Diane M. Simeone - US grants

Transforming growth factor- beta (TGFbeta) is a potent inhibitor of pancreatic cell proliferation. Disruption of the TGFBeta signaling pathway may be important in the development of pancreatic cancer. Recently, deleted in pancreatic carcinoma 4 (DPC4) was identified as a tumor suppressor gene that is inactivatated in 50% of pancreatic tumors. DPC4 (also known as Smad4) act as signaling molecules in TGFBeta-related signaling pathways. Many human pancreatic cancer cell lines are refractory to the growth inhibitory effects of TGF-beta. In this proposal, we will examine the role of Smad genes in TGFBeta- mediated growth regulation in the pancreas and in the development of pancreatic cancer. We have hypothesized that: 1) Smad genes are required for growth inhibition and regulation of differentiation by TGFBeta in pancreatic acinar and duct cells; 2) TGFBeta modulates Smad acitivity by interacting with other signaling pathways, including the MAP kinaase signaling pathway; 3) dominant negative Smads stably transfected into immortalized pancreatic cells will induce tumorigenesis; and 4) functional inactivation of Smad gnes in the pancreas in a transgenic mouse model results in uncontrolled cell proliferation. It is likely that the results of the proposed studies will reveal valuable insight into TGFBeta- mediated growth regulatory mechanism in the pancreas and the role of Smad proteins in neoplastic transformation of the pancreas.

Pancreatic cancer is one of the major unsolved problems in general surgery today. Pancreatic cancer has a dismal 5 year of less than 5%, and while the safety of surgery has improved, surgical treatment has not had a significant imapct on survival. Only a better understanding of the basic biology of pancreatic tumorigenesis will allow effective treatment of this disease. TGFbeta is an important regulator of pancreatic growth and differentiation. Recently, deleted in pancreatic carcinoma 4 (DPC4) was identified as a tumor suppressor gene that is inactivated in 50% of pancreatic tumors. DPC4 (also known as Smad4) is a signaling molecule in TGFbeta -related signaling pathways. Many human pancreatic cancer cell lines are refractory to growth inhibitory effects of TGFbeta. While the TGFbeta signaling pathway has been studied in several cultured cell lines, TGFbeta affects a wide variety of cellular responses and significant differences exist in this in this complex signaling pathway in different cells. The intracellular signaling pathways through which TGFbeta acts to generate cellular responses in the pancreas remain largely undefined. The pancreas, with its high incidence of mutations in this pathway and the likely importance of Smads in pancreatic, is arguably one of the most important tissues in which to investigate this pathway. The current research proposal is designed to investigate, on a fundamental level, TGFbeta signaling and the role of Smads in pancreatic acinar cells. We have hypothesized that: 1) Smad genes are required for growth inhibition and regulation of differentiation by TGFbeta in pancreatic in acinar cells; 2) MAP kinases interact with TGFbeta/Smad signaling; 3) TGFbeta- mediated induction of transcription factors in pancreatic acinar cells is regulated by both the Smad and MAP kinase signaling pathways; and 4) functional inactivation of Smad4 results in abnormal proliferation and dedifferentiation in the pancreas in vivo. These studies will help to elucidate the molecular events by which TGFbeta regulates growth and differentiation in normal pancreatic tissues and will likely reveal crucial insight into the mechanisms important in neoplatstic in the pancreas. This grant proposal is submitted for the Small Grants Program for K08 Recipients-NIH K08 DK02637-01.

