knockdown on YAP/TAZ activity in prostate cancer cells ................... 17 3.1.2. Verification of WNT, TFG-β and TEAD pathway downregulation ....................................... 17 3.1.3. Generation of a heat map for YAP/TAZ gene signature in prostate cancer cells with Hsp27 loss of function .................................................................................................................................... 18 3.1.4. Correlation of Hsp27 expression and YAP/TAZ target genes in human prostate cancer tissues 20 3.1.5. The effect of Hsp27 knockdown in lung and breast cancer cells ........................................ 22 3.2. Hsp27 is required for YAP activation and nuclear translocation in cancer cells ......................... 24 3.2.1. Investigating the effect of Hsp27 on YAP phosphorylation in vitro .................................... 24 3.2.2. Investigating the effect of Hsp27 on YAP phosphorylation in human tumor samples ....... 26 3.2.3. The effect of Hsp27 expression on YAP nuclear localization .............................................. 27 3.3. Hsp27 expression regulates YAP/TAZ cytoplasmic sequestration .............................................. 30 3.4. Hsp27 regulates the Hippo pathway by affecting core kinase components .............................. 32 3.4.1. The effect of Hsp27 knockdown on LATS1 and MOB1 ....................................................... 32 3.4.2. The effect of Hsp27 overexpression on LATS1 and MOB1 ................................................. 33 vi  3.4.3. The effect of Hsp27 expression on MST1, the core Hippo kinase ...................................... 33 3.4.4. The effect of antisense oligonucleotide OGX-427 on the Hippo pathway ......................... 35 3.5. Hsp27 forms a complex with MST1 and facilitates proteasomal degradation of ubiquitinated-MST1 36 3.5.1. Hsp27 forms a complex with MST1 in cancer cells ............................................................. 36 3.5.2. Hsp27 regulates the proteasomal degradation of MST1 .................................................... 37 3.5.3. The effect of Hsp27 on MST1 is not reciprocal ................................................................... 38 3.5.4 Hsp27 knockdown does not affect Hsp70 levels ................................................................ 38 3.6. Hsp27 negatively regulates the Hippo tumor suppressor pathway: A schematic summary ...... 39 4. Discussion ............................................................................................................................................ 41 Bibliography ................................................................................................................................................ 47 Appendix ..................................................................................................................................................... 52 Appendix 1 .............................................................................................................................................. 52    vii  List of Tables Table ‎1.1. Core components of the Hippo pathway in human and fruit fly. ................................................ 6 Table ‎3.1. List of YAP/TAZ target genes used to generate the heat map. .................................................. 19    viii  List of Figures  Figure ‎1.1. General structure of small heat shock proteins. ......................................................................... 2 Figure ‎1.2. Structure of Hsp27. ..................................................................................................................... 3 Figure ‎1.3. Schematic of Hippo tumor suppressor pathway. ....................................................................... 7 Figure ‎1.4. IPA analysis of top pathways affected in si Hsp27 PC3 cells compared to si Scr. ..................... 11 Figure ‎1.5. Previously reported regulation of TGF-β, BMP, WNT and ILK pathways by YAP/TAZ .............. 11 Figure ‎3.1. Hsp27 regulates YAP/TAZ transcriptional activity and downstream targets in Prostate Cancer. .................................................................................................................................................................... 18 Figure ‎3.2. Gene expression profiling reveals that Hsp27 knockdown decreases YAP/TAZ transcriptional activity. ........................................................................................................................................................ 20 Figure ‎3.3. : Expression of Hsp27 correlates with YAP/TAZ target genes in prostate cancer clinical samples. ...................................................................................................................................................... 21 Figure ‎3.4. Hsp27 regulates YAP/TAZ transcriptional co-activity in Lung and Breast Cancer cell lines. ..... 22 Figure ‎3.5. Hsp27 regulates YAP/TAZ target genes in Lung and Breast Cancer cell lines. .......................... 23 Figure ‎3.6. Hsp27 is required for YAP activation in vitro. ........................................................................... 25 Figure ‎3.7. Low expression of Hsp27 is correlated with higher YAP phosphorylation in human tumors. . 26 Figure ‎3.8. High expression of Hsp27 is correlated with lower YAP phosphorylation in human tumors. .. 27 Figure ‎3.9. Hsp27 knockdown decreases YAP nuclear translocation. ........................................................ 28 Figure ‎3.10. Hsp27 expression increases YAP nuclear translocation. ......................................................... 29 Figure ‎3.11. Hsp27 knockdown induces cytoplasmic sequestration of YAP/TAZ. ...................................... 30 Figure ‎3.12. Hsp27 overexpression decreases cytoplasmic sequestration of YAP/TAZ. ............................ 31 Figure ‎3.13. Hsp27 knockdown activates the Hippo pathway kinases. ...................................................... 32 Figure ‎3.14. Hsp27 overexpression inactivates the Hippo pathway kinases. ............................................. 33 Figure ‎3.15. Hsp27 expression regulates MST1 at protein level. ............................................................... 34 Figure ‎3.16. Hsp27 expression does not affect MST1 transcription. .......................................................... 34 Figure ‎3.17: Hsp27 regulation of MST1 mRNA in Lung and Breast Cancer cells. ....................................... 35 Figure ‎3.18. OGX-427 activates the Hippo tumor suppressor pathway. .................................................... 35 Figure ‎3.19.  Endogenous Hsp27 forms a complex with MST1 in cancer cells. .......................................... 36 Figure ‎3.20.  Hsp27 facilitates the proteasomal degradation of MST1 in cancer cells. .............................. 37 Figure ‎3.21. Hsp27 facilitates the proteasomal degradation of MST1 in cancer cells. ............................... 38 Figure ‎3.22. Hsp27 knockdown does not affect Hsp70 expression in Prostate, Lung or Breast Cancer. ... 39 ix  Figure ‎3.23: Proposed mechanism of negative regulation of the Hippo tumor suppressor pathway by Hsp27. ......................................................................................................................................................... 40    x  List of Abbreviations AKT Protein kinase B ANKRD1 Ankyrin repeat domain-containing protein 1 AR Androgen receptor ARHGAP29 Rho GTPase Activating Protein 29 ATP Adenosine triphosphate AXL Gene code for Tyrosine-protein kinase receptor UFO BMP Bone morphogenetic proteins CHIP C terminus of Hsc70-interacting protein CRPC Castration-resistant prostate cancer CTGF Connective tissue growth factor CYR61 Cysteine-rich angiogenic inducer 61 EGFR Epidermal growth factor receptor EMT Epithelial mesenchymal transition FGF2 Fibroblast growth factor 2 GLI2 Zinc finger protein GLI2 GPCR G-protein coupled receptor HSF Heat shock factor HSP Heat shock protein Hsp27  Heat shock protein 27 kDa Hsp60 Heat shock protein 60 kDa Hsp70 Heat shock protein 70 kDa Hsp90 Heat shock protein 90 kDa HSPB1 Heat shock protein beta-1, gene code for Hsp27 ILK Integrin-linked kinase IP Immunoprecipitation IPA Ingenuity pathway analysis LATS1/2 Large tumour suppressor kinases 1 and 2 xi  MOB1A/B Mps-one binder kinase activator 1 A and B MST1/2 Mammalian STE20-like protein kinase 1 and 2 NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells PI3 kinase Phosphatidylinositol-4,5-bisphosphate 3-kinase p27Kip1 Cyclin-dependent kinase inhibitor RAF or c-RAF proto-oncogene serine/threonine-protein kinase RPPA Reverse phase protein lysate microarray SAV1 Salvador homologue 1, WW45 Scr Scrambled sHSP Small heat shock protein siRNA Small interfering RNA SMADs Contraction of Sma and Mad (Mothers against decapentaplegic) family of transcription factors STAT3 Signal transducer and activator of transcription 3 STK4 Serine threonine kinase 4, gene code for MST1 STR Short tandem repeat TAZ Transcriptional co-activator with PDZ-binding motif TCF T cell factor family of transcription factors TCGA The cancer genomic atlas TEADs TEA domain containing family of transcription factors , also known as TEF Transcription enhancer factor TGF-β Transforming growth factor beta  WT Wild type WW45 Another name for SAV1 WWTR1 Gene code for TAZ YAP Yes Associated protein µM Micro molar nM Nano molar xii  Acknowledgments First and foremost, I would like to express my deepest appreciation to my supervisor, Dr. Amina Zoubeidi, for giving me the opportunity to be a part of such a great team, regardless of my different educational background. I would like to deeply thank her for encouraging me, granting me the scientific independence in this journey and believing in me, even when I didn’t believe in myself. This work was not possible without her constant guidance and supervision.  I would like to thank my supervisory committee, Dr. Vincent Duronio and Dr. Christopher Ong for their insightful comments and suggestions that helped me improve the research presented here. I also appreciate the time and consideration Dr. Zakaria Hmama put into reading and critically reviewing my thesis as my external examiner. Furthermore, I extend my deepest gratitude to Dr. Michael Cox, not only for his time and effort to convene my defence session, but also for being such an amazing scientist and never holding back on the constructive criticism.    