Wei-Xing Zong - US grants

DESCRIPTION (provided by applicant): This project focuses on studying how proapoptotic Bcl-2 family proteins Bax and Bak regulate apoptosis in response to ER stress, and extends the study to determine how excitotoxic neuronal cell death is regulated using Bax/Bak doubly deficient model system. ER stress triggers the unfolded protein response (UPR), which ultimately results in apoptosis. It remains unclear how signals from ER stress is transduced to initiate apoptosis. Bax and Bak play a fundamental role in initiating apoptosis. Preliminary studies have suggested that in addition to their presence at the mitochondrial outer membrane, Bax and Bak also localize to the ER and initiate apoptosis. The following areas will be addressed to study the mechanisms involved in the initiation of apoptosis by Bax and Bak from the ER: (a) Transcriptional and post-translational regulation of both anti-apoptotic and proapoptotic multi-domain Bcl-2 family proteins and, modification of the BH3-only proteins, e.g., change of localization, phosphorylation, protease cleavage, and transcriptional regulation, in response to ER stress, (b) Involvement of intracellular Ca2+ and proteases that may mediate cell death in response to ER stress, (c) Determining whether Bax/Bak can induce ER leakage by looking at the release of ER lumenal proteins into cytosol. The second aim is to characterize excitotoxic neuronal cell death using Bax/Bak doubly deficient cells. Excitotoxic cell death has been implicated in human neurodegenerative diseases and brain tumor invasion. The existence of both apoptotic and necrotic forms of cell death makes it complicated to study the mechanisms involved. Deficiency in both Bax and Bak blocks mitochondrial apoptotic pathways, yet neural progenitor cells isolated from Bax/Bak-deficient mice are sensitive to NMDAand amyloid B (AB)-induced cell death. Thus, mechanisms involved in excitotoxic cell death can be studied in Bax/Bak-deficient cells without the complexity resulting from the death amplification effect of mitochondria. Since excitotoxic cell death shares features with the ER-mediated cell death, such as the perturbation of intracellular Ca2+ homeostasis, I plan to study excitotoxic cell death by: (a) Isolating and culturing cerebellar granule cells and establishing immortalized NPC lines, (b) Characterizing cell death induced by NMDA and Ap in Bax/Bak-deficient cells to determine whether these cells die with characteristic apoptotic or necrotic features, (c) Determining the involvement of the acid-sensitive ionic channels (ASICs) or poly(ADP-ribose) polymerase (PARP) in excitotoxic cell death using Bax/Bak-deficient cells, and study aspartyl and calpain proteases that may be involved in Bax/Bak-independent cell death, (d) Studying the role of intracellular Ca2+ in excitotoxic neuronal cell death.

DESCRIPTION (provided by applicant): A paradox in medical oncology has long existed that although the majority of human cancers have acquired a deficiency in apoptosis, certain chemotherapeutic agents such as DNA alkylating agents remain the most effective means of treating cancer patients by inducing cancer cell death. This suggests that alternative cell death pathways may be involved. These may include necrosis and autophagic cell death. One fundamental difference between cancer and normal cells is their biochemical metabolism. Tumor cells display an abnormal propensity for growth and proliferation, thus are in net need of energy source for biosynthesis. This may render cancer cells more susceptible to the perturbation of cell metabolism. Several oncoproteins, such as c- myc, Akt, and Ras, have been shown to promote cell growth by regulating cell metabolism, and thus may prime cells to cell death induced by bioenergetic failure. We propose to explore the hypothesis that targeting cellular metabolism can be a strategy to kill cancer cells that often have crippled apoptosis machinery. We will also study whether and how certain oncoproteins such as c-myc, Akt, and Ras may differentially affect cell metabolism and render cells susceptible to the perturbation of cell metabolism, and study how tumor cells may respond to metabolic stress by inducing autophagy. We will: 1) Study the hypothesis that cell death can be induced in apoptosis-deficient cells by metabolic perturbation resulting from DNA alkylating damage. Our preliminary data indicates that necrosis can be induced by DNA alkylating damage as a result of the inhibition of glycolysis, which is caused by the NAD depletion resulting from the activation of a nuclear enzyme PARP. We will further examine this theory in vitro and in vivo, and will study the pro-inflammatory response triggered by this non-apoptotic cell death. 