DESCRIPTION (provided by applicant): Recent evidence indicates that perturbations in the TGFbeta signaling pathway plays a crucial role in the development of surgical pancreatic disease, including pancreatic cancer and pancreatitis. However, the mechanisms of TGFbeta signaling in the human pancreas are largely unknown. This lack of basic information is a major obstacle to rational treatment of human pancreatic diseases, and is reflected in the current empiric, and often ineffective treatment of human exocrine pancreatic cancer and pancreatitis. Recently, we have identified a novel signaling pathway for TGF[3 in the pancreas whereby the obligate second messengers of the TGFbeta signaling pathway, the Smads, activate protein kinase A (PKA) in pancreatic acinar cells. This may represent an important mechanism of crosstalk within the acinar cell. Our preliminary data demonstrates a novel interaction of Smads and the regulatory subunit of PKA within pancreatic acinar cells, and also suggests that PICA may mediate growth inhibitory responses induced by TGFbeta. Therefore we hypothesize that TGF's physiological effects on growth are mediated by Smads and their interactions with the PKA signaling pathway. This research proposal is designed to investigate the molecular basis for the interaction of TGFI3 signaling molecules with the PKA signaling pathway. The experiments will address the hypothesis that Smads3 and 4 directly bind to and activate PKA by a previously unidentified, cAMP-independent mechanism, and will determine interaction domains of Smads 3 and 4 and the regulatory subunit of PKA. In addition, we will investigate the role of PKA in TGFbeta-mediated growth inhibition in pancreatic acinar cells. The proposed research integrates physiological studies with novel molecular strategies, an approach likely to reveal important new insights. The long term goal of this project is to gain a detailed understanding of TGF signaling mechanisms in the pancreas that may be of benefit in the treatment and/or prevention of human pancreatic disease.

DESCRIPTION (provided by applicant): Recent evidence indicates that perturbations in the TGFbeta signaling pathway plays a crucial role in the development of surgical pancreatic disease, including pancreatic cancer and pancreatitis. However, the mechanisms of TGFbeta signaling in the human pancreas are largely unknown. This lack of basic information is a major obstacle to rational treatment of human pancreatic diseases, and is reflected in the current empiric, and often ineffective treatment of human exocrine pancreatic cancer and pancreatitis. Recently, we have identified a novel signaling pathway for TGF[3 in the pancreas whereby the obligate second messengers of the TGFbeta signaling pathway, the Smads, activate protein kinase A (PKA) in pancreatic acinar cells. This may represent an important mechanism of crosstalk within the acinar cell. Our preliminary data demonstrates a novel interaction of Smads and the regulatory subunit of PKA within pancreatic acinar cells, and also suggests that PICA may mediate growth inhibitory responses induced by TGFbeta. Therefore we hypothesize that TGF's physiological effects on growth are mediated by Smads and their interactions with the PKA signaling pathway. This research proposal is designed to investigate the molecular basis for the interaction of TGFI3 signaling molecules with the PKA signaling pathway. The experiments will address the hypothesis that Smads3 and 4 directly bind to and activate PKA by a previously unidentified, cAMP-independent mechanism, and will determine interaction domains of Smads 3 and 4 and the regulatory subunit of PKA. In addition, we will investigate the role of PKA in TGFbeta-mediated growth inhibition in pancreatic acinar cells. The proposed research integrates physiological studies with novel molecular strategies, an approach likely to reveal important new insights. The long term goal of this project is to gain a detailed understanding of TGF signaling mechanisms in the pancreas that may be of benefit in the treatment and/or prevention of human pancreatic disease.

DESCRIPTION (provided by applicant): Pancreatic cancer is a deadly disease characterized by late diagnosis, aggressive invasion of surrounding tissues, early metastasis, and resistance to therapy. There is an urgent need to develop highly effective, mechanism-based therapies to improve survival in these patients. We have recently found that the majority of human pancreatic adenocarcinomas specifically over-express the gene for Ataxia-Telangiectasia Group D Associated (ATDC). The ATDC gene was initially described in association with the genetic disorder ataxia-telangiectasia (AT) but was later found not to be the gene responsible for that disorder, and it's function remained unknown. We have identified ATDC as a novel DNA damage response gene that confers a survival advantage to pancreatic cancer cells when exposed to chemotherapy. We have shown that following DNA damage, ATDC traffics to the nucleus, is phosphorylated in response to gemcitabine and localizes to DNA repair foci. Loss of ATDC results in increased sensitivity to gemcitabine-induced apoptosis and a defect in downstream cell cycle checkpoint signaling. We have also found that high levels of ATDC confer a growth advantage to pancreatic cancer cells both in vitro and in vivo. The ATDC-mediated stimulation of cell proliferation may be due to enhancement of the beta-catenin pathway since overexpression of ATDC increases beta-catenin mediated transcription. We demonstrate that ATDC interacts with the HIT family protein HINT1, a negative regulator of the beta-catenin pathway, and we hypthesize that ATDC stimulates beta-catenin-mediated proliferation by sequestering HINT1. In this proposal, we will explore the following specific aims: 1) To examine the role of ATDC in the ATR-mediated DNA damage response. 2) To assess if ATDC's tumor promoting ability is linked to stimulation of the beta-catenin pathway through interactions with the HIT1 family protein HINT1. 3) To analyze the efficacy of targeting ATDC as a therapeutic modality in a pre-clinical, primary human pancreatic cancer orthotopic xenograft model. PUBLIC HEALTH RELEVANCE: We propose that ATDC is a promising novel therapeutic target in pancreatic cancer because it's inactivation may lead to both reduced tumor growth and sensitization to chemotherapy.