I am very thankful to all members of the Vancouver Prostate Centre (VPC), this new family of mine, for accepting me and helping me through this journey. I am grateful to all my friends and lab-mates at VPC, especially the Zoubeidi laboratory members, for their helpful comments and suggestions during this project. My mind is filled with beautiful memories of all of you.  I also like to thank all my family and friends here in Vancouver which with their invaluable support and friendship helped me feel at home. I particularly thank Dr. Anousheh Zardan for introducing me to Amina. I wouldn’t be here if it wasn’t for her. In addition, I would like to write a few special words of appreciation: Jennifer L. Bishop: You have been more than a colleague, more than a friend and more like family to me. I thank you from the bottom of my heart for your thoughtful comments during this project, for showing me the light at the end of the tunnel and cheer me up in my gloomy days. It is an honor and pleasure to work with you. xiii  Daksh Thaper: I am in awe of your beautiful and kind heart. No words can describe how grateful I am to have you as my friend, my partner in crime. You taught me everything I know in the lab and you put up with my craziness, with my laughter and cries. Thank you for listening to my silly ideas and constant nagging and thank you for introducing me to Butter Chicken, really. You have been my best friend since the first day I joined this lab and I hope that our friendship grows only stronger.  And of course my deepest thanks to my family: My dear sister, Saba: You are my inspiration, you always have been. Words can’t describe the level of admiration I have for you, I don’t understand how you can be great at everything, but that’s who you are, you are simply amazing. Thank you for always pushing me forward. My dear brother, Hamid: I have always looked up to you. You taught me to be strong and courageous. You taught me that I can conquer everything and anything, I just have to have a little sense of humor! You encouraged me through every step of this path and I thank you for that. My extended family, Mahsa, Ario and Payman: Thank you for your help and understanding and thank you for cheering me up when I was hopeless. Life is always better when I hear your words of encouragement. My parents in law, Mansoureh and Mehdi: I am deeply thankful to you. Your endless love and understanding means the world to me.  My dearest parents, Sima and Abolghassem: I am nothing, without your unconditional love and support. Thank you for always believing in me and giving me confidence to move forward. You are my role models and I hope I can make you proud. And at last, the love of my life, Hooman: You know better than anybody that this work was not possible without you. You are the sweetest, the most understanding and the most selfless person I’ve ever met. Thank you, my love, for standing by my side, for being the comfort of my life and for all the sacrifices you have made for us.   xiv  Dedication   To my dearest parents, For their endless love and support... & To Hooman, My soulmate…   ردپ به میدقت میزعز درام و ...ناشغیدر یب ینابیتشپ و ها تبحم سپا به و مرسمه و تسود نیرتهب به میدقت نموه  1  1. Introduction 1.1. Heat Shock Proteins  1.1.1. Biology, function and transcription Heat shock proteins (HSPs) are highly conserved molecular chaperones with indispensable roles in protein homeostasis, transport processes and signal transduction. Although expressed under normal conditions, the expression of HSPs is upregulated in response to different cellular stresses, including but not limited to, hyperthermia, hypoxia, nutrient deprivation, oxidative stress, UV light and hormone- or chemo-therapy. Stress conditions result in abnormal cellular mechanisms such as protein misfolding and aggregation which if not corrected will eventually lead to cell apoptosis; therefore, HSPs are upregulated and can assist cell survival via multiple mechanisms. Firstly, HSPs catalyze the proper folding of misfolded proteins and avoid their aggregation. Secondly, HSPs associate with key modulators of the apoptotic machinery and interfere with the programmed cell death at different stages. There is tremendous evidence on how heat shock proteins like Hsp27, Hsp70 and Hsp90 conduct their anti-apoptotic effects. Thirdly, HSPs are involved in and regulate protein degradation machinery (ATP- and ubiquitin-dependent proteasome systems as well as autophagy machinery), providing either the stability or the proteasomal degradation of selected proteins under stress conditions [1-3].  Mammalian HSPs are classified in to 4 major families based their electrophoretic characteristics (molecular weight). Heat shock protein families are Hsp90, Hsp70, Hsp60 and the small HSPs like Hsp27 and αB-crystallin. While the activities of high molecular weight HSPs are ATP-dependent, the small HSPs act in an ATP-independent manner. Transcription of HSPs is primarily regulated by heat shock transcription factors (HSFs), mainly HSF1, which senses protein damage and increases the transcription of chaperones [4, 5]. However aside form stress induction, the expression of HSPs is regulated by cell cycle and in response to growth factors, where the need for HSPs increases significantly [5]. As a result, HSPs are ubiquitously induced in 2  malignancies where uncontrolled proliferation, hypoxia and cytotoxic reagents put cancer cells in constant exposure to cellular stress [6]. 1.1.2. Small Heat Shock Proteins  Small Heat Shock Proteins (sHSPs) are ATP-independent low molecular weight chaperones (between 12-43 kDa) that are defined by a conserved α-crystallin domain, a sequence of approximately 90 amino acid residues which is constrained by less conserved amino- and highly variable carboxy-terminal extensions [7]. Figure 1.1 shows a general structure of sHSPs. In addition to α-crystalline, some sHSPs have one or two more domains which are located in N-terminus preceding α-crystalline domain [8].  Figure ‎1.1. General structure of small heat shock proteins.  All sHSPs are structurally characterized by an α-crystalline domain.  The primary role of sHSPs is to bind denaturing proteins and prevent their aggregation. In order to function properly, almost all sHSPs need to dimerize and then oligomerize into multimeric structures. The α-crystallin domain is responsible for dimerization of sHSPs [8, 9]. The amino (N)-terminal extension is thought to influence higher-order oligomerization, subunit dynamics and chaperoning, and finally the carboxy (C)-terminal domain is flexible and amorphous and is important in chaperon activity and solubility [10, 11].  Extensive studies in recent years showed that some of sHSPs are upregulated in diseases, especially in cancers, where they play important part in inhibition of cell death pathways, and modulation of pro-survival signaling networks [12].  1.1.3. Heat Shock Protein 27  Among the stress chaperons, Hsp27 (HSPB1), an ATP independent sHSP has been extensively studied due to its crucial involvement in diverse physiological and pathological cellular 3  processes. Human Hsp27 consists of 205 amino acid residues forming a α-crystallin domain, a flexible C-terminal tail and a N-terminal domain with WDPF motif which is crucial for oligomerization [13]. Like other HSPs, Hsp27 is regulated by HSF1 at the transcription level; however its function is also thought to be regulated by posttranslational modification including phosphorylation on multiple sites. Ser15, Ser78 and Ser82 have been characterized as the main phosphorylation sites of Hsp27 (Figure 1.2) [14]. Hsp27 is phosphorylated by various kinases in response to different stimuli including some growth hormones and cytokines [1]. Hsp27 phosphorylation controls its oligomerization and ultimately its functions. Larger oligomers are mostly involved in protein folding and the prevention of protein aggregation while smaller oligomers are responsible for ubiquitination and proteasomal degradation of client proteins [27].   Figure ‎1.2. Structure of Hsp27. In addition to the α-crystalin domain, Hsp27 has a WDPF domain in the N terminus. Hsp27 is phosphorylated on multiple sites mainly Serine 15, Serine 78 and Serine 82.   Hsp27 is expressed in almost all human tissues in normal conditions however its expression dramatically increases under certain pathological conditions such as cancer [1]. Under stress conditions, Hsp27 levels increase to prevent protein aggregation and facilitate elimination of misfolded proteins [15]. More importantly, Hsp27 has been reported to modulate apoptosis and interact with its regulators such as procaspase‐3 and cytochrome C to inhibit cell death [16, 17]. 1.1.4. Hsp27 in human cancer Due to the wide range of intra- and extracellular insults which malignant cells are constantly subjected to, Hsp27 is overexpressed across different cancers like prostate, breast, ovarian and gastric [18-22] and this overexpression has been associated with poor prognosis 4  and chemo-resistance [23-27]. Hsp27 contributes to cancer progression via different mechanisms; it is a major anti-apoptotic and pro-survival chaperone which plays crucial parts in tumorigenesis [28-30], increasing proliferation by facilitating cell cycle progression [31] and enhancing migration and invasion via multiple mechanisms [32, 33]. In addition, Hsp27 also participates in the maintenance of cancer stem cells [34, 35] which are also associated with aggressive tumors and poor prognosis [36].  1.1.5. Hsp27 and oncogenic signaling pathways The pleiotropic roles of Hsp27 underscore its position at hubs of cell signal cascades across multiple cancers. Hsp27 has been shown to cooperate closely with oncogenic signaling pathways like transforming growth factor beta (TGF-β) [37], WNT/β-catenin  [32, 38], STAT3 [33] and NF-κB [39]. Hsp27 stabilizes and activates AKT, the infamous oncogene in the PI3 kinase and insulin signaling pathway [28, 30]. Additionally, Hsp27 is required for trafficking and transcriptional activity of sex steroid receptors including the androgen receptor (AR), the central oncogenic driver in prostate cancer progression [40, 41]. Importantly, modulation of these pathways by Hsp27 has been linked to therapy resistance in different cancers [23-25].  Since Hsp27 is central to many upregulated signaling pathways in cancer, it is an attractive therapeutic target; Hsp27 inhibition may concurrently suppress several pathways implicated in cancer advancement and more importantly overcome resistance to hormone- and chemo-therapies.  