Alan P. Fields - US grants

The protein kinase C enzyme family is involved in a number of cellular functions, including the control of cellular proliferation and differentiation. Human erythroleukemia (K562) cells express three PKC isotypes, alpha PKC, betaII PKC and zeta PKC. These cells can be induced to differentiate along the megakaryocytic pathway upon exposure to the PKC activator phorbol myristate acetate e (PMA). Expression of the three PKC isotypes are differentially regulated during PMA-induced cytostasis and differentiation. Alpha and zeta PKC levels increase significantly and beta II PKC levels fall dramatically upon PMA treatment. Therefore, alpha and zeta PKC levels correlate with PMA-induced cytostasis and differentiation, whereas beta II PKC levels correlate with the proliferative capacity of the cells. In order to explore isotype-specific function, we assessed the effect of overexpression and anti-sense inhibition of expression of the alpha and beta II PKC isotypes on the proliferation and differentiation potential of k562 cells. Over expression of alpha PKC leads to a gene dose-dependent decrease in the rate of proliferation and increased responsiveness to PMA whereas anti-sense inhibition of alpha PKC expression leads to relative resistance to PMA-induced cytostasis. Conversely, overexpression of betaII PKC leads to relative resistance to PMA-induced cytostasis whereas anti-sense inhibition of beta II PKC expression blocks K562 cell proliferation. these results indicate that alpha PKC is directly involved in the cytostatic and differentiative effects of PMA whereas betaII PKC is required for K562 cell proliferation. Through the use of domain switch alpha beta II PKC chimera, we have identified regions on alpha and beta II PKC required for isotype-specific function. The goals of this proposal are to i) determine the role of zeta PKC in K562 cell proliferation and differentiation through overexpression and anti-sense inhibition of expression of the enzyme, ii) define minimal molecular determinants capable of mediating the isotype specific functions of alpha and beta II PKC in-vivo through expression of chimeric alpha beta II PKC in K562 cells and iii) identify and characterize cellular proteins involved in isotype-specific signal transduction in K562 cells through interactive cloning strategies. Completion of these aims will provide important new insight into the molecular basis for PKC isotype specific function in-vivo and identify molecular targets for PKC isotype-specific signalling. Furthermore, since anti-proliferative and differentiation therapy is an important modality for the clinical treatment of human leukemia, the proposed studies may identify molecular targets i human leukemic cells of therapeutic importance.

Colon carcinogenesis is a complex, multi-step process involving progressive changes in intestinal epithelial cell proliferation, differentiation and programmed death. Our long-term goal is to understand the role of protein kinase C (PKC) isozymes in intestinal epithelial cell biology and colon carcinogenesis. Several lines of evidence suggest that the PKC betaII isozyme (PKC betaII) is involved in colon carcinogenesis. First, PKC betaII levels and activity are elevated in colon carcinomas compared to normal colonic epithelium. Second, PKC betaII is involved in colon carcinoma cell proliferation in vitro. Third, components of a high fat diet can potently stimulate intestinal epithelial cell PKC betaII activity and promote colon carcinogenesis. Based on these findings, and our preliminary studies, we hypothesize that PKC betaII is directly involved in colon carcinogenesis. To directly test this hypothesis, we developed transgenic mice that express elevated PKC betaII levels in the intestinal epithelium. In preliminary studies, these mice exhibit evidence of colonic epithelial hyperproliferation and an increased susceptibility to carcinogen-induced colon cancer. In this application we propose four specific aims to: 1) characterize three transgenic PKC betaII lines for transgene copy number, tissue distribution, expression level and activity of the PKC betaII transgene, and for changes in intestinal epithelial cytokinetics; 2) assess whether transgenic PKC betaII mice exhibit increased susceptibility to carcinogen-induced colon cancer; 3) determine whether a high fat diet enhances the susceptibility of transgenic PKC betaII mice to colon cancer; and 4) determine whether elevated PKC betaII expression synergizes with loss-of-function mutation of the APC tumor suppressor gene in promoting intestinal tumorigenesis in the APCmin mouse model. These studies will allow the first direct analysis of the role of PKC betaII in colon cancer in two relevant animal models of the human disease.