Pancreatic cancer is a deadly disease characterized by late diagnosis, aggressive invasion of sun'ounding tissues, eariy metastasis, and resistance to therapy. The molecular basis of pancreatic cancer is incompletely understood. We have recently found that the majority of human pancreatic adenocarcinomas specifically over-express the gene for Ataxia-Telangiectasia Group 0 Complemented (ATDC). The ATDC gene was initially described in association with the genetic disorder ataxia-telangiectasia (AT) but was later found not to be the gene responsible for that disorder, and it's function remained unknown. We have found that high levels of expression of endogenous ATDC confer a growth advantage of pancreatic cancer cells both in vitro and in vivo by stabilization of beta-catenin. We have also identified ATDC as a novel DNA damage response gene that confers a survival advantage to pancreatic cancer cells exposed to iradiation therapy (RT) or the chemotherapeutic drug gemcitabine which are agents used for standard care of pancreatic cancer patients. We show that ATDC traffics to the nucleus and that loss of ATDC results in radioresistant DNA synthesis and a defect in downstream cell cycle checkpoint activation signaling. In this proposal, we will investigate the molecular mechanisms by which ATDC functions in the response to the combination of ionzing gemcitabine and RT. The experiments will test the hypothesis that ATDC is an important stress response regulator in both ATM- and ATR-mediated signaling cascades. Furthermore, we will analyze the effect of targeting ATDC in combination with gemcitabine and RT as a therapeutic modality in pancreatic cancer using a xenograft mouse model and immunoliposomes canning ATDC-targeting shRNA. The results from these preclinical animal studies will be used as a guide in the development of a clinical trial where ATDC will be targeted in pancreatic cancer cells prior to standard treatment with gemcitabine and RT. We propose that ATDC is a promising novel therapeutic target for both slowing the growth of pancreatic tumors as well as making them more susceptible to treatment with the combination of gemcitabine and RT.

DESCRIPTION (provided by applicant): Bladder cancer is a common and deadly disease, but the molecular events leading to its development are incompletely understood. We have recently identified a novel oncogene, Ataxia-Telangiectasia Group D Complementing (ATDC), which drives initiation and progression of bladder tumors in transgenic mice. These tumors are indistinguishable from their human counterparts. ATDC expression is elevated (about 70%) in human invasive bladder cancers and correlates with poorer survival after chemotherapy. In other tumor types, ATDC binds p53, modulates DNA damage responses and up-regulates beta-catenin signaling. In preliminary data, we find that ATDC expression is induced by inflammation, associated with decreased expression of PTEN and RB1, two important tumor suppressor genes implicated in bladder tumorigenesis, and show that ATDC promotes methylation and silencing of the PTEN promoter through upregulation of DNMT3a. We hypothesize that ATDC is a crucial determinant of formation, progression and the cytotoxic response in bladder cancer. To better understand the role of ATDC in bladder cancer, we propose the following studies: AIM 1: To characterize the role of ATDC in bladder cancer initiation, progression and metastasis using novel transgenic mouse models and determine the role of inflammation in inducing ATDC expression in normal urothelium. AIM 2: To characterize the role of PTEN, RB1 and p53 in ATDC-mediated bladder tumor formation and progression using both human and mouse model systems. AIM 3: To determine if ATDC expression in bladder cancer drives progression to invasive disease. AIM 4: To determine if ATDC expression promotes bladder tumor resistance to chemotherapy. These studies will characterize a novel mechanism of bladder tumor development and a valuable transgenic model of bladder cancer. We will also use existing human cell lines and primary human tumor samples to elucidate the molecular mechanisms by which ATDC induces bladder cancer and mediates resistance to chemotherapy. These studies are significant because understanding ATDC's oncogenic activity in bladder cancer may lead to new prognostic biomarkers and improved therapeutic approaches in humans.