1.1.6. Targeting Hsp27  The ultimate goal of translational cancer research is to identify molecular mechanisms that contribute to tumor progression and to develop novel and effective therapies that can target these mechanisms to improve patient outcome. However, although we have a relatively thorough understanding of how molecules such as Hsp27 contribute to cancer, targeting such molecules remains difficult. For example, designing small molecule inhibitors for Hsp27 is more difficult compared to some other HSPs like Hsp70 and Hsp90, due to the lack of an ATP binding 5  domain. As an alternative, Hsp27 suppression is achieved by using antisense oligonucleotides, which prevent its expression. Antisense oligonucleotides are synthetic single stranded nucleic acid structures that bind to RNA and interfere with gene expression by altering RNA function [42]. OGX-427 (Apatorsen), a second-generation antisense oligonucleotide for Hsp27, is currently in phase II clinical trials for prostate, bladder and lung cancers (ClinicalTrials.gov, NCT01454089, NCT01829113, and NCT01120470). Studies suggest that knockdown of Hsp27 using OGX-427 suppresses prostate tumour growth and sensitizes prostate cancer cells to chemo‐therapy [40, 43, 44]. OGX-427 also sensitize non-small cell lung cancer cells to Erlotinib and chemotherapy [45]. 1.2. Hippo Tumor Suppressor Pathway 1.2.1. Core components The Hippo tumor suppressor pathway is an evolutionarily conserved pathway which was first characterized in Drosophila melanogaster. Using genetic screens in fruit flies, a series of genes were isolated that when inactivated, allowed excessive tissue growth in developing wings or eyes. These proteins make up the core components of the Hippo tumor suppressor pathway and their function is highly conserved in mammals and D. melanogaster.  The mammalian Hippo pathway’s core consists of: Mammalian STE20-like protein kinase 1 and 2 (MST1/2), Large tumour suppressor kinases 1 and 2 (LATS1/2) and adaptor proteins WW45 (SAV1) and Mps-one binder kinase activator 1 A and B (MOB1A/B). The role of these kinases and scaffold proteins is to phosphorylate and inactivate Yes associated protein (YAP) and Transcriptional co-activator with PDZ-binding motif (TAZ), two co-activators which are able to partner with oncogenic transcription factors such as TEA domain-containing sequence-specific transcription factors (TEAD1-4) [46, 47]. Other transcription factors that use YAP and TAZ as their co-regulators are: SMADs (TGF-β signaling), β-catenin/TCF (WNT pathway), RUNXs (blood and bone formation), PAX3 (neural crest formation), KLF4 (terminal differentiation of goblet cells in colon), p63/p73 (apoptosis) [48-53]. Table 1.1 compares core components of the Hippo pathway in D. melanogaster and in humans.  6  Table ‎1.1. Core components of the Hippo pathway in human and fruit fly. Homo sapiens Gene name Human Gene name Drosophila melanogaster MST1/2* STK3/ STK4 HPO (HIPPO) WW45 (SAV1) WW45 SAV (SALVADOR) LATS1/2* LATS1/2 WTS (WARTS) MOB1 (A AND B) MOBKL1A, MOBKL1B MATS YAP/TAZ YAP1, WWTR1 YKI (YORKI) TEAD1-4 TEAD1-4 SD (SCAPOLLED) * Functions of MST1 and MST2 as well as LATS1 and LATS2 to inactivate YAP are redundant [54]. 1.2.2. Biology and function in mammals Growing interest to this pathway is driven by tremendous evidence showing the fundamental role of this pathway in organ growth, stem cell renewal and tumor suppression. The activation of the Hippo tumor suppressor pathway begins with the activation of MST1/2 which in turn activates LATS1/2 by phosphorylating it on T1079. Scaffold protein SAV1 tethers MST1/2 to LATS1/2 for optimal interaction. MOB1A and MOB1B potentiate the kinase activity of LATS1/2. LATSs are responsible for phosphorylation of the transcriptional co-activators YAP and its paralogue TAZ. Phosphorylation of YAP and TAZ results in their cytoplasmic sequestration with the 14.3.3 proteins and prevents their nucleus translocation. When the Hippo cascade is deregulated, absence of YAP/TAZ phosphorylation allows for their translocation to the nucleus leading to activation of pro-survival and metastatic pathways such as TGF-β/SMAD, WNT/β-Catenin and ILK/TEADs pathways [46, 55].  Figure 1.3 shows a schematic view of the Hippo kinase cascade. 7   Figure ‎1.3. Schematic of Hippo tumor suppressor pathway. When active, a series of phosphorylation events lead to phosphorylation of YAP and TAZ and their cytoplasmic sequestration with 14.3.3 proteins. When the Hippo pathway is inactive, the kinase cascade is not functional; therefore, YAP/TAZ are free to translocate into nucleus and regulate the activity of their partner transcription factors.     1.2.3. Upstream regulators of the Hippo pathway  Although the core components of this pathway appear to form a simple linear cascade, recent work has led to the realization that in fact the Hippo pathway is part of an interconnected signaling web which allows the cell to elicit the appropriate response to an external stimulus. Several cellular processes provide input into this catalytic torrent including mechanical compression [56], cell-cell contact [57], cellular polarity [58, 59], as well as signaling pathways such as G-protein coupled receptor (GPCR) [60], TGF-β [61], WNT [62], Notch [63], EGFR [64] and ILK [55].  8  1.2.4. Hippo tumor suppressor pathway in cancer The Hippo tumor suppressor pathway regulates and is regulated by cellular mechanisms that if defective, can lead to tumorigenesis. Inactivation of the Hippo pathway correlates with poor patient outcome, increase of migration, invasion and metastasis [65]. 1.2.4.1. Cell proliferation The Hippo pathway restricts cell proliferation, the most fundamental deregulated feature of cancer cells. Cell proliferation can be controlled via several parameters such as cell-cell contact, loss of which one of the hallmarks of cancer [66]. Defective function of the Hippo components leads to hyper-activation of YAP and TAZ causing ectopic cell proliferation [46, 67]. For example, YAP overexpression causes ovarian and prostate cancer cells to overcome contact-inhibition [57, 68].  1.2.4.2. Cell survival Aside from increasing proliferation, downregulation of the Hippo tumor suppressor pathway causes insensitivity to apoptosis. Studies in cancer cell lines showed YAP overexpression promotes resistance to apoptosis induced by chemotherapeutic agents [68, 69]. This could be one of the mechanisms by which, deregulation of the Hippo pathway leads to therapy resistance.   1.2.4.3. Treatment-resistance Inactivation of the Hippo tumor suppressor pathway results in activation of YAP and TAZ which are known to affect tumor cells’ resistance to chemotherapy. A recent study evaluated YAP1 to be a major regulator of resistance to RAF (a proto-oncogene serine/threonine-protein kinase) inhibitor.  Silencing of YAP1 increased cell sensitivity to RAF inhibitors across different tumor models—lung, melanoma, colon, thyroid and pancreatic cancer cell lines [70]. 1.2.4.4. Stem cell features It is well established that some cancer cells have stem cell capabilities including extensive replicative potential and loss of mature differentiation markers. These cancer stem cells are 9  hypothesized to persist in tumors as a distinct population; they could cause relapse and metastasize, giving rise to new tumors. Several studies have linked components of the Hippo pathway to stem cell features. YAP and TAZ can promote pluripotency characteristics of embryonic stem cells [71]. Gene expression profiling studies have shown that YAP1 and the TEAD transcription factors are enriched in multiple types of stem cells [72]. Also TAZ regulates mesenchymal stem cell differentiation [49, 73]. Mesenchymal cells are more invasive and therefore, YAP and TAZ hyperactivity might promote tumorigenic potential by enhancing stem-cell-like properties.   1.3. Hypothesis Formation The Hippo pathway has been shown to cross talk with a number of other molecular pathways commonly altered in human cancers. Similarly, Hsp27 acts as a central hub connecting networks that promote tumor growth and progression. While there is extensive overlap between the cell survival, anti-apoptotic and metastatic pathways that Hsp27 and Hippo components may regulate, a relationship between the two has not yet been described. 1.3.1. Gene expression profiling as a method to paint a global picture of cellular function Gene expression profiling quantifies expression patterns of genes in the cell. It is a particularly useful tool in studying cancer cells, where DNA microarrays in combination with statistical analysis have enabled researchers to determine the important genes in tumorigenesis or build classifiers based on expression profiling for many types of cancer such as breast, lung and prostate [74]. The oncogenic capacities of Hsp27 present scientists with a vast puzzle of cellular pathways interconnectedness. Our lab has been working extensively on tumorigenicity of Hsp27 in prostate cancer; we have shown that Hsp27 assists prostate cancer progression by increasing cell proliferation and survival, enhances migratory and invasive properties of prostate cancer cells and promotes AR signaling, the key player of this disease [28, 32, 33, 40]. Although these studies helped us put pieces of the puzzle of Hsp27 oncogenicity together, however, to better picture and understand its roles, we performed a gene expression 10  microarray in small interfering RNA (siRNA) Hsp27 treated PC3 prostate cancer cells and analyzed the results using Ingenuity Pathway Analysis (IPA).  1.3.2. Ingenuity Pathway Analysis Ingenuity Pathway Analysis (IPA) is a web-based software application that allows analysis of large datasets like microarray-based gene expression. IPA places genes into pathways based on previous published information and therefore by analyzing altered genes, it can be used to identify the most affected pathways. Unbiased comparison of the gene expression signature in siScrambled (siScr) versus siHsp27 treated samples using IPA verified downregulation of major signaling pathways where Hsp27 has been shown to play a role (Figure 1.4). Importantly, as illustrated in Figure 1.5, the most highly downregulated signaling pathways, including TGF-β, BMP, WNT/β-Catenin and ILK all utilize YAP/TAZ as transcriptional co-activators [48, 53, 55]. However, a link between YAP/TAZ and Hsp27 had not been established.   11   Figure ‎1.4. IPA analysis of top pathways affected in si Hsp27 PC3 cells compared to si Scr.  Pathway analysis shows knocking down Hsp27 in PC3 cells affects these pathways the most.    