The protein kinase C enzyme family is involved in a number of cellular functions, including the control of cellular proliferation and differentiation. Human erythroleukemia (K562) cells express three PKC isotypes, alpha PKC, betaII PKC and zeta PKC. These cells can be induced to differentiate along the megakaryocytic pathway upon exposure to the PKC activator phorbol myristate acetate e (PMA). Expression of the three PKC isotypes are differentially regulated during PMA-induced cytostasis and differentiation. Alpha and zeta PKC levels increase significantly and beta II PKC levels fall dramatically upon PMA treatment. Therefore, alpha and zeta PKC levels correlate with PMA-induced cytostasis and differentiation, whereas beta II PKC levels correlate with the proliferative capacity of the cells. In order to explore isotype-specific function, we assessed the effect of overexpression and anti-sense inhibition of expression of the alpha and beta II PKC isotypes on the proliferation and differentiation potential of k562 cells. Over expression of alpha PKC leads to a gene dose-dependent decrease in the rate of proliferation and increased responsiveness to PMA whereas anti-sense inhibition of alpha PKC expression leads to relative resistance to PMA-induced cytostasis. Conversely, overexpression of betaII PKC leads to relative resistance to PMA-induced cytostasis whereas anti-sense inhibition of beta II PKC expression blocks K562 cell proliferation. these results indicate that alpha PKC is directly involved in the cytostatic and differentiative effects of PMA whereas betaII PKC is required for K562 cell proliferation. Through the use of domain switch alpha beta II PKC chimera, we have identified regions on alpha and beta II PKC required for isotype-specific function. The goals of this proposal are to i) determine the role of zeta PKC in K562 cell proliferation and differentiation through overexpression and anti-sense inhibition of expression of the enzyme, ii) define minimal molecular determinants capable of mediating the isotype specific functions of alpha and beta II PKC in-vivo through expression of chimeric alpha beta II PKC in K562 cells and iii) identify and characterize cellular proteins involved in isotype-specific signal transduction in K562 cells through interactive cloning strategies. Completion of these aims will provide important new insight into the molecular basis for PKC isotype specific function in-vivo and identify molecular targets for PKC isotype-specific signalling. Furthermore, since anti-proliferative and differentiation therapy is an important modality for the clinical treatment of human leukemia, the proposed studies may identify molecular targets i human leukemic cells of therapeutic importance.

The U.S. PI has a long-standing interest in the various aspects of protein kinase C (PKC) isozyme signaling within cells. The PI has previously investigated in detail one particular pKC isozyme, PKC B2, and has generated a considerable amount of knowledge regarding the mechanisms surrounding activation of this isozyme. The present parent project for this FIRCA application by the PI has now shifted to explore the functional characterization of an atypical PKC isozyme, PKCi, in human leukemia cell survival. In examining factors responsible for the nuclear activation of PKC B2 prior to mitosis, the PI's laboratory isolated a nuclear PI-PLC from G2 phase nuclei. Preliminary experiments using antibodies to known PI-PLC isozymes indicated that this nuclear PI-PLC did not correspond to any of the previously identified PI-PLC isozymes. The foreign collaborator, while working with regenerating rat hepatocytes, also observed the presence of a novel nuclear PI-PLC activity involved in cell cycle regulation. Given these common findings and interests, and given the foreign collaborator's expertise in the biochemical analysis of phosphatidylinositol-metabolizing enzymes, a new collaborative effort has arisen between these investigators. Therefore, the main thrust of this proposal is to identify this novel nuclear PI-PLC activity. In addition, recent data suggests that a phosphatidylinositol 3-kinase (PI3K) signaling system exists within the nucleus and that this nuclear PI3K signaling system is distinct from the well described cytoplasmic-cell membrane PI3K system. The novel nuclear PI3K pathway is responsible for PKB activation. These preliminary results suggest that PI3K and PKB activation may play a direct physiologic role in nuclear events associated with cellular proliferation and cell cycle progression. The second and third specific aims therefore propose to expand and confirm these preliminary results of the roles of nuclear PI3K and PKB in these cellular events.

DESCRIPTION (provided by applicant): Colon cancer is the second leading cause of cancer death in the United States. It arises by a multi-step process involving progressive changes in signaling pathways that regulate epithelial cell proliferation, differentiation and survival. PKCbetaII induces hyperproliferation in the colon, and is required for early steps in colon carcinogenesis in vivo. PKCbetaII induces resistance to the growth inhibitory effects of TGFbeta in rat intestinal epithelial (RIE) cells through activation of a novel PKCbetaII->Cox-2->TGFbetaRII signaling axis. PKCbetaII also induces an invasive phenotype in RIE cells through activation of a PKCbetaII->Ras/Mek->PKCI/Rac1 signaling pathway. The overall hypothesis to be tested in this proposal is that PKCbetaII is required for the transformed phenotype in human colon cancer cells. Aim 1 will test the hypothesis that PKCbetaII confers TGFbeta resistance on human colon cancer cells by activating the PKCbetaII->Cox-->?TGFbetaRII signaling axis. Aim 2 will test the hypothesis that PKCbetaII is important for anchorage-independent growth and invasion of human colon cancer cells in vitro, and for tumorigenicity and metastasis in vivo. PKCbetaII expression will also be assessed in human colon cancers and compared with clinical outcome. Aim 3 will test the hypothesis that PKCII is required for K-Ras-mediated colon carcinogenesis in vivo using two complementary transgenic K-Ras mouse models crossed to PKCbeta KO mice. Aim 4 will test the hypothesis that PKCbetaII and PKC/I collaborate to promote colon carcinogenesis in vivo. The role of PKC/I in PKCbetaII-mediated colon carcinogenesis will be assessed in transgenic PKCbetaII mice crossed to transgenic mice expressing kinase-deficient PKC/I. Synergism between PKCbetaII and PKC/I in colon cancer progression, invasion and metastasis will be assessed in transgenic PKCbetaII mice crossed to mice expressing constitutively-active PKC/I. These studies will determine the role of PKCbetaII in human colon cancer cell transformation, the signaling mechanisms by which PKCbeta/II contributes to cellular transformation, and the relationship between PKCbetaII, PKC? and oncogenic K-Ras in promoting colon carcinogenesis in vivo. They will also assess PKCbetaII as a potential therapeutic target and prognostic marker in colon cancer, and develop new genetic models of colon cancer that may be suitable for studying all stages of colon carcinogenesis from initiation to invasive carcinoma in vivo.