2). Study the role of autophagy in cancer cells treated with chemotherapeutic agents. As an important cellular response to nutrient starvation and stress, autophagy has been shown to have opposite effects on cell survival and cell death. These opposing effects of autophagy may on one hand contribute to cancer cell death, on the other hand, to cancer cell resistance to therapy. We will study in this Aim whether and how DNA alkylating damage can induce autophagy, and how autophagy interplays with other forms of cell death. 3). Study the hypothesis that oncoproteins such as c-myc, Ras, and Akt can affect cell metabolism and prime cancer cells to die from bioenergetic failure. c-myc, Ras, and Akt oncoproteins are involved in cell growth, proliferation, and death. These proteins have been shown to regulate cell metabolism thus promoting cancer cell anabolic processes, however maybe through different mechanisms. We plan to express specific oncogenes in genetically defined murine cells as well as human cancer cells to study how they may differentially affect cellular metabolism, with respect to their ability to prime cancer cells to die of metabolic perturbation. PUBLIC HEALTH RELEVANCE: A major strategy for treating cancer is to selectively induce cancer cell death. Most human cancers evolved as a result of the loss of ability to die by apoptosis, and have acquired specific needs for cell metabolism. The overall goal of this project is to study how non-apoptotic cell death can be induced by chemotherapy, and by the inhibition of cell metabolism, thus targeting cell metabolism can be harnessed to treat cancer patients by inducing cancer cell specific death.

DESCRIPTION (provided by applicant): The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3'-hydroxyl group of phosphatidylinositol (PIs) and phosphoinositides. The generated phospholipids are critical signaling molecules. Based on substrate specificity and sequence homology, PI3Ks are grouped into three classes: Class I, Class II, and Class III. In vivo, Class I PI3Ks are believed to preferentially phosphorylate PtdIns(4,5)P2 to generate PI(3,4,5)P3, a pivotal signaling molecule that activates multiple downstream signaling cascades, including the Akt/TOR pathway. Class III PI3K is composed of a sole member, Vps34, that converts PtdIns to PI(3)P. Vps34 is the only PI3K reported to be evolutionarily conserved from yeast to mammals. An important cellular process controlled by PI3Ks is autophagy, which is involved in many physiological and pathological conditions. The current dogma is that in metazoans, autophagy requires PI(3)P, the product of Class III PI3K Vps34. On the contrary, autophagy is inhibited by PI(3,4,5)P3, the product of Class IA PI3Ks, that mediates activation of the Akt/mTOR pathway. However, the direct role of PI3Ks, especially that of the Class IA PI3Ks, in autophagy remains unclear. Using p110a and p110¿ conditional knockout mice, we have recently shown that the Class IA p110¿ isoform is a positive regulator of autophagy, both in cell culture and in vivo. p110¿ promotes autophagy by activating Vps34 kinase activity and the generation of the autophagy- essential phospholipid PI(3)P. This autophagy-promoting function of p110¿ is independent of its catalytic activity. These findings prompt us to propose the central hypothesis that the Class IA p110¿ subunit positively regulates autophagy acting as a molecular scaffold. In this proposal, we plan to study the molecular mechanisms underlying the autophagy-promoting function of p110¿, and to explore its biological roles. Based on our preliminary data, we propose that p110¿ may promote autophagy by activating the small GTPase Rab5, which has been recently shown to activate Vps34 and promote autophagy. We also hypothesize that p110¿ changes its subcellular localization and autophagy-promoting activity in response to trophic factor deprivation. Moreover, although it is well recognized that the Class III PI3K Vps34 plays an essential role in autophagy in yeast, its role in mammals remains elusive. Surprisingly, a recent report showed that autophagosomes still form in Vps34-null sensory neurons, suggesting that the molecular and physiological role of Vps34 in mammalian autophagy needs to be re-examined. Our recent study indicates a molecular connection between p110¿ and Vps34. Hence in this proposal, we will also use tissue-specific Vps34 knockout mice to study Vps34 and its interplay with p110¿ in regulating autophagy. Completion of this project will uncover the novel function of p110¿ as a molecular scaffold to promote autophagy both at basal state and in response to trophic factor availability, and define the role of Vps34 in autophagy in mammals. This will help our understanding to the roles of PI3Ks in regulating cellular homeostasis, metabolism, and their involvement in human diseases such as cancer.