Figure ‎1.5. Previously reported regulation of TGF-β, BMP, WNT and ILK pathways by YAP/TAZ  12  1.3.3. Hypothesis Gene expression profiling of PC3 prostate cancer cells showed that transient knock down of Hsp27 downregulates pathways dependent on YAP and TAZ. Since YAP and TAZ are predominantly inhibited by the Hippo pathway, we hypothesized that Hsp27 negatively regulates the Hippo tumor suppressor pathway.   13  2. Materials and Methods 2.1. Cell culture  Prostate cancer PC3 and lung cancer cell line A549 were purchased from the American Type Culture Collection (ATCC) and authenticated by isoenzymes analysis in 2008 and short tandem repeat (STR) profile in 2013 respectively. Both cell lines were maintained in RPMI media supplemented with 10% fetal bovine serum (FBS, Invitrogen-Life Technologies). Breast cancer cell line MDA-MB-453 (ATCC) and HSF1 knock-out murine embryonic fibroblasts (MEF-HSF1-/-) (a kind gift from I. J. Benjamin, University of Utah) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies) containing 10% FBS.  2.2. Transfections  2.2.1. Small interfering RNA transfection PC3, A549 and MDA-MB-453 were transfected with 20 nM Hsp27 siRNA (siHsp27) or control siRNA (siScr) using OligofectAMINE (Invitrogen-Life Technologies, Inc.) in Opti-MEM (Gibco) following the manufacturer procedures.  2.2.2. Plasmid transfection for overexpression pHR-CMV Empty and wild type Hsp27 vectors (WT) were used as previously described [28]. Cells were seeded at 5 X 105 cells and 106 cells in 10 cm2 dishes for siRNA and plasmid transfections respectively. Overexpression of MST1 (STK4) was achieved using pJ3H-MST1 plasmid purchased from Addgene (ID: 12203).  2.3. Gene Expression profiling  For differential expression profiling of siScr and siHs27 treated PC3 cells, total RNA was extracted using TRIzol® and the quality of RNAs were measured using Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA). Samples were prepared following Agilent’s One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling v6.0. An input of 100ng of total RNA was used to generate Cyanine-3 labeled cRNA.  Samples were then hybridized on Agilent 14  SurePrint G3 Human GE 8x60K Microarray (AMDID 028004) and arrays were scanned with the Agilent DNA Microarray Scanner at a 3um scan resolution. Data was processed with Agilent Feature Extraction 11.0.1.1. and processed signal was quantile normalized with Agilent GeneSpring 12.0. Normalized log2 values of YAP/TAZ target genes were between siHsp27 and siScr treated cells. All microarray profiling analyses were carried out in triplicate. RNA quality control as well as gene profiling was done by Dr. Collins laboratory at Vancouver Prostate Centre, Vancouver, British Columbia, Canada. 2.4. Western blotting  RIPA lysis buffer was mixed with protease and phosphatase inhibitors and was used to lyse total proteins as previously described [40]. Nuclear/ cytoplasmic fractions were extracted using the CelLyticTM NuCLEARTM Extraction Kit (Sigma-Aldrich, St Louis, MO) according to manufacturer’s protocol. 2.5. Immunoprecipitation Immunoprecipitation was performed using ImmunoCruz™ IP/WB Optima B System (Santa Cruz) based on the manufacturer’s guideline. 2 µg of primary antibody, or immunoglobulin G (IgG) was used for immunoprecipitation and control respectively. Blots were incubated overnight at 4 °C with designated primary antibodies at 1:1000 dilution, unless noted otherwise. Proteins were visualized using the Odyssey system (Li-Cor Biosciences) and densitometric analysis was performed using ImageJ software (National Institutes of Health, USA). 2.6. Reagents and antibodies  Antibodies against YAP (#4912), YAP/TAZ (# 8418), phospho-YAP S127 (# 4911), LATS1 (# 3477, 1:200), phospho-LATS1 Thr1079 (# 9159, 1:200), MOB1 (#3863), phosphor-MOB1T35 (#8699), MST1 (#3682) were purchased from Cell Signaling, USA, anti-14:3:3 ε (sc-393177), TAZ (sc-48805), Ubiquitin (sc-8017) and Lamin B1 (sc-377001) were from SantaCruz Biotechnology. Anti-LATS1 ab70562 (abcam) and anti-MOB1A/B sc-161867 (Santa Cruz) were used in the 15  mouse cell line with 1:500 dilution. Anti-Hsp27 (1:5000) was from ENZO lifesciences. MG132 was from Millipore (#474791). 2.7. Immunofluorescence PC3 and MEF-HSF1-/- cells were grown and transfected with siScr/siHsp27 and mock/Hsp27 respectively, in 10cm2 plates. Then the cells were trypsinized and plated in 12 well plates at 5000 cells/cm2 on coverslips submerged in RPMI+FBS 10% for 24 hours. Cells were then fixed and the Immunofluorescence was performed as we previously reported [40] using antibodies against YAP/TAZ (1:500) and 14.3.3 epsilon (1:200). DAPI was used to visualize nuclei and then the pictures were taken by 63x objective lens using Zeiss Axioplan II fluorescence microscope. Results are representative of random pictures taken from three independent experiments. 2.8.  Quantitative real time PCR (qRT-PCR) TRIzol® reagent (Invitrogen) was used to extract total RNA from cultured cells. 2 μg of total RNA was reversed transcribed using random hexamers (Applied Biosystems) as previously reported [33]. q-RT PCR amplification of cDNA was performed using the following primer pairs (sequences listed in table 2): Hsp27, YAP1, WWRT1, STK4, CTGF, CYR61, GLI2, FGF2, ARHGAP29, ANKRD1, GAPDH with FastStart Universal SYBR Green Master (ROX; Roche Applied Science) on the ABI PRISM 7900 HT Sequence Detection System. Target gene expression was normalized to GAPDH levels. The results represent three independent experiments with each sample run in triplicate. 2.9. Luciferase assay For TEAD, TCF and SMAD transcriptional activity, 2 x 104 cells were plated in triplicate in six-well plates and transfections were carried out using TransIT®-2020 (Mirus Bio.) and 9 µg of indicated luciferase reporter for each plate: pGL3-OT for TCF [32], 8xGTIIC-luc for TEAD and SBE4-Luc for SMAD1-4. TEAD and SBE4 luciferase reporter were purchased from Add gene (IDs: 34615 and 16495 respectively). Luciferase activities were measured 48 hours after using the Dual-Luciferase Reporter Assay System (Promega) and a microplate luminometer, Tecan Infinite® 200 PRO (Tecan, Männedorf, Switzerland).  The signal of firefly luciferase was 16  normalized to the total protein concentration of each well with the control condition set as one. All experiments were carried out in triplicate wells and repeated three times each triplicate well. 2.10. Public data mining Two Publically available databases were used in this study: 2.10.1. CBioportal for Cancer Genomics  This website allows the researcher to visualize, download and analyze large-scale cancer studies such as The Cancer Genome Atlas (TCGA) studies for different cancers [75]. TCGA provisional studies (2015) for lung adenocarcinoma and invasive breast cancer were analyzed in order to illustrate the correlation between mRNA expression of HSPB1 (based on z score) and the score of phospho-YAP S127 in human tumor samples. Z-score for mRNA expression data is the number of standard deviations away from the mean of expression in the reference population. This measure is useful to determine whether a gene is up- or down-regulated relative to the normal samples or all other tumor samples. 2.10.2. The UCSC Xena Genome Browser (http://genome.ucsc.edu/) This website is developed and maintained at the University of California Santa Cruz (UCSC) not only makes large-scale studies available for further analysis but also allows the researcher to visualize and analyze his/her data in the context of larger datasets [76]. Xena software was used to visualize the clinical correlation between mRNA expression of HSPB1 and following YAP/TAZ target genes in the prostate cancer TCGA dataset. Gene names: TAGLN, CCND2, AXL, CYR61, MET, FGF2, CAV1, RND3, IFI16, SNRPG, IFRD1, ID2, RHOU, MYOF, NT5E, CTGF, THBS1. 2.11. Statistical analyses  Data are representatives of three independent experiments and are expressed as mean ± standard error of the mean (SEM). P-values were calculated using Student t-test to compare control and treated groups and p-values less than 0.05 were considered statistically significant (*P< 0.05; **P< 0.001; ***P< 0.001).  17  3. Results 3.1. Identification of Hsp27 as a possible regulator of YAP/TAZ activity 3.1.1. The effect of Hsp27 knockdown on YAP/TAZ activity in prostate cancer cells Hsp27 is known to regulate multiple molecular pathways that contribute to prostate cancer progression; however an investigation of a global gene expression pattern regulated by Hsp27 in prostate cancer cells has not been reported. In order to identify novel pathways affected by Hsp27 expression, we performed a gene expression microarray in small interfering RNA (siRNA) Hsp27 treated PC3 prostate cancer cells. As illustrated in Figure 1.5 and Figure 1.6, the gene expression signature in siScrambled (siScr) versus siHsp27 treated samples showed downregulation of major signaling pathways that utilize YAP/TAZ as transcriptional co-activators including TGF-β, BMP, WNT/β-Catenin and ILK  However a link between YAP/TAZ and Hsp27 had not been established. 3.1.2. Verification of WNT, TFG-β and TEAD pathway downregulation Confirming IPA pathway analysis results, we showed that the transcriptional activities of TCF, SMAD1-4 and TEAD1, well-established readouts of YAP/TAZ activity [48, 53, 55] were significantly decreased in PC3 cells after Hsp27 knockdown compared to control (Figure 3.1A).                                     *** *** *** 00.20.40.60.811.2si Scr si Hsp27 si Scr si Hsp27 si Scr si Hsp27TCF SMAD1-4 TEAD1Relative luciferase activity [Type a quote from the document or the summary of an interesting point. You can position the text box anywhere in the document. Use the Drawing Tools tab to change the formatting of the pull quote text box.] A 18                                     Figure ‎3.1. Hsp27 regulates YAP/TAZ transcriptional activity and downstream targets in Prostate Cancer. (A) Relative activity of TCF, SMAD1-4 and TEAD1 assessed by luciferase assay in si Hsp27 PC3 cells compared to si Scr (=1), Graph represents pooled data from three independent experiments. (B) Relative mRNA expression of Hsp27 and YAP target genes in si Hsp27 PC3 cells compared to si Scr (=1). Graph is a representative of three independent experiments. To further validate the regulation of YAP and TAZ by this chaperone, we tested the effect of siHsp27 on the expression of well-known YAP/TAZ target genes including CTGF, CYR61, ANKRD1, GLI2, FGF1 and ARHGAP29 expression in PC3 cells using qRT-PCR. Inhibition of Hsp27 resulted in significant downregulation of these genes, but not YAP and TAZ (WWTR1) themselves (Figure 3.1B), suggesting that Hsp27 modulates YAP and TAZ activity but not their expression. 