DESCRIPTION (provided by applicant): Colon cancer is the second leading cause of cancer death in the United States. It arises by a multi-step process involving progressive changes in signaling pathways that regulate epithelial cell proliferation, differentiation and survival. PKCbetaII induces hyperproliferation in the colon, and is required for early steps in colon carcinogenesis in vivo. PKCbetaII induces resistance to the growth inhibitory effects of TGFbeta in rat intestinal epithelial (RIE) cells through activation of a novel PKCbetaII->Cox-2->TGFbetaRII signaling axis. PKCbetaII also induces an invasive phenotype in RIE cells through activation of a PKCbetaII->Ras/Mek->PKCI/Rac1 signaling pathway. The overall hypothesis to be tested in this proposal is that PKCbetaII is required for the transformed phenotype in human colon cancer cells. Aim 1 will test the hypothesis that PKCbetaII confers TGFbeta resistance on human colon cancer cells by activating the PKCbetaII->Cox-->?TGFbetaRII signaling axis. Aim 2 will test the hypothesis that PKCbetaII is important for anchorage-independent growth and invasion of human colon cancer cells in vitro, and for tumorigenicity and metastasis in vivo. PKCbetaII expression will also be assessed in human colon cancers and compared with clinical outcome. Aim 3 will test the hypothesis that PKCII is required for K-Ras-mediated colon carcinogenesis in vivo using two complementary transgenic K-Ras mouse models crossed to PKCbeta KO mice. Aim 4 will test the hypothesis that PKCbetaII and PKC/I collaborate to promote colon carcinogenesis in vivo. The role of PKC/I in PKCbetaII-mediated colon carcinogenesis will be assessed in transgenic PKCbetaII mice crossed to transgenic mice expressing kinase-deficient PKC/I. Synergism between PKCbetaII and PKC/I in colon cancer progression, invasion and metastasis will be assessed in transgenic PKCbetaII mice crossed to mice expressing constitutively-active PKC/I. These studies will determine the role of PKCbetaII in human colon cancer cell transformation, the signaling mechanisms by which PKCbeta/II contributes to cellular transformation, and the relationship between PKCbetaII, PKC? and oncogenic K-Ras in promoting colon carcinogenesis in vivo. They will also assess PKCbetaII as a potential therapeutic target and prognostic marker in colon cancer, and develop new genetic models of colon cancer that may be suitable for studying all stages of colon carcinogenesis from initiation to invasive carcinoma in vivo.

DESCRIPTION (provided by applicant): This application seeks support for a biennial scientific conference series entitled 'Lipid Signaling Pathways in Cancer'. The conferences are sponsored by the Federation of the American Societies for Experimental Biology (FASEB) as part of their biennial Summer Research Conference (SRC) Series and are co- organized by Dr. Alan P. Fields of the Mayo Clinic College of Medicine and Dr. Marcelo Kazanietz of the University of Pennsylvania School of Medicine. The next conference will be held at the Carefree Resort in Carefree, Arizona on July 19-24, 2009 with subsequent conferences scheduled for summer of 2011 and summer 2013. Support provided through funding of this application will be used to partially finance the costs of registration fees and travel expenses for junior investigators, including graduate students and post-doctoral fellows, invited to present their work at these conferences. The conference is held over the course of 4 days, with more than 30 invited leaders in the field attending to present the latest breakthroughs in this highly relevant area of cancer research. Recent exciting developments in the field of lipid signaling will be presented that have reinforced the concept that lipid mediators play key roles in the initiation and progression of cancer. For example, tumor-specific genetic alterations in proteins involved in phosphoinositide and PKC signaling have been found in many human cancers, highlighted the importance of these lipid signaling pathways in tumorigenesis. Exciting findings in PI 3-K/Akt/mTOR, PKC, DAG-dependent GTPases, lipid modifications of Ras family GTPases, and sphingomyelinase signaling and the role of these pathways in tumorigenesis will also be discussed. Many of these lipid signaling pathways are attractive candidates for development of novel therapeutic strategies for cancer treatment. Indeed, novel therapeutics that target several of these lipid signaling pathways are currently being evaluated in the clinic. Particular emphasis will therefore be placed on translational studies of lipid signaling pathways that may lead to promising new clinical cancer treatments. Given the rapid developments in this fertile research area, a biennial meeting schedule is appropriate. No other regularly scheduled conference focuses on this burgeoning area, and this conference series is poised to become a major forum for effective scientific interchange that will serve to facilitate advances in this field over the next five years. Public Health Relevance: Lipid signaling pathways function to allow cells to communicate with and respond to their environment, and in turn to function appropriately. In human cancer, these lipid signaling mechanisms are often dysregulated, either by inherited or somatic genetic mutations, or as a result of epigenetic changes in expression or activity of the genes encoding key components of lipid signaling pathways. A major focus of this project is to provide a forum to discuss advances in basic knowledge of lipid signaling mechanisms and how this knowledge is being translated into novel therapeutic strategies that will impact clinical oncology practice.

