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The impact of oncogenic kras on redox balance to support cellular transformation and tumorigenicity Lim, Jonathan Kah Meng 2018

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THE IMPACT OF ONCOGENIC KRAS ON REDOX BALANCE TO SUPPORT CELLULAR TRANSFORMATION AND TUMORIGENICITY by  Jonathan Kah Meng Lim  B.Sc., Simon Fraser University, 2012  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  January 2018  © Jonathan Kah Meng Lim, 2018 ii  Abstract Activating mutations in KRAS are found in ~90% of pancreatic cancers, ~40% of colorectal cancers, and ~30% of non-small cell lung cancers.	   To date no effective therapies exist for cancer patients of this genetic subset, driving an impetus to develop novel therapeutic agents that target KRAS or downstream effectors of KRAS. The impact of oncogenic KRAS on the intracellular redox balance and its contribution to tumorigenicity is still controversial. Many studies have reported that oncogenic RAS enhances intracellular reactive oxygen species (ROS) levels, while recent major work by several groups described that oncogenic RAS drives antioxidant programs, which are necessary to mediate tumorigenicity. It is therefore critical to further explore the role of oncogenic KRAS on redox balance and its impact on cellular transformation and tumorigenicity. To this end, I utilized whole transcriptome profiling in normal and oncogenic KRAS-transformed cells to identify redox pathways regulated by oncogenic KRAS to support tumorigenicity. Whole transcriptome analysis revealed that the Cystine/Glutamate Transporter, xCT had the highest positive fold change in KRAS-transformed cells in response to exogenous oxidative stress. xCT is responsible for the cellular uptake of cystine, the rate-limiting precursor in the synthesis of glutathione (GSH), which is the major intracellular antioxidant. As such, I postulated that oncogenic KRAS signaling promotes transcriptional upregulation of xCT to support cellular transformation and tumorigenicity by preventing oxidative stress. Notably, inhibition of xCT in KRAS-transformed cells exacerbates oxidative stress causing cell death and also impaired cellular transformation and tumorigenicity, providing the first evidence that xCT is a downstream effector of oncogenic KRAS signaling. In addition, I found clinical iii  evidence for the upregulation of xCT in subsets of cancer with activating mutations in KRAS and for the association of high xCT expression with poorer patient outcome. Finally, I delineated a novel mechanism of xCT activation involving the cooperative interaction between ETS1, which lies downstream of the RAS-MAPK signaling cascade, and ATF4, a known regulator of xCT. Overall, my findings demonstrate that oncogenic KRAS signaling modulates cellular redox balance by upregulating xCT expression to promote transformation and tumorigenicity.  iv  Lay Summary The cancer-causing gene KRAS is associated with some of the most aggressive and fatal cancers. Unfortunately, no effective therapies exist for targeting KRAS-associated cancers, driving the need for new drugs. In my studies I discovered that KRAS increases xCT, a transporter that brings into the cell the amino acid cystine, a building block for a major cellular antioxidant that detoxifies free radicals known as ROS. I hypothesized that KRAS increases xCT levels to support cancer formation. Indeed, blocking xCT in KRAS-expressing cells caused an accumulation of ROS and suppressed their ability to form tumors in mice. I also uncovered a mechanism explaining how xCT is increased by KRAS. Overall, my studies extend current understanding of how KRAS impacts antioxidants and ROS. Namely, that KRAS increases xCT to prevent damaging levels of ROS in cancer cells, supporting tumor growth. This presents a new therapeutic target for the treatment of KRAS-driven cancers.  v  Preface The conception and design of the research project encompassed in this thesis dissertation was developed by Dr. Poul Sorensen, Dr. Gabriel Leprivier, and I. The contents of this thesis dissertation are novel and I was responsible for the set up and analysis of 80% of all experiments. The whole transcriptome microarray in Section 2.3.3 of Chapter 2 was performed by the Centre for Translational and Genomic Science (CTAG, Vancouver, Canada). I was responsible for the culturing, treatment, and collection of cells and for the analysis of transcriptomic data. The FASu experiment in Section 2.3.5 was performed in collaboration with Milena Colovic from Dr. Paul Schaffer’s laboratory. The subcutaneous injections, tumor volume measurements, and harvesting of tumors from the mouse xenograft experiment in Chapter 3 was performed by Alberto Delaidelli, a fellow PhD student in the Sorensen Laboratory. Dr. Haifeng Zhang, a postdoctoral fellow in the Sorensen Laboratory, performed the Western Blot in Figure 3A., and the ETS-1 overexpression experiments in Section 4.3.4 of Chapter 4. In addition, the bioinformatics analyses of publicly available clinical datasets in Section 3.3.5 of Chapter 3 was conducted by Dr. Gian Luca Negri, a postdoctoral fellow in the Sorensen Laboratory. I was responsible for the culturing, treatment, and the collection of cells for the metabolite analysis. All animal experiments underwent ethical approval from the Animal Care Committee of the University of British Columbia (A16-0050; A16-0050-A001). A version of Chapter 2 is currently in preparation for publication as a review article. Additionally, the experimental studies contained within Chapters 2, 3, and 4 will constitute a manuscript that is currently in preparation. vi  Table of Contents  Abstract ........................................................................................................................................ ii	  Lay Summary ............................................................................................................................. iv	  Preface ...........................................................................................................................................v	  Table of Contents ...................................................................................................................... vi	  List of Tables .............................................................................................................................. xi	  List of Figures ........................................................................................................................... xii	  List of Abbreviations .............................................................................................................. xvi	  Acknowledgements .............................................................................................................. xviii	  Chapter 1: Introduction .............................................................................................................1	  1.1	   RAS Signaling in Normal and Cancer Cells ............................................................... 1	  1.1.1	   RAS proteins in normal cells .......................................................................... 1	  1.1.1.1	   Signaling upstream of RAS ............................................................................ 2	  1.1.1.2	   RAS effectors and downstream signaling .................................................... 4	  1.1.2	   Oncogenic RAS signaling in cancer cells ....................................................... 7	  1.1.2.1	   Clinical significance of RAS mutations ....................................................... 10	  1.1.2.2	   Biology of cancers harboring RAS mutations ............................................ 11	  1.1.2.3	   Therapeutic targeting of oncogenic RAS signaling .................................. 15	  1.2	   Cellular Transformation and its Impact on Redox Balance .................................... 20	  1.2.1	   Reactive oxygen species (ROS) in normal and cancer cells ........................ 20	  1.2.1.1	   ROS producing systems ............................................................................... 22	  1.2.1.2	   ROS scavenging systems ............................................................................ 23	  vii  1.2.1.3	   Function of ROS in biological systems ....................................................... 25	  1.2.1.4	   Oxidative stress in cancer cells ................................................................... 27	  1.2.2	   Therapeutic targeting of cancer cells via ROS-mediated mechanisms ........ 31	  1.3	   Hypothesis and Significance ....................................................................................... 34	  1.4	   Research Objectives .................................................................................................... 35	  Chapter 2: Transformation with Oncogenic KRAS Modulates Cellular Redox Balance via Upregulation of Cystine/Glutamate Transporter xCT Expression ........37	  2.1	   Background and Rationale .......................................................................................... 37	  2.2	   Methods .......................................................................................................................... 45	  2.3	   Results ............................................................................................................................ 51	  2.3.1	   Transformation with oncogenic KRAS leads to protection against oxidative stress   ..................................................................................................................... 51	  2.3.2	   Transformation with oncogenic KRAS leads to decreased intracellular ROS levels   ..................................................................................................................... 52	  2.3.3	   Transformation with oncogenic RAS enhances the upregulation of the light-chain subunit of the Cystine/Glutamate Transporter, xCT, in vitro .......................... 55	  2.3.4	   Transformation with oncogenic KRAS enhances the upregulation of the light-chain subunit of the Cystine/Glutamate Transporter, xCT, following exposure to known oxidative stress inducers ............................................................................. 57	  2.3.5	   Transformation with oncogenic KRAS enhances xCT activity following exposure to oxidative stress .................................................................................... 59	  2.3.6	   Transformation with oncogenic KRAS enhances intracellular GSH levels ... 61	  viii  2.3.7	   Expression of the heavy-chain subunit of the Cystine/Glutamate Transporter, CD98, is unaltered following transformation with oncogenic KRAS and exposure to exogenous oxidative stress ..................................................................................... 63	  2.3.8	   Transformation with oncogenic RAS leads to the upregulation of the light-chain subunit of the Cystine/Glutamate Transporter, xCT, in vivo .......................... 64	  2.3.9	   Inhibition of oncogenic KRAS reduces xCT expression ............................... 66	  2.3.10	  xCT supports the oncogenic KRAS-mediated protection against oxidative stress through the enhancement of intracellular GSH levels .................................. 67	  2.4	   Discussion ...................................................................................................................... 74	  Chapter 3: Oncogenic KRAS Upregulates xCT Expression to Support Transformation and Tumorigenicity ....................................................................................79	  3.1	   Introduction .................................................................................................................... 79	  3.2	   Methods .......................................................................................................................... 85	  Histopathology, immunohistochemistry, and tissue microarrays (TMAs) .................. 87	  3.3	   Results ............................................................................................................................ 89	  3.3.1	   Oncogenic KRAS is unable to transform xCT deficient cells ........................ 89	  3.3.2	   Genetic inhibition of xCT impairs transformation by oncogenic KRAS ......... 91	  3.3.3	   Pharmacological inhibition of xCT impairs cellular transformation by oncogenic KRAS ..................................................................................................... 92	  3.3.4	   Genetic inhibition of xCT impairs tumorigenicity of cells harboring oncogenic KRAS   ..................................................................................................................... 94	  3.3.5	   Increased expression of xCT occurs in clinical specimens of oncogenic KRAS-expressing tumors and is associated with poor prognosis ........................... 98	  ix  3.4	   Discussion .................................................................................................................... 102	  Chapter 4: Oncogenic KRAS-Mediated Upregulation of xCT Expression is Dependent on RAS-MAPK Signaling to ETS-1 ................................................................106	  4.1	   Introduction .................................................................................................................. 106	  4.2	   Methods ........................................................................................................................ 109	  4.3	   Results .......................................................................................................................... 111	  4.3.1	   Gene Set Enrichment Analysis (GSEA) of transcriptomic data reveals that ETS-1 target genes are upregulated in oncogenic KRAS-transformed cells ........ 111	  4.3.2	   ETS-1 transcription factor mediates oncogenic KRAS-dependent upregulation of xCT to modulate intracellular ROS levels ..................................... 112	  4.3.3	   MEK inhibition but not AKT inhibition ablates oncogenic KRAS-mediated upregulation of xCT ............................................................................................... 115	  4.3.4	   Ectopic overexpression of ETS-1 promotes upregulation of xCT ............... 116	  4.3.5	   ETS-1 expression and activity are not induced by oxidative stress ........... 118	  4.3.6	   Gene Set Enrichment Analysis (GSEA) reveals that ATF4 target genes are upregulated in oncogenic KRAS-transformed cells treated with exogenous ROS 118	  4.3.7	   Genetic inhibition of ATF4 ablates oncogenic KRAS-mediated upregulation of xCT  ................................................................................................................... 121	  4.3.8	   ETS-1 and ATF4 cooperatively enhance xCT expression ......................... 124	  4.3.9	   NRF2 potentially cooperates with ETS-1 and ATF4 to enhance xCT expression ............................................................................................................. 128	  4.4	   Discussion .................................................................................................................... 134	  Chapter 5: Conclusion and Future Directions ................................................................139	  x  References ................................................................................................................................147	   xi  List of Tables Table 1: The cystine/glutamate transporter, SLC7A11 (xCT), is most prominently upregulated in 3T3 KRAS cells following exogenous H2O2 treatment ............................ 56 Table 2: Gene Set Enrichment Analysis (GSEA) of transcriptomic data reveals that ETS-1 target genes enriched for in 3T3 KRAS cells as compared to 3T3 MSCV cells ...... ………………………………………………………………………………………………….113 Table 3: Gene Set Enrichment Analysis (GSEA) of transcriptomic data reveals that ATF4 target genes are enriched for in 3T3 KRAS cells treated with H2O2 relative to basal conditions ............................................................................................................ 122  xii  List of Figures Figure 1: Signaling upstream and downstream of RAS ................................................... 8 Figure 2: Intracellular ROS producing and scavenging mechanisms  ........................... 25 Figure 3: Transformation with oncogenic KRAS or EN leads to the protection of NIH3T3 cells against exogenous H2O2 treatment ........................................................................ 53 Figure 4: Transformation with oncogenic KRAS leads to decreased intracellular ROS and decreased protein oxidation under exposure to oxidative stress ............................ 54 Figure 5: Oncogenic RAS induces the upregulation of xCT mRNA .............................. 58 Figure 6: Oncogenic KRAS enhances the upregulation of xCT mRNA in cells following exposure to oxidative stress inducers, diethyl-maleate (DEM) and menadione (MEN) . 60 Figure 7: Oncogenic KRAS enhances 18F-5-fluoroaminosuberic acid (FASu) uptake in cells following exposure to H2O2 and DEM .................................................................... 62 Figure 8: Transformation with oncogenic KRAS and EN enhances intracellular GSH levels .............................................................................................................................. 63 Figure 9: Expression of CD98 is unaltered following stable expression of oncogenic KRAS and exposure to exogenous oxidative stress ...................................................... 65 Figure 10: Lung and colon tumor specimens from transgenic mice expressing oncogenic KRAS display higher xCT mRNA levels ........................................................ 66 Figure 11: KRAS-specific siRNAs reduce xCT expression in human cancer cell lines . 67 Figure 12: xCT-specific siRNAs resensitize 3T3 KRAS cells to exogenous H2O2 treatment ........................................................................................................................ 69 Figure 13: xCT-specific siRNAs increase intracellular ROS and decrease GSH in 3T3 KRAS cells ...................................................................................................................... 71 xiii  Figure 14: Loss of xCT increases intracellular ROS and decreases GSH in KRASV12-transformed MEFs, leading to increased susceptibility to exogenous oxidative stress .. 73 Figure 15: Ectopic overexpression of xCT in non-transformed 3T3 MSCV cells protects them against exogenous H2O2 treatment ....................................................................... 74 Figure 16: Oncogenic KRAS is unable to transform xCT deficient MEFs ..................... 91 Figure 17: RNA-mediated silencing of xCT reduces anchorage independent growth of mutant KRAS cells ......................................................................................................... 93 Figure 18: Erastin treatment reduces anchorage independent growth of mutant KRAS cells ................................................................................................................................ 95 Figure 19: Expression of xCT-specific shRNAs reduces the tumorigenicity of 3T3 KRAS cells and prolongs survival of mice ................................................................................. 96 Figure 20: shRNA-mediated knockdown of xCT induces significant necrosis in vivo ... 97 Figure 21: shRNA-mediated knockdown of xCT decreases GSH and increases DHE staining in vivo ................................................................................................................ 97 Figure 22: High xCT mRNA expression is correlated with the KRAS mutant subtype in lung cancer ..................................................................................................................... 99 Figure 23: Genes co-expressed with xCT in lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and colorectal adenocarcinoma (COAD) show enrichment for the “MEK_UP.V1_UP” signature ............................................................ 99 Figure 24: High xCT expression is correlated with significantly decreased overall survival in lung, AML, papillary renal cell carcinoma, and hepatocellular carcinoma ... 101 Figure 25: Gene Set Enrichment Analysis (GSEA) of transcriptomic data reveals that ETS-1 target genes enriched for in 3T3 KRAS cells as compared to 3T3 MSCV cells 113 xiv  Figure 26: ETS-1 mediates the upregulation of xCT by oncogenic KRAS .................. 114 Figure 27: MEK inhibition but not AKT inhibition ablates oncogenic KRAS-mediated upregulation of xCT ...................................................................................................... 117 Figure 28: Ectopic expression of ETS-1 promotes the upregulation of xCT ................ 119 Figure 29: ETS-1 expression and activity are unchanged under H2O2 treatment ....... 120 Figure 30: Gene Set Enrichment Analysis (GSEA) of transcriptomic data reveals that ATF4 target genes are enriched for in 3T3 KRAS cells treated with H2O2 relative to basal conditions ............................................................................................................ 122 Figure 31: ATF4 expression is induced in NIH 3T3 MSCV, EN, and KRAS cells following H2O2 treatment, while ETS-1 activity and expression remain unchanged ..... 123 Figure 32: ATF4-specific siRNA expression in 3T3 KRAS cells abrogates the induction of xCT following H2O2 treatment ................................................................................... 125 Figure 33: Combined siRNA-mediated inhibition of ETS-1 and ATF4 has an additive effect on the reduction of xCT mRNA levels ................................................................ 126 Figure 34: Combined siRNA-mediated inhibition of ETS-1 and ATF4 reduces anchorage independent growth mediated by oncogenic KRAS ................................... 127 Figure 35: ETS-1 and ATF4 synergistically promote the upregulation of xCT by activating the promoter region of the xCT gene ........................................................... 129 Figure 36: NRF2 expression is induced following transformation with EN or oncogenic KRAS, but remains unchanged following H2O2 treatment ............................................ 130 Figure 37: ETS-1 or ATF4-specific siRNA expression in 3T3 KRAS cells does not alter NRF2 or HO-1 expression ............................................................................................ 132 xv  Figure 38: NRF2-specific siRNA expression in 3T3 KRAS cells does not alter ETS-1 or ATF4 expression, but decreases the induction of xCT expression by ATF4 overexpression or exposure to H2O2 ............................................................................ 133 Figure 39: ETS-1 and ATF4 synergistically promote the upregulation of xCT, while NRF2 provides an additive contribution to this upregulation ........................................ 135 Figure 40: Oncogenic KRAS transcriptionally upregulates xCT via cooperative action of ETS-1 and ATF4 to promote transformation and tumorigenicity by preventing ROS overload ........................................................................................................................ 146 xvi  List of Abbreviations  CM-H2DCFDA: 5-(and-6)-chloromethyl-2’, 7’-dichlorodihydrofluorescein diacetate, acetyl ester DT: Dityrosine EGFR: Epidermal growth factor receptor EN: ETV6-NTRK3 ERA: Erastin GCL: Glutamate cysteine ligase GPX: Glutathione peroxidase GR: Glutathione reductase GS: Glutathione synthetase GSH: Reduced glutathione GSSG: Oxidized glutathione GTP: guanine triphosphate HMEC: Human mammary epithelial cells H2O2: Hydrogen peroxide KO: knockout MEF: Mouse embryonic fibroblasts NAC: N-acetyl cysteine NADPH: nicotinamide adenine dinucleotide phosphate NSCLC: non-small cell lung cancer NOX: NADPH oxidase xvii  PDAC: Pancreatic ductal adenocarcinoma PI: Propidium iodide RNAi: RNA interference ROS: Reactive oxygen species RTK: Receptor tyrosine kinase WT: wildtype xCT: Cystine/glutamate transporter or system xc– transporter, light subunit   xviii  Acknowledgements  I would like to thank my senior supervisor Dr. Poul Sorensen for the opportunity to conduct research in his laboratory, and for his confidence in affording me a high level of autonomy and ownership to forge an interesting research project. Without him this project would not have been possible. In addition, I would like to thank my supervisory committee members Dr. Haydn Pritchard, Dr. Andrew Weng, Dr. Andrew Minchinton, and Dr. Will Lockwood for their support and guidance throughout the course of my PhD work. I owe particular thanks to Dr. Gabriel Leprivier for his patient mentorship, for providing coherent answers to my countless questions, for enlarging my vision of science, and for helping me develop deeper thought. I offer my gratitude to fellow lab members past and present; this project is the sum of all your help, support, and invaluable intellectual exchange. Special thanks go to Dr. Barak Rotblat, Dr. Naniye Cetinbas, Dr. Sean Minaker, Dr. Haifeng Zhang, Dr. Gian Luca Negri, Dr. Rohoullah Mousavizadeh, Alberto Delaidelli, Arash Samiei, Amy Li, Jordan Cran, and everyone else who has offered help in one way or another. I offer my enduring gratitude to my parents as well as to my sisters, for the part they have played and continue to play in every endeavor of my life, scientific or otherwise. I am who I am today because of them. And finally I thank my wife Priscilla for her unceasing support, her words of assurance, and her incredible patience as hours in the lab culminate into weeks, months and years. To her I dedicate this thesis.  1  Chapter 1: Introduction  1.1 RAS Signaling in Normal and Cancer Cells 1.1.1 RAS proteins in normal cells The RAS proteins are members of a large superfamily of highly homologous, membrane-localized guanine triphosphate (GTP)-binding proteins, known as small GTPases, and are approximately 21 kDa in size. They have essential roles in modulating several central signaling pathways that control normal cellular growth and proliferation. In humans, three RAS genes encode four distinct proteins: HRAS, NRAS, and two alternative splice variants of the KRAS gene, known as KRAS4A and KRAS4B. All four proteins are widely expressed, with KRAS being ubiquitous in almost all cell types. Further, although they share 85% amino acid sequence identity and function in similar ways, they exhibit subtle differences in molecular function. For instance, knockout studies have shown that the deletion of Kras gene results in embryonic lethality, but the deletion of Nras or Hras, either alone or in combination, had no impact on normal development in the mouse (1).    The proper functioning of RAS proteins requires them to be post-translationally modified in order for their localization to the inner surface of the plasma membrane. When RAS is newly synthesized it exists in the cytosol. The first step in the post-translational modification of RAS is the Farnesyltransferase (FTase)-mediated transfer of a 15-carbon isoprenoid chain from farnesyl pyrophosphate (FPP) to a cysteine residue on the carboxy terminus (2, 3). This enables RAS to associate with intracellular membranes via the farnesyl group. Following isoprenylation, the CAAX endopeptidase 2  removes three of the amino acids (AAX) from the carboxyl terminus, which is then methylated by a methyltransferase. For HRAS and NRAS specifically, the final step is the addition of two palmitoyl long-chain fatty acid groups to a cysteine residue upstream of the farnesylated carboxyl terminus cysteine residue, stablilizing the interaction with the membrane. KRAS is not palmitoylated as its stabilization to the plasma membrane is mediated by lysine residues also near the carboxyl terminus that interact with negatively charged lipid head groups. It is also worth noting that KRAS and NRAS (but not HRAS), particularly when FTase is absent, can be geranylgeranylated by geranylgeranyltransferase (GGTase), in which a 20-carbon isoprenoid group is added as an alternative to farnseylation (4). Given the importance of localizing RAS proteins to the plasma membrane in order for its activation of downstream signaling, the enzymes involved in their post-translational modification presented very attractive targets for therapeutic intervention, which I will discuss in later sections.  1.1.1.1 Signaling upstream of RAS RAS proteins serve as transducers that relay signals from receptor tyrosine kinases on the cell surface to intracellular effector pathways. As small GTPases, they cycle between ‘on’ and ‘off’ conformations that are conferred by the binding of GTP or GDP, respectively. Purified RAS proteins display a low level of GTPase activity in vitro, whereby the conversion of GTP to GDP is slow; and also possesses a low rate of nucleotide exchange with surrounding medium in which bound GDP is replaced by GTP (5). In the cell, the transition between both states are catalyzed by two enzymes - GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). 