3.1.3. Generation of a heat map for YAP/TAZ gene signature in prostate cancer cells with Hsp27 loss of function  To further investigate the impact of Hsp27 on YAP/TAZ targets, a comprehensive list of YAP/TAZ target genes was compiled [73, 77, 78] (Table 3.1).   00.20.40.60.811.21.4HSP27CTGFCYR61ANKRD1GLI2FGF2ARHGAP29YAP1WWTR1si Scr si Hsp27Relative mRNA expression B 19  Table ‎3.1. List of YAP/TAZ target genes used to generate the heat map. ACSL4 CDH2 EPB41L2 IDI1 MSMO1 RAB3B SMAD6 ADAMTS1 CENPF ERRFI1 IFI16 MYBL1 RGS4 SNAI2 ADAMTS12 CHST9 ESM1 IFIT2 MYC RHOU SNAPC1 ADAMTS5 CLDN1 EXPH5 IFRD1 MYOF RIMKLB SNORA75 ADRB2 COX6C F2RL1 IL8 NID2 RND3 SNRPG AHNAK CPA4 F3 INSIG1 NT5DC3 S1PR1 SOX9 AMPH CRIM1 FBXW11 IRS1 NT5E SCD SP1 ANXA1 CSNK2A1 FGF2 ITGBL1 OPN3 SCD5 SQLE ANXA3 CTGF FRZB JPH1 PCLO SCHIP1 STXBP1 AOX1 CTNNAL1 FSCN1 KIT PDCD1LG2 SCML1 SYT14 AREG CTNNB1 FST KRT5 PDP2 SDPR TFPI2 ARHGAP29 CYP1B1 FSTL1 LCP1 PHLDA1 SEMA3C TGFA ARHGEF28 DAAM1 GADD45A LGALSL PITX2 SERPINB7 TGFB2 ASAP1 DAB2 GCNT4 LHFP PMAIP1 SERPINE1 THBS1 AXL DDAH1 GGH LMBRD2 PMP22 SGK1 TMEM154 BCL2 DIXDC1 GLI2 LPIN1 PRICKLE1 SHROOM3 TMEM27 BDNF DUT GLS LRP6 PRRG1 SLC16A6 TOP2A BICC1 ECT2 GPCPD1 LUM PRSS23 SLC2A3  CAV1 EMP1 HEXB MACC1 PSAT1 SLIT2  CCL28 EMP2 HMGCS1 MDFIC PSG5 SMAD1  CCND2 ENC1 HMMR MET PTPN14 SMAD3  CD55 EP300 ID2 MID1 PYGO1 SMAD5    Microarray expression of each target gene in siScr versus siHsp27 treated PC3 cells was compared. In accordance with our initial in vitro results, more than 70% of YAP/TAZ targets were downregulated in siHsp27 samples compared to control (Figure 3.2).       20                                              Figure ‎3.2. Gene expression profiling reveals that Hsp27 knockdown decreases YAP/TAZ transcriptional activity.  Microarray heat map showing expression of YAP/TAZ target genes in si Hsp27 PC3 cells compared to si Scr. Normalized log2 values were used to generate the heat map. (to see values please refer to Appendix 1)  3.1.4. Correlation of Hsp27 expression and YAP/TAZ target genes in human prostate cancer tissues These in vitro findings were supported by clinical data showing that expression of Hsp27 (HSPB1) and a subset of these YAP/TAZ target genes were positively correlated in the publicly available TCGA database for prostate cancer tissues [76]. Figure 3.3 shows gene expression of YAP/TAZ targets stratified by the expression of Hsp27 in the same patient (Figure 3.3, The Cancer Genome Atlas, https://genome-cancer.ucsc.edu/).  YAP/TAZ  Target Genes siScr siHsp27 17 3 Normalized log2 values 21             Figure ‎3.3. : Expression of Hsp27 correlates with YAP/TAZ target genes in prostate cancer clinical samples.  mRNA expression of Hsp27 (HSPB1) and YAP/TAZ target genes obtained from the Prostate Cancer TCGA data set show a positive correlation.  Taken together, our data suggest that Hsp27 knock-down induces a decrease in the activity of YAP/TAZ cooperating transcription factors leading to a downregulation of their targets, a hypothesis supported by both in vitro model and human tumor samples.    22  3.1.5. The effect of Hsp27 knockdown in lung and breast cancer cells Similar to what we had illustrated in PC3 prostate cancer cells, we investigated if knocking down Hsp27 negatively affects YAP activity in other cancer types. For that we used a luciferase assay to measure transcription activity of TCF, SMAD1-4 and TEAD1 in A549 lung cancer and MDA-MB-453 triple negative breast cancer cells (Figure 3.4).                                                                     Figure ‎3.4. Hsp27 regulates YAP/TAZ transcriptional co-activity in Lung and Breast Cancer cell lines. Relative transcriptional activity of TCF, SMAD1-4 and TEAD1 assessed by luciferase assay in (A) si Hsp27 A549 and (B) MDA-MB-453 cells compared to si Scr (=1), Graph represents pooled data from three independent experiments.  Similar to what we observed in PC3 cells, the transcriptional activity of TCF, SMADs and TEAD1 decreases upon Hsp27 siRNA treatment in A549 and MDA-MB-453 cells. *** *** *** 00.20.40.60.811.2si Scr si Hsp27 si Scr si Hsp27 si Scr si Hsp27TCF SMAD1-4 TEAD1*** *** *** 00.20.40.60.811.2si Scr si Hsp27 si Scr si Hsp27 si Scr si Hsp27TCF SMAD1-4 TEAD1A549 Relative luciferase activity A B MDA-MB-453 Relative luciferase activity 23  Next we confirmed that YAP/TAZ target genes decreased upon Hsp27 knockdown in lung and breast cell lines while the transcription levels of YAP and TAZ are not affected (Figure 3.5).               Figure ‎3.5. Hsp27 regulates YAP/TAZ target genes in Lung and Breast Cancer cell lines. Relative mRNA expression of Hsp27, YAP, TAZ (WWTR1) and YAP/TAZ target genes in (A) si Hsp27 A549 and (B) MDA-MB-453 cells compared to si Scr (=1). Graphs are representatives of three independent experiments for each cell line.  00.20.40.60.811.21.4HSP27CTGFCYR61ANKRD1GLI2FGF2ARHGAP29YAP1WWTR1A549si Scr si Hsp2700.20.40.60.811.21.4HSP27CTGFCYR61ANKRD1GLI2FGF2ARHGAP29YAP1WWTR1MDA-MB-453si Scr si Hsp27Relative mRNA expression A Relative mRNA expression B 24  3.2. Hsp27 is required for YAP activation and nuclear translocation in cancer cells 3.2.1. Investigating the effect of Hsp27 on YAP phosphorylation in vitro Our results suggested that Hsp27 affects YAP activity without affecting its transcription (Figure 3.1B). Post-translationally, YAP activity is inhibited by phosphorylation on S127 which prevents its nuclear translocation [46]. As shown in Figure 3.6A, siRNA inhibition of Hsp27 lead to increased S127 phosphorylation of YAP in PC3 prostate, A549 lung and MDA-MB-453 triple negative breast cancer cells. Reciprocally, Hsp27 overexpression decreased the inhibitory phosphorylation of YAP on S127 in PC3 and A549 cells compared to control (Figure 3.6B). We further tested this phenomenon using a mouse embryonic fibroblast cell line that lack heat shock factor protein 1 (HSF 1) (MEF-HSF1-/-), a transcription factor required to initiate transcription of all HSPs; these cells therefore do not express Hsp27 and provide a useful model to study the effects of Hsp27 overexpression on the Hippo pathway. In accordance with our observations in PC3 and A549 cells, phosphorylation of YAP on S127 decreased upon Hsp27 plasmid transfection (Figure 3.6B) in MEF-HSF1-/- cells.   25                                                                                                                                                                                                                         Figure 3.6:      00.511.522.5si Scr siHsp27 si Scr siHsp27 si Scr siHsp27PC3 A549 MDA-MB-453pYAP/YAPsiHsp27 si Scr Hsp27 Vinculin YAP p-YAP S127 Breast Cancer MDA-MB-453Lung Cancer A549Prostate Cancer PC3 siHsp27   si Scr siHsp27 si Scr Normalized Densitometry A549 PC3 Hsp27 Mock Hsp27 Vinculin YAP p-YAP S127 Hsp27 Mock Hsp27 Mock MEF-HSF1 -/- Normalized Densitometry 00.20.40.60.811.2Mock Hsp27 Mock Hsp27 Mock Hsp27PC3 A549 MEF-HSF1 -/-pYAP/YAPA B Figure ‎3.6. Hsp27 is required for YAP activation in vitro. (A,B) Protein expression of p-YAP S127, YAP, Hsp27 and Vinculin in PC3, A549, MDA-MB-453 and/or MEF-HSF1-/- cells transfected with (A) si Hsp27 or si Scr or (B) Hsp27 or vector control (mock). Densitometry shows fold expression of p-YAP S127 compared to total YAP (=1).  26  3.2.2. Investigating the effect of Hsp27 on YAP phosphorylation in human tumor samples To validate the clinical relevance of our findings, we interrogated the RPPA (reverse phase protein lysate microarray) score of p-YAP S127 in Hsp27 overexpressing tumors found in the cBioportal repository (www.cbioportal.org) [75]. Figure 3.7 illustrates statistically significant up-regulation of p-YAP S127 in lung (Figure 3.7 left) and breast (Figure 3.7 right) tumor samples that have lower Hsp27 (EXP<1) compared to samples with unaltered Hsp27.            Figure ‎3.7. Low expression of Hsp27 is correlated with higher YAP phosphorylation in human tumors.   RPPA score of p-YAP S127 in Lung Adenocarcinoma and Invasive Breast Carcinoma in tumors with Hsp27 (HSPB1) expression less than 1 z-score obtained from TCGA data sets. (For the definition of z score please refer to 2.10.1)  The opposite trend was observed in tumors that have higher Hsp27 expression (EXP>1); lung (Figure 3.8 left) and breast (Figure 3.8 right) cancer tissue samples with over-expression of Hsp27 showed significantly lower phosphorylation on YAP S127 (Figure 3.8).  RPPA score (YAP1 [pS127]) Lung Adenocarcinoma  (TCGA, Provisional) N= 578 samples  p-value: 0.0421869467 Breast Invasive Carcinoma  (TCGA, Provisional) N= 1104 samples P-value: 0.0003983248 27             Figure ‎3.8. High expression of Hsp27 is correlated with lower YAP phosphorylation in human tumors.   RPPA score of p-YAP S127 in Lung Adenocarcinoma and Invasive Breast Carcinoma in tumors with Hsp27 (HSPB1) expression more than 1 z-score obtained from TCGA data sets. (For the definition of z score please refer to 2.10.1)  These data combined with our in vitro findings further support a role for Hsp27 in the negative regulation of the Hippo pathway in human cancer.  3.2.3. The effect of Hsp27 expression on YAP nuclear localization Phosphorylation of YAP on S127 prevents its translocation to the nucleus. Based on our results showing Hsp27 affects p-YAP levels (Figure 3.6), we investigated the localization of YAP upon changes in Hsp27 expression. In accordance with our results showing Hsp27 knockdown increases YAP S127 phosphorylation, immunofluorescence showed extensive cytoplasmic localization of YAP in PC3 upon Hsp27 knockdown compared to siScr treated cells, in which YAP remained mostly nuclear (Figure 3.9 top). This phenomenon was also captured by western blot RPPA score (YAP1 [pS127]) Lung Adenocarcinoma (TCGA, Provisional) N= 578 samples p-value: 0.0421869467 Breast Invasive Carcinoma  (TCGA, Provisional) N= 1104 samples P-value: 0.0006086191 28  of samples which recieved Hsp27 siRNA (+), compared to control (-) (Figure 3.9 bottom). Laminb1 was used as a loading control for nuclear fraction.                 Figure ‎3.9. Hsp27 knockdown decreases YAP nuclear translocation.  (Top) Immunofluorescence of total YAP (green) and the nucleus (DAPI, blue) in PC3 cells transfected with  si Hsp27 or si Scr. Scale bar: 50 µm. (Bottom)  Western blots show nuclear and cytoplasmic levels of total YAP, Vinculin and Laminb1 in siRNA knockdown of Hsp27. Reciprocally, in MEF-HSF1-/- cells, immunofluorescence showed nuclear localization of YAP increased after Hsp27 over-expression compared to mock transfected cells (Figure 3.10 siScr YAP DAPI Merge siHsp27 PC3 Cells 29  top), and cyto/nuclear fractionation of MEF-HSF1-/- cells clearly demonstrated the translocation of YAP upon Hsp27 overexpression (+), compared to control (-) (Figure 3.10 bottom).                  Figure ‎3.10. Hsp27 expression increases YAP nuclear translocation.   (Top) Immunofluorescence of total YAP (green) and the nucleus (DAPI, blue) in PC3 cells transfected with Mock or Hsp27 plasmid. Scale bar: 50 µm. (Bottom) western blots show nuclear and cytoplasmic levels of total YAP, Vinculin and Laminb1 in overexpression of Hsp27.  Taken together, these findings indicate that expression of Hsp27 controls YAP activity by affecting its phosphorylation and subsequent nuclear translocation. Mock HSP27 MEF-HSF1 -/- Cells YAP DAPI Merge 30  3.3. Hsp27 expression regulates YAP/TAZ cytoplasmic sequestration  Phosphorylation of YAP and its paralogue TAZ lead to their sequestration in the cytoplasm by the 14.3.3 proteins [46]. Concordantly, we observed that Hsp27 silencing resulted in not only increased cytoplasmic sequestration, but also in significant co-localization of YAP/TAZ with 14.3.3 in PC3 cells (Figure 3.11A). An increase in interaction between 14.3.3 and TAZ was also observed upon Hsp27 siRNA treatment (Figure 3.11B).     si Scr siHsp27 DAPI 14.3.3 ε YAP/TAZ Merge PC3 A B    siHsp27 Input siScr 14-3-3 ε IP:TAZ IgG TAZ Hsp27 Vinculin 14-3-3 ε Total proteins PC3 Figure ‎3.11. Hsp27 knockdown induces cytoplasmic sequestration of YAP/TAZ. (A) Immunofluorescence of 14.3.3 (red), YAP/TAZ (green) and the nucleus (DAPI, blue) in  PC3 cells transfected with si Hsp27 or si Scr. Scale bar: 50 µm. (B) Protein expression of 14.3.3 ε, total TAZ, Hsp27 and Vinculin after TAZ immunoprecipitation in PC3 cells transfected with si Hsp27 or si Scr. 31  Reciprocally, YAP/TAZ were more nuclear and less sequestered with the 14.3.3 proteins upon Hsp27 overexpression in MEF-HSF1-/- cells (Figure 3.12A). This decreased interaction between TAZ and 14.3.3 proteins was again observed by immunoprecipitation in MEF-HSF1-/- cells after Hsp27 overexpression (Figure 3.12B).                    Together, these findings suggest that Hsp27 regulates YAP and TAZ activity through inhibition of phosphorylation, decreasing their sequestration by 14.3.3 proteins and promoting their translocation to the nucleus. MEF-HSF1 -/- Mock Hsp27 WT DAPI 14.3.3 ε YAP/TAZ Merge A B Input Mock Hsp27 14-3-3 ε IgG TAZ IP:TAZ Vinculin Hsp27 14-3-3 ε Total proteins MEF-HSF1 -/- Figure ‎3.12. Hsp27 overexpression decreases cytoplasmic sequestration of YAP/TAZ.  (A) Immunofluorescence of 14.3.3 (red), YAP/TAZ (green) and the nucleus (DAPI, blue) in MEF-HSF1-/- cells transfected with Hsp27 or vector control (mock). Scale bar: 50 µm. (B) Protein expression of 14.3.3 ε, total TAZ, Hsp27 and Vinculin after TAZ immunoprecipitation MEF-HSF1-/- cells transfected with Hsp27 or vector control (mock). 32  3.4. Hsp27 regulates the Hippo pathway by affecting core kinase components 3.4.1. The effect of Hsp27 knockdown on LATS1 and MOB1 Our results indicate that Hsp27 modulates YAP phosphorylation status, which is known to be controlled by the Hippo pathway through a core kinase cascade involving LATS1, MOB1 and MST1; however, a relationship between these kinases and Hsp27 has never been described. To examine the effect of Hsp27 in regulating the phosphorylation of YAP in more detail, PC3, A549 and MDA-MB-453 cells were treated with Hsp27 siRNA and functional changes in the Hippo pathway core components were examined. YAP is directly phosphorylated and restrained by LATS1, which is active when it is phosphorylated on T1079 [79]. As shown in Figure 3.13, knocking down Hsp27 resulted in increased p-LATS1 (T1079) across three cell lines, while the total protein remained unchanged. Looking further upstream in the Hippo pathway, we observed that phosphorylation of MOB1 on T35, which is required for activation of LATS1 [80]  was also increased in siHsp27 treated samples compared to siScr (Figure 3.13).                                                                                                         Figure ‎3.13. Hsp27 knockdown activates the Hippo pathway kinases. Protein expression of Hsp27, p-LATS1 T1079, total LATS1, p-MOB T35, total MOB1 and Vinculin in PC3, A549 and MDA-MB-453 cells transfected with control or Hsp27 siRNA. Densitometry shows fold expression of p-LATS1 T1079 compared to total LATS1 (=1) and p-MOB1 T35 compared to total MOB1 (=1).   33  3.4.2. The effect of Hsp27 overexpression on LATS1 and MOB1  Reciprocally, we observed that Hsp27 overexpression In PC3 and MEF-HSF1-/- cells drastically decreased phosphorylation of LATS1 (T1079) and MOB1 (T35) in both cell lines (Figure 3.14).    Figure ‎3.14. Hsp27 overexpression inactivates the Hippo pathway kinases. Protein expression of Hsp27, p-LATS1 T1079, total LATS1, p-MOB T35, total MOB1 and Vinculin in PC3 and MEF-HSF1-/- cells transfected with control or Hsp27 plasmid. Densitometry shows fold expression of p-LATS1 T1079 compared to total LATS1 (=1) and p-MOB1 T35 compared to total MOB1 (=1).  3.4.3. The effect of Hsp27 expression on MST1, the core Hippo kinase Finally, we examined the effects of Hsp27 modulation on MST1, the mammalian homologue of Drosophila’s Hippo kinase that is directly responsible for phosphorylation of LATS1 and MOB1 [80]. In accordance with changes observed in pLATS1 and pMOB1, we found that Hsp27 loss of function increased MST1 total protein (Figure 3.15 left), while overexpression of Hsp27 decreased total MST1 across multiple cell lines (Figure 3.15 right). Importantly however, RT-PCR analysis of MST1 mRNA in these conditions showed no change in transcription (Figure 3.16).    34                         Figure ‎3.15. Hsp27 expression regulates MST1 at protein level.  Protein expression of Hsp27, MST1 and Vinculin in PC3, A549, MDA-MB-453 and/or MEF-HSF1-/- cells transfected with si Hsp27 (left panel) or Hsp27 plasmid (right panel) compared to control (si Scr or Mock transfected cells).                                                                           Figure ‎3.16. Hsp27 expression does not affect MST1 transcription.  Relative mRNA expression of MST1 in PC3 cells transfected with si Hsp27 or Hsp27 plasmid compared to control (si Scr or Mock transfected cells=1). Graph is a representative of three independent experiments.  Levels of MST1 mRNA were also tested in lung and breast cancer cell lines upon Hsp27 knockdown (Figure 3.17). These data clearly demonstrate that Hsp27 inhibits the Hippo tumor suppressor pathway by regulating MST1 at the protein level. PC3 cells 35                                Figure ‎3.17: Hsp27 regulation of MST1 mRNA in Lung and Breast Cancer cells. Relative mRNA expression of MST1 (STK4) in si Hsp27 A549 and MDA-MB-453 cells compared to si Scr (=1), Graph represents pooled data from three independent experiments.  3.4.4. The effect of antisense oligonucleotide OGX-427 on the Hippo pathway OGX-427 is an antisense oligonucleotide designed for Hsp27 and is already in phase 2 clinical trials for different cancers. In order to verify our findings on YAP phosphorylation we tested the effects of OGX-427 on PC3 and A549 cell lines. Similar to our results in Figure 3.6 and Figure 3.15 left, knockdown of Hsp27 by OGX-427 increases p-YAP S127 and MST1 respectively (Figure 3.18).               Figure ‎3.18. OGX-427 activates the Hippo tumor suppressor pathway.  Protein expression of Hsp27, p-YAP S127, YAP, MST1 and Vinculin in PC3 and A549 cells transfected with scrambled oligonucleotide or OGX-427. Densitometry shows fold expression of MST1 in OGX-427 treated cells compared to control (=1)  36  3.5. Hsp27 forms a complex with MST1 and facilitates proteasomal degradation of ubiquitinated-MST1 3.5.1. Hsp27 forms a complex with MST1 in cancer cells In an effort to understand the exact mechanism by which Hsp27 regulates protein levels of MST1, we first performed immunoprecipitation (Co-IP) to unveil protein-protein interactions between MST1 and Hsp27. We found that endogenous Hsp27 formed a complex with MST1 in PC3 cells (Figure 3.19A, top). This interaction was confirmed with the reciprocal immunoprecipitation (Figure 3.19A, bottom) and was also observed in A549 cells (Figure 3.19B).                                      Figure ‎3.19.  Endogenous Hsp27 forms a complex with MST1 in cancer cells.  (A) Protein expression of Hsp27, MST1 and IgG after MST1 (top) and Hsp27 (bottom) immunoprecipitation in PC3 cells. (B) Protein expression of Hsp27, MST1 and IgG after MST1 immunoprecipitation in A549 cells.   37  3.5.2. Hsp27 regulates the proteasomal degradation of MST1  Since Hsp27 can enhance the proteasomal degradation of ubiquitinated proteins [34, 39, 81] and MST1 degradation is ubiquitin-mediated [82, 83] we tested if Hsp27 overexpression or silencing affects levels of ubiquitinated MST1. By pulling down MST1 and blotting for ubiquitin, we observed that overexpression of Hsp27 in PC3 cells lead to decreased amount of ubiquitinated MST1 (Figure 3.20A, left) while knocking down Hsp27 resulted in increased levels of ubiquitinated MST1 (Figure 3.20A, right). Moreover, a decrease in ubiquitinated MST1 was observed in MEF-HSF1-/- cells upon Hsp27 introduction (Figure 3.20B). These findings demonstrate that Hsp27 binds to MST1 and facilitates the proteasomal degradation of ubiquitinated MST1.                              Figure ‎3.20.  Hsp27 facilitates the proteasomal degradation of MST1 in cancer cells. (A) Protein expression of ubiquitin and MST1 after MST1 immunoprecipitation in Hsp27 plasmid (left) or si Hsp27 (right) transfected PC3 cells treated with MG132. (B) Protein expression of MST1 and ubiquitin after MST1 immunoprecipitation in MEF HSF1-/- cells transfected with Hsp27 or vector control (mock).  38  3.5.3. The effect of Hsp27 on MST1 is not reciprocal To better understand the interaction between Hsp27 and MST1, we overexpressed MST1 in PC3 cells and looked at the protein changes by western blotting. In harmony with what was reported previously [84] MST1 overexpression resulted in YAP inactivation in PC3 cells displayed by increased p-YAP S127, however no change was observed in the expression of Hsp27 (Figure 3.21A), suggesting that the effect of Hsp27 on MST1 is not reciprocal. We further confirmed inactivation of YAP by demonstrating decreased transcriptional activity of YAP’s transcriptional partners: TCF, SMAD1-4 and TEAD1 (Figure 3.21B).   