? DESCRIPTION (provided by applicant): Pancreatic adenocarcinoma (PDA) is a deadly disease characterized by late diagnosis, aggressive invasion of surrounding tissues, and early metastasis. Patients with stage 1A disease have a 30% 5 year survival rate following resection, highlighting that PDA metastasizes early in patients, however, the molecular mechanisms driving early metastasis in PDA are not understood. We have recently found that the majority of human pancreatic adenocarcinomas have elevated expression of the Ataxia-Telangiectasia Group D Associated (ATDC) gene and that ATDC has an oncogenic function in pancreatic cancer through Wnt pathway activation and ?-catenin stabilization. Using newly created genetically engineered mouse models of pancreatic cancer, we have now identified a previously unknown role for ATDC in pancreatic cancer, where ATDC is upregulated during the early PanIN stage of tumorigenesis, and in the presence of oncogenic Kras, accelerates PanIN progression and the development of invasive and metastatic tumors. ATDC upregulates CD44 in mouse and human PanIN lesions through a ?-catenin-induced mechanism, resulting in the induction of an epithelial to mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. Knockdown of ATDC blocks invasion in mouse and human PDA cells and this effect is mediated through ?-catenin and CD44. In this proposal, we hypothesize that ATDC is a proximal regulator of EMT and drives progression and metastasis of pancreatic cancer, thus serving as an important therapeutic target. To dissect the role of ATDC in pancreatic cancer, we will: 1) investigate the role of ATDC in pancreatic tumorigenesis using novel genetically engineered mouse models; 2) characterize the molecular mechanisms by which ATDC induces an invasive phenotype in PDA; and 3) determine the mechanism of ATDC upregulation in pancreatic cancer.