DESCRIPTION (provided by applicant): Inhibition of protein degradation is an emerging anti-cancer strategy. The proteasome inhibitor Bortezomib has been approved by FDA for treatment of multiple myeloma, and is being trialed in numerous cancers. However, the underlying molecular mechanisms remain elusive. Moreover, it has been unclear whether certain molecular signatures can be used to predict the outcome of proteasome inhibitor-based therapy. We recently reported that proteasome inhibitors can induce an intracellular aggregation and activation of caspase-8 and subsequent apoptosis. This caspase-8 activation is mediated by its association with a ubiquitin-binding protein SQSTM1/p62 and an autophagy-related protein microtubule-associated protein light chain 3 (LC3). These findings prompt us to hypothesize that there exists a previously uncharacterized cell death mechanism that involves protein aggregate formation and intracellular activation of caspase-8. Along this direction, our additional preliminary results show that p62 itself undergoes ubiquitination. This novel modification of p62 may play a critical function in mediating aggregate formation and apoptosis. We also reported that an endogenous serine/cysteine protease inhibitor SerpinB3 (also termed squamous cell carcinoma antigen 1, SCCA1) may function as a molecular signature for predicting efficacy with proteotoxicity-based anti-cancer therapy. SCCA1 is an intracellular serpin that inhibits lysosomal proteases. SCCA1 is believed to limit cellular damage resulting from unscheduled activation of lysosomal protease that is detrimental to the cell, hence may contribute to tumorigenesis and chemo-resistance. Studies including those recently from my laboratory have demonstrated that elevated SCCA1 expression is associated with poorer prognosis in numerous advanced human cancers such as squamous cell carcinomas of lung, head and neck, and esophagus, as well as hepatocellular carcinoma and breast carcinoma. Indeed, at the molecular level, we found that SCCA1 protects cells from lysosomal injury induced by DNA alkylating damage and oxidative stress. On the other hand, we also found that SCCA1 promotes apoptosis in response to proteotoxic stress. Therefore, SCCA1 on one hand may confer resistance to chemotherapy by protecting cells against lysosomal injury, on the other hand, it may sensitize cancer cells to proteotoxicity. This proposal is designed to understand the molecular mechanisms underlying the anti-tumor effect of proteasome inhibitors, and to determine whether certain molecular changes in cancer cells such as elevated expression of LC3 or SCCA1 can confer tumor cell sensitive to proteotoxic agents in vivo. We propose three molecularly and clinically related Specific Aims: 1) Characterize the activation of caspase-8 upon the inhibition of proteasome degradation. 2) Study the mechanisms through which p62 regulates aggregate formation and caspase-8 activation. 3) Examine the hypothesis that certain molecules such as LC3 and SCCA1 can sensitize tumors to proteotoxic agents in vivo. Accomplishing this project will have a general impact on the understanding of the molecular basis for proteotoxicity-based anti-cancer therapy, as well as on the physiological relevance of protein aggregates in many pathological conditions. At the clinical level, it may establish LC3 or SCCA1 as a molecular signature for suggesting treatment with proteotoxic agents.