Figure ‎3.21. Hsp27 facilitates the proteasomal degradation of MST1 in cancer cells.  (A) Protein expression of Hsp27, MST1, p-YAP S127, total YAP and Vinculin in PC3 cells transfected with MST1 (WT MST1) and vector control (mock). (B) Relative activity of TCF, SMAD1-4 and TEAD1 assessed by luciferase assay in PC3 cells transfected with MST1 compared to vector control (mock=1). Graph represents pooled data from three independent experiments.  3.5.4 Hsp27 knockdown does not affect Hsp70 levels MST1 is reported to be regulated by Hsp70 [83], therefore we investigated the effect of Hsp27 siRNA on levels of Hsp70. Figure 3.22 shows that knocking down Hsp27 does not affect protein expression of Hsp70. 39   Figure ‎3.22. Hsp27 knockdown does not affect Hsp70 expression in Prostate, Lung or Breast Cancer.  Protein expression of Hsp70, Hsp27 and vinculin in PC3, A549 and MDA-MB-453 cells transfected with si Hsp27 or si Scr.  3.6. Hsp27 negatively regulates the Hippo tumor suppressor pathway: A schematic summary Based on our accumulated in vitro data, we propose the following mechanism on how Hsp27 negatively regulates the Hippo tumor suppressor pathway. Overexpression of Heat shock protein 27 in cancer cells inactivates the Hippo tumor suppressor pathway via facilitating the degradation of ubiquitinated MST1 resulting in disruption of the Hippo kinase cascade. Degradation of MST1 protein results in decreased LATS1 and MOB1 phosphorylation/activation leading to decreased YAP/TAZ phosphorylation/inactivation, and ultimately increased YAP/TAZ nuclear localization. Translocation of YAP and TAZ from cytoplasm to nucleus drives transcription of genes associated with malignant cell phenotypes (Figure 3.23).    40   Figure ‎3.23: Proposed mechanism of negative regulation of the Hippo tumor suppressor pathway by Hsp27.  (Pathway On) The Hippo tumor suppressor pathway is activated by the core kinase MST1, which phosphorylates LATS1 and MOB1, leading to YAP/TAZ phosphorylation and their sequestration in the cytoplasm by 14.3.3 proteins. This prevents YAP/TAZ nuclear translocation and transcription of genes associated with malignant phenotypes such as those downstream of SMADs, TCF and TEADs. (Pathway Off) Hsp27 binds MST1 and promotes proteasomal degradation of ubiquitinated MST1. This prevents phosphorylation of LATS1 and MOB1, and phosphorylation of YAP/TAZ, which allows their nuclear translocation and their transcriptional activation of genes associated with malignant phenotypes such as those downstream of SMADs, TCF and TEADs.    41  4. Discussion The ability of cancer cells to survive in an environment filled with cellular stress inducers like hypoxia, toxic radicals, DNA damaging reagents, glucose deprivation, etc. depends on how they respond to these stimuli; In the context of such toxic stress, cancer cells activate survival pathways including overexpression of heat shock proteins, especially Hsp27.  Extensive investigation of Hsp27 expression in human tumors has shown that Hsp27 is elevated across different cancers and is associated with aggressive and treatment-resistant malignancies. Research driven by our laboratory and others showed that Hsp27 promotes cell proliferation and tumor growth in different cancers including prostate and bladder [85, 86] and that targeting Hsp27 not only reduces tumor progression, but also opposes chemotherapy resistance in prostate and lung cancer [44, 45, 85]. Moreover, we showed that Hsp27 drives β-catenin-mediated Epithelial Mesenchymal Transition (EMT) that is required for invasion and metastasis in prostate cancer [32, 33]. EMT is a process by which epithelial cells gain migratory and invasive properties and can initiate metastasis. Finally, our efforts to decipher the roles of Hsp27 in cancer led us to perform a gene expression profiling in prostate cancer cells with Hsp27 loss of function.     Using an un-biased approach, we found that in prostate cancer PC3 cells, transient knockdown of Hsp27 reduces the gene signature of several pathways including WNT/β-catenin, TGF-β/SMADs and ILK signaling. Detailed examination of gene transcription activities in these pathways revealed that they all share YAP and TAZ as transcriptional co-activators [48, 53, 55]. YAP and TAZ intertwine closely with other oncogenic pathways and are key modulators in organ size, cell proliferation and apoptosis by regulating the activity of different transcription factors such as β-catenin/TCF, SMAD1-4 and TEAD1-4. Numerous studies have shown that YAP and TAZ dysregulation contributes to cancer development and progression [87]; interestingly increased expression of YAP/TAZ, their nuclear localization, as well as elevation of their target genes, are reported in many types of cancers and are known to be involved in malignant phenotypes such as enhanced cell proliferation, EMT and drug resistance. For example, activation of YAP1 is highly associated with poor prognosis and treatment resistance in colorectal cancer [88] and 42  promotes migration and invasion in prostate cancers cells [89]. In addition, 90% of metaplastic (a subtype of triple negative) breast cancers with EMT morphology stained positive for nuclear TAZ [90]. YAP and TAZ have also been associated with progression and metastasis in lung cancer [91] and their direct transcription targets, CTGF and AXL, were linked to EMT and drug resistance in this disease [92]. Comparison of pro-tumorigenic characteristics of YAP/TAZ transcriptional co-activators and Hsp27 suggested the possibility of intersection between their oncogenic pathways; however a functional relationship between the two has not been established yet.  Therefore, we compiled an elaborate list of YAP/TAZ-dependent genes regardless of the conventional pathways they were attributed to and investigated their changes when Hsp27 is lost in PC3 cells. We found that Hsp27 knockdown significantly attenuated the gene signature of YAP/TAZ without affecting YAP and TAZ transcript levels. To validate the clinical relevance of our findings we interrogated TCGA data of prostate cancer tumors for mRNA expression of YAP/TAZ target genes in tumors with Hsp27 upregulation or downregulation. Similar to our in vitro results, the positive correlation between the expression of Hsp27 and YAP/TAZ target genes was also observed in 568 prostate tumor samples, suggesting the clinical relevance of this association. This prompted us to look at YAP/TAZ regulation more closely. YAP/TAZ activity and protein levels are predominantly controlled by the Hippo tumor suppressor pathway. First discovered in Drosophila melanogaster, the Hippo pathway has a fundamental role in organ growth control, stem cell function, regeneration and tumour suppression [65]. The ability of this evolutionary conserved pathway to inhibit proliferation and promote apoptosis has fascinated cancer researchers in the past decade. Recent work has led to the realization that in fact the Hippo pathway is part of an interconnected web which allows the cell to elicit the appropriate response to an external stimulus [93]. In fact, decreased activity of the Hippo pathway is associated with poor outcome across multiple cancer types. Similarly, numerous studies on cancer cell survival emphasized on the essential role of small heat shock proteins and have shown that Hsp27 acts as a central hub connecting networks that promote tumor growth and progression [1]. While there is extensive overlap between the cell survival, anti-apoptotic and metastatic pathways that Hsp27 and the Hippo components may 43  regulate, a relationship between the two has not been described yet. Here, for the first time, we report that Hsp27 negatively regulates the Hippo tumor suppressor pathway across different cancers.   Mechanistic investigations revealed that Hsp27 regulates YAP by decreasing the inhibitory phosphorylation of YAP on Serine 127, an established read out for Hippo pathway’s tumor suppressive activity. Interestingly, this phenomenon was observed across three different cancers, suggesting a pan-cancer mechanism for regulation of the Hippo pathway by Hsp27. Strikingly, protein-based patient data analysis of 578 lung and 1104 breast adenocarcinoma tumor samples from TCGA demonstrated the same pattern where tumors with higher expression of Hsp27 showed lower p-YAP (S127) and samples with reduced expression of Hsp27 had a higher score for p-YAP (S127). These findings suggest that the interaction between Hsp27 and the Hippo pathway we see in vitro also occurs in human samples. We then demonstrated that the functional consequence of Hsp27 overexpression and the subsequent decrease in p-YAP, is the reduced cytoplasmic retention and increased nuclear translocation of this transcriptional co-activator, allowing it to interact with pro-tumorigenic transcription factors. Most importantly, we follow the effects on YAP phosphorylation through the Hippo kinase cascade and report for the first time that downregulation of this tumor suppressor pathway is dependent on Hsp27 negative regulation of the core Hippo kinase, MST1.  MST1 (STK4) is a multifunctional kinase with tumor suppressive roles and is considered an independent prognostic factor in different cancers, where its reduction or loss of expression is associated with poor prognosis [94-96]. For example, a study on more than 1000 colorectal cancer samples showed that loss of cytoplasmic MST1 expression (and not transcriptional changes) was an independent adverse prognostic factor in this disease [94]. In prostate adenocarcinoma, MST1 protein, but not mRNA, expression is significantly downregulated compared to paired normal tissue, pointing to the importance of post-translational modifications of this kinase in prostate cancer [97]. Another independent study revealed that levels of MST1 also decreased with progression of the disease to CRPC [96]. These findings, combined with our previous work showing that transition from hormone naïve prostate cancer 44  to CRPC is accompanied by an increase in the expression of Hsp27 [33], suggest that Hsp27 may control MST1 in CRPC progression.  