3  GAPs promote the inactivation of RAS by accelerating RAS-mediated GTP hydrolysis to GDP, while GEFs activate RAS by stimulating the exchange of GDP for GTP. Therefore, the activation state of RAS and its downstream effector pathways are largely determined by the balance between GAPs and GEFs. The two most extensively studied RAS-GEFs are SOS1 and SOS2, initially identified as mammalian homologues of the son of sevenless gene product in Drosophila, and its involvement in RAS regulation genetically characterized in Drosophila, Caenorhabditis elegans, as well as mammalian tissue culture systems (6). The model for RAS regulation begins with the upstream activation of receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) or the platelet-derived growth factor receptor (PDGFR) by their respective ligands - EGF and PDGF. The autophosphorylation of the RTK creates a binding site for the SH2 domain of an adaptor protein called the growth factor receptor-bound protein 2 (GRB2). GRB2 then binds SOS through its SH3 domain, recruiting it to the plasma membrane and within close proximity to RAS. The interaction between SOS and RAS thus leads to the enhanced exchange of GDP for GTP on RAS and its subsequent activation. Although this described model is the most commonly accepted, there are minor variations in certain stages of signal transduction. Firstly, other receptor types such as G-protein-coupled receptors (GPCRs) can activate RAS through stimulation of GEFs (7). Secondly, another adaptor protein SHC can mediate the interaction between RTKs and GRB2. Thirdly, there is also strong evidence for the activation of RAS by non-receptor tyrosine kinase signaling. For instance, the non-RTK v-Src is known to activate RAS by phosphorylating SHC, leading to the recruitment of the GRB2/SOS complex and consequently RAS activation (8). In addition, there are 4  several other RAS-related GEFs that are subjected to a different mode of regulation (9). It is also worth noting that the plasma membrane is not the exclusive platform on which RAS regulates signaling, as there have been studies showing that it can be tethered to the Golgi and endoplasmic reticulum where it engages other signaling pathways (10, 11). The diversity in how RAS can be activated not only alludes to its critical role in regulating cell proliferation, but also highlights the challenge in therapeutically targeting RAS, given the alternate mechanisms of activation that exists.    1.1.1.2 RAS effectors and downstream signaling The first effector of RAS to be characterized in mammalian cells is the RAF family of serine/threonine kinases. This family of kinases consists of ARAF, BRAF, and RAF1 (also known as CRAF). GTP-bound RAS is active by virtue of its increased affinity for its effectors and thus interacts with RAF, recruiting it to the plasma membrane and contributing to its catalytic activation (12, 13). The formation of a RAS-RAF complex and the activation of RAS involves the phosphorylation and dephosphorylation of several positive and negative regulatory sites in RAF, although the exact mechanistic processes are not well understood. It is however now well appreciated that RAF dimerization is a critical determinant of RAF activation (14). Activated RAF then phosphorylates and activates the mitogen-activated protein kinase kinases 1 and 2 (MEK1 and MEK2) that in turn phosphorylate and activate the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (extracellular signal-regulated kinases). Activated ERK 1 and 2 regulate a whole range of cellular events, with substrates that comprise of cytosolic as well as nuclear proteins, given that activated 5  ERK can enter the nucleus. One of the ERK substrates that have received the most attention are the E26-transcription factor (ETS) family of proteins, which include ETS-1, ETS-2, ELK1, SAP1, SAP2, E1Af, PEA3, PU1 and others (15). Given their involvement in a wide variety of biological functions such as cellular differentiation, cell cycle regulation, cell migration, angiogenesis, cell proliferation, and apoptosis, this signaling cascade was the prototype for other plasma-membrane-to-nucleus signal transduction pathways (16). For instance, ERK phosphorylates a member of the ETS family known as ELK1, which regulates the expression of FOS (17). In addition ERK can also phosphorylate c-JUN leading to the formation of FOS-JUN heterodimers, which is the process by which the AP-1 transcription factor is activated. Notably, AP-1 is a key regulator of D-type cyclins that enable cells to progress through the G1 phase and into the S phase of the cell cycle (18). The RAF signaling cascade is thus perceived as a key regulator of cell cycle progression. The second best-characterized effector of RAS is phosphoinositide 3-kinase (PI3K), which plays a critical role in RAS-mediated cell survival and growth. The activation of PI3K involves its recruitment to the plasma membrane by receptor tyrosine kinase interaction followed by conformational changes as a result of the interaction of its catalytic subunit with GTP-loaded RAS (19, 20). Activated PI3K phosphorylates phosphatidylinositol (4,5)-biphosphate (PIP2), converting it into phosphatidylinositol (3,4,5)-triphosphate (PIP3). PIP3 functions as a second messenger which binds to the pleckstrin homology (PH) domain of a large number of enzymes thereby controlling the activity of many downstream pathways. PI3K activation can stimulate PDK1 (3-phosphoinositide-dependent protein kinase), which in turn can activate various 6  members of the AGC protein kinase family such as PKCs, RSK, and also AKT (21). In addition, PI3K activation leads to the activation of GTP-binding protein RAC, which is a RHO family protein involved in the regulation of the actin cytoskeleton (22). Much attention has been paid to the direct activation of AKT/PKB by PI3K. PI3K-AKT signaling regulates a host of proteins, some of which are components of the cell death machinery (23, 24). For instance, AKT protects cells from cell death by inhibiting the pro-apoptotic Bcl-2 family members BAD and BAX (25-27). Similarly, AKT phosphorylates the pro-death caspase- caspase-9, which results in the inhibition of its catalytic activity (28). In addition, Akt can also exert pro-survival function by inhibiting the Forkhead family of transcription factors (FoxO) through phosphorylation, which promotes their association with 14-3-3 proteins, thereby causing their cytoplasmic retention and inability to induce the gene expression of apoptotic factors such as BIM and FAS ligand (29). The best-studied downstream substrate of PI3K-Akt signaling is another serine/threonine kinase called mammalian target of rapamycin (mTOR), a master regulator of protein translation (30). Akt activates mTOR through two means: either directly phosphorylating and activating it, or by phosphorylating and inactivating TSC2 (tuberous sclerosis complex 2), which normally inhibits mTOR through Rheb (Ras homolog enriched in brain). mTOR then signals to S6 kinase/ribosomal protein S6 or 4EBP-1/eIF-4E to promote ribosomal biogenesis and mRNA translation of proteins involved in many molecular pathways including cell survival, growth, and metabolism. Another group of effectors of RAS are three exchange factors for the RAL proteins: RAL guanine nucleotide exchange factor (RALGDS), RALGDS-like gene (RGL/RSB2) and RGL2/RLF. These proteins catalyze the activation of RAL by RAS, 7  resulting in the activation of phospholipase D1 (PLD1), which may be involved in endosomal trafficking and cell migration, as well as the activation of RAL-binding protein 1 (RALBP1), which is also involved in receptor-mediated endocytosis (31). Further, the RALGDS pathway also contributes to the inhibition of the FoxO family, similar to the action of AKT (32). Phospholipase Cε is another downstream effector of RAS, whose main role is to catalyze the hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and inositol triphosphate, which are important second messengers that lead to the activation of PKC and mobilization of calcium from intracellular stores, an integral step in calcium signaling (33). The signaling cascades upstream and downstream of RAS are summarized in Figure 1.   1.1.2 Oncogenic RAS signaling in cancer cells Oncogenic RAS signaling in cancer cells can occur as a result of genetic alterations to several classes of proteins involved in the RAS pathway. The genetic alterations that have received the most attention are mutations that occur on the RAS genes themselves. Activating point mutations in RAS are well established as the most common oncogenic event in cancer, being found in about 30% of all human tumors, most frequently in KRAS (~85%), followed by NRAS (~11%) and HRAS (~3%) (34). The inactivation of RAS activity by GAP-mediated hydrolysis of GTP is the predominant target of these mutations, compromising the GTPase activity of RAS, and these mutations almost always occur on residues G12, G13 and Q61. This results in the accumulation of GTP-bound RAS and constitutive activation of RAS-dependent effector  8   Figure 1: Signaling upstream and downstream of RAS. RAS activation is controlled by its cycling between GTP- and GDP-bound state. The replacement of GDP with GTP is catalyzed by GEF while the hydrolysis of bound GTP is catalyzed by GAP. The best studied mechanism involves autophosphorylation of RTK from growth factor signaling, followed by the recruitment of GEF-SOS to the plasma membrane through the adaptor protein GRB2 and SHC. Once in its active state, RAS can interact with several downstream effectors, including but not limited to RAF, RALGDS, PI3K, and PLCε. Several of the downstream signaling cascades are highlighted above.       9  pathways. Another source for the oncogenic activation of RAS is through the loss of GAP. The most common example of this is the loss of neurofibromin, NF1 (35). The loss of one copy of NF1 causes neurofibromatosis type I, an inherited autosomal dominant disorder that manifests in a large number of benign skin or nerve tumors called neurofibromas, as well as other symptoms including scoliosis, mental disabilities, and epilepsy. Homozygosity for the NF1 gene inactivation is believed to drive malignancy due to incessant activation of RAS signaling.   Oncogenic RAS signaling also occurs through overexpression of growth factor-receptor tyrosine kinases due to chromosomal translocation, mutation or amplification of these genes. Examples of these include alterations in EGFR and HER2 (ERBB2) that drive many cancer cell types not limited to lung cancer, breast cancer, ovarian cancer, and glioblastomas (36). RAS effectors constitute another group of proteins whose genetic alterations can result in oncogenic RAS activation. BRAF is a frequent target of activating mutation or amplification, found in about 70% of melanomas and 15% of colon carcinomas (37). The PI3K pathway is also commonly activated in cancer as a result of point mutations or amplification of the p110𝛼 gene, which is a subunit of the PI3K enzyme (38). Less direct activation of this pathway also occurs via the activating mutations of AKT2, as well as deletion of the tumor suppressor gene PTEN (phosphatase and tensin homologue) (39).  PTEN is lost in 30-40% of all human tumors, which is the second most frequent alteration on a tumor suppressor gene after TP53.  10  1.1.2.1 Clinical significance of RAS mutations  Genetic alterations in RAS and RAS oncogenic signaling underlie several of the most aggressive cancer types, including pancreatic cancer, colon cancer, and lung cancer. Pancreatic ductal adenocarcinoma (PDAC) for instance, the predominant form of pancreatic cancer, has an overall survival rate of 8% in the U.S. (40). This is in spite of the advancements made in understanding the biology of the disease and tremendous effort to improve patient prognosis. Notably, mutant-activated KRAS is present in >90% of PDAC and is understood to be the most frequent and earliest genetic alteration, as it is found in >90% of neoplastic precursor lesions called PanINs (pancreatic intraepithelial neoplasia) (41, 42). The fact that the annual number of deaths is still very close to the number of newly diagnosed cases underscores the importance of developing novel and innovative strategies to target oncogenic KRAS and downstream signaling. Similarly, mutant-activated KRAS is present in 30-40% of colorectal tumours and close to 20% of patients with non-small cell lung cancer (43). In lung cancer, the mutation is associated with poorer prognosis in the metastatic setting and confers a high risk of recurrence. Many genetically-engineered mouse models have been developed to recapitulate the in vivo biological manifestations of oncogenic RAS signaling in pancreatic, colorectal, and lung cancer (44). These have not only highlighted the potent cancer-driving role of mutant-activated RAS, but also afforded a wealth of information regarding the biology of these diseases (discussed in the following section), that will undoubtedly translate into therapeutic strategies that have meaningful clinical benefit.   11  1.1.2.2 Biology of cancers harboring RAS mutations RAS mutations are genetic alterations that occur early in tumor progression. Multiple genetically engineered mouse models of RAS-driven cancers have been developed and have demonstrated the potency of mutant RAS in driving tumorigenesis, in addition to recapitulating the biological characteristics of human tumors harboring RAS mutations (44). One of the phenotypes induced by oncogenic RAS is the promotion of uncontrolled proliferation, unsurprising given that the role of RAS proteins in normal growth and development is to relay upstream mitogenic stimuli to downstream effector pathways. Indeed, shortly after the identification of RAS, seminal studies demonstrated that even in the absence of growth signaling, oncogenic HRAS expression was sufficient to drive quiescent cells into the G1 and through to S phase of the cell cycle (45, 46). In spite of the known ability of oncogenic RAS to circumvent growth factor receptor function in activating downstream mitogenic pathways, the role of these growth factor receptors were not completely supplanted as it was also shown that RAS could establish an autocrine loop through the transcriptional upregulation of growth factors or growth factor receptors in order to enhance cellular proliferation (47-49). These include epidermal growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HBEGF), and transforming growth factor-α (TGFα). Further, it was shown that HRASG12V promotes the upregulation of integrins to enhance proliferation while downregulating other integrin subunits that support cellular quiescence (50, 51). As earlier mentioned, oncogenic RAS signaling also culminates in several transcription factors such as AP-1 being upregulated, which trigger the expression of cyclin D to promote progression through the cell cycle.  12  In parallel with the promotion of uncontrolled proliferation, oncogenic RAS activates multiple mechanisms that suppress apoptosis. As earlier described, both the RAS-RAF-MEK-ERK and RAS-PI3K-AKT pathways can converge upon regulators of apoptosis such as caspase-9, BCL-2 and BIM/FAS ligands (25-28). In addition, oncogenic RAS can also epigenetically silence FAS gene expression as an anti-apoptotic mechanism, although the exact effector pathway remains to be elucidated (52, 53). Another anti-apoptotic pathway of RAS involves NF-κB, which is a transcription factor downstream of RAC or AKT signaling, which can promote the transcription of anti-apoptotic proteins such as the inhibitor of apoptosis proteins (IAPs) (54-56).  Another hallmark of oncogenic RAS that has garnered wide attention lately is its ability to reprogram metabolic pathways of cancer cells in order to support the elevated growth requirements of cancer cells. A well-recognized mechanism by which this occurs is through RAS-mediated activation of both MAPK and PI3K signaling, which leads to the induction of mTOR activity (57). In addition to its regulation on cell growth, mTOR also plays critical roles in energy and nutrient metabolism by stimulating anabolic processes that lead to the synthesis of primary building blocks such as proteins, nucleotides and lipids (58). Furthermore, oncogenic RAS can enhance the cellular capacity for glucose transport by increasing the transcription of the glucose transporter GLUT1, and in parallel promotes the glycolytic processing of glucose by modulating key enzymes in the glycolytic pathway including hexokinase, phosphofructokinase, and lactate dehydrogenase (59-62). More recent work has shown that oncogenic RAS not only stimulates glycolysis, but also causes the shunting of glycolytic intermediates into specific anabolic pathways. One of the major mechanisms by which it does so is 13  through the RAF/MEK/ERK effector pathway, leading to the upregulation of MYC, which in turn activates the hexosamine biosynthesis pathway (HBP) as well as ribose biogenesis through the pentose phosphate pathway (PPP), generating NADPH for macromolecule biosynthesis and ROS detoxification (63). Another key metabolic role of oncogenic RAS is by shunting the flux of glutamine away from the canonical glutamate dehydrogenase (GLUD1) pathway, which converts glutamine-derived glutamate into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid cycle (TCA) (64). Instead, by increasing the expression of aspartate aminotransferase (GOT1), glutamine-derived aspartate is converted into oxaloacetate, which is subsequently converted into malate and then pyruvate, leading to increases in the NADPH/NADP+ ratio and maintaining cellular redox state. On this note, oncogenic RAS plays a role in maintaining redox balance, not only in altering NADPH availability, but also through the upregulation of NRF2, which is widely regarded the master regulator of antioxidant response (65). Alternatively, there is also evidence that oncogenic RAS can redirect glucose and glutamine into anabolic pathways through its regulation on NRF2 activity (65, 66). The role of NRF2 in oncogenic RAS signaling will be discussed further below. Given the enhanced shuttling of metabolic intermediates into anabolic pathways, RAS-driven cancers have also acquired adaptations to ensure adequate availability of biosynthetic precursors. One such adaptation is by elevating basal autophagy, the process of self-consumption that generates energy and biosynthetic precursors particularly critical for organelle homeostasis (67, 68). Another involves the utilization of extracellular fuel sources through macropinocytosis, a highly conserved endocytic process by which extracellular proteins are internalized into the cells through large 14  vesicles called macropinosomes, yielding amino acids that can then enter central carbon metabolism and support cellular growth requirements (69).  Cancers that harbor activating mutations in RAS can also extensively remodel their microenvironment to facilitate adequate supply of oxygen and nutrients to cells within the tumor (70). Through several effector pathways, oncogenic RAS-transformed tumor cells can induce pro-angiogenic growth factors such as vascular endothelial growth factor A (VEGFA), stimulate the release of proteases such as matrix metalloproteinase 2 and 9 (MMP2 and MMP9) and urokinase–type plasminogen activator (uPA) to remodel the surrounding extracellular matrix (ECM), and promote the recruitment of macrophages and neutrophils through the production of inflammatory cytokines, all of which drives the pro-angiogenic program towards the development of tumor vasculature (71-73). Besides remodeling the microenvironment, many tumors that contain RAS mutations also have the propensity to metastasize to distant sites (70). Oncogenic RAS directly contributes to the enhanced motility and invasion of cancer cells by modulating the organization and stability of the actin cytoskeleton and microtubules, while inducing alterations to cell-cell and cell-matrix interactions by disrupting E-cadherin and β-catenin functioning (74-77). Collectively, these changes establish the invasive and migratory phenotype that can enhances tumor metastasis.  Finally, tumors that contain oncogenic RAS are also known to activate responses which allow them to evade the immune system (70). Studies have shown that oncogenic RAS reduces surface-expression of antigen-presenting major histocompatibility complexes (MHC) on cancer cells to decrease their immunogenicity (78-81). This is mediated primarily through disruptions in the functionality of 15  components of the antigen-processing machinery such as antigen peptide transporters TAP1 and TAP2, as well as proteasome subunits LMP2 and LMP7 (82, 83). Furthermore, oncogenic RAS-driven cancers can attenuate adaptive immune responses of the host by recruiting immunosuppressive regulatory T-cells (TRegs) or myeloid-derived suppressor cells (MDSCs) to the tumor site, a process that can gradually lead to complete evasion of immune surveillance (84, 85). The mechanisms by which MDSCs exert their immunosuppressive effects are not yet fully understood, but a strong inverse correlation between the prevalence of MDSCs and CD8+ T cells in the immune infiltrate suggests one explanation in which MDSCs negatively influence T-cell trafficking or T-cell proliferation within the tumor (84). Meanwhile TRegs suppress effector T-cell activation, proliferation, as well as cytokine production including IL-10 and transforming growth factor-β (86).  1.1.2.3 Therapeutic targeting of oncogenic RAS signaling Genetic alterations of RAS are considered an early event in the onset of tumorigenesis. Nonetheless, there is considerable evidence that sustained expression of oncogenic RAS is necessary to support tumor maintenance. RNA interference-mediated silencing of RAS was shown to impair in vitro growth of RAS-mutant human cancer cell lines and ablation of KRAS in mouse models of lung adenocarcinoma and pancreatic cancer leads to dramatic tumor regression (63, 87, 88). Thus, the most direct way to target oncogenic RAS would be to target the activating mutation itself. This approach however has been generally perceived as a highly challenging, if not impossible task. Indeed, RAS proteins bind GTP/GDP with picomolar affinity and thus 16  targeting oncogenic RAS with nucleotide analogs is inconceivable given that high GTP concentrations make competition impossible (43). Moreover, crystallographic studies have revealed structures of RAS that show an absence of allosteric regulatory sites suitable for targeting with small molecules. As such, in the 1990s, the main direction for the therapeutic targeting of oncogenic RAS is the targeting of post-translational modifications of RAS to interfere with its subcellular localization. Given that farnesylation is the first step in the post-translational modification of RAS, which is critical for the stable localization to the plasma membrane and subsequent activation, this was an obvious early target for the design of rational therapies against RAS (89, 90). Several pharmaceutical companies led the efforts of identifying farnesyltransferase inhibitors (FTIs) with the use of high-throughput screening of compound libraries. Early validation of these FTIs showed that they effectively inhibited farnesylation of HRAS in cell culture, and disrupted growth of mammary tumors in a HRAS-driven transgenic mouse model (91). Moreover, the FTIs caused little overall toxicity, solidifying its potential in the treatment of human cancers harboring oncogenic RAS mutations. Disappointingly, the early promise of FTIs was not achieved in the context of clinical trials and the initial success of the mouse models failed to be recapitulated in human patients (92, 93). This was due to the fact that although HRAS is exclusively modified by farnesyltransferase, KRAS and NRAS can both be alternatively modified by geranylgeranyltransferase, and thus even in the absence of farnesylation were able to carry out their biological activity (94, 95). The subsequent logical attempt to inhibit KRAS and NRAS by inhibiting both farnseyltransferase and geranylgeranyltransferase in combination also failed as this resulted in very serious toxicity (96). Given that the 17  vast majority of RAS-driven tumors have KRAS and NRAS mutations, and very few HRAS mutations, the general interest in further pursuit of strategies that target RAS membrane association steadily declined. There have been several other approaches in targeting post-translational modifications of RAS. These approaches include developing inhibitors against the CAAX-processing enzymes- RAS-converting enzyme 1 (RCE1) and isoprenylcysteine carboxymethyltransferase (ICMT), as well as inhibiting palmitoylation and depalmitoylation of RAS (97-100). Even though these inhibitors show some potential, the potential for off-target effects of these inhibitors on non-RAS substrates diminish enthusiasm for this approach and their potential clinical value largely remains unresolved. When it became clear that FTIs did not live up to their promise, and with the advent of the genomics era in which mutations in kinases were beginning to be well-appreciated as mediators of oncogenesis, therapeutic strategies for targeting RAS steadily moved downstream. Following the discovery that the RAF-MEK-ERK pathway was an effector pathway of RAS, several RAF kinase inhibitors were developed with the assumption that inhibiting the MAPK pathway would be equivalent to inhibiting RAS itself. Paradoxically, these inhibitors rather than blocking RAF kinase activation led to enhanced RAF and ERK activation (101-103). This was attributed to the fact that some of the first generation RAF inhibitors seemed to promote RAF dimerization and thus its activation. Efforts to develop MEK and ERK inhibitors were also largely unsuccessful because the early perception that the MAPK kinase cascade is a linear, unidirectional signaling pathway is incorrect and it is now recognized that this cascade has multiple inputs and outputs, feedback mechanisms, as well as hundreds of substrates of ERK1 18  and 2, resulting in a very small therapeutic window (34, 104). For instance, one main problem is the resistance of RAS-mutant cancer cells to MEK and ERK inhibitors due to the upregulation of RTK activity or amplification of upstream activators that enhance flux through the pathway to restore or increase ERK activation, or simply lead to the activation of the alternate PI3K-AKT effector pathway (105, 106). Inhibitors of the PI3K-AKT cascade have also not fared much better due to feedback mechanisms (107). The combination of these PI3K inhibitors with MAPK inhibitors may hold therapeutic value, but still requires extensive clinical evaluation. Other downstream effectors of RAS such as RAL proteins, RAC1, and phospholipase Cε are potential candidates for therapeutic targeting and investigation using mouse models lend support to this notion (108, 109). Nevertheless, considerable challenges remain as the recent disappointments with effector inhibition underscore the importance of thorough pre-clinical and clinical evaluation before we can be confident about their therapeutic efficacy. Another recent approach is the identification of potential targets that have synthetic lethal interactions with oncogenic RAS. Several screens using siRNA or shRNA libraries to identify synthetic lethal interactors with mutant RAS have uncovered exploitable vulnerabilities of KRAS-mutant cell lines without having to target KRAS itself. These include genes that encode proteins involved in a range of cellular processes including cell cycle – survivin, cell survival – WT1 and BCL-XL, and collaborative transcriptional programmes – GATA2 and SNAIL2 (110-114). However, this relatively new approach has thus far seen disappointing clinical results and several problems still need to be addressed before we see the use of synthetic lethal targets across broader contexts (115).  19  Interestingly, while most approaches have moved downstream, Shokat and colleagues recently developed compounds that interact with KRASG12C mutant protein, which is the most frequent RAS mutation in non-small cell lung cancer, specifically in its GDP form (34, 116). This revolutionary strategy seems counterintuitive given the perception that oncogenic RAS mutants are constitutively locked in its GTP-bound state. However, it was shown that codon 12 RAS mutants still retain measurable GTPase activity: even though they are not amenable to GAP-mediated GTP hydrolysis, which renders GTP hydrolysis to be slow, the rate of GDP to GTP exchange is also slow, and thus as these two states approach steady state, the levels of GTP- and GDP-bound RAS are comparable, presenting an opportunity for targeting the GDP-bound state and trapping RAS in this off state. As predicted, these compounds effectively blocked SOS1-mediated nucleotide exchange leading to reduction in binding of RAS to RAF, and they selectively killed cancer cells that harbor the specific G12C mutation. In spite of the progress made in targeting the mutant form of KRAS, KRASG12C represents a small subset of cancers compared to the complete spectrum of RAS mutations (~12%) (117). It is conceivable that compounds that interact with G12D or G13D mutant forms using a similar approach can be identified in the near future. However, the binding affinity of these compounds would have to be constantly improved before they can achieve clinical application and with any drug, innate or acquired resistance to these compounds is a possibility that is yet to be determined.  With the recent expansion in our understanding of the biology of cancers that harbor RAS mutations, so has expanded the potential repertoire for targeting RAS. The fact that RAS-driven tumors are critically dependent on metabolic adaptations presents 20  several attractive therapeutic targets. For instance, due to the fact that RAS-tumors are dependent on elevated levels of basal autophagy to provide energy and biosynthetic precursors, autophagy inhibitors such as chloroquine and hydroxychloroquine (HCQ) are currently being tested in clinical trials against multiple cancer types including RAS-driven pancreatic cancer (34, 118, 119). In addition, as earlier described, oncogenic KRAS regulates the flux of glutamine into anabolic pathways and so inhibitors of glutaminase, the enzyme responsible for glutamine to glutamate conversion, are making their way into early phase clinical trials (120). A recurrent theme arising from these studies also includes the modulation of redox balance by oncogenic RAS. Indeed, several studies have pointed towards oncogenic KRAS promoting a metabolic shift towards pathways that generate NADPH and the hallmark study by DeNicola and colleagues present a role of NRF2 in mitigating oxidative stress to support RAS-mediated tumorigenicity (65). The therapeutic targeting of cancer cells using reactive oxygen species (ROS)-mediated approaches is not a new concept and processes that maintain redox balance therefore represent tractable therapeutic opportunities for targeting RAS-driven cancers as well, given their high dependency on these processes. I will discuss the role of ROS in cancer cells in the following section and the impact of oncogenic RAS on redox homeostasis in the following chapter.  