DESCRIPTION (provided by applicant): Our long-term goals are to elucidate protein kinase C (PKC) signaling mechanisms that contribute to cancer and translate these mechanistic insights into better prognostic and treatment strategies. In previous funding periods, we discovered that PKC? is an oncogene in non-small cell lung cancer (NSCLC) the leading cause of cancer death in the United States, elucidated a major oncogenic PKC? signaling pathway, and developed a therapeutic agent that targets oncogenic PKC? signaling that is currently being evaluated in the clinic. During the current funding period we showed that: 1) PKC? forms an oncogenic PKC?/Par6 signaling complex in the cytoplasm of NSCLC cells that is necessary for cell proliferation and invasion in vitro, and tumor formation in vivo; 2) the guanine nucleotide exchange factor (GEF) Ect2 binds the PKC?/Par6 complex and activates Rac1, a key downstream effector of this complex; 3) PKC? regulates the intracellular location and oncogenic activity of Ect2 through direct binding and phosphorylation; 4) matrix metalloproteinase 10 (Mmp10) is a critical downstream effector of the PKC?/Ect2/Par6/Rac1 signaling axis that is required for NSCLC cell proliferation and invasion in vitro, and Kras-mediated lung tumorigenesis in vivo; and 5) both PKC? and Mmp10 are required for Kras-mediated transformation of bronchio-alveolar stem cells (BASCs), putative lung tumorinitiating cells (TICs) in vivo. Our preliminary studies indicate that: 1) the PKC?/Ect2/Par6/Rac1/Mmp10 signaling axis maintains a tumor-initiating cell phenotype in NSCLC cells characterized by stem-like behavior and enhanced tumorigenicity; 2) a significant pool of cellular Ect2 localizes to the nucleolus in a PKC?- dependent manner where it regulates ribosomal RNA (rRNA) transcription; 3) PKC? transcriptionally activates cell autonomous hedgehog (Hh) signaling in NSCLC tumor-initiating cells; and 4) PKC? regulates recruitment of the stem cell pluripotency factor Sox2 to th promoter region of the gene encoding Hedgehog Acyl Transferase (HHAT), an enzyme that catalyzes a key step in the production of Hh ligand. Based on these data, we hypothesize that: 1) PKC?-mediated transformation involves regulation of Ect2 nucleolar localization and pre-ribosomal RNA synthesis; 2) Ect2 signaling is required for Kras-mediated BASC transformation and lung tumorigenesis in vivo; 3) PKC? maintains a lung tumor-initiating cell phenotype, at least in part, through Sox2-mediated induction of HHAT transcription and activation of a cell autonomous Hh signaling axis; and 4) HHAT, a PKC?-dependent transcriptional target, plays a key role in lung tumor-initiating activity in vivo. These hypotheses will be tested through completion of four interrelated specific aims to: 1) determine the mechanism by which PKC? and Ect2 regulate ribosomal RNA transcription; 2) assess the role of Ect2 in Kras-mediated lung tumorigenesis; 3) determine the mechanism by which PKC? regulates hedgehog acyl-transferase (HHAT) expression; and 4) assess the role of HHAT in lung tumorigenesis.