PROJECT SUMMARY: The overarching goal of this proposal is to uncover a previously uncharacterized molecular function of c-Myc (hereafter referred to as Myc), a proto-oncoprotein that is frequently amplified in breast cancer and many other types of human cancer. Recently, we found that Myc overexpression leads to elevated expression of glutamate-ammonia ligase (GLUL) and interestingly, this Myc-induced GLUL is not through the direct transactivation by Myc, rather it involves promoter demethylation of the GLUL gene. We further found that the demethylation is dependent on increased expression of thymine DNA glycosylase (TDG), which is a direct Myc transcriptional target. These results suggest an unexpected role of Myc in promoting glutamine synthesis, and intriguingly, suggest a previously unidentified molecular function of Myc in activating gene expression by regulating DNA methylation. This prompts us to form the hypothesis that Myc can regulate gene expression via the modulation of DNA methylation. We propose two Specific Aims to study this hypothesis. In Aim 1, we plan to identify Myc-induced DNA methylation and gene expression profiles by whole genome bisulfite sequencing (WGBS) and RNA-Seq using various breast cancer cell lines with stable or inducible expression or knock-down of Myc. We will first prepare cell cultures with different treatments and extract genomic DNA and total cellular RNA for the next-generation sequencing (NGS). After NGS sequence reads are obtained, we will perform the bioinformatics analysis to (1) identify and annotate differentially methylated regions (DMR) in WGBS, with focus on gene promoters; (2) identify differentially expressed genes (DEG) in RNA-seq, using the same samples for DMR; and (3) rank Myc ?epigenetic targets? using integrated bioinformatics analysis of DMR and DEG, and identify potential biological pathways preferentially affected by Myc through epigenetic regulation. We expect to discover specific ?epigenetic targets? of Myc in various breast cancer cell lines. In Aim 2, we plan to validate the identified methylation profiles using traditional molecular techniques and examine their biological relevance. We will first use the quantitative PCR and focal bisulfate sequencing on specific gene promoters to validate the ?epigenetic targets? targets of Myc to be found in Aim 1. We will also examine the expression of TDG and the Myc epigenetic targets in various breast cancer cell lines. Furthermore, we will examine the expression patterns of TDG and validated epigenetic Myc targets by IHC using de-identified breast cancer clinical tissue samples, and correlate them with histopathological characteristics and clinical outcomes. If successful, this project will uncover DNA demethylation as a novel mechanism for Myc regulated gene expression and oncogenesis. In the long run, the knowledge gained from this study will help with the understanding of cancer etiology and shed light on the development of novel therapeutics.

SUMMARY Dysregulated metabolism has long been recognized as a key hallmark of neoplastic disease. One of the major metabolic changes in tumor cells is increased glutamine (Gln) usage via glutaminolysis: Gln is deaminated by glutaminase (GLS) to glutamate (glutamic acid, Glu), which is converted by glutamate dehydrogenase (GLUD) to a-ketoglutarate (aKG) to enter the tricarboxylic acid (TCA) cycle for anaplerosis (replenishment of metabolic intermediates for energy production or biosynthesis). It is well recognized that oncogenic c-Myc (hereafter referred to as Myc) enhances Gln usage by directly transactivating the expression of Gln transporters SLC1A5 and SLC7A5/SLC3A2, and by increasing GLS1 expression via transcriptional suppression of the GLS1 repressor micro RNAs (miR)-23a/b. Pharmacologically targeting GLS1 is being actively pursued as an anti- cancer approach, although thus far with little success. On the other hand, several recent studies, including those from our laboratory, point to the importance of Gln synthesis, at least in certain cell/tissue types. Gln is synthesized de novo by condensation of Glu and ammonia, catalyzed by the enzyme Gln synthetase (GS, also known as glutamate ammonia ligase, GLUL). Using stable isotope-based metabolite tracing, we recently reported that this synthesized Gln is not used via glutaminolysis to fuel the TCA cycle; rather it is used for several TCA-independent anabolic processes including biosynthesis of nucleotides and transport of essential amino acids. Importantly, elevated expression of GS promoted cell survival under Gln limitation; inhibition of GS led to decreased cell proliferation and increased cell death upon Gln limitation, and slowed xenograft tumor growth. Moreover, we recently reported that Myc can induce the expression of GS in a number of cancer cell lines. We also found a positive correlation between Myc activation and GS expression several mouse models and in human patient samples. These findings lead us to propose the following hypothesis: oncogenic Myc, at least in certain cell/tissue types, upregulates GS expression to promote Gln production and its anabolic usage (away from the TCA cycle), thereby facilitating oncogenesis. We propose two Specific Aims to study this hypothesis: 1) Study the metabolic and cell biological consequences, and the regulation of increased GS expression in the context of Myc activation; and 2) Determine the in vivo role of GS in Myc- driven metabolic reprogramming and oncogenesis. If accomplished, this study will provide a novel dimension to understanding the functions of dysregulated Myc in cancer cells and a potential new target for treatment of Myc-driven tumors.