1.2 Cellular Transformation and its Impact on Redox Balance 1.2.1 Reactive oxygen species (ROS) in normal and cancer cells ROS are defined as oxygen-containing, chemically reactive molecules consisting of two main groups: free radicals that contain one or more unpaired electrons in their 21  outer molecular orbitals, and non-radical molecules that do not have unpaired electrons but are highly reactive and can be converted to radical ROS (121, 122). Examples of free radicals include superoxide (O2•‒), hydroxyl radicals (•OH), and nitric oxide (NO), while non-radical ROS include hydrogen peroxide (H2O2), peroxynitrite (ONOO‒), and hydroxide ion (OH‒). Superoxide is considered as the primary ROS, which can be rapidly dismutated by superoxide dismutase (SOD) yielding hydrogen peroxide. In the presence of Fe2+ or Cu2+ ions, a process called the Fenton reaction can further convert hydrogen peroxide into hydroxyl radical. In parallel, superoxide and hydrogen peroxide can also react to form hydroxyl radical. These different ROS species have different levels of reactivity and also capabilities for diffusion. Hydroxyl radical is extremely reactive but does not diffuse beyond its site of formation while superoxide is very active and displays limited diffusion, particularly through membrane channels. On the other hand, hydrogen peroxide readily diffuses through membranes and along with its labile nature, makes it an ideal candidate for a signaling molecule in biological systems.  ROS are well-recognized for playing dual roles for both deleterious and beneficial effects in cellular systems, and the modulation of ROS homeostasis is fundamental in maintaining normal cellular function (123). The beneficial effects of ROS occur at low to moderate concentrations, where they play a crucial role in various signaling pathways to support cellular proliferation and differentiation, or to engage stress-response pathways to ensure cell survival.  In contrast, the overproduction of ROS results in oxidative stress, a deleterious condition that is characterized by damage to cellular structures such as DNA, proteins, and lipids, which can gradually lead to the onset of disease 22  states including cancer and neurodegeneration. As such, tight regulation of both ROS-producing and ROS-scavenging systems is critical for cellular homeostasis.  1.2.1.1 ROS producing systems There are various sources of ROS including the environment, which can generate ROS from pollutants, heavy metals, drugs, and radiation, or they can be produced from within the cell. Endogenous ROS are principally derived as by-products of oxygen that is consumed in several biological processes, or produced directly by a number of enzymes. The mitochondrion is considered a major source of ROS as electron leakage from the electron transport chain (ETC) may react with molecular oxygen to form the highly reactive superoxide free radical (124). Superoxide formed in the mitochondria may be released into the mitochondrial intermembrane space, or may diffuse into the cytosol via voltage-dependent anion channels (VDACs). The endoplasmic reticulum (ER) constitutes another source of ROS production as it provides an oxidizing environment that favors disulphide bond formation and protein folding. In phagocytes as well as in some cancer cells, ROS, or more specifically superoxide, is produced by the NADPH oxidase complex not as a byproduct, but rather as the primary function of the enzymatic system (125, 126). The latter evolved primarily as an essential component of the innate immune response against invading microorganisms, known as the respiratory burst. There are six homologs of the transmembrane catalytic subunit of the NADPH oxidase complex: NOX1-5 and DUOX1 and 2. NOX1-3 mainly generate superoxide, while NOX4, DUOX1, and DUOX2 are capable of producing hydrogen peroxide directly. Interestingly, NOX1-3 require RAC1 for activation, which is a member 23  of the RHO subclass of the RAS superfamily of GTPases and is involved in RAS signaling.  Apart from host defense, the NOX family of enzymes execute a wide array of biological functions depending on their subcellular localization, which range from cellular signaling, regulation of gene expression, and cellular differentiation, to cell migration, apoptosis, and senescence (127). Other sources of endogenous ROS include enzymes such as xanthine oxidase (XO), lipoxygenases (LOX), peroxisomes, and cytochrome P450.  1.2.1.2 ROS scavenging systems The balance between production and scavenging of ROS is of critical importance as the accumulation of ROS in cells can result in disruption of biological function due to irreversible damage of DNA, proteins, and lipids. The cellular redox balance is maintained by a battery of antioxidant systems that is comprised of enzymatic antioxidants such as SOD, catalase, peroxiredoxins (Prxs), thioredoxins (Trx), and glutathione peroxidases (GPx), as well as non-enzymatic antioxidants, which include Vitamin C (ascorbic acid), Vitamin E (α-tocopherol), glutathione (GSH), carotenoids, and flavonoids (121). GSH is the most abundant non-enzymatic antioxidant in the cell. It is synthesized in the cytosol by the sequential action of glutamate-cysteine ligase (GCL) and glutathione synthetase (GS). The enzymatic step catalyzed by GCL involves the formation of γ-glutamylcysteine from glutamate and cysteine, while GS catalyzes the formation of GSH from γ-glutamylcysteine and glycine. While intracellular glutamate is readily available in the cell, intracellular cysteine concentration approximates the Km value of GCL for cysteine and thus is the rate-limiting step for the synthesis of GSH 24  (128). Several key factors that regulate cysteine availability include the membrane transport of cysteine via the neutral system ASC (alanine, serine, cysteine-preferring) transporter, membrane transport of cystine via the system xc– transporter, and the transsulfuration pathway. Once GSH is synthesized in the cytosol, where it mediates key redox reactions, it can also be transported into the mitochondria or nucleus where it serves to maintain a redox state critical for DNA repair and gene expression. More significantly, the main protective roles of GSH in the cell include being a cofactor in enzymatic systems, the regeneration of vitamin C and E to their active forms, as well as the direct scavenging hydroxyl radical and singlet oxygen. Oxidized glutathione (GSSG) accumulates in the cell as a result of GSH function, and thus the ratio of GSH/GSSG reflects the redox buffering capacity of the cell.  The various enzymatic systems are crucial in scavenging different ROS (123). As earlier described, superoxide generated by the mitochondria or NADPH complex is rapidly converted to H2O2 by SODs. Catalase further degrades H2O2 to water and oxygen. Glutathione peroxidases can also eliminate H2O2 using reduced glutathione (GSH) as a cofactor, converting it into oxidized glutathione (GSSG). Oxidized glutathione can be converted back to its reduced form by glutathione reductase, a process which in turn consumes NADPH. H2O2 can also be scavenged by peroxiredoxin which results in the oxidation and subsequent inactivation of peroxiredoxin. Thioredoxin then acts as an immediate electron donor and converts peroxiredoxin back to its active form. Finally, thioredoxin is reduced back to its active form by thioredoxin reductase, using NADPH. The various ROS producing and scavenging systems are highlighted in Figure 2. 25  1.2.1.3 Function of ROS in biological systems Cell signaling is composed of all signal transduction processes that transmit extracellular signals from hormones, growth factors, cytokines, and neurotransmitters into various functional elements within the cell. These signaling processes induce a myriad of biological activities that constitute normal cell functioning. ROS play a major physiological role in several aspects of cellular signaling. Firstly, cytokines and growth factor receptor signaling can activate ROS production, which can then function as second messengers to relay the signals to downstream effectors (129, 130). This is due to the fact that ROS can induce tyrosine phosphorylation of several protein tyrosine kinases, in some cases through reversible inactivation of protein tyrosine phosphatases (PTP). Furthermore, serine/threonine kinases have been shown to stimulate ROS production and conversely, ROS can also regulate serine/threonine kinases of the MAPK family (130). Finally, the role of ROS in cell signaling extends downstream to the regulation of nuclear transcription factors, including AP-1, NF-ΚB, p53, and HIF-1, thus influencing a myriad of cellular processes (129). One of these cellular processes is the oxidative or respiratory burst in neutrophils and macrophages, which is characterized by a rapid release of ROS to degrade internalized bacteria and foreign particle, a key step in the defence against environmental pathogens (125). In addition, ROS play an important role in endothelial cells, cardiac myocytes, and vascular smooth muscle cells through their involvement in multiple intracellular signaling pathways in these non-immune cells (130). ROS are also crucial mediators of cell adhesion through their induction by cytokines and bacterial lipopolysaccharides and involvement in the regulation of focal adhesion kinase, and are 26   Figure 2: Intracellular ROS producing and scavenging mechanisms. Several sources of intracellular ROS include the mitochondrial electron transport chain (Mito-ETC), NOX complexes (NOX), the endoplasmic reticulum (ER), xanthine oxidase (XO), lipoxygenases (LOX), peroxisomes, and cytochrome P450. Superoxide (O2•‒) produced by these systems is converted to H2O2 via the action of superoxide dismutases (SOD). Hydroxyl radical (•OH) can be formed from the Fenton reaction. Various antioxidant mechanisms exist to scavenge H2O2 including catalase (CAT), peroxiredoxins (PRX) and glutathione peroxidase (GPX). The action of GPX involves the converstion of reduced glutathione (GSH) to its oxidized form (GSSG). GSH is produced by a stepwise biosynthetic pathway catalyzed by Glutamate-cysteine-ligase (GCL) and Glutathione synthetase (GS), which uses Cysteine (reduced form of Cystine), Glutamate, and Glycine as precursors. The uptake of cystine is mainly attributed to the system xc– transporter.     27  therefore implicated in embryogenesis, cell growth and differentiation, and wound repair (131, 132). Amongst various other processes, apoptosis or programmed cell death, which is critical not just in pathological conditions but also for the proper development and integrity of biological systems, is dependent on ROS and mitochondrial signaling, as these mechanisms are triggered by irreversible oxidative damage to the mitochondria, and to intracellular lipids, proteins and DNA (133).   1.2.1.4 Oxidative stress in cancer cells A seminal study in 1981 first established a link between ROS and cellular transformation through the demonstration that insulin increased tumor cell proliferation by elevating intracellular H2O2 levels (134). Since then, many studies have shown that various types of cancer cells display elevated levels of ROS, compared to their normal counterparts. It was also reported that cancer cell lines and clinical tumor specimens exhibit higher levels of oxidative damage, as evidenced by oxidized DNA (8-oxo-D-guanosine) and lipid peroxidation (135, 136). Evidence that has accumulated over recent years suggests that the onset of oxidative stress during cancer progression can be attributed to several mechanisms including activation of oncogenes, loss of p53 and resultant antioxidant control, metabolic reprogramming, and mitochondrial dysfunction (137-140). Interestingly, in spite of the deleterious role that oxidative stress plays in most contexts, mounting evidence suggests that elevated ROS has a pro-tumorigenic role. Indeed, elevated ROS stress in cancer is correlated with aggressiveness of disease and poorer patient outcome (136, 141). Previous studies have shown that ectopic expression of the oncogene MYC leads to the upregulation of mitochondrial 28  genes and subsequent increase in ROS production (142, 143). Similarly, ectopic RAS expression leads to increased ROS levels as a result of increased NADPH oxidase (NOX) expression and NOX complex activity. ROS can then function downstream of the activation of these oncogenes in part through the induction of a vicious cycle in which ROS promotes widespread genetic instability that involves activation of other oncogenes, inactivating mutations in tumor suppressor genes, and mutations in the mictochondrial genome, contributing to malignancy and further exacerbating ROS production (121). Further reports suggest that ROS can also enhance signaling cascades downstream of receptor tyrosine kinases (RTKs) or oncogenes through the inactivation of protein tyrosine phosphatases (PTPs) (144). For instance, phosphatases such as PTP1B and PTEN are known to be selectively targeted by NOX-derived ROS, and this could largely be explained by the proximity of NOX complexes to these PTPs (145). In line with the role of ROS in normal vascular regulation, compelling evidence suggests that ROS are also a decisive factor in the regulation of angiogenesis or neo-vascularization in tumors (146). It has been shown that generation of H2O2 by NOX2 and NOX4 promotes endothelial cell survival and proliferation (147). In parallel, ROS also promotes vascular endothelial growth factor (VEGF) receptor expression and activation through the induction or stabilization of the transcription factor HIF-1α (146, 148). Angiogenesis and the development of metastasis are two intrinsically linked processes. There is significant experimental evidence for the existence of positive feedback loops between ROS and growth factor activation (VEGF, PDGF, HGF) as well as between ROS and integrin signaling, which is a key contributor to cancer cell 29  invasion leading to metastasis (149). Alternatively, ROS participates in cancer cell motility and invasion through proteolytic degradation of glycosaminoglycan (GAG) and other components of the extracellularmatrix such as through the induction of various matrix metalloproteases (MMPs) (149). Again, RAC1 function and NOX-derived ROS have been frequently implicated in invasion and metastasis given their role in the formation of invadopodia and focal adhesions (145). It is clear that cancer cells maintain high levels of ROS to support tumorigenicity, yet above a certain threshold, ROS can be cytotoxic and lead to cell death or senescence phenotypes. Accordingly, the fine tuning of ROS generation and ROS scavenging is extremely crucial for cancer cell growth and survival. There is a growing body of evidence to show that cancer cells in many cases acquire adaptive mechanisms to combat oxidative stress that arises as a tumor progresses and in some cases also possess intrinsic antioxidant response mechanisms that confer them the ability to do so, the absence of which impedes their ability to drive malignant progression. For instance, it has been observed that a subset of cancer stem cells in human and mouse mammary tumours contain lower levels of ROS, display less DNA damage, and are less susceptible to irradiation relative to their non-tumorigenic counterparts (150). These traits were associated with an enhanced capacity for scavenging free radicals, which was particularly owed to an increase in the expression of GSH biosynthesis genes such as GCLM and GSS. Similarly, another group found that genetic loss of GCLM resulted in the inability of mice to form mammary tumors (151). Studies in c-MYC-driven melanoma cells revealed that c-MYC controlled the expression of glutamate-cysteine ligase (GCL), and knockdown of c-MYC resulted in the depletion of reduced GSH by 30  more than 60% followed by apoptosis (152). In addition, the antioxidant enzyme SOD1 appeared to be critical for cancer cells, as small molecule inhibition of SOD1 reduced the growth of lung adenocarcinoma cells lines (153). Truitt and colleagues recently demonstrated that cancer cells hijack an eIF4E-dependent translation program that is selectively enriched for mRNAs involved in oxidative stress response to support cell survival and fuel oncogenic transformation (154). As mentioned, cellular transformation with oncogenes such as RAS and MYC leads to the upregulation of the master regulator of antioxidant response, NRF2, and cancer cells also undergo extensive metabolic reprogramming to ensure adequate availability of the non-enzymatic antioxidant NADPH (63, 65). More recently, the role of NRF2 in malignancy was further extended as Chio and colleagues showed that NRF2 directly stimulates mRNA translation of pro-survival transcripts by maintaining the reduced state of specific cysteine residues in translational regulatory proteins (155). They also demonstrated that NRF2-dependent redox regulation promotes EGFR autocrine signaling through AKT in oncogenic KRAS-expressing cells to fuel cap-dependent translation initiation. Findings from several other groups also demonstrate that in human breast tumors, PI3K-AKT signaling that is dysregulated either through oncogenic activation or estrogen stimulation results in the upregulation of NRF2 gene targets associated with GSH biosynthesis to support resistance to oxidative stress, initiation of tumor spheroids, and anchorage-independent growth (156).  Notably, the constitutive activation of NRF2 itself, or the inactivation of its repressor KEAP1 by somatic mutation, are found in about 15% and 10% of patients with lung cancer respectively (157). Altogether, these studies indicate that malignant transformation relies on the induction of ROS to promote pro-31  tumorigenic processes through redox-sensitive signaling pathways, and yet are critically dependent on adaptive or intrinsic ROS-scavenging pathways to mitigate oxidative damage that can impede tumor progression. This dependence on ROS-clearing mechanisms thus reveals a vulnerability that represents a tractable therapeutic strategy.   1.2.2 Therapeutic targeting of cancer cells via ROS-mediated mechanisms The achievement of a sufficient therapeutic window that will allow the elimination of cancer cells while leaving normal cells unharmed is vital in the treatment of cancer. In cancer cells, the elevation of ROS generation and therefore increased intrinsic oxidative stress confers them a crucial dependence on ROS-detoxification mechanisms and basal antioxidant capacity. On the other hand, normal cells have a low level of basal ROS and have the ability to mobilize antioxidant pathways that maintain redox homeostasis. It is therefore conceivable that oxidative insults induced by ROS-producing agents or by compounds that inhibit key antioxidant programs in the cell could preferentially elevate ROS levels in cancer cells above a tolerable threshold, without affecting normal cells (121). The idea of targeting the vulnerability of tumor cells based on their differential redox state relative to normal cells is not a novel concept. Platinum-based antineoplastic agents (for example, cisplatin, carboplatin, and oxaliplatin), anthracyclines (for example, doxorubicin, epirubicin, and daunorubicin), as well as ionizing radiation all generate substantial increases in ROS levels to induce apoptosis in tumor cells (121, 158). Several other agents such as Arsenic Trioxide (As2O3), paclitaxel and docetaxel, vincristine, and anti-folates also trigger the production of superoxide radicals by interfering with the function of the electron transport chain, 32  ultimately resulting in cell death (121). Although some of these ROS-inducing agents have shown clinical efficacy in some settings, general toxicity remains a limiting factor, and in many cases low clinical response or resistance to these drugs were reported. All this alludes to the importance of garnering more insights into the different redox vulnerabilities of different subsets of cancers, so as to identify the pros and cons of each redox-modulating strategy and enable the development of effective therapeutic agents. Given the recent surge in studies that have revealed various intrinsic antioxidant pathways hijacked by cancer cells, or adaptive mechanisms employed to counteract oxidative stress, the feasibility of redox-modulating therapeutic strategies has gained traction. In the recent years, by using high-throughput small molecule screens, several groups have identified a number of chemical compounds that selectively induce high levels of ROS in tumor cells as a means to kill them. Among these is Lanperisone, a derivative of tolperisone, which is used as a muscle relaxant to treat muscle spasms arising from neurological disorders (159). Lanperisone causes the non-apoptotic cell death of oncogenic KRAS-expressing cells independently of cell cycle phase or protein translation, and by selectively inducing high levels of ROS. The exact mechanism by which Lanperisone initiates ROS production is unclear, but functional studies suggests that it occurs through the interaction of Lanperisone with voltage-gated ion channels. Several of the compounds identified from these high-throughput screens were known to cause oxidative-stress mediated cell death in cancer cells by interfering with GSH availability or GSH-dependent scavenging mechanisms. For instance, benzyl isothiocyanate (BITC) and β-phenylethyl isothiocyanate (PEITC) can conjugate with GSH to deplete the intracellular GSH pool (160). Another drug Piperlongumine, which is 33  a naturally occurring alkaloid present in the Piper Longum pepper plant, has been shown to increase ROS levels by modulating GSTP1 activity, leading to cell death and tumor growth suppression in xenograft-based mouse models (161). As mentioned, inhibition of SOD1 using the small molecule LCS-1, reduced the growth of lung adenocarcinoma cells lines and is currently being pursued as a therapeutic approach (153). Finally, 1-buthionine-S,R-sulfoximine (BSO), a classical drug known to inhibit GCL, has also been investigated for decades and is still currently undergoing extensive clinical testing in combination with other anticancer drugs (162). The modulation of cysteine levels, given that it is a precursor for the biosynthesis of GSH, has also been pursued as a GSH-dependent anticancer strategy. Indeed, Sulfasalazine, an anti-inflammatory drug that is currently FDA-approved for the treatment of arthritis was found to specifically inhibit xCT activity – the light chain subunit of the system xc– transporter responsible for cystine uptake (163-168). Unfortunately, the use of Sulfasalazine clinical trials has seen low response rates coupled with severe toxicities, and more potent derivatives are currently being developed (169, 170). Recently, another inhibitor of xCT called Erastin was discovered (171-173). Interestingly, Erastin was first identified as a RAS-selective lethal (RSL) compound referring to its selective lethal effect on RAS mutant cell lines and studies have described that the type of cell death induced by Erastin and other RSLs was characterized by unique morphological, biochemical, and genetic features distinct from those of apoptosis or necrosis (174). This novel mode of cell death was termed ferroptosis, in accordance with the fact that the increased levels of ROS associated with cell death could be prevented by iron chelation or inhibition of iron uptake. Compared to 34  Sulfasalazine, Erastin was a much more potent inhibitor of xCT and effectively reduced cystine uptake and intracellular GSH levels (171, 172).  Various other promising anticancer drugs have been developed based on the modulation of ROS and oxidative stress in cancer cells. Among these are inhibitors of enzymes involved in thioredoxin metabolism, glucose metabolism, and glutamine metabolism. As the exact nature of the impact of oxidative stress on cancer initiation and progression is elucidated, and further mechanistic insight into the role of cellular antioxidants in different subtypes of cancer is acquired, I predict that the described inhibitors, along with novel ones that remain to be identified will present new vistas for better-tailored and efficacious anticancer treatments.   1.3 Hypothesis and Significance Although substantial research has been directed towards targeting oncogenic RAS and understanding the biology of RAS-driven cancers, the concept of targeting redox-based vulnerabilities within this genetic subset of cancers is still relatively novel. Indeed, the impact of RAS on redox homeostasis and its contribution to transformation and tumorigenesis remain controversial. Furthermore, most studies exploring ROS regulation by oncogenic RAS have relied on hypothesis-driven approaches that are based on established knowledge of cell-intrinsic antioxidant mechanisms, rather than an unbiased, hypothesis-generating approach. As such, unexpected contributory mechanisms that may be important for RAS-mediated oncogenesis may remain undiscovered. 35  To attain my objective of elucidating novel signaling mechanisms that are employed by oncogenic RAS to regulate intracellular redox balance, I will utilize hypothesis-generating experimental systems to identify novel protein candidates that are up- or down-regulated by oncogenic RAS under oxidative stress. I hypothesize that the analysis of global transcriptome data in mutant-activated KRAS-expressing cells under oxidative stress will highlight specific genes that constitute the redox-modulatory profile of oncogenic RAS. In later chapter studies, I discover that the cystine/glutamate transporter, xCT, is upregulated by oncogenic KRAS signaling under oxidative stress and I hypothesize that xCT is necessary for oncogenic KRAS-mediated malignant transformation and tumorigenicity. Overall, I expose a novel redox-based vulnerability in oncogenic RAS-driven cancers and predict that the targeting of xCT could be an important therapeutic strategy that will lead to better clinical outcomes in patients of this genetic subset.   1.4 Research Objectives The studies in this thesis were designed to test the above hypothesis and introduced in three research chapters that seek to delineate the impact of oncogenic RAS on redox-related mechanisms, and the contribution of these mechanisms to transformation and tumorigenesis. In Chapter 2 I discuss the current literature on the regulation of redox balance by oncogenic RAS and demonstrate that transformation with oncogenic KRAS leads to a decrease in overall intracellular ROS levels through the upregulation of the cystine/glutamate transporter, xCT. In Chapter 3, I introduce xCT function in normal physiology as well as in the context of cancer, and proceed to 36  investigate the role of xCT in oncogenic KRAS-mediated transformation and tumor formation.  Finally, in Chapter 4, I describe the currently known mechanisms that govern xCT expression, and then investigate the RAS-mediated signaling pathways and mechanisms that promote the upregulation of xCT to support oncogenic transformation.   37  Chapter 2: Transformation with Oncogenic KRAS Modulates Cellular Redox Balance via Upregulation of Cystine/Glutamate Transporter xCT Expression  2.1  Background and Rationale Much of the early literature implicating oncogenic RAS and the cellular redox environment has supported a role of elevated ROS levels as a driver of tumorigenesis. Irani and colleagues (137) first showed that ectopic expression of HrasV12 in NIH3T3 fibroblasts leads to the production of large amounts of superoxide. They demonstrated that this is correlated with the progression of cells through the cell cycle in a RAC dependent manner. Further investigation determined that oncogenic RAS increases superoxide production by upregulating Nox1 transcription through the MAPK pathway (175). In addition, suppression of Nox1 expression abrogates superoxide generation and prevents oncogenic RAS-transformed phenotypes, including anchorage-independent growth and morphological changes, while strongly suppresses RAS-induced tumor formation in vivo, altogether suggesting that NOX1-mediated ROS production is necessary to support RAS transformation and tumorigenesis. Alternatively, it was proposed that KRAS-signaling can directly mediate NOX1 activation independently of stimulating Nox1-transcription (176). Specifically, it was demonstrated that KrasV12 activates MAPK p38 to induce 3-phosphoinositide-dependent protein kinase 1 (PDPK1), in turn activating protein kinase C-theta (PKCtheta). This latter kinase catalyzes the phosphorylation and activation of the p47phox Nox1 subunit, 38  through its translocation to the plasma membrane. This facilitates the signaling cascade for ROS generation. More importantly, they showed that inhibition of p38, PDPK1, PKCthetha, p47phox, or NOX1 suppresses KRAS-induced ROS generation and cellular transformation as displayed by soft agar colony formation and tumor formation assays. Thus these data confirm, albeit through a different mechanism, that KRAS activates NOX1-dependent ROS production, which is necessary to support KRAS-driven tumor growth. Similarly, a recent study established that oncogenic RAS-induced ROS formation is dependent on RAC1 and NOX4, a homolog of NOX1, as demonstrated in normal human fibroblasts and in an HrasV12 transgenic zebrafish model (177). This study further demonstrated that pharmacological inhibition of NOX4 prevents HrasV12-mediated hyperproliferation and DNA damage response activation. Similar effects were observed by scavenging ROS generation with N-acetyl cysteine (NAC), arguing that the role of NOX4 in supporting oncogenic RAS transformation is directly related to its ROS-producing function. In addition, NOX4 levels were found to be increased during pancreatic cancer progression in a KrasG12D-driven mouse model of pancreatic cancer, highlighting a link between high NOX4 expression and advanced stage of a KRAS-driven tumor type (177). In contrast to these studies, Maciag and colleagues (178) found that oncogenic KRAS causes an increase in the level of intracellular ROS while superoxide levels remain unchanged, eliminating the involvement of NOX in this system. It was postulated that KRAS regulates cyclooxygenase-2 (COX-2), an enzyme whose activity releases prostaglandin-E2 and produces H2O2 as a by-product. Specifically, they found that COX-2 protein expression and activity is significantly elevated in mutant KrasV12 mouse lung 39  epithelial cells and that treatment with a COX-2 inhibitor results in a concentration-dependent reduction in ROS. More importantly, they observed that KrasV12-induced ROS generation leads to a significant increase in DNA single strand breaks in a COX-2 dependent manner. This is of relevance at advance stages of cancer, as there is usually a high occurrence of DNA damage accompanied by elevated levels of ROS, due in part to a vicious cycle in which ROS promotes DNA damage and genetic mutations due to defective DNA repair, which leads to further redox changes and finally resulting in more malignancy (179). It is interesting to speculate the reason as to why previous studies showed that oncogenic RAS increases superoxide levels while this paper did not. On one hand this could be attributed to specific cell lines used (previous papers expressed mutant RAS in mouse fibroblasts, while this one used lung epithelial cells). On the other, it is possible that in parallel to the elevation of superoxide levels, oncogenic RAS may also be activating superoxide dismutase (SOD) (180), which rapidly converts superoxide to H2O2 thereby causing superoxide levels to appear unchanged. Interestingly, besides the direct activation of ROS-producing enzymes such as NOX and COX-2, RAS oncogenic signaling can also promote an oxidant environment by repressing antioxidant molecules. A study showed that NrasD13-induced ROS up-regulation is accompanied by transcriptional repression of the Sestrin gene family (181), while over-expression of sestrins interferes with the ROS increase. This is in line with the role of sestrins (SESN1, 2, and 3), which play a crucial role in the regeneration of cytosolic peroxiredoxins, the enzymatic antioxidants involved in the decomposition of endogenously produced H2O2. Functionally, the increase in intracellular ROS was shown to cause chromosome instability, as evidenced by an increase in DNA oxidation 40  and the number of chromosome breaks, which may contribute to oncogenic RAS transformation (182).  