DESCRIPTION (provided by applicant): The goals of this project are to elucidate protein kinase C (PKC) signaling mechanisms that contribute to lung cancer initiation and maintenance and translate these mechanistic insights into better prognostic and treatment strategies. We recently identified the atypical PKC? gene PRKCI as a human oncogene in the lung. Furthermore, we find that the mouse PKC? gene Prkci is required for the very earliest steps of lung tumorigenesis induced by oncogenic Kras in vivo. Specifically, Prkci is necessary for Kras-mediated transformation of bronchio-alveolar stem cells (BASCs), the putative cell of origin of Kras-mediated lung tumorigenesis. Genetic disruption of Prkci, or treatment with the PKC9 inhibitor aurothiomalate (ATM), blocks Kras-mediated expansion and morphological transformation of BASC in vitro and in vivo, and lung tumor growth in vivo. Preliminary results suggest that Kras-, Prkci-mediated expansion of BASCs involves induction of the polycomb gene Bmi1. Bmi1 is an epigenetic chromatin modifier/transcriptional regulator implicated in cancer stem cell identity and self-renewal. Like Prkci, Bmi1 is necessary for Kras-mediated BASC expansion and lung tumorigenesis. Based on these preliminary data, we hypothesize that Prkci drives Kras-mediated BASC transformation and lung tumorigenesis, at least in part, through activation of the Bmi1 signaling pathway. We further hypothesize that a similar PKC?-Bmi1 signaling axis plays a critical role in the maintenance, expansion and tumorigenic potential of human lung cancer stem cells. Finally, we hypothesize that ATM will effectively inhibit the self-renewal and tumorigenic potential of human lung cancer stem cells. We will test these hypotheses through completion of two interrelated specific aims. In Aim 1 we will assess the role of the PKC?-Par6-Ect2-Rac1 signaling axis in BASC transformation and dissect the mechanism of crosstalk between PKC? and Bmi1 signaling in these cells. In Aim 2 we will determine the role of PKC? and Bmi1 signaling in the self-renewal and tumor initiating activity of lung cancer stem cells. We will develop and characterize a panel of lung cancer stem cell lines from primary lung tumors. Completion of these studies will provide important new mechanistic insight into oncogenic PKC? signaling in putative lung cancer stem cells, enhance our understanding of Kras-mediated lung tumorigenesis, and assess the efficacy of a novel therapeutic agent that targets a critical oncogenic pathway in lung cancer stem cells. These studies have important implications for PKC? as a therapeutic target and for the use of ATM as a novel therapeutic for the treatment of lung cancer. PUBLIC HEALTH RELEVANCE: Lung cancer is the number one cause of cancer death in the United States. Protein kinase C??(PKC?) is an oncogene, prognostic marker and therapeutic target in lung cancer. This project will elucidate PKC? signaling mechanisms that drive lung cancer stem cell growth and tumor-initiating activity, and determine the efficacy of a novel PKC? inhibitor, aurothiomalate, to block lung cancer stem cell growth and tumorigenicity. Finally, this project will generate novel lung cancer stem cell resources from human lung cancer patients. These tissue resources will be invaluable in the assessment of novel therapeutics targeting these deadly cells.

DESCRIPTION (provided by applicant): More than 200,000 new cases and 160,000 deaths occur from lung cancer annually in the US, most from non-small cell lung cancer (NSCLC). The dismal outlook for patients with advanced NSCLC has prompted a search for more effective treatment strategies. New approaches are targeted against growth factors (EGF, VEGF), gene translocations (EML4-ALK), and oncogenes (RAS), but are applicable most commonly only to non-squamous subtypes of NSCLC. The most promising targeted therapy for squamous histology has had recent significant setbacks when trials of the IGF monoclonal antibody figitumumab failed to improve outcomes compared to chemotherapy alone and was associated with excess toxic deaths in the treatment arm. Since SCC patients account for ~40% of all NSCLC patients, there is a pressing need for more effective treatments. We demonstrated that the atypical PKC isozyme, PKCi, is an oncogene in NSCLC. PKCi is over-expressed in NSCLC cell lines and primary tumors, and PKCi expression may predict poor survival in NSCLC patients. The PKCi gene PRKCI is amplified in ~70% of SCC and PRKCI amplification drives elevated PKCi expression in these tumors. Genetic disruption of PKCi signaling blocks transformed growth of SCC cells in vitro and in vivo. We recently identified a small molecule PKCi inhibitor, aurothiomalate (ATM), which exhibits potent anti-tumor activity in NSCLC cells, particularly SCC cells harboring elevated PKCi expression as a result of PRKCI amplification. ATM disrupts the protein-protein interaction between PKCi and Par6, thereby inhibiting oncogenic PKCi signaling. ATM is FDA-approved for rheumatoid arthritis making it an attractive candidate for clinical development as an anti-tumor agent. We have established a safe dose for ATM in a phase I dose escalation clinical trial in advanced NSCLC patients. In preclinical studies, ATM exhibits synergistic anti-tumor activity against NSCLC tumor growth when combined with the mTOR inhibitor rapamycin. Based on these results, we hypothesize that ATM will be a safe and effective treatment for advanced SCC expressing elevated PKCi when used in combination with the mTOR inhibitor RAD001. This hypothesis will be tested in two interrelated specific aims. In Aim 1 we will conduct a phase IB/II clinical trial to assess the safety and efficacy of combined therapy with ATM and RAD001 in the maintenance of advanced NSCLC patients. In Aim 2 we will assess tumor and circulating blood biomarkers of PKCi and mTOR signaling as predictors of response to combined ATM/mTOR therapy. Successful completion of these aims will provide proof of principle for use of combined PKCi and mTOR targeted therapy in lung cancer and characterize candidate biomarkers that may be useful in identifying patients most likely to respond to this therapy. PUBLIC HEALTH RELEVANCE: Lung cancer is the number one cause of cancer death in the United States with a five year survival rate of approximately 15%. Protein kinase C9 (PKC9) is an oncogene and therapeutic target in lung cancer and a novel small molecule PKC9 inhibitor aurothiomalate (ATM) exhibits potent anti-tumor activity against lung cancer, especially when combined with an mTOR inhibitor. This project will assess the safety and efficacy of combined inhibition of PKC9 with ATM and mTOR with RAD001 in the maintenance therapy of patients with advanced squamous cell carcinoma of the lung, and assess the ability of surrogate markers of PKC9 and mTOR signaling to predict response to therapy.