PROJECT SUMMARY: The Class IA phosphatidylinositol 3-kinases (PI3Ks) p110a and p110b isoforms are ubiquitously expressed and respond to membrane receptor signals. They PI(3,4,5)P3, which acts as a signaling molecule to activate downstream signaling pathways including the Akt/mTOR pathway. Despite their similarities, two isoforms have distinct biochemical and biological features, one of which is that p110b has a kinase-independent function. We have reported that p110b positively regulates the small GTPase Rab5 in a manner that is independent of its catalytic activity. p110b, through a direct binding and as a molecular scaffold, keeps Rab5 in its ?active? GTP- bound form by preventing Rab5 from interacting with its GTPase-activating protein (GAP), which inactivates Rab5 by hydrolyzing its GTP to GDP. Rab5 is well known for regulating endocytosis. Our preliminary data strongly indicate that p110b can regulate endocytic turnover of cell surface nutrient transporters such as glucose transporter 1 (GLUT1) in response to growth factor deprivation. We found that genetic ablation or shRNA silencing of p110b decreased the level of Rab5-GTP. This resulted in increased cell surface GLUT1 that led to increased glucose utilization of cell lines and promoted their oncogenic transformation. These findings lead us to propose the hypothesis that p110b, as a molecular scaffold, activates Rab5 and endocytic turnover of nutrient transporters. We further propose that this novel function of p110b regulates membrane trafficking and cell metabolism and homeostasis. We have designed two Specific Aims to test this hypothesis and establish its biological relevance. Aim 1: Test the hypothesis that p110b facilitates Rab5-mediated endocytic turnover of nutrient transporters. We will use FL5.12, MEFs, HK-2, and MCF10A cells to determine that p110b deficiency can lead to the stabilization of nutrient transporters, and to determine if the failure of endocytic turnover is the result of Rab5 inactivation. Aim 2. Determine the molecular and cell biological consequence of the stabilized nutrient transporters in p110b-deficient cells (with a focus on GLUT1). We will characterize the metabolic features associated with the nutrient transporter stabilization, and characterize the growth/proliferation signaling in p110b-deficient cells. We will also determine the physiological effects of the p110b-Rab5 interaction in vivo. !