A large part of recent RAS investigation has shifted towards the regulation of RAS on mitochondrial metabolism. Several lines of evidence also suggest that the effect of KRAS transformation on mitochondrial metabolism is associated with increased ROS generation. For instance, Weinberg et al (183) showed that the major site of KRAS-induced ROS generation is the Qo site of mitochondrial complex III and that this mitochondria-derived ROS is critical for oncogenic KRAS-driven cell proliferation and anchorage-independent growth. Furthermore, Liou and colleagues (184) elucidated a signaling pathway linking KRAS-induced mitochondrial ROS generation to the formation of pancreatic precancerous lesions. Their data showed that KrasG12D-induced mitochondrial ROS leads to the activation of protein kinase D1 (PKD1) and subsequently NF-kB. This initiates the transcription of epidermal growth factor receptor, EGFR, and its ligands EGF and TGFalpha and their sheddase ADAM17, together leading to the autocrine activation of EGFR signaling, which drives the de-differentiation of acinar cells to a duct-like progenitor phenotype and progression to pancreatic precancerous lesions known as pancreatic intraepithelial neoplasias (PanINs).  Taken together, these studies provide insight on how oncogenic RAS can drive intracellular oxidant programs to modulate essential molecular pathways, including cell proliferation, de-differentiation to a progenitor phenotype, genetic instability, and cellular transformation in a way that supports the initiation and progression of RAS-mediated tumorigenicity.  41  Largely due to the battery of studies that outline the role of RAS in activating oxidant programs to drive tumorigenesis, coupled with prospective studies that suggest an association between dietary antioxidants and a decreased risk for developing cancer (185, 186), cellular antioxidant programs have for the most part been an unappreciated mediator of oncogenesis, and to the contrary have been largely considered to have tumor suppressive roles. Using functional proteomic approaches, several initial studies provided evidence that KRAS-transformed cells display an upregulation of several antioxidant enzymes including peroxiredoxin 3, thioredoxin peroxidase, and catalase, which correlates with higher intracellular glutathione (GSH) as well as enhanced detoxification and apoptosis resistance in response to H2O2 or formaldehyde (187, 188). Another study showed that KRAS-transformation in prostate epithelial cells results in an upregulation of gamma-glutamyltransferase 2 (GGT2), an enzyme involved in the maintenance of glutathione homeostasis (189). In this study, the authors found that KRAS-mediated GGT2 activation also confers resistance to H2O2-induced apoptosis and that GGT2 expression is dependent on the ERK pathway. These studies laid the foundation for an important study by DeNicola et al (65), which demonstrated that endogenous KrasG12D expression leads to an increase of the antioxidant capacity (indicated by increase of the reduced to oxidized glutathione ratio [GSH/GSSG]) linked to a decrease of intracellular ROS levels. The mechanism supporting endogenous KrasG12D reduction of ROS relies on the control of the transcription factor NRF2. Specifically, the authors showed that endogenous KrasG12D, via the RAF-MEK-ERK-JUN pathway, transcriptionally activates NRF2 and NRF2 target genes (Hmox1, Nqo1, Gclc, and Ggt1) in cells, genetically engineered 42  mouse models (GEMMs) of pancreatic and lung cancer, and in human pancreatic cancer. More importantly, it was shown in vivo that NRF2 deficiency attenuates the reduction in ROS due to KrasG12D and causes a significant reduction in tumor volume and tumor cell proliferation in oncogenic KRAS mouse models of pancreatic and lung cancer, highlighting the role of NRF2 and antioxidant transcriptional program in KrasG12D-initiated tumorigenesis and proliferation. It is worth noting that ROS detoxification by enzymatic antioxidants is a biochemical process that consumes GSH and ultimately NADPH, given that NADPH is required to reduce oxidized glutathione and is thus the predominant source of reducing power. Thus, generation and maintenance of intracellular NADPH levels is vital for redox homeostasis. A recent study revealed that oncogenic KRAS promotes a constant supply of NADPH by reprogramming glutamine metabolism via the transcriptional upregulation of aspartate transaminase (GOT1) (64). They showed that pancreatic ductal adenocarcinoma (PDAC) cells and tumors are critically dependent on this non-canonical, GOT1-mediated metabolic pathway of glutamine that leads to the cytosolic conversion of glutamine-derived aspartate into oxaloacetate (OAA), malate and then pyruvate. This pathway increases the NADPH/NADP+ ratio and thereby maintains redox balance to sustain PDAC tumor growth. Another study showed that in mouse embryonic fibroblasts (MEFs) and lung cancer cell lines as well as advanced lung tumors, mutant KrasG12D allelic copy gains (KrasG12D/G12D) leads to a reprogramming of glucose metabolism marked by increased channeling of glucose-derived metabolites into the tricarboxylic acid (TCA) cycle and glutathione biosynthesis, leading to enhanced NADPH and GSH levels as well as ROS detoxification (190). KrasG12D copy gain and 43  associated upregulation of antioxidant capacity also drives malignant progression and metastatic potential in lung cancer cells and lung tumors in vivo. Conversely, the rate of tumor cell proliferation was reduced by treatment with the GSH biosynthesis inhibitor BSO in vivo. The capacity of oncogenic KRAS to rewire metabolic networks is not limited to glycolytic or glutamine pathways but also lipid biosynthetic processes. Recently, a group reported that mutant KRAS promotes the cellular uptake, accumulation, and beta-oxidation of fatty acids in lung cancer cells as well as lung tumors through the upregulation of Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3) (191). This has relevance for antioxidant generation as fatty acid oxidation generates acetyl CoA, which is metabolized to produce NADPH (192), especially under conditions of glucose scarcity. Therefore, it is possible that oncogenic KRAS-driven fatty acid oxidation could support NADPH generation and contribute to intracellular antioxidant capacity, although this mode of NADPH maintenance remains to be defined. Altogether, these data from several studies outline the role of oncogenic RAS in enhancing intracellular antioxidant capacity and drive the notion that antioxidants are in fact supportive of oncogenic RAS-mediated tumorigenicity by way of their effect on molecular pathways such as adaptation to oxidative stress, apoptotic resistance, proliferation, and cellular transformation. Nonetheless, as outlined, a large proportion of literature is also supportive of a role of oncogenic RAS as an inducer of ROS and oxidative stress to promote tumorigenesis. In the context of this discrepancy, in this chapter I decided to reinvestigate the impact of oncogenic RAS expression on intracellular levels of ROS in the basic cell model of NIH 3T3 fibroblasts and their 44  susceptibility to exogenous oxidative stress. Furthermore, the investigations in this chapter will be approached using a global, non-biased technique of whole transcriptome profiling, such that the initial approach is hypothesis-generating in contrast to many previous studies that relied on hypothesis-driven approaches, in order to identify novel targets and novel insights into mechanisms of redox modulation by oncogenic RAS. In addition, while many previous studies only use the expression of RAS as a single variable to assess the impact on ROS, none of them model oxidative stress conditions in parallel.  In light of the plethora of antioxidant pathways present in the cell to combat oxidative stress, this represents a possibility of missed candidate genes downstream of oncogenic RAS signaling that are worthy of subsequent biochemical validation and eventual therapeutic targeting. Therefore, in my assays I use exogenous hydrogen peroxide at sub-lethal doses on cells to model oxidative stress, in an attempt to identify novel targets that are upregulated by oncogenic RAS not only in ambient conditions but also in response to oxidative stress. Using these approaches, I implicate for the first time that transformation with oncogenic KRAS leads to upregulation of the Cystine/Glutamate Transporter, xCT, as a mechanism for the modulation of redox balance by RAS. In addition, I show that xCT supports the protection of oncogenic KRAS-transformed cells against oxidative stress leading to cell death.     45  2.2 Methods  Cell culture and hydrogen peroxide (H2O2) treatment NIH 3T3 fibroblasts were obtained from ATCC (Rockville, MD) and maintained in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) supplemented with 10% bovine calf serum and propagated by passaging 1:5 when plates reached 80-90% confluence. xCT-/- (KO) and xCT+/+ (WT) mouse embryonic fibroblasts (MEFs) were a kind gift from Hideyo Sato (Niigata University, Niigata, Japan) and routinely cultured in DMEM supplemented with 10% FBS.  xCT-/- MEFs were also supplemented with 50µM of 2-mercaptoethanol (Sigma-Aldrich). H2O2 treatment was carried out by making a 10mM working solution from hydrogen peroxide solution 30% (Sigma-Aldrich) and then treating cells in 6–well format.  Compounds Erastin was purchased from Tocris Bioscience. Diethyl-maleate (DEM), Menadione (MEN), N-acetylcysteine (NAC) and Glutathione (GSH) were purchased from Sigma-Aldrich.  Plasmids and retro-/lentiviral infection Retroviral expression vectors pMSCVpuro-KRASV12, pMSCVpuro-ETV6-NTRK3, and the empty vector pMSCVpuro were used for generation of stable cell lines as previously described (193). The pMSCVpuro-KRASV12 vector was constructed by subcloning the BamHI-SalI fragments from the pBabe-puro-KRASV12 open reading frame into the BglII-46  XhoI sites of pMSCVpuro. Infections of the NIH 3T3 cells were performed using retroviral-packaging Phoenix A cells transfected with target vector. Phoenix A cells grown in antibiotic free media were transfected with 3:1 target plasmid to X-tremeGENE HP (Roche) reagent ratio following the manufacturer’s instructions (Sigma-Aldrich) and incubated at 37°C. 48 and 72 hours post-transfection, medium containing retroviral particles was collected, filtered and used to infect NIH 3T3 in the presence of Polybrene (Sigma-Aldrich). The lentiviral plasmid pReceiver-LV151 containing the ORF of human SLC7A11/xCT was purchased from GeneCopoeia.  HEK293T cells grown in antibiotic free media were transfected with lentiviral envelope and packaging vectors pVSVG and psPAX2, as well as target plasmid with X-tremeGENE HP reagent (Roche) following the manufacturer’s instructions (Sigma-Aldrich) and incubated at 37°C. 48 and 72 hours post-transfection, medium containing lentiviral particles was collected, filtered and used to infect NIH 3T3 cells or MEFs in the presence of Polybrene (Sigma-Aldrich). Infected cells were selected using appropriate antibiotic selection for 48 hours to a week, depending on the target plasmid.  Western blot analysis Western blot analysis was performed on samples lysed in EBC buffer (50mM Tris-HCL, pH 7.4, 120mM NaCl, 0.5% NP40, 1mM EDTA, 2.5mM MgCl2) with the addition of PhosSTOP and Complete Mini protease inhibitors (Roche) using standard procedures.  Pan-Ras antibody was purchased from EMD Millipore. TrkC (EN) and Actin antibodies were from Santa Cruz.  47  Cell death assay Cell death analyses were performed using propidium iodide (PI) staining. Both detached and attached cells were harvested, centrifuged, washed, and resuspended in cold PBS containing 1µg/ml PI (Life Technologies) to form a single cell suspension. Data was collected using a BD FACSCalibur flow cytometer in FL-3 channel to measure PI levels. At least 10,000 events were recorded for each replicate and analyzed using FlowJo software.  Glutathione assay Cell were seeded into 96-well plates and allowed to attach overnight following which they were treated with 200µM of H2O2 for 6 hours. Cells were then collected and cellular concentrations of GSH were then quantified using the GSH/GSSG-Glo assay kit, according to the manufacturer’s protocol (Promega). Luminescence was measured using a plate reader (Molecular Devices) and concentrations of GSH were calculated against concurrently run standards provided in the kit.   ROS DCFDA assay ROS were measured using the general ROS indicator CM-H2DCFDA (5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester; Life Technologies). Cells were seeded in 6-well plates in triplicates. Cells were then incubated with 10µM CM-H2DCFDA for 30 minutes following which they were harvested, centrifuged, washed, and resuspended in cold PBS containing 1µg/ml PI. Data was collected using a BD FACSCalibur flow cytometer with FL-1 channel to quantify DCFDA levels and FL-3 48  channel to quantify PI levels. At least 10,000 events were recorded for each replicate and analyzed using FlowJo software.  Analysis of protein oxidation by dityrosine Protein oxidation was measured by standard western blotting techniques on protein lysates using an antibody specific for dityrosine (Adipogen Life Sciences). Dityrosine staining was quantified by ImageJ and normalized to loading control staining.  Transcriptome microarray expression profiling and Gene Set Enrichment Analysis Affymetrix GeneChip Mouse Gene 1.0 ST Arrays were processed at the Centre for Translational and Applied Genomics (CTAG; Vancouver, Canada). Briefly, biotin-labeled cRNA was prepared from total RNA and hybridized to the Affymetrix arrays according to the manufacturer’s protocol (Affymetrix Inc, Santa Clara, CA). Gene-level normalization and signal summarization of array data was carried out using the Robust Multi-Array Average module in the Affymetrix Expression Console software package and subsequent differential gene expression analysis was performed using the Affymetrix Transcriptome Analysis Console (TAC) software (Affymetrix Inc, Santa Clara, CA) (194). Candidate genes were selected as differentially expressed based on at least 2-fold mean difference in log-transformed expression values between experimental groups, and t-tests were used to determine significance (p < 0.05). Gene Set Enrichment Analysis between experimental groups was performed as previously 49  described, using the Broad Institute software with default parameters, 1000 permutations of gene sets, and Signal2Noise metric for ranking of genes (195).  RNA isolation and quantitative RT-PCR For RNA isolation in mammalian cells, after indicated treatments cells were lysed and total RNA was extracted using RNeasy RNA extraction kit (Qiagen). RNA concentrations were determined by nanodrop and equal amounts of RNA were used to generate cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNA and Power SYBR Green PCR master mix (Applied Biosystems) were mixed with primers according to the manufacturer’s instructions and ran in an Applied Biosystems Quant Studio 6 Flex RT-PCR system. As an internal control, Gapdh or β-actin was amplified.  siRNA transfections Cells were transfected at ~30% confluency in 6-well plates with 25nM control siRNA (1:1 mix of non-targeting siRNA (Stealth RNAi negative control duplexes, medium GC Duplex; Invitrogen) and luciferase targeting siRNAs (Stealth RNAi siRNA Luciferase Reporter Control; Invitrogen)) or with 25nM of specific single-targeting siRNAs using the Lipofectamine RNAiMAX transfection reagent (Invitrogen). Unless otherwise indicated, cells were replenished with new media after 24 hours and harvested at 72 hours post-transfection.   50  18F-5-fluoroaminosuberic acid (FASu) uptake For uptake studies, cells were seeded into 24-well plates (1 × 105 cells/mL) such that confluency was achieved the next day. Each plate was washed 3 times with cold HEPES buffer, and then 0.148 MBq of 18F-FASu in 400 µL of HEPES buffer was added. Subsequently, the samples were incubated at 37°C with orbital shaking for the indicated duration. After incubation, supernatants were removed, cells were washed twice with cold HEPES buffer, 400 µL of 1 M NaOH were added to the cells, the NaOH lysate was collected 10 min later, and 25 µL of the lysate was taken to assess the protein concentration using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The activity in the solution and lysate was measured using a PerkinElmer Wizard 2480 γ-counter and normalized to the protein concentration.  Amplex glutamate release assay The release of glutamate from NIH 3T3 cells into the extracellular medium was detected using an Amplex Red glutamate release assay kit (Molecular Probes/Life Technologies Corp., Eugene, OR). Cells were seeded overnight at 70% confluency into 6-well plates. The next day, cells were washed twice in PBS and then incubated for 6 hours in Na+-containing, glutamine-free media (Cellgro/Corning) containing Erastin at 20µM. For siRNA experiments, cells were transfected with siRNA and 72 hours later, washed twice in PBS and then incubated for 6 hours in Na+-containing, glutamine-free media. 50 µl of medium per well was removed and transferred to a 96-well assay plate (Falcon) and incubated with 50 µl of a reaction mixture containing glutamate oxidase, L-alanine, glutamate-pyruvate transaminase, horseradish peroxidase, and Amplex Red reagents 51  as per the manufacturer's protocol. Fluorescence is measured using a plate reader (Molecular Devices) and glutamate release is normalized to protein concentration and control conditions.  Statistical analysis Student’s t-test was used to determine statistical significance unless otherwise state, with p < 0.05 considered significant.  2.3 Results  2.3.1 Transformation with oncogenic KRAS leads to protection against oxidative stress   As indicated earlier, I decided to reinvestigate the impact of oncogenic RAS expression on intracellular levels of ROS in the basic cell model of NIH 3T3 fibroblasts and their susceptibility to exogenous oxidative stress. To this end, I used the NIH 3T3 fibroblast cell line stably expressing mutant-activated KRASV12 (3T3 KRAS), ETV6-NTRK3 (3T3 EN) or the empty vector (3T3 MSCV). In parallel with KRAS-transformation I used ETV6-NTRK3, the congenital fibrosarcoma fusion oncogene previously shown by us to activate downstream RAS signaling, to provide insight as to whether the effects on intracellular ROS are due to mutant-activated KRAS itself or constitutive RAS signaling (196, 197). Both 3T3 KRAS and 3T3 EN cells were previously established to be transformed through anchorage independence assays and the expression of KRAS and EN was confirmed (Fig. 3A) (198). To assess the impact of oncogenic KRAS- and EN-52  transformation on susceptibility to exogenous ROS, I treated 3T3 MSCV, 3T3 EN, and 3T3 KRAS cells with 200 to 600μM of H2O2 for 16 hours and then quantified cell death by measuring propidium iodide (PI) incorporation into dead cells using flow cytometry. High levels of H2O2 treatment induced severe levels of cell death in the 3T3 MSCV cells (>60% at 600μM), while 3T3 KRAS and 3T3 EN cells were less susceptible to this treatment (Fig. 3B), even though they also showed statistically significant levels of cell death relative to untreated cells. Consistent with the literature, this finding provides evidence that oncogenic KRAS or active RAS signaling confer protection to cells against exogenous ROS. Based on this preliminary data, I speculate that oncogenic KRAS is able to protect cells against oxidative stress by either reducing intracellular ROS-generating pathways or by enhancing antioxidant capacity (65).    2.3.2 Transformation with oncogenic KRAS leads to decreased intracellular ROS levels  Since transformation with oncogenic KRAS leads to protection against exogenous H2O2, I postulated that these transformed cells might display decreased levels of intracellular ROS, as compared to control cells. Accordingly, I treated 3T3 MSCV and 3T3 KRAS or EN cells with a sub-lethal dose of H2O2 for 3 to 6 hours and measured ROS levels using the general ROS indicator CM-H2DCFDA followed by flow cytometry. As expected, 3T3 KRAS cells showed low levels of intracellular ROS under ambient conditions and continued to display low levels of ROS following treatment with exogenous H2O2, in contrast to 3T3 MSCV cells, which showed rapid accumulation of intracellular ROS (Fig. 4A). Similar effects were found in 3T3 EN cells (Figure 4B).  53    Figure 3: Transformation with oncogenic KRAS or EN leads to the protection of NIH3T3 cells against exogenous H2O2 treatment. A. Immunoblot analysis of NIH 3T3 MSCV, EN, and KRASV12 cells. B. Propidium iodide staining quantification using flow cytometry on NIH3T3 MSCV, EN, and KRASV12 cells following a 16 hour treatment with indicated concentrations of H2O2 (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05).   In addition, to specifically determine if the differences in ROS levels between oncogenic KRAS-transformed cells and control cells represent the presence or absence of oxidative stress at a functional level, I analyzed protein oxidation using the specific biomarker dityrosine. Tyrosine is one of the major targets of protein oxidation and dityrosine is a tyrosine dimer derived from tyrosyl radicals, which are formed following exposure to ROS. Indeed, 3T3 KRAS cells under 3 to 6 hours of exogenous H2O2 treatment showed lower levels of dityrosine relative to 3T3 MSCV cells, again supporting the notion that transformation with KRAS leads to a protection against oxidative stress (Fig. 4C). Further, these studies also show that exogenous H2O2 treatment can lead not only to an increase in intracellular ROS, but also an increase in protein oxidation as shown by dityrosine levels, providing a reliable in vitro model of  54   Figure 4: Transformation with oncogenic KRAS leads to decreased intracellular ROS and decreased protein oxidation under exposure to oxidative stress. A. DCFDA staining quantification using flow cytometry in NIH 3T3 MSCV and KRASV12 cells, and B. NIH 3T3 EN cells following treatment with 200µM H2O2 for the indicated times (n=3). C. Immunoblot analysis of the oxidative stress marker Dityrosine in NIH 3T3 MSCV and KRASV12 cells following treatment with 200µM H2O2 for the indicated times. Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).    oxidative stress. Notably, it appeared that at 6 hours of exogenous H2O2 treatment, both 3T3 KRAS and 3T3 MSCV cells begin to exhibit a decrease in dityrosine levels, alluding to a mechanism for ROS clearance. 55  2.3.3 Transformation with oncogenic RAS enhances the upregulation of the light-chain subunit of the Cystine/Glutamate Transporter, xCT, in vitro To investigate the expression profile response of oncogenically-transformed and non-transformed NIH 3T3 cells under oxidative stress, and to show that oncogenic KRAS expression modulates intracellular redox pathways, I performed whole transcriptome microarray analysis on 3T3 MSCV, 3T3 KRAS, and 3T3 EN cells using the Affymetrix GeneChip Mouse Gene 1.0 ST platform. To model oxidative stress, I used quantitative RT-PCR to optimize treatment conditions that would transcriptionally activate Nrf2 and several Nrf2 target genes without inducing cellular toxicity (data not shown). I determined the ideal conditions for activation of antioxidant response in NIH 3T3 cells to be a sub lethal dose of 200μM of H2O2 for 3 to 6 hours. From the differential gene expression profiling of oncogenically-transformed and non-transformed cells, I identified a list of top ten most highly upregulated genes following exogenous oxidative stress in 3T3 KRAS (and 3T3 EN) cells, as compared to 3T3 MSCV cells (Table 1). Among these genes, I observed several that are known to be involved in the endoplasmic reticulum (ER) stress response and unfolded protein response (UPR) pathway including CAR6, CHAC1, and TRIB3. This is not entirely surprising given that oxidative stress and ER stress are closely linked events and RAS signaling is known to modulate ER stress response and UPR pathways (199). The direct contribution of these genes toward the protection of 3T3 KRAS cells against oxidative stress is an interesting area for future investigation. Notably, the most prominently upregulated gene in the list was the solute carrier family 7 member 11; SLC7A11. The SLC7A11 gene encodes the light-chain subunit of the cystine/glutamate transporter (system xc–), also commonly  56  Table 1: The gene encoding the light-chain subunit of the cystine/glutamate transporter, SLC7A11 (xCT), is most prominently upregulated in 3T3 KRAS cells following exogenous H2O2 treatment. Differential gene expression profiling from Affymetrix GeneChip Mouse Gene 1.0 ST assays in NIH 3T3 MSCV and KRASV12 cells following 200µM H2O2 for 6 hours. The top 10 genes are ordered by positive fold change and ANOVA p-value denotes the significance value.   referred to as xCT. Given the well-established role of system xc– as a plasma membrane transporter that mediates the uptake of cystine and its critical involvement in the biosynthesis of GSH, I decided to focus on this gene and hypothesized that oncogenic KRAS promotes the upregulation of xCT as a protective mechanism against oxidative stress (200). To corroborate my findings from gene expression profiling, I additionally performed quantitative RT PCR on the same cell lines under the same conditions and confirmed the elevation of xCT transcript levels in 3T3 KRAS and EN cells relative to 3T3 MSCV cells (Fig. 5A). I similarly found that NIH 3T3 fibroblasts stably expressing mutant-activated HRASV12 exhibited higher levels of xCT mRNA following stimulation with exogenous H2O2 (Fig. 5B). In addition to these data, by interrogating a publicly available gene expression dataset (GSE2151), I found that in human mammary epithelial cells (HMECs) stably transfected with a panel of oncogenes, xCT mRNA is specifically upregulated in cells that express oncogenic RAS but not in those that 57  express other oncogenes such as β-Catenin, E2F3, or MYC, or in control GFP-expressing cells (Fig. 5C) (201). In addition to showing that oncogenic transformation with RAS leading to increased xCT expression is highly reproducible in several independent in vitro systems, this finding also suggests that the induction of xCT is specific to oncogenic RAS but not to malignant transformation in general.   2.3.4 Transformation with oncogenic KRAS enhances the upregulation of the light-chain subunit of the Cystine/Glutamate Transporter, xCT, following exposure to known oxidative stress inducers In the previous section, using a whole transcriptome microarray approach, I showed that transformation with oncogenic RAS leads to the upregulation of the light-chain subunit of the cystine/glutamate transporter (system xc–), xCT, following treatment with H2O2 as a model of oxidative stress. While this finding is consistent with the established role of the system xc– transporter in the cellular antioxidant response, interestingly, among the most highly upregulated genes in 3T3 KRAS cells following exposure to H2O2 were those involved in the endoplasmic reticulum (ER) stress response and unfolded protein response (UPR) pathway including CAR6, CHAC1, and TRIB3. Even though it is widely appreciated that oxidative stress can induce ER stress and vice versa, forming a vicious cycle, I wanted to ascertain that the effect of H2O2 on xCT expression in 3T3 KRAS cells is indeed associated with the induction of oxidative stress. To this end, I treated 3T3 KRAS cells with two known inducers of oxidative stress – diethyl-maleate (DEM) and menadione (MEN), and examined the effect on xCT expression. DEM is known to induce oxidative stress by decreasing GSH by direct  58   Figure 5: Oncogenic RAS induces the upregulation of xCT mRNA. A. Validation of differential gene expression profiling by quantitative RT-PCR for xCT transcript in NIH 3T3 MSCV, EN and KRASV12 cells following treatment with 200µM H2O2 for the indicated times. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to MSCV control conditions (n=3). B.  Quantitative RT-PCR for xCT transcript in NIH 3T3 MSCV and NIH3T3 stably expressing HRASV12 following treatment with 200µM H2O2 for the indicated times. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to MSCV control conditions (n=3). C. Expression levels of xCT in human mammary epithelial cells (HMEC) stably expressing control GFP or indicated oncogenes. Expression levels are expressed as log2 of mRNA levels. Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).     59  oxidation to GSSG, or by forming GSH conjugates through the action of glutathione S-transferase (202), whereas MEN causes acute generation of ROS through redox cycling (203). As shown in Figure 6, treatment with 100µM with DEM for up to 6 hours significantly induced xCT expression in cells with 3T3 KRAS cells showing markedly higher levels of xCT mRNA relative to 3T3 MSCV controls. Similarly, treatment with 20μM of MEN for up to 3 hours also induced xCT expression in cells with 3T3 KRAS showing significantly higher levels of xCT mRNA, as compared to controls. Thus I provide further evidence that transformation with oncogenic KRAS enhances the upregulation of xCT following exposure to oxidative stress.  2.3.5 Transformation with oncogenic KRAS enhances xCT activity following exposure to oxidative stress In previous sections I found that transformation with oncogenic RAS leads to the upregulation of the SLC7A11 gene, which encodes the light-chain subunit of the cystine/glutamate transporter (system xc–), xCT. The biological significance and role of system xc– in cancer will be discussed in the introduction of Chapter 2. Briefly, the system xc– transporter mediates the exchange of the anionic form of extracellular cystine for intracellular glutamate with a stoichiometry of 1:1 (200), and this function is primarily attributed to the activity of the xCT subunit. I have shown that transformation with oncogenic KRAS leads to enhanced induction of xCT expression following exposure to oxidative stress, as compared to control cells, although the studies shown have only been limited to the transcript level. This is due to the lack of a reliable, commercially available antibody against mouse xCT (204). Therefore, to verify that  60   Figure 6: Oncogenic KRAS enhances the upregulation of xCT mRNA in cells following exposure to oxidative stress inducers, diethyl-maleate (DEM) and menadione (MEN). Quantitative RT-PCR for xCT transcript in NIH 3T3 MSCV and NIH 3T3 KRASV12 cells following treatment with A. 100µM DEM or B. 20µM MEN for the indicated times. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to MSCV control conditions (n=2). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).  transformation with oncogenic KRAS indeed leads to enhanced xCT activity, I performed an in vitro uptake assay using the xCT-specific PET tracer 18F-5-fluoroaminosuberic acid (FASu) (205, 206). FASu was developed as a potent substrate of xCT with the goal of assessing its potential as a diagnostic tracer of oxidative stress via system xc– activity. The specificity of FASu for xCT has been thoroughly evaluated using a combination of in vitro uptake and inhibition studies as well as in an in vivo setting, and can therefore serve as an appropriate substitute for radiolabeled-cystine uptake studies. In fact, FASu may better represent xCT activity as opposed to radiolabeled-cystine, as cystine can also participate as a substrate for other neutral amino acid transporters, albeit with lower affinity than with xCT. To assess differences in xCT activity following exposure to oxidative stress, 3T3 KRAS and 3T3 MSCV cells were treated with either H2O2 or DEM for 6 hours before uptake measurements. Cells 61  were then washed, and incubated with FASu in the absence or presence of Erastin, a specific inhibitor against xCT (171). Cells were washed again after the uptake incubation period and radioactivity was analyzed using a scintillation counter. As shown in Figure 7, while all cells showed FASu uptake following exposure to H2O2 or DEM, 3T3 KRAS cells consistently showed significantly higher levels (>2-fold) of uptake relative to 3T3 MSCV controls. In addition, FASu uptake could be blocked with addition of Erastin, which blocks xCT activity, confirming the specificity of FASu for xCT. Altogether, this data suggests that oncogenic KRAS enhances xCT activity following exposure to oxidative stress, and as shown, this is correlated with increased xCT expression.  2.3.6 Transformation with oncogenic KRAS enhances intracellular GSH levels  Oncogenic KRAS-transformed cells display a lower susceptibility to oxidative stress leading to cell death, which is correlated with enhanced induction of xCT expression and xCT activity following exposure to oxidative stress. As mentioned, system xc– is a plasma membrane transporter that mediates the uptake of cystine via its xCT subunit. Once inside the cell, cystine is rapidly reduced to cysteine, which is the rate-limiting precursor in the synthesis of glutathione (GSH), thus highlighting the role of system xc– in GSH biosynthesis. To determine if enhanced xCT expression and activity in oncogenic KRAS-transformed cells is correlated with increased GSH levels, I compared GSH levels in 3T3 KRAS and 3T3 MSCV cells following exposure to H2O2 or DEM for 6 hours, using the GSH-Glo assay.  62             Figure 7: Oncogenic KRAS enhances 18F-5-fluoroaminosuberic acid (FASu) uptake in cells following exposure to H2O2 and DEM. FASu uptake in NIH 3T3 MSCV and NIH 3T3 KRASV12 cells following treatment with 200µM H2O2 or 100µM DEM for 6 hours, with or without blocking with Erastin (20µM). FASu radioactivity was normalized to protein concentration. Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).   Consistent with previous data, I found that 3T3 KRAS and 3T3 EN cells displayed higher levels of reduced GSH relative to 3T3 MSCV cells under ambient conditions (Fig. 8). In addition, while all cells showed a decrease in GSH levels following exposure to exogenous oxidative stress, 3T3 KRAS and 3T3 EN consistently had higher levels of GSH, as compared to 3T3 MSCV controls. Thus I provide further evidence that transformation with oncogenic KRAS (or EN) is associated with increased intracellular GSH levels, and I speculate that this is potentially explained by enhanced xCT expression and activity.  63            Figure 8: Transformation with oncogenic KRAS and EN enhances intracellular GSH levels. Quantification of reduced GSH levels using GSH-Glo assay (Promega) in NIH 3T3 MSCV, KRASV12 and EN cells following treatment with 200µM H2O2 or 100µM DEM for the 6 hours (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05).  2.3.7 Expression of the heavy-chain subunit of the Cystine/Glutamate Transporter, CD98, is unaltered following transformation with oncogenic KRAS and exposure to exogenous oxidative stress The system xc– transporter is a member of a family of heteromeric amino acid transporters (HAATs), which are composed of a heavy-chain subunit and a light-chain subunit coupled by a disulfide bridge. In the case of system xc–, the heavy-chain subunit is designated 4F2hc and is coupled to the light-chain subunit xCT. 4F2hc is a type II membrane glycoprotein (also known as CD98) found to be a shared counterpart among several amino acid transporters (207, 208). Its main role is in trafficking and serves to sequester the heterodimer to the plasma membrane. On the other hand, xCT is the 64  subunit that confers specificity for cystine, and the transport activity of system xc– has been shown to be exclusively dependent on xCT. To ascertain that the reported enhancement of FASu uptake and intracellular GSH levels, as well as the protection of cells transformed by oncogenic KRAS against oxidative stress is associated with the xCT subunit alone and not with CD98 expression, I treated 3T3 KRAS cells with 200μM of H2O2 for 6 hours and probed CD98 expression using immunobloting. As shown in Figure 9, neither expression of oncogenic KRAS or exposure to exogenous H2O2 results in altered CD98 expression. This provides evidence that the protection of 3T3 KRAS cells against oxidative stress, as well as the enhanced FASu uptake and increased intracellular GSH levels relative to control cells can be attributed to the xCT subunit of the system xc– transporter.  2.3.8 Transformation with oncogenic RAS leads to the upregulation of the light-chain subunit of the Cystine/Glutamate Transporter, xCT, in vivo To determine whether oncogenic KRAS induces the upregulation of xCT at a genetic level in vivo, mouse lung cancer specimens were evaluated. mRNA levels of xCT were assessed from whole transcriptome data of several genetically engineered mouse models of lung cancer, namely transgenic mice that expressed either an EGFR in-frame exon 19 deletion mutant, EGFR L858R mutant, KrasG12D mutant, or overexpression of c-Myc. Lung specimens from transgenic mice expressing KrasG12D mutant displayed higher levels of xCT mRNA compared to those from oncogenic c-Myc or normal lung tissue (Fig. 10A). Interestingly, elevated levels of xCT mRNA were also found in lung specimens from transgenic mice expressing either of the EGFR mutants,  65    Figure 9: Expression of CD98 is unaltered following stable expression of oncogenic KRAS and exposure to exogenous oxidative stress. A. Immunoblot analysis of NIH 3T3 MSCV, EN, and KRASV12 cells following exposure to 200µM of H2O2 for 6 hours (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05).  as compared to those from oncogenic c-Myc or normal lung tissue. In addition, I probed publicly available gene expression data (GSE50794) and found that in genetically engineered mouse models of colorectal cancer, tumors from mice that harbored Kras mutations had higher levels of xCT, as compared to wildtype samples (Fig. 10B).  Interestingly, xCT mRNA levels were also higher in tumors from transgenic mice that had mutations in BRAF. This suggests (along with gene expression profiling data in EN-expressing cells) that it is most likely activated KRAS signaling, rather than the mutant form of KRAS itself, which upregulates xCT expression. More importantly, this data also provides in vivo evidence to the fact that transformation with oncogenes that do not activate RAS signaling such as MYC, do not appear to upregulate xCT expression. 66   Figure 10: Lung and colon tumor specimens from transgenic mice expressing oncogenic KRAS display higher xCT mRNA levels. A. Expression levels of xCT in normal mouse lung (n=5), or in lung tumor specimens from transgenic mice expressing oncogenic c-Myc (n=5), EGFR deletion mutant (n=4), EGFRL858R mutant (n=4) or KRASG12D mutant (n=7). B. Expression levels of xCT in Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).   2.3.9 Inhibition of oncogenic KRAS reduces xCT expression To further establish the role of oncogenic KRAS in upregulating xCT in a more clinically relevant model, I investigated the effect of KRAS inhibition on xCT expression in human cancer cell lines known to harbor activating mutations of KRAS. Using the lung cancer cell line H460 and the colon cancer cell line SW620, I showed that siRNA-mediated knockdown of KRAS decreases xCT protein expression (Fig. 11A). Similarly, knockdown of KRAS in SW620 resulted in a decrease in xCT transcript levels both under ambient condition or following exposure to exogenous H2O2 (Fig. 11B). This lends further support that expression of oncogenic KRAS upregulates xCT expression. 67    Figure 11: KRAS-specific siRNAs reduce xCT expression in human cancer cell lines. A. Immunoblot analysis showing KRAS, xCT, phospho-ERK (p-ERK), total ERK, and loading control (HSC70) expression in H460 and SW620 cells 72 hours following transfection with KRAS-specific siRNAs. The image is representative of three independent experiments. B. Quantitative RT-PCR for xCT transcript in SW620 cells transfected with a KRAS-specific siRNA for 72 hours and treated with 200µM H2O2 for the indicated time. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to scrambled control conditions (n=3).   2.3.10 xCT supports the oncogenic KRAS-mediated protection against oxidative stress through the enhancement of intracellular GSH levels To obtain functional verification that xCT is indeed mediating the protection of oncogenic KRAS-transformed cells to oxidative stress by enhancing their capacity to decrease intracellular ROS, I decided to examine the effects of genetic inhibition of xCT in the cell line models previously described. I first demonstrated that two individual siRNAs against xCT were able to reduce xCT transcript levels by more than 50% (Fig. 12A). To ensure that this reduction in xCT transcript levels corresponds to decreased 68  xCT activity, I also performed an assay to measure glutamate release from cells, since xCT mediates the exchange of the anionic form of extracellular cystine for intracellular glutamate (171). Briefly, cells were transfected with siRNAs for 72 hours, incubated in glutamine free media for 6 hours, following which culture media was removed and assayed using the Amplex Red glutamate release assay kit (Molecular Probes). Indeed, both xCT-specific siRNAs decreased glutamate release into culture media by about 50%, relative to cells transfected with a scrambled control (Fig. 12B). This glutamate release could be attributed to the function of system xc– since treatment of scrambled control cells with Erastin also resulted in about 70% inhibition of glutamate release, confirming that the xCT-specific siRNAs reduced not only xCT transcript but also its activity. Notably, as shown in Figure 12C, these xCT-specific siRNAs effectively re-sensitized 3T3 KRAS cells to lethal doses of H2O2 over 16 hours, as indicated by propidium iodide (PI) incorporation analyzed by flow cytometry (Fig. 12C).  To establish that xCT is playing a role in supporting the oncogenic KRAS-mediated reduction of intracellular ROS, I also measured ROS levels using the ROS indicator CM-H2DCFDA following siRNA-mediated knockdown of xCT. As shown in Figure 13A, both xCT-specific siRNAs caused an increase in ROS levels under ambient conditions, relative to cells transfected with a scrambled control. Exposure to exogenous H2O2 caused ROS to increase in all cells, but cells with xCT knockdown showed significantly higher levels relative to controls. Notably, the increase in intracellular ROS due to xCT knockdown could be reversed with GSH, or another antioxidant N-acetyl-cysteine (NAC).  NAC has been shown to boost intracellular GSH levels as it can serve as a membrane-permeable cysteine precursor for GSH synthesis  69   Figure 12: xCT-specific siRNAs resensitize 3T3 KRAS cells to exogenous H2O2 treatment. A. Propidium iodide staining quantification using flow cytometry on NIH 3T3 KRASV12 cells that were transiently transfected with 25nM of control (scr) or xCT-directed siRNAs, grown in complete media for 48 hours, then treated with H2O2 for 16 hours at indicated concentrations (n=3). B. Quantitative RT-PCR for xCT transcript to validate siRNA-mediated knockdown of xCT. Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).  without the need of active transport (209). As mentioned, an important step in GSH synthesis involves the action of glutamate-cysteine ligase (GCL), and I found that inhibition of GCL using its inhibitor 1-buthionine-S,R-sulfoximine (BSO) rendered NAC supplementation unable to reverse the increase in intracellular ROS due to xCT 70  knockdown (NAC+BSO), suggesting that the role of xCT in supporting the protection of cells against oxidative stress is by providing cystine intermediates for the synthesis of GSH via GCL. In addition to ROS quantification, I performed identical xCT-knockdown experiments and measured intracellular GSH levels (Fig. 13B). Consistent with the levels of intracellular ROS, both xCT-specific siRNAs caused a decrease in GSH under ambient conditions, relative to cells transfected with a scrambled control. Exposure to exogenous H2O2 and BSO caused GSH to decrease in all cells, but cells with xCT knockdown showed significantly attenuated levels relative to controls. Notably, GSH levels could be rescued with NAC, but not in the presence of BSO, providing further evidence that xCT protects 3T3 KRAS cells against oxidative stress by enhancing cystine uptake leading to enhanced GSH synthesis through the action of GCL.   Encouraged by these findings, I also acquired mouse embryonic fibroblasts (MEFs) derived from xCT-/- (KO) mouse or ones derived from the xCT+/+ (WT) littermates (210). KRASV12 was ectopically expressed in these cells, and they were subjected to ROS and GSH quantification as well. As shown in Figure 14A, xCT-deficient cells (xCT-/- KRASV12) showed markedly higher levels of ROS (~2-fold) under ambient conditions relative to control cells (xCT+/+ KRASV12), which could be reversed by re-expressing xCT (+xCT KRASV12), or by incubation with 2-mercaptoethanol (2ME), GSH or NAC. 2ME is a reducing agent, which has been demonstrated to reduce extracellular cystine to cysteine, which can then be taken up by the cell via neutral amino acid transporters (described in Chapter 3). Similar to my findings with xCT-specific siRNAs, NAC again failed to reverse high ROS levels in xCT-/- KRASV12 in the presence of BSO (GCL inhibition). Moreover, xCT-/- KRASV12 cells displayed significantly 71   Figure 13: xCT-specific siRNAs increase intracellular ROS and decrease GSH in 3T3 KRAS cells. A. DCFDA staining quantification using flow cytometry in NIH 3T3 KRASV12 cells that were transiently transfected with 25nM of control (scr) or xCT-directed siRNAs, grown in complete media for 72 hours, then treated with GSH (5mM), NAC (5mM), BSO (100µM), NAC and BSO, or H2O2 (200µM) for 6 hours (n=3). B. Quantification of reduced GSH levels using GSH-Glo assay (Promega) in NIH 3T3 KRASV12 cells that were transiently transfected with 25nM of control (scr) or xCT-directed siRNAs, grown in complete media for 72 hours, then treated with NAC (5mM), BSO (100µM), NAC and BSO, or H2O2 (200µM) for 6 hours (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).     72  lower GSH levels, as compared to xCT+/+ KRASV12 cells, which were restored in +xCT KRASV12 cells, or following incubation with 2ME or NAC, but not NAC and BSO together (Fig. 14B). Finally, I also demonstrated that xCT-deficiency rendered cells susceptible to exogenous H2O2, but not in the presence of 2ME, NAC, and TROLOX (another antioxidant), or re-expression of xCT (Fig. 14C). Altogether, my data suggests that xCT supports the protection of cells against oxidative stress mediated by oncogenic KRAS, through its role in transporting cystine into the cell, leading to enhanced intracellular GSH levels.  Conversely, I attempted to validate these results by evaluating whether overexpression of xCT could render non-transformed 3T3 MSCV better able to survive exogenous oxidative stress. Consistent with my previous findings, I found that ectopic overexpression of xCT in these cells phenocopied the effect of KRAS-transformation, and the cells were less susceptible to lethal doses of exogenous H2O2 treatment (Fig. 14). In summary, these data suggest that the role of xCT in oncogenic KRAS-transformed cells is to support the protection against oxidative stress by increasing intracellular GSH leading to an enhanced capacity for ROS clearance.   73   Figure 14: Loss of xCT increases intracellular ROS and decreases GSH in KRASV12-transformed MEFs, leading to increased susceptibility to exogenous oxidative stress. A. DCFDA staining quantification using flow cytometry in xCT+/+ and xCT-/- (KRASV12-transformed) MEFs with or without 6 hour supplementation with 2ME, GSH, NAC or NAC and BSO, or reexpression of xCT (n=3). B. Quantification of reduced GSH levels using GSH-Glo assay (Promega) in xCT+/+ and xCT-/- (KRASV12-transformed) MEFs with or without 6 hour supplementation with 2ME, NAC or NAC and BSO, or reexpression of xCT (n=2). C. Propidium iodide staining quantification using flow cytometry in xCT+/+ and xCT-/- (KRASV12-transformed) MEFs following treatment with 200µM H2O2 for 16 hours with or without incubation with 2ME, NAC or Trolox, or reexpression of xCT (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).    74   Figure 15: Ectopic overexpression of xCT in non-transformed 3T3 MSCV cells protects them against exogenous H2O2 treatment. Propidium iodide staining quantification using flow cytometry on NIH 3T3 MSCV cells stably expressing control (EV) vector or xCT overexpression vector treated with indicated concentrations of H2O2 for 16 hours (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).   2.4 Discussion To date, many studies have attempted to investigate the impact of oncogenic RAS on redox balance using numerous cell lines and mouse models. In spite of these studies, the role of oncogenic KRAS in modulating intracellular ROS and its contribution to tumorigenicity is still controversial, with some reports showing RAS transformation as increasing cellular ROS to support tumorigenesis, and with others showing the converse. In this chapter, I first used NIH 3T3 fibroblasts to model KRAS transformation. NIH 3T3 cells are less genetically complex compared to cell lines derived from patient tumors and thus offer a relatively straightforward system to study the impact of KRAS transformation on redox homeostasis. In addition, while previous studies assessed the 75  impact of KRAS on cells at ambient conditions, in my assays I modeled oxidative stress conditions using exogenous hydrogen peroxide (H2O2) treatment. First, I demonstrated that in cells exposed to exogenous H2O2, ectopic expression of KRASV12 resulted in decreased susceptibility to cell death, which was associated with lower levels of intracellular ROS and protein oxidation, as compared to control cells. Second, by using a hypothesis-generating approach of whole transcriptome microarray analysis, my study appears to be the first in literature to identify the light-chain subunit of the cystine/glutamate transporter, xCT, as being upregulated by oncogenic KRAS in response to oxidative stress. While the treatment with exogenous H2O2 approach may be artificial and may not accurately recapitulate pathophysiological oxidative stress, I was able to demonstrate using two other known inducers of oxidative stress- diethyl maleate and menadione, that the induction of xCT transcript and activity (as measured by FASu uptake) was enhanced in KRASV12-transformed cells relative to control cells. In addition, through a combination of knockdown studies as well as the use of xCT-knockout cells lines, I provide evidence that the protection of KRASV12-transformed cells against oxidative stress is supported by the role of xCT in transporting extracellular cystine into the cell for the biosynthesis of reduced glutathione (GSH), which is the major intracellular antioxidant. Notably, knockdown of xCT (or xCT-deficiency in knockout cells) led to a decrease in cellular GSH and an increase in ROS, an observation that could be reversed upon administration of NAC (known to serve as an alternate source of cystine precursors), but not in the presence of BSO (an inhibitor of GCL, a crucial enzyme in GSH synthesis). In addition, the association between enhanced xCT transcript levels and oncogenic KRAS were recapitulated with two 76  human cancer cell lines, as well as in genetically engineered mouse models of oncogenic KRAS.  It is worth noting that in my studies, the effect of mutant KRAS transformation on cellular redox balance was consistent with the findings of DeNicola and colleagues (65). Namely, they demonstrated that cells expressing KRASG12D from an endogenous locus display augmented levels of GSH and lower levels of intracellular ROS. In their study, the authors additionally showed that when KRASG12D was expressed ectopically, to the contrary, this leads to a reduction of the GSH/GSSG ratio but to an increase in ROS. Such a discrepancy was explained by the ability of ectopic KrasG12D, but not of endogenous KrasG12D, to induce Nox transcription and NOX activity, which promotes ROS production. Their findings certainly raise doubts on earlier studies which mainly relied on ectopic overexpression approaches to illustrate that oncogenic KRAS promotes oxidant programs (such as NOX) on the basis that they may not accurately recapitulate tumor physiology, and that those findings are now being supplanted by more current models that use endogenous expression studies to show how oncogenic RAS promotes antioxidant responses (such as Nrf2). Interestingly, the assays I performed modeled RAS transformation using ectopic expression of KRASV12, HRASV12, and EN but revealed effects similar to the DeNicola study. This highlights the complexities of oncogenic RAS signaling and the conundrum of whether oncogenic RAS will increase or decrease intracellular ROS, which is difficult to define in every context. Nonetheless, the regulation of RAS on other downstream effectors may shed light on this problem. Namely, even though oncogenic RAS can activate more than 20 77  different downstream effectors, in any given cell type or context only some of these will be selectively activated resulting in distinct physiological outputs (211). Therefore, it is conceivable that similarly, oncogenic RAS can regulate a combination of both oxidant and antioxidant programs in concert or in succession in order to promote transformation and tumorigenesis, and the induction of these programs may in fact occur very early on in the transformation process such as in pre-neoplastic tissue. Moreover, it is now widely held that apart from just mutational status, the expression level of oncogenic RAS also defines phenotypic outcome (70). Several studies for instance have shown that endogenous expression of mutant KRAS alone in GEMMs fail to show any discernible pathological effect, unless coupled with chronic pancreatitis or the presence of inflammatory stimuli, which are necessary to amplify RAS signaling to an effective level (212, 213). In short, mutant RAS behaves more like a rheostat rather than a binary switch, and its expression levels can be enhanced by non-genetic events in order to effectuate downstream signaling (214). Taking all of this into account, I speculate that even though I ectopically express KRASV12, HRASV12, and EN, the level of their expression perhaps do not transmit a threshold of RAS signaling that is sufficient to activate NOX systems. Further, based on this understanding, I also speculate a possible scenario of cancer initiation in which the early genetic event of KRAS mutation results in cellular transformation in parallel with the activation of specific antioxidant pathways, such as the Nrf2 program. Later, as oncogenic RAS signaling is further amplified in transformed cells that experience inflammatory stimuli or stress conditions, this will allow RAS to activate downstream oxidant pathways such as NOX and COX-2, resulting in the accumulation of mutational events, further increasing genetic instability, 78  de-differentiation, and hyperproliferation, all of which are necessary for tumor progression. In these highly stressful conditions caused by excessive ROS levels, cells that already have an established strong antioxidant response will then be refractory to senescence and cell death, and will be able to support neoplastic growth (65, 215, 216). In this light, the absence of any of these antioxidant or oxidant programs will therefore be deleterious for tumor initiation and progression. In support of this notion, Liou and colleagues (2016) found that even though Nrf2 expression was increased in acinar cells and PanIN lesions driven by oncogenic KRAS, these pre-neoplastic structures still showed significant oxidative damage as indicated by 4-hydroxenonenol staining. So even though the activation of Nrf2 by oncogenic RAS signaling was present, this did not completely mitigate ROS levels (which can be considered tumor suppressive), but instead drove antioxidant responses to an extent that was sufficient to prevent cellular senescence or apoptosis, while allowing a threshold of intracellular ROS from oxidant programs to continue exerting other pro-tumorigenic effects.  It is evident that further studies to reconcile the contrasting effects of oncogenic RAS signaling on general cellular redox balance are warranted. However, I will not attempt to address this issue in my studies. Rather, I will focus on the role of xCT as a downstream effector of oncogenic KRAS-induced transformation, in hopes of characterizing novel therapeutic strategies against cancers of this genetic subset.  79  Chapter 3: Oncogenic KRAS Upregulates xCT Expression to Support Transformation and Tumorigenicity  3.1 Introduction In Chapter 1 I established that transformation with oncogenic KRAS leads to the protection of cells against oxidative stress. Using an unbiased, global, transcriptomic analysis approach, I uncovered that this was mediated by xCT, which is part of the system xc– transporter. The system xc– transporter was first described by Bannai and Kitamura (217) close to forty years ago upon discovering a cystine transport system in mammalian cultured cells whose activity was inhibited by glutamate. The activity of system xc– is Cl–-dependent and Na+-independent and its primary function is to mediate the exchange of the anionic form of extracellular cystine for intracellular glutamate with a stoichiometry of 1:1 (200). As mentioned, xCT is the subunit which confers specificity for cystine, and the transport activity of system xc– is exclusively dependent on xCT. As such, the terms system xc– and xCT are commonly used interchangeably, even though they are technically not identical.  