It is important to note that in prostate cancer MST1 also interacts with androgen receptor (AR), the main driver of this disease. Different studies showed that MST1 directly binds to AR and antagonizes AR transcriptional activity [98-100]. In addition, we previously showed that cooperative interactions between AR and Hsp27 facilitate AR transcriptional activity [40]. Taken together, these findings are encompassing evidence on how Hsp27 regulation of MST1 can be a player in prostate cancer progression; meaning Hsp27-dependent decrease of cellular MST1, a negative regulator of AR activity, may be another mechanism by which Hsp27 promotes AR activity. Even though these connections are important in an AR-driven model, we strongly believe that what we observe in our PC3 model (AR negative prostate  cancer cell line) as well as non-androgenic lung and breast cancer cells is independent of AR signaling and is more reflective of a pan-cancer phenomenon. Furthermore, our study provides mechanistic insight into how MST1 levels are decreased in aggressive cancers. Although MST1 and its tumor suppressive roles have been studied in multiple cancers, there is little known about the underlying mechanism by which MST1 is reduced, except that its degradation is ubiquitin-mediated through C terminus of Hsc70-interacting protein (CHIP) [82]. CHIP is a HSP co-chaperone with E3 ubiquitin ligase activity that promotes ubiquitination. The ubiquitination system brands proteins to be degraded by the 26S proteasome and HSPs, especially Hsp27, can promote the degradation of proteasomal client proteins. Containing an ubiquitin-like domain, Hsp27 directly binds to one of the subunits of 26S proteasome and enhances the catalytic activity of the 26S proteasome machinery in response to stressful stimuli [39]. Here for the first time, we show that Hsp27 forms a complex with MST1 and enhances its proteasomal degradation. This finding is in concordance with previous studies describing the role of Hsp27 in enhancing the degradation of other tumor suppressor proteins, I-κB (inhibitor of kappa B) and p27Kip1 (cyclin-dependent kinase inhibitor) [39, 81]. Moreover, our data compliment other results showing that another heat shock protein, Hsp70, regulates MST1 degradation [83]. This study by Ren et al. showed that overexpression of Hsp70 in stress conditions, such as chemotherapy, leads to degradation 45  of MST1 in PCa cells. Importantly however, we did not find that Hsp27 knockdown affects the expression of Hsp70, suggesting that there is an independent role for Hsp27 in MST1 regulation (Figure 3.22). Although Hsp27 and Hsp70 share several properties such as apoptosis inhibition, resistant induction and tumorigenesis enhancement, they are different in terms of how they regulate these processes. For example, Hsp27 is and ATP independent molecular chaperone where Hsp70 is ATP dependent. They also differ based on their interaction with the proteasomal machinery [101]. Parcellier et al. showed that although overexpression of Hsp70 can enhance proteasomal activity in response to stress, this effect does not depend on a direct interaction with ubiquitin chains, as it is the case with Hsp27 [39].  Comparison of our results to findings of Ren et al. further confirms that Hsp27 has a distinct mode of connection with the proteolytic machinery and that enhancement of client proteins’ degradation via Hsp27 is independent of Hsp70.  The novel link our data establish between Hsp27 and the Hippo pathway adds to the mechanisms by which Hsp27 may control tumor size, development and phenotype. For example, Hsp27 has been shown to affect tumor growth in different cancer models like breast and prostate [29, 40]. A study in breast cancer showed that high Hsp27 expression is linked to larger tumor volume, more proliferation and aggressive tumor phenotype. Xenografts originated from stably Hsp27 knock-downed cells were smaller and less angiogenic compared to the ones with normal Hsp27 expression. Also analysis of tissues from human patients suggested that patients with less Hsp27 expression have better survival rate [29]. Although justified through the role of Hsp27 in angiogenesis and increase of VEGF, the findings of this study raises the question whether Hsp27 contributes to breast cancer tumor size via other mechanisms like inactivation of the Hippo pathway, the most important pathway in organ size regulation, particularly because a recent study showed that YAP knockdown reduces VEGF transcripts in a breast cancer cell line [102]. The anti-apoptotic and cyto-protective roles of Hsp27 as well as the fact that it is ubiquitously overexpressed in different cancers, makes this chaperone an attractive target in cancer therapy. Indeed, multiple inhibitors of Hsp27 have been screened or developed. For 46  example, RP101 (Brivudine), an Hsp27 binding compound, is in phase II for pancreatic cancer [103].  Also depletion of Hsp27 using antisense technology in several preclinical animal models resulted in tumor regression [40, 43, 44]. OGX-427 (Apatorsen), a second-generation antisense oligonucleotide for Hsp27, is currently in phase II clinical trials for prostate, bladder and lung cancers (ClinicalTrials.gov, NCT01454089, NCT01829113, and NCT01120470). Our work suggests that especially in tumor types that depend heavily on inactivation of the Hippo pathway, inhibition of Hsp27 may be efficacious in preventing tumor progression. In summary, our study reveals an original role for Hsp27 in the negative regulation of the Hippo tumor suppressor pathway in cancer cells. For the first time we show that Hsp27 facilitates the proteasomal degradation of ubiquitinated MST1 and therefore interrupts the Hippo pathway kinase cascade. Consequently, YAP and TAZ oncoproteins are less phosphorylated, free to translocate into the nucleus and promote a malignant phenotype (Fiure 3.23). 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J Cancer Res Clin Oncol, 2011. 137(9): p. 1349-61.     52  Appendix Appendix 1 List of YAP/TAZ gene signature and respective normalized log2 values in si Scr and si Hsp27 treated PC3 cells. Gene si Scr si Hsp27 PSAT1 17.01364523 16.1642907 SMAD1 15.77624513 14.04485355 PDCD1LG2 15.41523747 13.03842473 MSMO1 14.44154268 13.5796855 SMAD6 14.09740834 13.86459485 SCHIP1 13.83917042 11.42588929 LGALSL 13.48697463 12.36662794 MACC1 13.38562382 11.33284504 UGCG 13.35147678 11.49528249 EPB41L2 13.13598257 10.42346027 PITX2 13.08989616 11.32308609 DAB2 12.73120113 9.442547146 CPA4 12.6546593 11.14469644 SGK1 12.45230107 10.3728517 GPCPD1 12.42749121 9.09193641 PYGO1 12.42169003 8.412915021 CENPF 12.25494483 7.459307484 NT5E 12.09808253 8.255554475 ANXA1 12.00732237 6.890668068 MID1 11.77436835 8.955273931 CHST9 11.55329618 11.39165048 CAV1 11.51225568 7.556603253 PCLO 11.43639944 7.630748029 ARHGAP29 11.43577666 6.087261559 ENC1 11.2918425 9.665932932 TGFA 11.22669414 10.22174955 PRSS23 11.17193986 7.576703769 SERPINB7 11.17093091 9.214762959 MYBL1 10.96697517 8.530321332 BCL2 10.94477655 6.761392428 SERPINE1 10.93055913 8.142799633 THBS1 10.92734813 9.371137888 RND3 10.84163479 5.350830328 53  Gene si Scr si Hsp27 CTNNB1 10.80275149 9.240144947 CTNNAL1 10.79686734 6.947355114 EP300 10.71243002 7.898946112 CRIM1 10.63554954 8.386907855 SHROOM3 10.41196544 8.431558379 FRZB 10.40232472 7.362189017 SLIT2 10.35295578 7.58824035 PTPN14 10.27103809 6.666341606 EMP2 10.2659359 9.57971603 SNAPC1 10.26458358 7.145387872 SCD5 10.22401325 7.076027669 MYOF 10.14549825 7.461591073 BICC1 10.10906223 7.341722295 TOP2A 10.08226216 4.789844276 CDH2 9.911701168 7.153638113 LHFP 9.883599282 6.750840879 OPN3 9.814634293 8.469460352 SNAI2 9.583307336 6.189412359 CCND2 9.554704646 7.913117608 ESM1 9.498819395 4.834771394 TGFB2 9.360629972 6.459271661 LRP6 9.21220502 5.925356234 RIMKLB 9.183667138 5.320722922 ID2 9.178417119 4.52703121 TSC22D2 9.177835418 5.627012519 MET 8.949237843 3.821837258 AHNAK 8.898692163 6.254458186 ECT2 8.806316323 3.835926993 GGH 8.804056141 4.43331799 LMBRD2 8.700756036 5.765337189 TMEM154 8.62234022 5.528784582 ARHGEF28 8.599561719 6.656254082 LUM 8.468867234 5.038729883 CCL28 8.467728622 6.398733417 ADAMTS12 8.441855426 6.549500705 ITGBL1 8.376938801 2.12801484 PRRG1 8.352987282 2.224120624 MDFIC 8.345931973 2.094483875 IFIT2 8.197555527 2.264627753 TMEM27 8.177187891 3.354978572 F3 8.115716005 6.928779886 GLS 8.045898437 3.696395629 54  Gene si Scr si Hsp27 PDP2 8.011860896 6.620456942 ACSL4 7.933785474 2.662645565 GADD45A 7.701168511 6.037925031 IFI16 7.622054602 2.050958104 FGF2 7.511950981 1.995879439 SDPR 7.462854366 4.965308957 CLDN1 7.239845759 5.320962217 FSTL1 7.083340184 5.046671197 SCML1 7.074799546 1.903302376 EXPH5 7.039980488 4.218589926 AXL 6.945869383 5.133898067 HMMR 6.936588828 2.002870378 PMAIP1 6.880130696 4.199617898 SEMA3C 6.734954858 1.896036602 PHLDA1 6.70416787 3.880824834 IRS1 6.253338848 3.421702896 DAAM1 6.192013225 2.476968698 FST 6.04905035 4.96256993 ASAP1 6.010676018 2.918076275 INSIG1 5.996509531 3.41756635 RHOU 5.991762634 3.120563863 BDNF 5.957348829 2.186108342 HMGCS1 5.844367976 2.159839427 IDI1 5.784216829 3.639358338 CYP1B1 5.763458318 3.870782342 AOX1 5.760320681 5.30405597 SNORA75 5.739108559 3.52797127 PSG5 5.650150278 2.265399203 JPH1 5.603233315 3.438913103 F2RL1 5.597450724 4.187126117 DIXDC1 5.574666968 3.871582299 SP1 5.564356308 3.687062782 SMAD5 5.327253844 4.402483257 LPIN1 5.295987629 2.184630145 PRICKLE1 5.184416781 4.49580554 SNRPG 5.179853968 2.054330556 DDAH1 5.169179416 2.620085155 FSCN1 5.131463136 5.427053199 DUT 4.8357267 2.088257542 EMP1 4.807664862 2.510870108 LCP1 4.683229889 2.353511348 RAB3B 4.467957492 3.081287668 55  Gene si Scr si Hsp27 CTGF 4.462551023 4.503400582 SMAD3 4.38810679 4.10214715 S1PR1 4.174602962 2.572433119 FBXW11 3.958380475 2.234871706 IFRD1 3.725963596 1.924834297 CD55 3.711125816 1.916858113 ANXA3 3.627528632 1.996193033 TFPI2 3.607848585 3.986906103 AMPH 3.573904048 2.29338142 TSC22D1 3.569048688 2.286186518 SYT14 3.565704521 1.962384886 ERRFI1 3.511753982 2.239459703 RGS4 3.509068722 2.251786479 SQLE 3.440047241 2.142507146 CSNK2A1 3.397488108 2.125744639 PMP22 3.382857986 2.074582587 GLI2 3.376551556 2.108634709 ADAMTS1 3.371090433 2.62306717 ADAMTS5 3.368382694 2.130463824 SLC2A3 3.366874704 2.221180596 ADRB2 3.351806111 2.072713945 HEXB 3.351496604 2.054180811 GCNT4 3.338375777 2.085630121 SCD 3.283995386 2.003441001 IL8 3.28391955 2.046099953 COX6C 3.261657177 2.024147335 NT5DC3 3.240491564 1.977508755 STXBP1 3.234171572 2.016912687 SLC16A6 3.226513616 2.035211092 KIT 3.222433331 2.037982755 AREG 3.202016281 2.010265667 KRT5 3.177729672 1.961008123 SOX9 3.137527623 1.921058386 MYC 3.105650424 1.882593394 UPK1B 3.088504261 1.863256292 NID2 3.086198317 1.863334638  

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