PROJECT SUMMARY Protein homeostasis (proteostasis) and reduction-oxidation (redox) balance are two tightly regulated and mutually associated molecular events. They play crucial roles in many physiological/pathological conditions including cancer. Disruption of proteostasis and redox balance is an effective approach to selectively kill cancer cells. For examples, proteasome inhibitors such as Bortezomib (Velcade) are highly effective in treating numerous cancers, and autophagy inhibitors are being actively pursued as anti-cancer therapeutics. Many clinically effective chemotherapeutic agents such as arsenic trioxide can induce oxidative burst and cell death, which is believed to contribute, at least in part, to their anti-cancer effectiveness. On the other hand, dysregulated proteostasis and redox homeostasis can contribute to oncogenesis by activating numerous pro- survival/growth signaling pathways. The seemingly paradoxical effects (pro- and anti-cancer) are generally thought to be accounted for by the intensity and duration of the stresses, although the precise underlying mechanisms remain largely elusive. The ubiquitin-binding protein, p62 (SQSTM1), among its numerous functions, critically regulates both proteostasis and redox balance, by sequestering certain proteins in aggregates and delivering them to autophagosomes for degradation. This sequestration function of p62 relies on its dimmerization via the hydrogen bond between lysine (K)7 and aspartate (D)69 residues. We recently reported that TRIM21 (Tripartite motif-containing protein 21), a RING domain-containing ubiquitin E3 ligase, directly interacts with and ubiquitylates p62 at K7 via K63-linkage, which abolishes the K7-D69 hydrogen bond and inhibits p62 oligomerization, aggregation, and sequestration functions. One of the client proteins sequestered by p62 is Keap1, a negative regulator of the antioxidant response that suppresses the antioxidant transcription factor Nrf2. TRIM21-mediated p62 K7 ubiquitylation leads to the failure of Keap1 sequestration and suppressed antioxidant response. Conversely, TRIM21-deficient cells display increased p62 oligomerization, protein aggregation, Keap1 sequestration, Nrf2 activation, and antioxidant response. In this project, we propose to study the hypothesis that TRIM21 functions as a stress-adaptation molecule and plays a crucial role in proteostasis and redox homeostasis, by ubiquitylating p62 and negatively regulating its sequestration function, for the underlying mechanisms and biological significance, with a main focus on anti- cancer therapy and oncogenesis. As TRIM21 expression is dysregulated and correlates with prognosis in numerous cancers, accomplishing this project will uncover TRIM21 as a new important regulator for cellular proteostasis and redox homeostasis, and will help reveal the role of TRIM21 in cancer development and therapy.

CANCER METABOLISM AND GROWTH PROGRAM PROJECT SUMMARY/ABSTRACT The Cancer Metabolism and Growth (CMG) Program has the overall goal to determine how oncogenic alterations regulate tumor cell metabolism, growth, proliferation, survival, and tumor-host interaction to facilitate disease progression. The ultimate aim is to identify new approaches to improve cancer treatment through innovative biochemical, molecular and biological research. In vivo approaches to address metabolic, physical and immunologic functions in cancer and state-of-the-art measurement of cancer metabolism are signature Program features that span the Rutgers/Princeton consortium. CMG provides the platform for productive, collaborative, and impactful science, and interfaces with the Cancer Center for the translation of that science, both bench to bedside and bedside to bench. CMG has 59 members from 26 Departments, 10 Schools, and 3 Universities. CMG research is well funded with $14.3M annual direct peer-reviewed grant support, $9.2M of which is cancer-focused (9 multi-PI), with $3M from the NCI (21 R01-equivalent/14 PIs). In the last funding period CMG members published more than 835 papers, 31% of which are collaborative (15% intra- and 22% inter-collaborative) with 21% top-tier journals and 50% collaborative with other institutions. In comparison to the last funding period, this represents an increase in both total and collaborative publications, and seven additional multi-PI grants. Impactful CMG cancer science includes the discovery that circulating lactate is a major supplier of carbon to the tricarboxylic acid (TCA) cycle in tumors, and that the folate pathway significantly contributes to NADPH production. How glutamine metabolism is critical for MYC-driven cancers, how mTOR signaling is controlled by nutrient availability, and how protein and lipid scavenging contribute to cancer growth, proliferation and survival were also discovered by CMG research. Examination of metabolic interactions between tumor and host revealed new mechanisms of metastasis, and how tumors physically interact with their local environment and the immune system. Program members discovered that metastasis represents corruption of normal developmental processes, that cell polarity and tissue/cytoskeletal tension in the tumor microenvironment alter oncogenic signaling via the Hippo and other pathways, and that nutrient scavenging, interferons and the removal of dead cells by efferocytosis alter the immune response to tumors. Translation of CMG research has led to clinical trials targeting metabolism, promoting apoptosis and activating anti-tumor immune responses. In turn, clinical observations have informed CMG research to model treatment, resistance and exceptional responders to identify underlying mechanisms and to improve therapy.