Although the primary function of the system xc– transporter is to transport cystine into the cell, the physiological function of this transport system is only defined largely from in vitro studies. Cultured cells cannot survive when the culture medium is devoid of cystine, and this observation is attributed to the depletion of intracellular glutathione (GSH), which as mentioned is the predominant endogenous antioxidant. This is due to the fact that cystine, once inside the cell, is rapidly reduced to cysteine, which is the 80  rate-limiting precursor in the synthesis of GSH. In general, cysteine is undetectable in normal culture medium since it is rapidly oxidized under normoxic conditions, and given that most cultured cells are unable to maintain appropriate levels of endogenous cysteine, the proper maintenance of GSH levels in cultured cells is critically dependent on the uptake of extracellular cystine (218). Thus, the primary function of system xc– is for the biosynthesis and intracellular maintenance of GSH, and consequently its expression is essential for survival at least for cells cultured in vitro. As a caveat, even though system xc– is expressed in almost all cultured mammalian cell lines, this may not reflect the native status of the cells. Even when cells do not display system xc– transporter activity in vivo, in many cases it is swiftly induced upon transfer into cell culture, indicative of the possibility that its expression may be an artifactual adaptation to circumvent cysteine deprivation in in vitro conditions (219-221). In contrast to somatic cells including fibroblasts, monocytes, endothelial cells, and macrophages that induce system xc– expression upon culture, cells such as lymphocytes and neutrophils are defective in system xc– activity even in vitro and require a reducing agent such as β-mercaptoethanol (β-ME) to be supplemented in regular culture media (221). In this case, the reducing agent reacts with cystine in the extracellular milieu to form a mixed disulfide, releasing cysteine, which can then be taken up by cells via neutral amino acid transporters including system ASC or LAT-2 (large amino acid transporter 2) (208).  A second important function of system xc– is the maintenance of a cystine/cysteine redox cycle. In this scenario, cystine is taken up into the cell by system xc– and reduced to cysteine, which in turn, alongside being used for GSH synthesis, is also released into the extracellular milieu via neutral amino acid transporters. There, 81  cysteine is rapidly oxidized to cystine and once again taken up by system xc–. This cystine/cysteine cycle reaches steady-state conditions and during cell culture, cysteine gradually becomes detectable in the culture medium and the cystine to cysteine ratio begins to resemble that of plasma (222). Evidence for this cystine/cysteine redox cycle maintaining a reduced microenvironment over the membrane was provided by xCT overexpression studies using Burkitt’s Lymphoma (BL) cells (223). BL cells display a limited uptake capacity for cystine and are thus highly susceptible to oxidative stress-induced cell death, a phenotype that is reversed upon stable overexpression of xCT. This finding was initially hypothesized to be attributable to the fact that higher cystine uptake mediated by system xc– would initiate an increase GSH biosynthesis. However, it was found that xCT overexpression and resultant cystine availability barely affected the intracellular GSH pool. In contrast, extracellular cysteine concentrations were boosted, and this was shown to be responsible for the rescuing effect (223). More strikingly, even in cells with knockout of γ-glutamylcysteine synthetase (γ-GCS), the enzyme responsible for GSH synthesis, stable expression of xCT and high intracellular and extracellular cysteine levels are sufficient to sustain survival in the absence of exogenous GSH or NAC (224, 225). As such, sustained system xc– activity may drive a highly efficient cystine/cysteine redox cycle that maintains a reducing extracellular environment, in some cases independently of GSH function.   System xc– activity has been demonstrated in numerous cultured cancer cell lines including hepatoma cells (226-228), lymphoma cells (165), glioma cells (163), as well as breast (168, 229), ovarian (230), lung (166), prostate (164), gastrointestinal (231), colon (232), and pancreatic (167) cancer cells. In fact, early evidence for a role of system xc– 82  in cancer progression came from the rat Nb2 lymphoma model, in which sublines that were clonally developed from the parent line had increasing number of chromosomal alterations and displayed various phenotypic property changes including circumvention of the requirement of growth supplements in culture media and increased metastatic ability (233). Strikingly, while the parental Nb2 cell line was critically dependent on cysteine supplementation in the culture medium, the emergence of system xc– expression in the Nb2-SFJCD1 subline resulted in augmented cystine uptake and growth autonomy (234). These studies provided headway for the therapeutic targeting of system xc– with the discovery that sulfasalazine, an anti-inflammatory drug, potently inhibited cystine uptake via system xc–(165). Since then, various studies have explored the use of cystine starvation or sulfasalazine treatment to arrest the proliferation of cancer cell lines or inhibit tumor growth (164, 166-168, 230, 235). Encouraged by the promising findings of these studies, a phase 1/2, prospective, double-blind randomized clinical trial was initiated to test the efficacy of sulfasalazine in the treatment of malignant gliomas. Unfortunately, this study had to be discontinued due to a lack of clinical responses as well as toxic side effects from treatment and it was concluded that sulfasalazine was not to be recommended for the routine treatment of cancer patients (169, 170). In light of this unsuccessful therapeutic approach, some progress has been made toward developing more potent analogs of sulfasalazine (236), as well as more potent small molecule compounds that specifically target system xc– (171, 173, 237).  More recently, there has been a resurgence of interest in targeting system xc– or xCT, with many studies elucidating novel mechanisms of how xCT can contribute to cancer progression and resistance. Ishimoto et al. showed that a variant of the adhesion 83  molecule CD44 (CD44v) contributes to ROS resistance of gastrointestinal cells by interacting with xCT and stabilizing it to the plasma membrane (231). Furthermore, treatment with sulfasalazine suppressed CD44-dependent tumor growth in a xenograft model and significantly enhanced the effect of the ROS-inducing anticancer drug cisplatin (231). A study by another group showed that while chronic lymphocytic leukemia (CLL) cells display a limited capacity to transport cystine for GSH synthesis due to a low expression level of xCT, neighbouring bone marrow stromal cells could effectively import cystine and then release cysteine into the microenvironment for uptake by CLL cells (238). They also showed that the protection of stromal cells on CLL cells by elevating GSH levels could essentially be abolished by blocking cystine uptake by stromal cells with sulfasalazine treatment (238). In another recent study by Timmerman and colleagues, it was found that a subgroup of triple-negative breast (TNBC) cancers that are dependent on glutamine supplementation are also critically dependent on xCT function, the inhibition of which reduces GSH levels, increases ROS and as a result attenuates the growth of TNBC tumors in addition to sensitizing TNBC cells to chemotherapy (229). Similarly, in estrogen-receptor-positive breast cancer cells, insulin-like growth factor 1 (IGF-1) activates the expression of xCT to promote cell proliferation and blocking xCT activity with sulfasalazine sensitizes cells to inhibitors of IGF-1 receptor (239). In further support of the notion that xCT plays a crucial role in tumor progression, a hallmark study by Jiang and others recently showed that p53 inhibits cystine uptake by acting as a transcriptional repressor of xCT expression, and thus sensitizes cells to ferroptosis, a non-apoptotic form of cell death, as a mode of tumor suppression (216). Consistent with this finding, they found that xCT is highly 84  expressed in various human tumors, while its overexpression inhibits ROS-induced cell death and mitigates p53-mediated tumor suppression of xenograft models.  In spite of the confluence of findings which illustrate a role for xCT in cancer, there is no available data describing oncogenic RAS as driving xCT expression to support tumor progression. Furthermore, there is a dearth of information whether the upregulation of xCT is an adaptive process to a tumor microenvironment characterized by high levels of oxidative stress, or whether an early and intrinsic transformation-associated mechanism drives its expression. I have shown that oncogenic transformation with KRASV12 upregulates the expression of xCT to mitigate ROS levels and thus to an extent show that xCT activation in cancer cells is not an adaptive process but rather an early event in oncogenesis. Motivated by the findings of Jiang and colleagues which suggest that p53-mediated repression of xCT is a tumor suppressive mechanism, in this chapter I seek to show that oncogenic KRAS conversely promotes the early activation of xCT to support cellular transformation (216). Indeed, I provide novel evidence that the upregulation of xCT is necessary for transformation and show through in vitro anchorage-independence assays and tumor xenograft models that the inhibition of xCT not only abrogates malignant transformation but also in vivo tumor growth. Moreover, I lend further support to these findings by extracting information from publicly available clinical datasets to show that xCT expression is strongly associated with mutant-activated KRAS expression and poorer clinical outcomes in patients.       85  3.2 Methods  Cell culture and H2O2 treatment NIH 3T3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) supplemented with 10% bovine calf serum and propagated by passaging 1:5 when plates reached 80-90% confluence. xCT-/- (KO) and xCT+/+ (WT) mouse embryonic fibroblasts (MEFs) were routinely cultured in DMEM supplemented with 10% FBS.  xCT-/- MEFs were also supplemented with 50µM of 2-mercaptoethanol (Sigma-Aldrich). H460 cell line was maintained in RPMI 1640 media supplemented with 10% FBS. H2O2 treatment was performed as described in Section 2.2 of Chapter 2.  Compounds Erastin was purchased from Tocris Bioscience. N-acetylcysteine (NAC) was purchased from Sigma-Aldrich.  Soft agar colony formation assays Cells were plated in 6-well plates at 8,000 cells per well in DMEM, 10% bovine calf serum in a top layer of 0.25% agar added over a base layer of 0.4% agar in DMEM and 10% bovine calf serum. Cells were fed every other day with 2-4 drops of corresponding medium onto the top layer and colonies were allowed to form over the course of 2-3 weeks. Where indicated, the antioxidant N-acetyl cysteine (Sigma-Aldrich) was added to cultures at a concentration of 0.5mM to inhibit reactive oxygen species. After 2-3 weeks 86  at 37°C, colonies were counted using ImageQuant LAS4000 and ImageQuantTL software (GE) as per manufacturer’s guidelines. All assays were performed in triplicate.  siRNA transfections Cells were transfected at ~30% confluency in 6-well plates with 25nM control siRNA (1:1 mix of non-targeting siRNA (Stealth RNAi negative control duplexes, medium GC Duplex; Invitrogen) and luciferase targeting siRNAs (Stealth RNAi siRNA Luciferase Reporter Control; Invitrogen)) or with 25nM of specific single-targeting siRNAs using the Lipofectamine RNAiMAX transfection reagent (Invitrogen). Unless otherwise indicated, cells were replenished with new media after 24 hours and harvested at 72 hours post-transfection.  shRNA lentiviral transfections xCT-targeting shRNA lentiviral vectors and control vector were purchased from GeneCopoeia. shRNA lentiviral transfections were performed as described in Section 2.2 of Chapter 2.  Tumorigenicity assay Aliquots of 0.5 x 106 cells were resuspended in 200 µL of PBS and injected subcutaneously into the flanks of 5-6 week old female Nu/Nu immunodeficient mice using standard procedures. Starting from day 13 after injection, mice euthanasia was required when tumors exceeded humane practice guidelines (500 mm3). Mice were evaluated for tumor growth every two days until the experimental endpoints. Tumor 87  were measured with a caliper and volumes were estimated using the following formula: tumor length x (tumor width)2x π/6 mm3. All animal experiment underwent ethical approval from the Animal Care Committee of the University of British Columbia (A16-0050; A16-0050-A001).  Histopathology, immunohistochemistry, and tissue microarrays (TMAs) For histopathology of mouse tissues, samples were formalin-fixed for 48  h, paraffin embedded, and sectioned at 5  µm. For each sample, sections were stained using standard hematoxylin and eosin (H&E) protocols. Extent of necrosis was manually quantified using Adobe Photoshop on 5 pictures (× 10 magnification, total surface: 1.61  mm2) randomly acquired on each HE section. Areas of structurally damaged tissue with no viable tumor cell nuclei were considered necrotic. For TMAs, four 1  mm diameter cores for each sample were punched from the paraffin block in non-necrotic areas and used for a TMA construction. The recipient block was subsequently sectioned at 5  µm thickness and IHC was performed using the following antibodies. IHC was quantified by for staining intensity (0–3) and percentage of positive cells (0–100%). For each sample, the H-score was calculated as staining intensity × percentage of positive cells. When cores derived from the same sample showed a different H-score, the highest score was considered for the analysis.   88  DHE measurements in tumor tissue To measure reactive oxygen species (ROS) in mouse tissues, snap-frozen organs were cut into 10 µm sections on a cryostat at −20 °C, washed with ice-cold buffer, mounted on precooled cover slides with DAKO mounting medium with DAPI, and stained with 10 mM dihydroethidium (DHE) for 30 min. Representative sections from independent tissues were imaged with a Zeiss Axioplan2 microscope and analyzed using ImageJ mean density calculation. Bioinformatics analyses expression profiling log2 transformed xCT mRNA data from GSE39582 was accessed using R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). log2 transformed xCT mRNA data from GSE72094 was IRON normalized and subjected to Wilcoxon rank test (240). Spearman correlation was computed on RSEM gene normalized data obtained from FireBrowse (Firehose), for LUAD, LUSC and COAD cohorts. Next I defined xCT associated genes, for every cohort, as those having a correlation index higher than 99 percentile. I then computed hypergeometric test of the xCT associated genes for the oncogenic signatures set (MSigDB release v6.0). Only three signatures showed significance, after correcting for multiple testing (qvalue<0.05), in all three cohorts.  Kaplan-Meier analysis For lung cancer, gene expression data and overall survival information from GEO (Affymetrix microarrays only), EGA and TCGA were accessed using Kaplan Meier 89  Plotter Software (241). The data was automatically handled by a PostgreSQL server, which integrates gene expression and clinical data simultaneously. To analyze the prognostic value of xCT, the patient samples were split into two groups according to median expression. The hazard ratio with 95% confidence intervals and logrank P value are calculated. In addition, TCGA RSEM normalized gene expressions and clinical annotations were retrieved from Firebrowse. Kaplan-Meier curves were computed with survival package (Therneau T (2015). A Package for Survival Analysis in S. version 2.38) and log-rank test, in R software. Samples were split, based on xCT expression, in High (higher than third quartile) or Low (lower than third quartile).  Statistical analysis Student’s t-test was used to determine statistical significance unless otherwise state, with p < 0.05 considered significant.   3.3 Results  3.3.1 Oncogenic KRAS is unable to transform xCT deficient cells Modulation of ROS levels directly influence the intracellular redox state and thus can have a profound impact on critical physiological or oncogenic processes. Based on available published literature as well as the studies conducted in Chapter 2, I postulated that the upregulation of xCT by oncogenic KRAS is a process that supports cellular transformation and tumorigenicity by mitigating the deleterious effects of ROS. In order 90  to test this, I assessed the ability of oncogenic KRAS to transform mouse embryonic fibroblasts (MEFs) derived from xCT-/- (KO) mouse or ones derived from the xCT+/+ (WT) littermates using an in vitro soft agar colony formation assay. The soft agar colony formation assay is a well-established method that is considered to be one of the most stringent in vitro tests for malignant transformation in cells, and is based on the principle that transformed cells are able to grow independently of a solid surface, referred to as anchorage independence (242). I found that xCT KO MEFs stably expressing mutant-activated KRASV12 failed to form colonies in soft agar, while their WT counterparts could grow under such conditions (Fig. 16). Similarly, re-expression of xCT in the KO MEFs restored the ability of cells to grow under anchorage independent conditions. This suggests that xCT plays a crucial role in supporting anchorage-independent growth, an indicator of malignant transformation. Given the role of xCT in cystine uptake and GSH availability, I wondered if either the circumvention of cystine uptake using 2-mercaptoethanol (2ME) or the antioxidant NAC could rescue the ability of the KRASV12-expressing KO MEFs to proliferate under anchorage independence. As expected, supplementation with either of these compounds could rescue the ability of the xCT KO MEFs to form colonies in soft agar. These results suggest that oncogenic KRAS upregulates xCT to support cellular transformation.     91   Figure 16: Oncogenic KRAS is unable to transform xCT deficient MEFs. Soft agar colony formation assay as a measure of oncogenic transformation upon overexpression of KRASV12 in xCT+/+ (WT) and xCT-/- (KO) MEFs with or without 2-ME or NAC rescue, or rescue with xCT re-expression. Colony formation is shown as a percentage relative to WT control conditions. Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).  3.3.2 Genetic inhibition of xCT impairs transformation by oncogenic KRAS To corroborate my findings from the xCT KO MEFs, I further investigated the effect of xCT inhibition on malignant transformation as displayed through anchorage independence, using an RNAi approach. To this end, I generated stable 3T3 KRAS cell lines ectopically expressing either control scramble shRNA or two individual shRNAs against xCT and reduced xCT expression was confirmed both under ambient condition and following treatment with H2O2 (Fig. 17A). Stable knockdown of xCT reduced the ability of 3T3 KRAS cells to form colonies in soft agar by more than 70% (Fig. 17B). This phenotype was reversed if cells were supplemented once every two days with 92  media containing 5µM GSH. In addition, the ability to form colonies was also restored in one of the sh-xCT cell lines when supplemented with 5µM NAC in the agar, but not in the presence of BSO. Moreover, I showed that this observation was generalizable to a human lung cancer cell line that carries mutant-activated KRAS- H460, as cells that were transfected with two individual xCT-specific siRNAs exhibited reduction in soft agar colony formation by at least 60% (Fig. 17C). Consistent with 3T3 KRAS cells, addition of the antioxidant NAC also partially restored the ability of xCT knockdown cells to form colonies under anchorage independence, suggesting that the role of xCT in supporting malignant transformation could potentially be explained by its role in contributing cystine for GSH biosynthesis.  3.3.3 Pharmacological inhibition of xCT impairs cellular transformation by oncogenic KRAS As mentioned, it has recently been shown that Erastin is a highly potent and selective inhibitor of xCT (171). Accordingly, I asked whether pharmacological inhibition of xCT using sub-lethal doses of Erastin could prevent malignant transformation in oncogenic KRAS-expressing human cancer cell lines, as indicated using soft agar colony formation assays. Indeed, I found that treatment with 20µM of Erastin was sufficient to impair the proliferation of H460 over 96 hours, without inducing cell death. Importantly, the presence of Erastin in soft agar assays strongly inhibited the ability of H460 cells to form colonies (Fig. 18A). This observation was also recapitulated in 3T3 KRAS cells, which failed to grow under these anchorage independent conditions (Fig. 18B). Moreover, as previously observed, addition of NAC or overexpression of xCT was  93   Figure 17: RNA-mediated silencing of xCT reduces anchorage independent growth of mutant KRAS cells. A. Quantitative RT-PCR for xCT transcript to validate siRNA-mediated knockdown of xCT (n=3). B. Soft agar colony formation assay as a measure of oncogenic transformation on NIH3T3 KRASV12 cells transiently transfected with 25nM of control (scr) or xCT-directed siRNAs with or without GSH, NAC, or NAC and BSO supplementation (n=2). C. Soft agar colony formation assay on the NSCLC cell line H460 transiently transfected with 25nM of control (scr) or xCT-directed siRNAs, with or without NAC rescue (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005). 94  able to overcome the growth inhibitory effect of Erastin under these conditions, suggesting that this effect could be attributed to the loss of xCT activity. In line with my previous data, these findings provide further evidence that the inhibition of xCT reduces malignant transformation by oncogenic KRAS, as displayed by the inability of Erastin-treated cells to grow under anchorage independent conditions. In addition, my findings highlight the potential suitability of Erastin for clinical treatment of oncogenic KRAS-driven cancers.  3.3.4 Genetic inhibition of xCT impairs tumorigenicity of cells harboring oncogenic KRAS  Thus far I have demonstrated that the genetic or pharmacological inhibition of xCT impairs oncogenic KRAS-mediated malignant transformation in vitro, as displayed using an anchorage-independence assay. To determine the relevance of these findings in vivo, 3T3 KRAS cells expressing scrambled control or xCT-specific shRNA were subcutaneously implanted in nu/nu immunodeficient mice using standard procedures. As expected, tumor xenografts established from 3T3 KRAS cells expressing xCT-specific shRNA were dramatically impaired in their growth, as compared to their counterparts that were established from control scramble cells (Fig. 19A). Significantly, this was accompanied by dramatically improved survival of mice bearing tumors with xCT knockdown compared to control tumors (Fig. 19B).    IHC analysis on tumor sections showed no effects of xCT knockdown on apoptosis as measured by cleaved caspase-3 staining (Fig. 20A). Moreover, I observed that xCT knockdown tumors had quantitatively larger areas of necrosis than control 95   Figure 18: Erastin treatment reduces anchorage independent growth of mutant KRAS cells. Soft agar colony formation assay as a measure of oncogenic transformation on (A) H460 cells and (B) NIH3T3 KRASV12 cells treated with indicated concentrations of Erastin, with or without NAC rescue or rescue with xCT overexpression (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).  counterpart tumors (Fig. 20B). To ascertain whether xCT plays a role in regulating anti-oxidative stress response in vivo, I measured ROS via dihydroethidium (DHE) staining as well as antioxidant levels by quantitating tumor GSH levels. In line with my findings in vitro, xCT knockdown tumor tissue exhibited reduced GSH levels compared to control tumors (Fig. 21A). As shown in Figure 21B, I also observed an increase in DHE staining in one of the xCT knockdown tumors relative to control tumors. While DHE staining follows a similar trend in the second group of xCT knockdown tumors, the data did not reach significance due to variability in measurements (p=0.07). Altogether, these in vivo findings lend further support to my previous data, suggesting that xCT plays a role in supporting oncogenic KRAS-mediated tumor growth and that this is correlated with regulation of ROS and GSH synthesis.  96                        Figure 19: Expression of xCT-specific shRNAs reduces the tumorigenicity of 3T3 KRAS cells and prolongs survival of mice. A. Tumor xenograft volumes of NIH3T3 KRASV12 cells stably expressing control (scr) or xCT-specific shRNA as shown after subcutaneous implantation in nu/nu immunodeficient mice. Error bars indicate SEM for n=8. *p<0.0005 B. Percent overall survival for mice inoculated with NIH3T3 KRASV12 cells stably expressing control (scr) or xCT-specific shRNA. P values were calculated using two-way ANOVA test and found to be significant *p<0.0001.   97    Figure 20: shRNA-mediated knockdown of xCT induces significant necrosis in vivo. Quantitation of A. Cleaved caspase-3-positive cells by IHC and B. the percentage amount of necrosis on H&E sections from tumor xenografts established from NIH3T3 KRASV12 cells stably expressing control (scr) or xCT-specific shRNA. Error bars indicate SD for n=8 (*p < 0.05, **p < 0.01, and ***p < 0.005).     Figure 21: shRNA-mediated knockdown of xCT decreases GSH and increases DHE staining in vivo. A. Quantitation of A. GSH levels from tissue and B. DHE staining from cryosections of tumor xenografts established from NIH3T3 KRASV12 cells stably expressing control (scr) or xCT-specific shRNA. Error bars indicate SD for n=3 (*p < 0.05, **p < 0.01, and ***p < 0.005). 98  3.3.5 Increased expression of xCT occurs in clinical specimens of oncogenic KRAS-expressing tumors and is associated with poor prognosis To extend my findings to primary human tumors, I focused on lung and colon adenocarcinoma, as activating mutations of KRAS are common in these tumors, and because expression data were publicly available. Both colon (GSE39582) and lung (GSE72094) cancer datasets that were publicly available revealed that xCT expression was upregulated in the mutant KRAS-expressing subsets samples, relative to the wildtype KRAS-expressing samples (Fig. 22) (243, 244). In addition, to further establish the link between oncogenic KRAS and xCT expression, I wanted to ascertain that the genes positively correlated with xCT in lung and colon cancer showed significant enrichment for genes associated with oncogenic KRAS signature. To this end, I determined the top 1% genes positively co-expressed with xCT in lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and colorectal adenocarcinoma within “The Cancer Genome Atlas” (TCGA) and performed a hypergeometric test against a pre-defined oncogenic signatures gene set from the Broad Institute Molecular Signatures Database (MSigDB; C6 collection). Indeed, genes co-expressed with xCT in all three datasets showed significant enrichment for the “MEK_UP.V1_UP” signature, which represents genes upregulated following stable over-expression of constitutively active MEK (Fig. 23). This is congruent with the rest of my findings considering that MEK is a well-established downstream effector of KRAS signaling. This also sheds lights on the possibility that the upregulation of xCT downstream of oncogenic KRAS is primarily mediated by the canonical RAF/MEK/ERK signaling arm.  99   Figure 22: High xCT mRNA expression is correlated with the KRAS mutant subtype in lung cancer. A. log2 transformed xCT mRNA data from GSE39582, accessed using R2: Genomics Analysis and Visualization Platform, show significantly higher expression in KRAS mutated samples (KRAS Mut) of colon adenocarcinoma compared to wild type (KRAS WT). B. IRON normalized, log2 transformed xct mRNA data from GSE72094 show significantly higher expression in KRAS mutated samples of lung adenocarcinoma compared to wild type (Wilcoxon rank test).    Figure 23: Genes co-expressed with xCT in lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and colorectal adenocarcinoma (COAD) show enrichment for the “MEK_UP.V1_UP” signature. A hypergeometric test was performed on the top 1% of genes co-expressed with xCT in TCGA datasets against the MSigDB oncogenic signatures collection of gene sets. “MEK_UP.V1_UP” signature represents genes upregulated following stable over-expression of constitutively active MEK. The pvalue for each dataset is shown after correcting for multiple testing (qvalue<0.05), in all three cohorts. 100  Furthermore, high xCT expression was correlated with significantly decreased overall patient survival in lung adenocarcinoma (Fig.24A). This strongly indicates that therapies based on the inhibition of xCT may be effective in the treatment of this disease. Interestingly, I also discovered that xCT expression was a prognostic indicator for poorer overall survival in renal cell carcinoma, hepatocellular carcinoma, and acute myeloid leukemia (AML) (Fig. 24B,C,D). This could be attributed to fact that while oncogenic KRAS activation can upregulate xCT, the tumor suppressor p53 also represses xCT as a means for tumor suppression and the correlation between high xCT expression and poor overall survival may be generalizable to tumors in general that have inactivating mutations in p53 (216). I also attempted to correlate xCT expression with survival specifically in patient subsets with tumors that harbored activating mutations in KRAS and found that while some datasets supported my hypothesis, the data did not reach significance. This again could possibly be explained by the presence or absence of p53 inactivating mutations, indicating that further genetic stratification of the clinical data may be required to see the effect of xCT on survival within the subset of tumors with activating mutations in KRAS. Together, these data confirm that activating mutations in KRAS is correlated with high xCT expression. In addition, given the fact that high xCT expression is a prognostic indicator for poorer survival, the targeting of xCT in tumors expressing oncogenic KRAS presents a viable clinical strategy. 