DESCRIPTION (provided by applicant): Paclitaxel is one of the most commonly prescribed cancer drugs and serves an important role in the treatment of a variety of malignancies. The paclitaxel-induced acute pain syndrome (PIAPS) occurs after paclitaxel administration. Its seminal feature is refractory, untreatable pain. Even opioids are ineffective. Although PIAPS typically remits by day 7, patients suffer from refractory pain one-third of their time on chemotherapy. Our group was the first to prospectively characterize this syndrome from a clinical standpoint, but the pathophysiology of this syndrome remains unknown and remarkably understudied. We hypothesize that the atypical protein kinase C (PKC) isoenzymes, PKC iota and zeta, are key mediators of the PIAPS and that the gold compound auranofin, an inhibitor of of PKC iotal and zeta, prevents or palliates this syndrome. Indeed, 1) the PKC family has been implicated in a host of other musculoskeletal pain syndromes; 2) our preclinical data demonstrate that paclitaxel results in PKC iota activation; and 3) our preclinical and clinical dat also show that auranofin, a drug used to treat autoimmune arthritis, inhibits PKC iota and zeta. Hence, we propose the following specific aims: 1) To measure quantitatively the mRNA expression of PKC iota and zeta in the muscle of patients before and after paclitaxel administration and to determine whether PKC iota or zeta overexpression are associated with the development of the PIAPS (as assessed by patients' completion of the Brief Pain Inventory). 2) To conduct a pilot, randomized controlled trial to test whether auranofin prevents or palliates the PIAPS. Thirty patients (15 per arm) will be randomly assigned to either auranofin (one 6 mg dose) the day after paclitaxel versus placebo. Daily pain scores (Brief Pain Inventory) will be compared between auranofin- and placebo-exposed arms. These two independent but integrated aims -- which include two independent groups of patients for each aim -- will provide long-overdue insight into the pathophysiology of the PIAPS and will potentially position us to mount a future, definitive randomized controlled trial with auranofin to prevent or palliate the PIAPS.

PROJECT SUMMARY ? Project 6 Ovarian cancer remains the deadliest gynecological cancer, underscoring the need for better treatment options. Our published work demonstrates that PKC?, a PKC isoform not targeted by PKC inhibitors previously tested in the clinic, is an oncogene in ovarian and non-small cell lung cancer (NSCLC), where it plays critical roles in tumor initiation and progression. PKC? is also genetically activated in the most common serous ovarian tumor subtype: ~80% of high grade serous ovarian tumors exhibit PRKCI copy number gains associated with elevated PKC? expression. Interestingly, our published and preliminary data demonstrate that PKC? drives tumorigenesis in the ovary by a distinct, novel mechanism not observed in other tumors. Specifically, our preliminary studies indicate that: 1) ovarian cancer cells are ?addicted? to PKC? such that genetic or pharmacologic inhibition of PKC? leads to loss of ovarian cell growth and viability; 2) PKC? maintains a chemoresistant phenotype in ovarian cancer cells characterized by stem-like behavior, enhanced resistance to cisplatin and enhanced tumorigenicity; 3) PKC? activates a novel oncogenic PKC?- SOX2-HIPPO/YAP1 signaling pathway in these chemoresistant tumor-initiating cells (TICs); and 4) a novel small molecule PKC? inhibitor developed in collaboration with a pharmaceutical partner inhibits proliferation and viability of both bulk tumor cells and chemoresistant TICs. Based on these data we hypothesize that: 1) PKC? maintains a chemoresistant phenotype, in part, through activation of HIPPO/YAP1 signaling; 2) a novel PKC? inhibitor will effectively inhibit this pathway and block ovarian cancer cell transformed growth; 3) high grade serous ovarian cancers harboring PRKCI amplification and PKC? overexpression are ?addicted? to PKC? and will, therefore, be more responsive to PKC? inhibition than tumors without this genetic characteristic; 4) components of oncogenic PKC?-SOX2- HIPPO/YAP1 signaling can be developed as pharmacodynamic biomarkers of PKC? inhibition and possible predictive biomarkers of ovarian cancer sensitivity to PKC? inhibitor therapy; 5) a highly potent and specific PKC? inhibitor will effectively inhibit tumor PKC? and exhibit anti-tumor activity in a first-in-human therapeutic trial. These hypotheses will be tested through completion of three interrelated specific aims to: 1) dissect the mechanism by which PKC? regulates chemoresistant ovarian cancer cell behavior and assess the effect of PKC? inhibition on these cells; 2) assess the effect of PKC? inhibitor on primary serous ovarian cancer growth and validate biomarkers of response to PKC? inhibition using well validated patient-derived xenograft models; and 3) assess the ability of a highly potent and specific PKC? inhibitor to inhibit PKC? clinically in a first-in-human phase I clinical trial. Collectively, these studies will provide the first rigorous test of the hypothesis that PKC? can be targeted for therapeutic benefit in ovarian cancer.

? DESCRIPTION (provided by applicant): Lung cancer is the leading cause of cancer death in the US, exhibiting a dismal five year survival rate of ~16%, underscoring the need for new therapeutic approaches. The recent development of targeted therapeutics that effectively treat lung cancer subtypes harboring specific driver mutations in EGFR, MET and EML4-ALK have made important inroads in treating these specific subsets of lung cancer. Despite these key advances, treatment options for mutant KRAS-driven lung adenocarcinoma (KRAS LADC), the most prevalent form of lung cancer, remain limited. We have identified protein kinase C? (PKC?) as an oncogene in KRAS LADC and lung squamous cell carcinoma (LSCC), the two major forms of non-small cell lung cancer (NSCLC). PKC? functions to maintain a tumor-initiating cell (TIC) phenotype in both of these NSCLC tumor types. Surprisingly however, our published and preliminary studies demonstrate that PKC? drives a TIC phenotype in KRAS LADC and LSCC through distinct signaling pathways. Preliminary data demonstrate that: 1) PKC? drives a KRAS LADC TIC phenotype by activating expression of the pluripotent stem factor NOTCH3; 2) PKC? activates NOTCH3 expression by recruiting the ELF3 transcription factor to the NOTCH3 promoter; 3) PKC? phosphorylates ELF3 at Ser68 to regulate ELF3 occupancy and activation of the NOTCH3 promoter; 4) a newly identified, highly potent and selective PKC? inhibitor inhibits LADC TIC cell behavior in vitro. Based on these data, we hypothesize that: 1) PKC? regulates ELF3 promoter occupancy on NOTCH3 and other gene targets involved in maintaining a LADC TIC phenotype; 2) the novel PKC?-ELF3-NOTCH3 signaling axis drives Kras-mediated LADC initiation and progression in mouse models of Kras LADC; 3) our novel, potent and highly selective PKC? inhibitor will exhibit anti-tumor activity in KRAS LADC, and PKC?-ELF3-NOTCH3 signaling intermediates will be useful predictive and pharmacodynamic biomarkers of response. These hypotheses will be tested in three interrelated specific aims to: 1) assess the role of PKC?-mediated ELF3 phosphorylation in NOTCH3 promoter occupancy, NOTCH3 expression and LADC TIC behavior; 2) assess the role of the PKC?- ELF3-NOTCH3 signaling axis in mutant Kras-mediated lung tumor initiation and progression; and 3) assess the potential of a novel highly potent and selective PKC? inhibitor as a therapeutic strategy for treatment of KRAS LADC. Successful completion of these aims will: 1) provide new mechanistic insight into the newly- identified PKC?-ELF3-NOTCH3 signaling axis; 2) assess the importance of this pathway in the maintenance of a LADC TIC phenotype; 3) identify novel targets for PKC?-ELF3-dependent transcriptional regulation; 4) assess the importance of the PKC?-ELF3-NOTCH3 signaling axis in LADC tumor initiation and maintenance; and 5) assess the utility of a newly-developed, highly potent PKC? inhibitor in the treatment of KRAS LADC. Given the poor clinical outcome of patients with KRAS LADC, and the dearth of therapeutic options, these studies may have widespread impact on the clinical management of these patients.