101   Figure 24: High xCT expression is correlated with significantly decreased overall survival in lung, AML, papillary renal cell carcinoma, and hepatocellular carcinoma. A. Gene expression and clinical annotations for lung cancer was accessed using Kaplan Meier Plotter Software (241) and samples were split based on median xCT expression. The p value was calculated based on log-rank test. TCGA RSEM normalized gene expressions and clinical annotations for B. AML, C. papillary renal cell carcinoma, and D. hepatocellular carcinoma were retrieved from Firebrowse. Kaplan-Meier curves were computed with survival package (Therneau T (2015). A Package for Survival Analysis in S. version 2.38) and log-rank test, in R software. Samples were split, based on xct expression, in High (higher than third quartile) or Low (lower than third quartile). 102  3.4 Discussion xCT serves a pivotal role as the primary transporter for the cellular uptake of cystine, the rate-limiting precursor for the biosynthesis and intracellular maintenance of GSH. As such, its expression is critical for survival at least for cells cultured in vitro. In Chapter 2, I demonstrated that oncogenic KRAS leads to a decrease in intracellular ROS and increase in GSH levels, and this is associated with an enhancement of xCT expression and activity, which protects transformed cells against oxidative stress. Prompted by these findings, in this chapter I asked whether the upregulation of xCT supports oncogenic KRAS-mediated malignant transformation through its role in oxidative stress response. The ability of cells to grow in soft agar is the gold standard for defining cellular transformation in vitro that has been routinely used for decades (242). Indeed, I showed that oncogenic KRAS failed to transform cells that are completely devoid of xCT expression, as displayed through the inability of these cells to grow under anchorage independent conditions. Furthermore, I demonstrated that genetic inhibition of xCT via an shRNA-mediated approach also impaired the ability of 3T3 KRAS and H460 cells to form colonies in such conditions. Although it is not technically feasible to quantitate cellular ROS and GSH in the context of soft agar assays as extraction of cells from agar would yield insufficient cell numbers for such assays and also introduce variability from processing (245), there is recurring evidence that growth under such conditions leads to high ROS levels and can be reversed with administration of antioxidants (246, 247). Notably, the reduction of growth of KRAS cells due to xCT inhibition could be reversed by the administration of GSH or NAC, but not NAC in the presence of BSO, signifying that the impairment of anchorage independence 103  could be attributed to the role of xCT in GCL-mediated GSH synthesis, possibly to decrease cellular ROS levels that may be deleterious for cellular transformation. To provide further evidence for the role of xCT in supporting in vivo tumor growth, I established tumor xenografts from 3T3 KRAS cells stably expressing xCT-specific shRNAs to assess their tumorigenicity in immunocompromised mice. Strikingly, the knockdown of xCT in 3T3 KRAS cells strongly impaired their ability to form tumors in mice, as compared to their counterparts that expressed control scramble shRNA. Moreover, consistent with my in vitro findings, xCT knockdown resulted in decreased GSH levels in vivo compared to control tumor tissue consistent with an increase in DHE staining as a readout for tumor ROS levels. This was also associated with larger areas of necrosis in xCT knockdown tumors relative to control counterpart tumors, suggesting that decreased GSH and higher ROS levels due to xCT knockdown may impair tumor growth due to necrosis. In addition, I found that higher xCT expression was associated with the mutant KRAS subtype of lung and colon adenocarcinoma, and that xCT expression strongly correlated with significantly decreased overall survival in patients, regardless of KRAS mutational status. Thus, I show for the first time that xCT is a potential downstream mediator of oncogenic KRAS-driven cellular transformation and tumorigenicity. These results are congruent on several levels with the findings of Jiang and colleagues, who showed that p53-mediated transcriptional repression of xCT is critical for ROS-induced ferroptosis and tumor suppression. Firstly, this may explain why xCT expression is correlated with poorer outcome regardless of KRAS mutational status- even in cancers with wildtype KRAS, p53 may be inactivated and therefore to a certain extent, xCT may 104  be upregulated and play a role in supporting tumor growth. Secondly, activating mutations in KRAS are often concomitant with the loss of p53. Given their opposing effects on xCT expression, this further alludes to the importance of xCT to support malignant transformation and tumorigenicity by reducing the deleterious effects of ROS. This model is also reminiscent of the opposing effects that KRAS and p53 have on NF-κB activity, with RAS activating this signaling network and p53 repressing it. Future studies into the relative contribution of KRAS activation and p53 inactivation towards the upregulation of xCT and whether their mutational status can predict sensitivity to xCT inhibition are warranted. However, based on the results of my studies and others, I speculate that the subtype of cancers that may be most amenable to therapeutic targeting of xCT are the ones with concomitant activating mutations in RAS and loss of p53.   Another interesting question arising from my studies is the role of xCT in vitro and in vivo. Loss-of-function studies in mice have shown that even though xCT is vitally important for the survival of cells in vitro due to its critical role in maintaining intracellular GSH levels and modulating cysteine/cysteine redox balance, it is dispensable in mammalian development. Namely, xCT-/- (KO) mice are healthy in appearance and fertile. The only difference compared to wildtype (WT) littermates was that xCT KO mice had higher levels of cystine concentration and lower levels of GSH levels in the plasma. The exact mechanism by which xCT KO mice compensate for the loss of xCT is still unclear. In these studies I found that absence of xCT prevented the growth of oncogenic KRAS-expressing cells under anchorage independent conditions, unless accompanied by supplementation with either the reducing agent 2-ME or the antioxidant 105  NAC. However, in contrast to the dispensability of xCT in a physiological context, I demonstrated that xCT was crucial for the growth of tumors. This is likely due to the fact that under malignancy-associated conditions, tumor cells potentially undergo high levels of metabolic and oxidative stress. With an intrinsic mechanism in place to upregulate xCT and thus increase the capacity to buffer ROS through GSH availability, oncogenic KRAS-transformed cells are conferred an ability to survive under these stressful conditions. However, I speculate that this may also render them more reliant on xCT, as compared to normal cells, and therefore more sensitive to xCT inhibition. Taken together, my data strongly suggest xCT as a potential candidate for therapeutic targeting in mutant KRAS tumors. 106  Chapter 4: Oncogenic KRAS-Mediated Upregulation of xCT Expression is Dependent on RAS-MAPK Signaling to ETS-1  4.1 Introduction The results obtained from the studies in Chapter 2 and 3 established that oncogenic KRAS induces xCT expression to enhance cellular antioxidant capacity as a process that supports transformation and tumorigenicity.  The expression of xCT is inducible by diethyl maleate (248), cystine deprivation(249), oxygen, ROS-inducers such as hydrogen peroxide and sodium arsenite (250), lipopolysaccharide (251), and oxidized low density lipoprotein (252), among various other stimuli. Several cis-acting transcriptional regulatory elements in the 5’ flanking region of the xCT gene have been described. One of these regulatory elements is the electrophile response element (EpRE) or Antioxidant Response Element (ARE) present in the promoter region of xCT. The consensus EpRE/ARE sequence has been extensively characterized and shown to be essential for the coordinated expression of many antioxidant/detoxification enzymes including NAD(P)H:quinine oxidoreductase (NQO1) and glutathione S-transferase (GST). In addition, this consensus binding sequence was demonstrated to be principally recognized by NRF2 following its induction by electrophilic agents such as diethyl maleate, hydroquinone, and arsenite. Specifically, Sasaki and colleagues showed using cells derived from NRF2-deficient mice that the induction of xCT by electrophilic agents is mediated by NRF2 (253). Furthermore, the overexpression of NRF2 in astrocytes 107  resulted in an increase in the expression of xCT alongside a coordinated upregulation of enzymes involved in glutathione biosynthesis (gamma-glutamylcysteine synthetase, glutathione synthase), utilization (GST, glutathione reductase), and export (multidrug resistance protein 1) (254). Under basal conditions, NRF2 is usually sequestered in the cytoplasm by the Kelch-like ECH-associated protein-1 (KEAP1). Upon oxidation of its cysteine residues, KEAP1 dissociates from NRF2, allowing NRF2 to translocate into the nucleus where it binds EpRE/ARE sequences leading to the transcriptional activation of antioxidant genes. Given the link between oncogenic KRAS and upregulation of NRF2, in this chapter I additionally explored the role of NRF2 in mediating the activation of xCT by oncogenic KRAS. In addition to the EpRE/ARE elements, the promoter region of xCT also contains two amino acid response elements (AARE) just downstream in the 3’ flanking region of the functional EpRE. The consensus AARE sequence is recognized by Activating Transcription Factor 4 (ATF4) belonging to the ATF/CREB (cyclic AMP-response element binding protein) family of basic region-leucine zipper (bZip) transcription factors.  The basic region of ATF4 confers its ability to bind DNA, and the leucine zipper domains allow ATF4 to form dimers with a number of proteins such as Jun, Fos and C/EBP proteins. One of the primary mechanisms by which ATF4 is regulated is through translational control by the eukaryotic initiation factor 2α (eIF2α). Under stress conditions, the phosphorylation of eIF2α results in the global suppression of protein translation, while it selectively activates the translation of particular mRNA, of which ATF4 is one. Notably, two of the known eIF2α kinases are protein kinase R-like kinase (PERK), which mediates the cellular response to endoplasmic reticulum stress and 108  oxidative stress, and general control nonderepressible-2 (GCN2), which mediates the cellular response to amino acid stress. As such, ATF4 plays a major role in the integrated response towards various cellular stressors. Through co-immunoprecipitation and mammalian two-hybrid assays, there have been several lines of evidence that ATF4 and NRF2 interact with each other and cooperatively upregulate the expression of heme oxygenase-1 (HO-1) as well as xCT (255, 256). Even though ATF4 and NRF2 have been established as transcriptional activators of xCT, literature points to the fact that transcription factors typically act in concert with other proteins to form complexes on DNA and there are thus possibly other transcription factors or enhancers that may bind to other regulatory elements within the xCT gene, or that may work in concert with ATF4/NRF2 heterodimers as a complete transcriptional complex. As described in the previous chapter, it was only recently discovered that p53 serves as a transcriptional repressor of xCT as a novel mechanism for tumor suppression (216). There is also recent evidence that the NF-E2-p45-related factor 1 (NRF1) can bind to the EpRE/ARE region and transcriptionally repress xCT expression (257). Thus, in this chapter I investigate the mechanisms that mediate the transcriptional upregulation of xCT downstream of oncogenic KRAS. While my previous transcriptome profile studies have focused on identifying candidate genes as downstream effectors of mutant KRAS-mediated oncogenic transformation, the current chapter studies involves the analysis of whole transcriptome data in a broader manner to elucidate the specific effector pathways and the transcription factors that contribute to the activation of xCT downstream of oncogenic KRAS signaling. Indeed, I present the RAF/MEK/ERK transcription factor ETS-1 as a novel regulator of xCT gene expression, 109  and provide strong evidence using luciferase reporter assays to show that ETS-1 and ATF4 interaction integrates RAS pathway signaling and ROS signaling to cooperatively induce xCT expression.   4.2 Methods  Cell culture, siRNA transfection, H2O2 treatment Cell culture and siRNA transfections were performed as described in Section 3.2 of Chapter 3.   ROS DCFDA and Cell Death assays ROS DCFDA and cell death assays were performed as described in Section 2.2 of Chapter 2.  Plasmids pSG-ETS-1 plasmid was a kind gift from Lawrence McIntosh (University of British Columbia, Vancouver, Canada). pRK-ATF4 plasmid was a gift from Yihong Ye (Addgene plasmid #26114). pxCT-pro-WT-Luc and pxCT-pro-mt2-Luc plasmids were kindly provided by Junsei Mimura (Hirosaki University, Hirosaki, Japan). To construct the pxCT-pro-mt-1-Luc plasmid, a GGàAA mutation was introduced into the ETS-1 consensus sequence (TGAGGAAGCT) of the pxCT-pro-WT-Luc plasmid using site-directed mutagenesis. This was carried out using the Express Mutagenesis service provided by GenScript (NJ, USA). 110  Compounds Akt inhibitor VII was from Calbiochem. PD184352 and NAC were from Sigma-Aldrich.  Gene Set Enrichment Analysis Gene Set Enrichment Analysis between experimental groups was performed as previously described in Section 2.2 of Chapter 2.  Western blot analysis Western blot analysis was performed as described in Section 2.2 of Chapter 2. ETS-1, ATF4, and HSC70 antibodies were from Santa Cruz. Phospho-AKT and Phospho-ERK antibodies were from Cell Signaling. Phospho-ETS-1 antibody was purchased from Abcam. GRB2 antibody was from BD Biosciences.  RNA isolation and quantitative RT-PCR RNA isolation and quantitative RT-PCR were performed as described in Section 2.2 of Chapter 2.  Luciferase reporter assays HEK293 cells were seeded in 12-well plates and transfected with luciferase reporter constructs 24 hours later. Cells were harvested 48 hours post-transfection and luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega). Briefly, Passive Lysis Buffer was added to each well and the plates were then placed on a shaker for 15 minutes. Equal amounts of lysate and Luciferase Assay 111  Reagent II (LARII) were mixed in a 96-well plate and Firefly luciferase luminescence was analyzed using MolecularDevices SpectraMax Microplate reader. Afterwards, the same amount of Stop and Glo reagent as LARII was added to each well and Renilla luciferase luminescence was analyzed with the same plate reader. All samples were performed in triplicate and the final luciferase quantification was formulated as the ratio of Firefly luciferase to Renilla luciferase luminescence.  Statistical analysis Student’s t-test was used to determine statistical significance unless otherwise state, with p < 0.05 considered significant.  4.3 Results  4.3.1 Gene Set Enrichment Analysis (GSEA) of transcriptomic data reveals that ETS-1 target genes are upregulated in oncogenic KRAS-transformed cells To elucidate the regulatory signaling pathway mediating the upregulation of xCT by oncogenic KRAS, I applied GSEA using a publicly available dataset of annotated microarray experiments (“Transcription Factor Targets” dataset, Broad Institute) to identify gene sets that were upregulated following oncogenic KRAS transformation. GSEA using this annotated dataset was analyzed against the transcriptome profiles of 3T3 KRAS cells versus 3T3 MSCV cells under ambient conditions, as previously described in Table 1. Several of the gene sets that were most enriched for in 3T3 KRAS cells were those containing binding sites for transcriptional repressors such as YY1 112  transcription factor and Nuclear Respiratory Factor 1 (NRF1) (Table 2). In addition, genes regulated by p300, a general transcriptional co-activator, were also among gene sets most highly enriched for. Notably, the transcription factor ETS-1 gene set was among this list of most significantly enriched gene sets in 3T3 KRAS cells, as compared to 3T3 MSCV cells (Figure 25). This was an obvious target to pursue as a potential regulator of xCT due to the fact that ETS-1 is a well-described effector of the RAS-MAPK pathway. More importantly, the 5’ flanking promoter region of the xCT gene contains two consensus binding sites for ETS-1 (GGAA/T).   4.3.2 ETS-1 transcription factor mediates oncogenic KRAS-dependent upregulation of xCT to modulate intracellular ROS levels Given the potential role of ETS-1 as a transcription factor which mediates the upregulation of xCT by oncogenic KRAS, I wished to test whether inhibition of ETS-1 would abrogate the induction of xCT following exogenous oxidative stress treatment of 3T3 KRAS cells. As shown in Figure 26A, 3T3 KRAS cells treated with control scrambled siRNA induced xCT expression at the mRNA level following H2O2 treatment. In contrast, cells treated with ETS-1-specific siRNAs exhibited significant reduction in the induction of xCT mRNA under these conditions. ETS-1 down regulation was confirmed by Western blot analysis (Fig. 26B). Further, I found that siRNA-mediated knockdown of ETS-1 sensitized 3T3 KRAS to exogenous oxidative stress-induced cell death, as measured by PI (Fig. 26C), caused an increase in intracellular ROS levels as measured by the general ROS indicator DCFDA (Fig. 26D), and that this could be reversed by supplementation with NAC or ectopic overexpression of xCT (Fig. 26E). 113   Table 2, Figure 25: Gene Set Enrichment Analysis (GSEA) of transcriptomic data reveals that ETS-1 target genes enriched for in 3T3 KRAS cells as compared to 3T3 MSCV cells. GSEA of expression profiles of NIH3T3 KRASV12 versus MSCV cells analyzed against expression profiles generated as described in Table 1. ES denotes the Enrichment Score, while the p-value denotes the significance of the ES and FDR denotes the False Discovery Rate.   114    115  Figure 26: ETS-1 mediates the upregulation of xCT by oncogenic KRAS. A. Quantitative RT-PCR for xCT transcript in NIH3T3 KRASV12 cells transfected with control (scr) or ETS-1-specific siRNAs followed by treatment with 200µM H2O2 for the indicated times at 72 hours following transfection. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to scr control conditions (n=3). B. Immunoblot analysis confirming siRNA-mediated knockdown of ETS-1. C. Propidium iodide staining quantification using flow cytometry on NIH3T3 KRASV12 cells that were transiently transfected as described in A., grown in complete media for 48 hours, then treated with H2O2 for 16 hours at indicated concentrations (n=3). D. DCFDA staining quantification using flow cytometry in NIH3T3 KRASV12 cells transfected as described in A., followed by treatment with 200µM H2O2 for the indicated times (n=3). E. DCFDA staining quantification performed as described in D. using individual ETS-1-specific siRNA, with and without rescue with NAC or xCT overexpression. Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).  4.3.3 MEK inhibition but not AKT inhibition ablates oncogenic KRAS-mediated upregulation of xCT ETS-1 is a downstream effector of RAS-MAPK (RAS/RAF/MEK/ERK) signaling. Specifically, activated ERK phosphorylates ETS-1, which results in its translocation to the nucleus and subsequent activation of its target genes.  Accordingly, I next asked whether inhibition of this signaling cascade would abrogate the upregulation of xCT by oncogenic KRAS, and if inhibition of the alternate PI3K-AKT canonical signaling pathway, would have the same effects. To this end, I use the MEK inhibitor PD184352 and the AKT inhibitor VIII in 3T3 KRAS cells treated with exogenous H2O2 over a 6-hour period. Similar to Figure 5A, exogenous oxidative stress stimulates the expression of xCT in these cells but when cells are pre-incubated with PD184352, the induction of xCT is abrogated, consistent with a decrease in the activation of ETS-1 through phosphorylation (Fig. 27A,B). In contrast, pre-incubation with Akt Inhibitor VIII failed to 116  have any effect on the induction of xCT (Fig. 27C), suggesting that that the mechanism for upregulation of xCT is mediated through the RAS-MAPK pathway and not the PI3K-AKT pathway, a finding which is consistent with the fact that ETS-1 is a transcriptional activator of xCT.  4.3.4 Ectopic overexpression of ETS-1 promotes upregulation of xCT I next asked whether ectopic overexpression of ETS-1 could promote the upregulation of xCT. To answer this question, I used two human colorectal cancer cell lines HCT116 and DLD-1, which harbor mutant-activated KRAS but are devoid of endogenous ETS-1 expression, and showed that ectopic expression of ETS-1 in these cell lines could enhance the upregulation of xCT mRNA levels under H2O2 treatment relative to control cells (Fig. 28A,B). Notably, in HCT116 xCT expression was upregulated following ETS-1 expression even under ambient conditions. These findings provide further evidence that ETS-1 enhances the induction of xCT expression downstream of oncogenic KRAS signaling. In addition, to investigate whether the upregulation of xCT gene expression by ETS-1 is mediated through the xCT gene promoter, I obtained a human xCT gene promoter-luciferase reporter construct and analyzed its responsiveness to ectopic expression of ETS-1 by measuring luciferase activity in HEK293 cells. As shown in Figure 28D, increasing concentrations of ETS-1 construct resulted in a dose-dependent response in luciferase activity of the xCT gene promoter-luciferase construct. These results suggest that ETS-1 modulates human xCT gene expression through the promoter region of xCT gene. 117   Figure 27: MEK inhibition but not AKT inhibition ablates oncogenic KRAS-mediated upregulation of xCT. A. Quantitative RT-PCR for xCT transcript in NIH3T3 KRASV12 cells pre-treated with vehicle (-) or 2µM of the MEK inhibitor PD184352 (+) for 1 hour followed by treatment with 200µM H2O2 for the indicated times. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to scr control conditions (n=3). B. Quantitative RT-PCR for xCT transcript in NIH3T3 KRASV12 cells pre-treated with either 2µM PD184352 or 2µM Akt Inhibitor VIII for 1 hour followed by treatment with 200µM H2O2 for the indicated times (n=3). C. Immunoblot analysis from NIH3T3 KRASV12 cell lysates described in B. Where shown, data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).     118  These findings suggest that ETS-1 can enhance the induction of xCT downstream of oncogenic KRAS signaling and I speculate that this may support malignant transformation, consistent with the role of xCT in combatting oxidative stress.  4.3.5 ETS-1 expression and activity are not induced by oxidative stress While I have shown that xCT is a transcriptional target of ETS-1, my previous data also demonstrates that oncogenic KRAS signaling increases xCT expression to a much stronger degree under oxidative stress. As such, I questioned whether ETS-1 expression or activity could be modulated by oxidative stress. To this end, I treated 3T3 KRAS cells with sub-lethal doses of H2O2 and examined expression of ETS-1 or phospho-ETS-1 (activated ETS-1) using Western blotting. As shown in Figure 29, neither ETS-1 expression or activity are increased following H2O2 treatment, although their levels are significantly higher in 3T3 KRAS cells, as compared to 3T3 MSCV cells. This alludes to the possibility that ETS-1 is cooperating with another transcription factor that is a sensor of oxidative stress, to upregulate xCT downstream of KRAS signaling.  4.3.6 Gene Set Enrichment Analysis (GSEA) reveals that ATF4 target genes are upregulated in oncogenic KRAS-transformed cells treated with exogenous ROS I have demonstrated so far that ETS-1 mediates the upregulation of xCT expression downstream of oncogenic KRAS signaling. Nonetheless, ETS-1 is not itself a sensor of ROS and its expression or activity is not increased following H2O2 treatment. Because xCT expression is induced not solely by signaling downstream of oncogenic KRAS, but also in response to oxidative stress conditions, I investigated the 119   Figure 28: Ectopic expression of ETS-1 promotes the upregulation of xCT. Quantitative RT-PCR for xCT transcript in HCT116 cells (A.) and DLD-1 cells (B.) with or without ectopic ETS-1 expression and under treatment with 200µM H2O2 for the indicated times. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to scr control conditions (n=3). C. Immunoblot analysis confirming expression of ETS-1 under ambient conditions or treatment with indicated concentrations of H2O2 in HCT116 and DLD-1 cells. D. Luciferase activity from HEK293 cells transiently expresing a human xCT gene promoter-luciferase reporter construct and increasing concentrations of ETS-1 plasmid, using the Dual-Luciferase Reporter Assay System (Promega) (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).  120   Figure 29: ETS-1 expression and activity are unchanged under H2O2 treatment. Immunoblot analysis showing activated ETS-1 (p-ETS-1), total ETS-1, and loading control (HSC70) in NIH 3T3 MSCV and KRASV12 cells following 200µM of H2O2 for the indicated times. The image is representative of three independent experiments.  involvement of other possible transcription factors that might increase xCT expression in response to oxidative stress. Again I performed GSEA using the publicly available dataset of annotated microarray experiments (“Transcription Factor Targets” dataset, Broad Institute) and limiting the analysis to 3T3 KRAS cells in an attempt to identify gene sets that were upregulated following exogenous H2O2 treatment. To this end, GSEA was analyzed against the transcriptome profiles of 3T3 KRAS cells treated with H2O2 versus 3T3 KRAS cells under ambient conditions. GSEA revealed that the gene sets that were enriched for are primarily those controlled by transcription factors in the cyclic-AMP response element binding protein/Activating Transcription Factors (CREB/ATF) family including CREB, ATF2, ATF3, and ATF4 (Table 3). Notably, genes containing a binding site for the transcription factor ATF4 were among the top ten list of 121  most significantly enriched gene sets (Fig. 30). This was an obvious candidate to pursue as the xCT promoter region contains several AARE binding sites and ATF4 is a known regulator of xCT expression. To confirm the induction of ATF4 by ROS, I probed ATF4 expression levels in 3T3 MSCV, 3T3 EN, and 3T3 KRAS cells treated with exogenous H2O2 using Western blot analysis. Based on Figure 31, ATF4 levels were not detectable under ambient conditions and subsequently induced in all three cell lines following H2O2 treatment, but KRASV12 and EN-transformed cells did not appear to show elevated levels of ATF4. Interestingly, in 3T3 KRAS and 3T3 EN cells, ATF4, even though significantly induced at 3 hours of exposure to H2O2, begins to exhibit a gradual reduction in expression levels over time relative to 3T3 MSCV. Moreover, as shown before, phosphorylated-ETS-1 levels (active ETS-1) were higher in 3T3 EN and KRAS cells as compared to 3T3 MSCV cells, consistent with levels of phosphorylated-ERK and phosphorylated-MEK. These data provide further evidence that ETS-1 is upregulated by RAS-MAPK signaling, and while ATF4 is not upregulated downstream of this pathway, its levels are induced by H2O2.  4.3.7 Genetic inhibition of ATF4 ablates oncogenic KRAS-mediated upregulation of xCT The findings above prompted me to ask whether inhibition of ATF4 in my system could ablate the ROS-dependent upregulation of xCT mediated by oncogenic KRAS. As shown in Figure 32A, 3T3 KRAS cells treated with control scrambled siRNA induced xCT expression at the mRNA level following H2O2 treatment. In contrast, cells treated 122   Table3, Figure 30: Gene Set Enrichment Analysis (GSEA) of transcriptomic data reveals that ATF4 target genes are enriched for in 3T3 KRAS cells treated with H2O2 relative to basal conditions. GSEA of expression profiles of NIH3T3 KRASV12 cells under 200µM versus cells under ambient control conditions analyzed against expression profiles generated as described in Table 1. ES denotes the Enrichment Score, while the p-value denotes the significance of the ES and FDR denotes the False Discovery Rate.         123    Figure 31: ATF4 expression is induced in NIH 3T3 MSCV, EN, and KRAS cells following H2O2 treatment, while ETS-1 activity and expression remain unchanged. Immunoblot analysis showing ATF4, activated ETS-1 (phospho-ETS-1), total ETS-1, phospho-ERK, phospho-MEK, and loading control (HSC70) in NIH 3T3 MSCV, EN, and KRASV12 cells following 200µM of H2O2 for the indicated times. The image is representative of three independent experiments.       124  with ATF-4-specific siRNAs exhibited significant reduction in the induction of xCT mRNA under these conditions. ATF4 down regulation was confirmed by Western blot analysis (Fig. 32B).  