SUPPLEMENT PROJECT SUMMARY Lung squamous cell carcinoma (LSCC) is a major sub-type of lung cancer. LSCC tumors can be further sub- classified into four distinct molecular subtypes: primitive, classical, secretory and basal based on expression profiling. The LSCC molecular subtypes share similarities in gene expression profiles with distinct lung epithelial cell populations suggesting that the LSCC molecular subtypes may originate from different lung epithelial cell populations. Genomic analysis of human LSCC pre-neoplastic lesions and tumors demonstrates that 3q26 copy number gains (CNGs) and TP53 mutations are among the most prevalent early genetic alterations in LSCC, suggesting that these genetic events play an important role in the initiation and progression of LSCC. Furthermore, interrogation of The Cancer Genome Atlas (TCGA) LSCC database reveals that 3q26 CNGs are present in all four LSCC molecular subtypes suggesting that 3q26 CNGs may play a role in the molecular signaling pathways that drive the pathogenesis of these molecular subtypes. Our studies have shown that the genes SOX2, PRKCI and ECT2 are key targets of 3q26 CNGs that functionally collaborate in maintaining the transformed phenotype of human LSCC cells. Our more recent studies have demonstrated that overexpression of SOX2, PRKCI and ECT2, in the context of Trp53 loss, is sufficient to; 1) transform mouse lung basal stem cells (LBSCs) (a cell of origin for LSCC); and 2) drive formation of LBSC tumors with LSCC-like histopathology in vivo. Based on these observations, we hypothesize that co- overexpression of SOX2, PRKCI and ECT2 and Trp53 loss can cooperate to transform other putative LSCC cells of origin to develop LSCC-like tumors that exhibit signaling pathways observed in the four human LSCC molecular subtypes. This hypothesis will be tested through two interrelated specific aims designed to; 1) assess cooperative oncogenic activities of SOX2, PRKCI and ECT2 on the transformation of putative LSCC cells of origin; and 2) determine the mechanisms underlying the roles of SOX2, PRKCI and ECT2 in lung epithelial cell transformation and LSCC development. Successful completion of these aims will provide important mechanistic insight into the molecular heterogeneity of LSCC and offer novel pre-clinical models to identify and evaluate therapeutic targets in distinct molecular subtypes of LSCC.

Project Abstract Lung cancer is the number one cause of cancer death in the United States. Non-small cell lung cancer (NSCLC), which accounts for the majority (80%) of lung cancer diagnoses, is divided into two major sub-types, lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LUSC). New therapeutic strategies targeting major oncogenic drivers of LADC have led to improved response rates and patient survival. However, due to a lack of well-characterized, validated, and therapeutically-actionable oncogenic drivers, similar therapeutic advances for LUSC have not been forthcoming. Genomic analysis of primary human LUSC reveals that the most prevalent recurrent genetic alterations in LUSC are concurrent loss of TP53 and copy number gains (CNGs) at chromosome 3q26 (>85-90%). These genetic alterations are observed in both precancerous lesions and early-stage LUSC, indicating they are early promotive events in LUSC tumorigenesis. Our previous studies have demonstrated that the 3q26 genes, PRKCI, SOX2 and ECT2 are coordinately co-amplified and functionally collaborate to maintain the transformed phenotype of LUSC cells. PKC?, the protein product for PRKCI, directly phosphorylates and regulates the oncogenic function of SOX2 and ECT2 in LUSC cells. Furthermore, our preliminary data demonstrate that: 1) overexpression of SOX2, PRKCI and ECT2, in the context of Trp53 loss, is sufficient to transform mouse lung basal stem cells (LBSCs), a major cell of origin for LUSC, and drive formation of tumors with malignant LUSC characteristics; 2) PKC?-SOX2 signaling activates a master transcriptional program specifying lineage-restricted lung squamous transformation; 3) PKC?-ECT2 signaling functions to increase the proliferative potential of LUSC cells through control of MEK-ERK signaling and enhanced ribosome biogenesis; and 4) both human LUSC cell lines with 3q26 CNGs and SOX2, PRKCI and ECT2 transformed LBSCs are sensitive to the growth inhibitory effects of Auranofin (ANF), a potent PKC? inhibitor, and to small molecule inhibitors of oncogenic PKC?-SOX2 and PKC?-ECT2 driven effector pathways including Hedgehog, WNT, NOTCH, MEK-ERK and rRNA synthesis. Based on these data we hypothesize that: 1) PRKCI, SOX2 and ECT2 represent cooperative multigenic drivers of LUSC tumorigenesis; and 2) combined treatment with ANF and inhibitors of PKC?-SOX2 and PKC?-ECT2 effector pathways will synergistically inhibit transformed growth of LUSC tumors. These hypotheses will be tested through completion of two interrelated specific aims designed to: 1) evaluate the efficacy of novel drug combinations that target oncogenic PKC? effector pathways; and 2) characterize a novel genetically- tractable mouse model for PRKCI-driven LUSC. Successful completion of these studies will facilitate the design of novel therapeutic strategies to improve outcomes for patients with LUSC. Furthermore, our novel clinically-relevant genetically-engineered LUSC mouse model will enhance our understanding of LUSC biology, characterize LUSC initiation and progression from preneoplastic lesions to malignant LUSC, identify markers for early LUSC diagnosis and develop and test novel targeted therapies for improved treatment of LUSC.