4.3.8 ETS-1 and ATF4 cooperatively enhance xCT expression As mentioned, ETS-1 itself is not a sensor of ROS and its levels are unchanged under H2O2 treatment. On the other hand, I uncovered ATF4 as a regulator of xCT in the 3T3 KRAS system, but only in response to H2O2 treatment. Therefore, I wondered whether the coordinated input of both ETS-1, which transmits signals from upstream oncogenic RAS, and ATF4, which is activated in response to oxidative stress, are both necessary to promote the upregulation of xCT expression. To answer this question, I first performed siRNA-mediated combined knockdown of both ETS-1 and ATF4 and probed xCT mRNA levels. As shown in Figure 33, ETS-1-specific siRNA and ATF4-specific siRNA, each individually abrogated the induction of xCT under H2O2 treatment, but combined knockdown of both ETS-1 and ATF4 was able to further decrease xCT mRNA levels. This suggests that ETS-1 and ATF4 may potentially cooperate to enhance xCT expression. Functionally, I also wondered if similar to inhibition of xCT, combined inhibition of ETS-1 and ATF4 could also suppress the transformative phenotype in oncogenic KRAS-expressing cells. Indeed, I was also able to demonstrate that combined inhibition of ETS-1 and ATF4 phenocopied the effect of xCT inhibition by suppressing the ability of 3T3 KRAS cells to form colonies under anchorage independent conditions. This effect was reversed with either overexpression of xCT in these cells, or supplementation with NAC (Figure 34).  125     Figure 32: ATF4-specific siRNA expression in 3T3 KRAS cells abrogates the induction of xCT following H2O2 treatment. Quantitative RT-PCR for xCT transcript in NIH3T3 KRASV12 cells transfected with control (scr) or ATF4-specific siRNAs followed by treatment with 200µM H2O2 for the indicated times at 72 hours following transfection. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to scr control conditions (n=3). B. Immunoblot analysis confirming siRNA-mediated knockdown of ATF4. Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).          126    Figure 33: Combined siRNA-mediated inhibition of ETS-1 and ATF4 has an additive effect on the reduction of xCT mRNA levels. Quantitative RT-PCR for xCT transcript in NIH3T3 KRASV12 cells transfected with control (scr) or ETS-1-specific siRNA alone or ATF4-specific siRNA alone, or both in combination, followed by treatment with 200µM H2O2 for the indicated times at 72 hours following transfection. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to scr control conditions (n=3).          127     Figure 34: Combined siRNA-mediated inhibition of ETS-1 and ATF4 reduces anchorage independent growth mediated by oncogenic KRAS. Soft agar colony formation assay as a measure of transformation on NIH3T3 KRASV12 cells transiently transfected with 25nM of control (scr) or ETS-1-specific siRNA or ATF4-specific siRNA, or both combined, with or without NAC rescue or rescue with xCT overexpression. Colony formation is shown as a percentage relative to scr control conditions. Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005). (n=3).      128  To further extend my findings, I also generated human xCT gene promoter-luciferase reporter constructs with a point mutation in the ETS-1 binding site of the promoter, or with point mutations in the the two ATF4-responsive AARE binding sites. Using these constructs, I analyzed the luciferase activity under co-expression with ETS-1 plasmid alone, ATF4 plasmid alone, or ETS-1 and ATF4 plasmid together. My results reveal that in control conditions, combined ETS-1 and ATF4 ectopic expression has a synergistic effect on the luciferase activity of full-length xCT promoter reporter construct (Fig. 35). Notably, mutations in either the binding site of ETS-1 or the AARE consensus sites dramatically reduced luciferase reporter activity. Collectively, these results suggest that ETS-1 and ATF4 synergistically enhance the expression of xCT by increasing promoter activity of the xCT gene.  4.3.9 NRF2 potentially cooperates with ETS-1 and ATF4 to enhance xCT expression There are several lines of evidence demonstrating that xCT is a target of NRF2. It has also been shown that ATF4 and NRF2 interact with each other and cooperatively upregulate the expression of heme oxygenase-1 (HO-1) and xCT (255, 256). To ascertain the involvement of NRF2 in the regulation of xCT in these models, I first examined expression levels of NRF2 in 3T3 MSCV, 3T3 EN, and 3T3 KRAS cells under ambient conditions, or treatment with a sub-lethal concentration of H2O2. As shown in Figure 36, stable expression of either EN or KRAS increases NRF2 expression, but similar to ETS-1, these levels remain unchanged even under exogenous ROS treatment. This is consistent with the findings of DeNicola et al., who showed that stable  129   Figure 35: ETS-1 and ATF4 synergistically promote the upregulation of xCT by activating the promoter region of the xCT gene. Luciferase activity from HEK293 cells transiently expresing a human xCT gene promoter-luciferase reporter construct (WT), the same construct with mutation in the ETS-1 binding site (mt1), or the same construct with mutations in the two AAREs (mt2), with transient expression of ETS-1 plasmid alone, ATF4 plasmid alone, or combined and using the Dual-Luciferase Reporter Assay System (Promega) (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).          130   Figure 36: NRF2 expression is induced following transformation with EN or oncogenic KRAS, but remains unchanged following H2O2 treatment. Immunoblot analysis showing NRF2 and loading control (HSC70) in NIH 3T3 MSCV, EN, and KRASV12 cells following 200µM of H2O2 for the indicated times. The image is representative of three independent experiments.  expression of oncogenic KRAS led to increased transcription of NRF2 (65). However, the levels of NRF2 under exogenous oxidative stress were not probed. In addition, given the fact that the NRF2 can be upregulated by JUN and MYC downstream of the RAS-MAPK pathway, I wondered if NRF2 was possibly downstream of ETS-1 signaling. Using ETS-1 specific siRNAs, I found that inhibition of ETS-1 did not alter the expression of NRF2, or one of its gene targets heme oxygenase 1 (HO-1), whether under ambient conditions or H2O2-treated conditions (Fig. 37A). This provides evidence that even though ETS-1 and NRF2 can both be regulated by RAS-MAPK signaling, the modulation of xCT by either of these transcription factors fall under distinct pathways. Whether ETS-1 can compensate for NRF2-loss in the regulation of xCT and vice versa remains to be determined and warrants further study. Furthermore, ATF4-specific siRNA-mediated knockdown failed to alter NRF2 or HO-1 expression, whether under ambient conditions, or exogenous ROS, suggesting that in these model systems ATF4 also does not regulate NRF2 (Fig 37B). As mentioned, several studies have reported 131  ATF4 as a heterodimerization partner of NRF2, while a more recent study by DeNicola and colleagues in non-small cell lung cancer described the ability of NRF2 to control the expression of serine/glycine biosynthetic enzyme genes, which included ATF4 (255, 256, 258). In my data, there was no evidence to support these claims, as ATF4 expression was only upregulated under H2O2 induction and not under stable expression of oncogenic KRAS (Figure 31) in contrast to NRF2 levels which were augmented in transformed cells. To further investigate the possibility that the expression of ATF4 is downstream of NRF2 regulation, siRNA-mediated knockdown of NRF2 was employed in conjunction with lentiviral-mediated ectopic overexpression of ATF4 and expression levels of these proteins in 3T3 KRAS cells were probed in addition to xCT transcript (Figure 38). As expected, NRF2-specific siRNA expression resulted in a decrease of xCT transcript levels under ambient condition, and although xCT was induced following exposure to H2O2, this induction was also subsequently decreased with NRF2 knockdown. Notably, ATF4 expression levels were unchanged following NRF2 knockdown, whether under ambient condition or following exposure to H2O2 (Fig. 38B, long exposure of ATF4). Further, ATF4 overexpression, while able to partially rescue xCT expression following NRF2 knockdown, also markedly enhanced the induction of xCT at ambient conditions and under H2O2 in the absence of NRF2 knockdown. Altogether, these findings confirm the role of ATF4 in enhancing xCT expression and suggest that while ATF4 and NRF2 can both regulate xCT expression, the expression of ATF4 is not controlled downstream of NRF2. Moreover, while the knockdown of either ATF4 or NRF2 can decrease xCT expression, the fact that ATF4 overexpression  132   Figure 37: ETS-1 or ATF4-specific siRNA expression in 3T3 KRAS cells does not alter NRF2 or HO-1 expression. Immunoblot analysis showing expression of NRF2, HO-1, and loading control (HSC70) in NIH 3T3 KRASV12 cells transfected with control (scr) or ETS-1-specific siRNAs (A) or ATF4-specific siRNAs (B) followed by treatment with 200µM H2O2 for the indicated times at 72 hours following transfection. ETS-1 (A) and ATF4 (B) expression levels are also shown as validation of the knockdown. The image is representative of three independent experiments.      133   Figure 38: NRF2-specific siRNA expression in 3T3 KRAS cells does not alter ETS-1 or ATF4 expression, but decreases the induction of xCT expression by ATF4 overexpression or exposure to H2O2. A. Immunoblot analysis showing expression of NRF2, ETS-1, ATF4, and loading control (Actin) in NIH 3T3 KRASV12 cells with or without lentiviral-mediated ATF4 overexpression and transfected with control (scr) or NRF2-specific siRNAs followed by treatment with 200µM H2O2 for the indicated times at 72 hours following transfection. B. Quantitative RT-PCR for xCT transcript in NIH3T3 KRASV12 cells subjected to the same conditions as in A. Transcript levels were normalized to beta-actin (actb) and results are expressed as relative to scr control conditions (n=2).  134  partially rescues xCT expression following NRF2 knockdown suggests that while they fall under distinct signaling pathways, they possibly interact on the xCT promoter region and may enhance each other’s activation of the xCT promoter. To ascertain this, I analyzed the luciferase activity of the previously described xCT-promoter luciferase construct under co-expression with ETS-1 plasmid alone, ATF4 plasmid alone, NRF2 plasmid alone, or each of the two, or all three together. As shown before, ETS1 and ATF4 co-expression dramatically increased luciferase reporter activity, as compared to expression of each of those plasmids alone. However, either ETS-1 and NRF2 co-expression, or ATF4 and NRF2 co-expression resulted in only marginal increases in luciferase reporter activity, as compared to expression of each of those plasmids alone (Fig. 39). On the other hand, expression of all three plasmids together could amplify to an extent the luciferase reporter activity generated from ETS-1 and ATF4 co-expression, highlighting the ability of NRF2 to provide an additive effect on the synergistic regulation of xCT by ETS-1 and ATF4. Thus, these studies provide evidence that ETS-1 and ATF4 synergize to enhance the promoter activity of xCT, and NRF2 can contribute an additive effect to this regulation, possibly constituting part of a larger transcriptional complex, that may also include other transcription factors.  4.4 Discussion In this chapter I sought to delineate a mechanism by which oncogenic KRAS promotes the upregulation of xCT. To this end, I developed an unbiased approach and applied gene set enrichment analysis to whole transcriptome data. By analyzing whole  135   Figure 39: ETS-1 and ATF4 synergistically promote the upregulation of xCT, while NRF2 provides an additive contribution to this upregulation. Luciferase activity from HEK293 cells transiently expresing a human xCT gene promoter-luciferase reporter construct with transient expression of ETS-1 plasmid alone, ATF4 plasmid alone, NRF2 plasmid alone, two of each combined, or all three combined and using the Dual-Luciferase Reporter Assay System (Promega) (n=3). Data are reported as means ± SD with indicated significance (*p < 0.05, **p < 0.01, and ***p < 0.005).  sets of transcription factor targets that are enriched in 3T3 KRAS cells as compared to non-transformed 3T3 MSCV cells, I uncovered ETS-1 as a potential transcription factor  that mediates the upregulation of xCT by oncogenic KRAS. Through siRNA-mediated silencing, ectopic overexpression studies, and luciferase promoter activation assays, I provide compelling evidence that ETS-1 is a novel regulator of xCT, downstream of RAS-MAPK signaling. These studies have additionally demonstrated that xCT 136  expression is enhanced not just by KRAS transformation alone but also in response to exogenous oxidative stress. Given the fact that ETS-1 is not a sensor of ROS and its activity or expression remains unchanged in response to oxidative stress, I decided to perform a separate gene set enrichment analysis based on the hypothesis that other ROS-responsive transcription factors may be acting in concert with ETS-1. By analyzing transcription factor targets enriched for in 3T3 KRAS cells treated with H2O2 as compared to under ambient conditions, ATF4 was revealed to be another potential regulator of xCT. The role of ATF4 in activating xCT expression is known, and this was confirmed by quantifying xCT transcript levels following siRNA-mediated knockdown of ATF4. Interestingly, ATF4 levels were not detectable under ambient conditions but only induced in 3T3 cells following H2O2 treatment, and KRASV12 and EN-transformed cells did not appear to show elevated levels of ATF4 relative to MSCV cells. This led me to speculate that while ETS-1 enhanced the induction of xCT downstream of RAS signaling, ATF4 could explain the induction of xCT in response to oxidative stress, and the two transcription factors potentially regulate the xCT promoter in a co-operative manner. Accordingly, I was able to demonstrate using luciferase reporter activation assays that ETS-1 and ATF4 synergized to activate the xCT promoter. Counter-intuitively, in 3T3 KRAS and 3T3 EN cells, ATF4, even though significantly induced at 3 hours of exposure to H2O2, began to display a gradual reduction in expression levels over time relative to 3T3 MSCV. While I did not probe the dynamics of xCT expression levels beyond 6 hours of exposure to oxidative stress, this observation may not necessarily mean that a reduction in xCT expression would follow. Rather, because ATF4 co-operates with ETS-1 to activate the xCT promoter, it is conceivable that upon 137  exposure to oxidative stress, ATF4 expression is activated, where it translocates to the nucleus and interacts with ETS-1 on the xCT promoter region. It is widely appreciated that the ETS-domain family of transcription factors interact with a multitude of co-regulatory partners (259). These protein-protein interactions have important regulatory consequences for protein-DNA interactions of the dimerization partners, including conformational changes or the blocking of specific autoinhibitory sites allowing each transcription factors to mutually increase their DNA binding activities. For instance, ETS-1 stimulates DNA binding activity of AML-1 by binding to its NRDB domain (negative regulatory domain for DNA binding) (260, 261). I speculate that in a similar fashion, ETS-1 could possibly enhance DNA-binding to ATF4, thereby synergistically acting on the xCT promoter, and sustaining its activation, even while ATF4 expression levels gradually decrease due to possible feedback inhibition from the relieving of initial oxidative stress stimuli. While this is speculative at this point in time, it provides an avenue for future studies, which I will elaborate in the Conclusion chapter.  Finally, I also explored the contribution of NRF2 to ETS-1 and ATF4-mediated activation of xCT in my model system, given that previous literature has demonstrated xCT to be a target gene of NRF2, and ATF4 has been reported as both a direct transcriptional target and heterodimerization partner of NRF2. Evidence from my studies suggests that while NRF2 can enhance xCT expression, it does not regulate ETS-1 or ATF4 expression. In addition, based on luciferase activation studies, it appears that NRF2 can have an additive effect towards the synergistic activation of xCT promoter by ETS-1 and ATF4. My findings also do not exclude the possibility that ETS-1, NRF2, and ATF4 are all part a larger regulatory transcriptional complex.  138  Based on my data I propose a model for the induction of xCT downstream of oncogenic KRAS and in response to oxidative stress, which comprises of two signaling arms. The first arm involves the role of ETS-1, which transmits oncogenic KRAS activation signals from the RAF/MEK/ERK cascade. The other is the ROS-responsive arm in which ATF4 is activated in response to oxidative stress. Evidence from these studies suggests that ETS-1 and ATF4 synergistically induce the expression of xCT by activating the promoter region of the xCT gene. Previous studies have demonstrated that the ETS family of proteins can interact and work cooperatively with the bZIP family of transcription factors, but the exact nature of the protein-protein interaction between ETS-1 with ATF4 warrants further investigation. Notably, my work proposes a novel model in which signals from environmental stressors (in this case ATF4 activity in response to oxidative stress) can directly enhance the downstream effects of oncogenic signaling (ETS-1 signaling downstream of KRAS).   139  Chapter 5: Conclusion and Future Directions  The overall aim of this thesis was to characterize the impact of oncogenic KRAS on redox homeostasis and its contribution to malignant transformation and tumorigenesis, with the hopes of presenting a novel strategy for targeting redox-based vulnerabilities within this genetic subset of cancers. The research was divided into three main sections.  In the first section (Chapter 2), the effect of oncogenic KRAS expression on intracellular ROS and the response to oxidative stress was first investigated in a simple model of NIH 3T3 fibroblast cell line, which has no mutational background in contrast to established mouse or human cancer cell lines. Findings from the studies in Chapter 2 demonstrated that transformation with oncogenic KRAS led to a decrease in intracellular ROS levels, which corresponded with an increase in GSH levels, and conferred protection of cells against oxidative stress-induced cell death. Following this, I was interested in the mechanism underlying these observations and utilized a hypothesis-generating approach in the form of whole transcriptome microarray analysis to obtain a global view of genes that were activated or repressed following transformation with oncogenic KRAS and in response to oxidative stress. This approach enabled me to describe for the first time, that transformation with oncogenic KRAS leads to the upregulation of the light-chain subunit of the cystine/glutamate transporter, xCT. This finding was supported by the observation that oncogenic KRAS-transformed cells also showed higher xCT activity relative to control cells, as indicated using the xCT-specific FASu PET tracer. In addition, the association between mutant-activated 140  KRAS and enhanced xCT expression was recapitulated in several human cancer cell lines. These findings are in congruence with several recent studies that have demonstrated that oncogenic transformation leads to a reduction in intracellular ROS due to an upregulation of metabolic pathways that support the biosynthesis of antioxidants (64, 65). Paradoxically, high ROS levels and oxidative stress also constitute a well-recognized phenomenon in tumor cells, and are believed to be drivers of oncogenesis. Thus, one of the most intriguing but unanswered questions arising from my studies and studies of redox balance in cancer in general, is whether malignant transformation leads to an overall decrease or increase of intracellular ROS levels, and which of these two processes are essential to promote transformation and tumorigenicity. While these studies lend support to the former, it is important to recognize the complexities surrounding the process of cellular transformation as well as the dynamic nature of ROS which is difficult to capture from in vitro or in vivo studies. Therefore, I speculate that in reality, both ROS-inducing and ROS-clearing mechanisms may be employed by the cancer cell as it progresses from the initial step of transformation towards late-stages of tumor progression, and the disruption of either of these will disrupt the fine-tuning of redox balance leading to cancer cell death and tumor regression.  Indeed, in my studies, I found that genetic inhibition of xCT using knockout cell lines or siRNA-mediated knockdown of xCT rendered oncogenic KRAS-transformed cells susceptible to oxidative stress-induced cell death, which correlated with an increase in intracellular ROS levels and decrease in reduced GSH levels. Notably, administration of the antioxidant NAC could consistently restore GSH levels, but not in 141  the presence of BSO, which inhibits GSH production through GCL. Thus, these observations suggest that oncogenic KRAS enhances the induction of xCT to support the protection of cells against oxidative stress by upregulating cystine uptake, leading to enhanced GSH synthesis. Studies in the Chapter 3 were mainly driven by the question of whether the enhancement of xCT expression supports oncogenic KRAS-mediated cellular transformation and tumor growth. To this end, I employed the use of the soft agar colony formation assay, an in vitro assay routinely used to ascertain anchorage-independence, which is a defining phenotype of transformed cells (242). I discovered that genetic and pharmacological inhibition of xCT impaired growth of KRAS-expressing cells under anchorage independent conditions, which could be restored upon administration of GSH or NAC, but not NAC in the presence of BSO. Further, to corroborate my findings in vivo, tumor xenografts were established from mutant KRAS cells expressing xCT-specific shRNA. Notably, xCT knockdown impaired the growth of tumor xenografts, providing compelling evidence for the role of xCT in supporting KRAS-driven oncogenesis. In addition, immunohistochemical probing in tumour samples revealed that xCT knockdown tumors displayed extensive necrosis compared to control counterparts, which was associated with decreased GSH levels and increase ROS. Moreover, I obtained strong clinical evidence that oncogenic KRAS signaling was correlated with higher expression of xCT and that in general, xCT was a prognostic indicator for poorer overall survival in patients. These findings provide an interesting avenue for future work. Firstly, it would be interesting to test the utility of pharmacological xCT inhibitors, such as Erastin and 142  Sulfasalazine, in tumors with mutant-activated RAS. In my studies, I found that genetic inhibition of xCT dramatically impaired growth of xenograft tumors established from oncogenic KRAS-expressing NIH 3T3 cells. However, the use of xenograft models may not faithfully recapitulate various aspects corresponding to human disease, and genetically engineered mouse models specific to KRAS may serve as better predictors for clinical outcome. Therefore, a future direction from my studies may involve the use of a non-small cell lung cancer (NSCLC) mouse model - KrasLSL-G12D; p53frt/frt mice for instance, to test the therapeutic response to Erastin either alone or in combination with standard-of-care chemotherapeutics (carboplatin for NSCLC) (44). This will involve the establishment of maximum tolerated doses of Erastin, in vivo stability as well as other pharmacokinetic parameters including any alterations in drug metabolism due to drug-drug interactions. In addition, although Erastin was discovered through a screen as a result of its ability to cause selective lethality in cells overexpressing HRASG12V, there was no consistent difference between wild-type and mutated RAS cells (172). This may be explained by the ability of ectopic KrasG12D, but not of endogenous KrasG12D, to induce Nox transcription and NOX activity, which promotes overall ROS elevation (65). As such, it would also be interesting to test the efficacy of Erastin in sensitizing mutant RAS tumors towards oxidative stress inducers (262). A second interesting direction for future work focuses on the potential of xCT in PET applications toward diagnosis, prognosis and subsequent management, as well as monitoring response to therapy and recurrence. Alongside tumor glucose metabolism, amino acid metabolism is usually enhanced compared to normal tissue and in recent years, radiolabeled amino acids are gaining attention as an important class of PET 143  tracers. Currently there are several 18F-labelled amino acids that specifically target the system xc– transporter, such as 18F-FSPG, and 18F-Asu (FASu), which was used in my studies (263). Given that some evidence points towards the induction of xCT being an early event in RAS transformation and tumorigenesis, an xCT-targeted PET tracer such as FASu may have higher sensitivity in the detection of smaller lesions and also in specificity for the evaluation of tumors post-treatment (264). Given the literature supporting the role of xCT in cancer metastasis, xCT-targeted PET tracers may also improve detection of nodal and distant metastases, allowing better staging, and subsequent treatment planning (265). Further, if targeted inhibition of system xc– in RAS tumors prove efficacious, companion diagnostic xCT-targeted PET tracers may allow stratification of patients who will respond to treatment. Currently, the clinical use of glucose-based PET tracer 18F-FDG in intracranial malignancies is limited due to high uptake in normal brain (266). Hence, the use of FASu in the diagnosis, staging, and monitoring treatment response of these malignancies may circumvent current limitations of 18F-FDG, particularly in tumors with alterations in EGFR and NF1, which represent gliomas with active RAS signaling (267). Mechanistically, one of the most intriguing questions arising from my studies is how oncogenic KRAS transcriptionally upregulates xCT as a means to combat oxidative stress. Studies in the Chapter 4 sought to delineate this mechanism. Accordingly, I successfully defined ETS-1 to be a novel transcriptional regulator of xCT downstream of the RAS/RAF/MEK/ERK signaling cascade and showed that ETS-1 synergizes with ATF4, a known regulator of xCT that is specifically activated in response to oxidative stress. Specifically, genetic inhibition of ETS-1 or ATF4 abrogated the enhancement of 144  xCT induction in oncogenic KRAS-expressing cells following exogenous ROS treatments. Notably, combined inhibition of both transcription factors disrupted the ability of oncogenic KRAS-transformed cells to grow under anchorage independence conditions, an observation that could be partially reversed with overexpression of xCT. Additionally, I tested the role of NRF2 in the ETS-1 and ATF4 mediated induction of xCT and found that while it may contribute in an additive manner to the activation of the xCT gene promoter, NRF2 does not regulate ETS-1 or ATF4. Overall, these studies demonstrate that signals from environmental stressors (oxidative stress signaling) can amplify the signals downstream of oncogenic KRAS due to the synergistic interaction between transcription factors on the promoter region of a downstream effector, in this case, xCT. The proposed model for oncogenic KRAS-mediated transcriptional upregulation of xCT is illustrated in Figure 40.  A future direction from this work is first to demonstrate that ETS-1 indeed binds to its consensus sequence on the xCT promoter. This can be accomplished through a chromatin immunoprecipitation assay (ChIP). Upon optimization of this assay, it would be interesting to test whether siRNA-mediated knockdown of ATF4 would decrease the binding of ETS-1 to the promoter region of xCT, and vice versa. Second, it would be interesting to characterize the specific synergistic effect between ETS-1 and ATF4, if they interact directly or constitute a larger transcriptional complex. To this end, I will generate ETS-1 and ATF4 truncation mutant constructs and apply them in the context of co-immunoprecipitation assays to identify which domains are necessary for their interaction. These can be carried out in parallel with luciferase assays to obtain an output of xCT promoter activation. Also, these truncation constructs can also be 145  overexpressed in cells subjected to ChIP analysis to ascertain whether particular functional domains in ETS-1 and ATF4 can enhance the other’s protein-DNA interactions. In addition, besides ETS-1 and ATF4, GSEA analyses in Chapter 4 uncovered several other potential transcriptional regulators of xCT, such as CREB, CEBPdelta, AREB6, and MIF1, which may provide interesting avenues for future work. Taken together, this thesis established the regulation of a key aspect of cancer cell redox balance by oncogenic KRAS signaling. Namely, these results describe that oncogenic KRAS enhances the transcriptional upregulation of xCT as a means to support cellular transformation and tumorigenicity. Moreover, I provided evidence for a novel mechanism involving the cooperative action of ETS-1 downstream of the RAS-MAPK pathway, and ATF4 on the xCT promoter. Given that the therapeutic targeting of oncogenic RAS-driven cancers remains a significant challenge in the clinic, this thesis has contributed to the cancer cell redox field owing to the identification of a novel downstream effector of oncogenic KRAS. In addition, this thesis further solidifies the proposition that targeting the redox-based vulnerabilities of oncogenic RAS-driven cancers (in this case, xCT) rather than the oncogene itself, could hold potential as therapeutic strategy that will lead to better clinical outcomes in patients.       146   Figure 40: Oncogenic KRAS transcriptionally upregulates xCT via cooperative action of ETS-1 and ATF4 to promote transformation and tumorigenicity by preventing ROS overload. Schematic diagram to demonstrate that KRASV12 activates ETS-1 through RAS/RAF/MEK/ERK signaling.  ATF4 is induced by ROS and synergizes with ETS1 on the xCT promoter, amplifying RAS-MAPK signaling to xCT. 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