PROJECT 3: SUMMARY The major goal of this project is to study the molecular mechanisms that underlie the critical steps of bladder cancer (BC) progression: invasion and metastasis. Specifically, we will focus on how autophagy-related gene 7 (ATG7)-mediated autophagy signaling drives BC cell invasion in vitro and metastasis in vivo. During the last funding period, we found that basal-subtype muscle-invasive bladder cancer (MIBC) in mice induced by bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) markedly overexpress ATG7 and long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1), and have significantly upregulated autophagy. In stark contrast, knockin mice lacking the RING domain of XIAP, which are completely resistant to BBN-induced basal MIBCs, have markedly reduced autophagy. We also found that, during BBN-mediated bladder tumorigenesis, the RING domain of XIAP is essential for SNHG1 overexpression, and that ectopic expression of SNHG1 in vitro induces autophagy and promotes BC cell invasion accompanied by upregulated ATG7, MMP2 and MMP9. Furthermore, we showed that knockdown of ATG7 strongly inhibits autophagy, abolishes BC cell invasion and reduces the expression of basal MIBC marker KRT14. These data reveal a heretofore unknown role of autophagy in basal MIBC formation. Based on these data, we hypothesize that the upregulation of SNHG1 and ATG7 by the RING domain of XIAP, and the autophagic signaling that these molecules trigger play critical roles in the genesis and progression of basal MIBC. We will test this hypothesis in three Specific Aims. Aim 1 will define the regulatory circuitry in the SNHG1/ATG7/autophagy signaling axis that is operative in basal MIBC in vitro. Aim 2 will determine the biological effects of the SNHG1/ATG7/autophagy signaling on BC cell invasion in vitro and tumorigenesis and metastasis in vivo. Aim 3 will test the hypotheses that overexpression of SNHG1 in basal urothelial cells of transgenic mice promotes basal MIBC formation, and that ablation of ATG7 in these cells of knockout mice renders mice resistant to basal MIBC formation and progression. These complementary approaches will provide definitive evidence regarding the in vivo roles of SNHG1 and ATG7 in the formation and progression of basal MIBC. While invasion and metastasis are the main reasons of the high mortality caused by MIBC, very little is known about the principal molecules or pathways that drive these crucially important biological processes. Our proposed studies that are highly focused on an important, but poorly understood signaling pathway comprising SNHG1/ATG7/autophagy should yield critical information on not only the underlying mechanisms, but also novel prognostic biomarkers to differentiate MIBC subtypes and new druggable targets to treat this aggressive form of BC.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal human malignancy, typically diagnosed at an advanced stage and known to be largely unresponsive to chemotherapy and ionizing radiation. Recent genomic characterization of PDA reveals that between 20-25 % of PDA harbor recurrent mutations in genes, including BRCA1/2, PALB2, and ATM, which are critical for homologous recombination (HR), an important form of DNA repair. In many patients, these may be germline mutations. This subgroup of PDAs, termed HR-deficient PDA, has emerged as a defined biological entity associated with increased chemoresistance and a more aggressive disease course. The defects in HR observed in these tumors impart cells with a specific vulnerability to PARP inhibitors and platinum-containing therapy. Still, as observed in the case of many other targeted therapies, only a fraction of HR-defective patient tumors respond to PARP inhibition. More so, many patients that initially respond eventually often develop resistance and progress. Therefore, novel therapies which can be effective against HR- defective PDA, alone or in combination with PARP inhibitors or other combinatorial regimens, are urgently needed. We have recently determined that inactivation of the HR pathway is associated with overexpression of polymerase theta (Pol?, also known as POLQ) in PDA. POLQ is a key enzyme that regulates an alternative pathway of DNA repair, known as the alternative non-homologous end-joining (Alt-NHEJ) pathway. Data from our group indicates that in the setting of defective HR, Alt-NHEJ becomes a critical pathway responsible for the repair of DNA breaks. Furthermore, we show that POLQ inhibition in HR-defective tumor cells demonstrates a synthetic lethality phenotype, not observed in cells with intact HR. In this proposal, we present exciting new data that knockdown of POLQ is synthetically lethal in PDA cells deficient for Brca1, Brca2, Atm, and Palb2 genes. POLQ knockdown significantly inhibited growth of both Brca2- and Atm-deficient tumors cells in vivo. Further, POLQ knockdown significantly upregulated the cGAS-STING pathway in HR-deficient PDA and promoted T cell infiltration. Here, we plan to examine the unique role of POLQ in pancreatic cancer biology and its role as a novel therapeutic target in HR-defective pancreatic cancers. We will also evaluate the antitumor effect of combining POLQ inhibition with: i) current standard cytotoxic chemotherapies, ii) PARP inhibition, and iii) immunotherapy. An important goal of this proposal is to generate a set of data for proof-of-concept that targeting POLQ in a valuable therapeutic strategy in HR-defective pancreatic cancer, as POLQ inhibitors are currently in development for clinical use.

Summary Pancreatic ductal adenocarcinoma (PDA) is the 5th leading cancer diagnosis in the USA and is highly lethal. There are no effective means to prevent or delay PDA onset and few effective treatment options exist once transformation has occurred. Bacterial dysbiosis is emerging as an accomplice to carcinogenesis in extra- pancreatic malignancies such as colon and liver cancer. Nevertheless, the gut microbiome has not been clearly implicated in carcinomas remote from the intestinal lumen or its drainage. There are tangential data, however, that support an association between PDA and gut bacteria. For example, the oral microbiome in patients with PDA has been found to substantially differ from control subjects. Further, our laboratories have obtained critical preliminary data which suggest that the intestinal microbiome may be an important regulator of PDA development in genetically predisposed hosts: (i) We found that intraluminal gut bacteria can directly access the pancreas; (ii) The relative abundance of select bacterial taxa was higher in the gut of PDA patients compared with healthy individuals; (iii) Specific pattern recognition receptors (PRRs), which transduce inflammation in response to microbial pathogens, are highly expressed in the PDA tumor microenvironment (TME) and activation of these receptors accelerates tumorigenesis whereas mice deficient in select PRRs have slower progression of PDA; (iv) Endo-luminal administration of pathogenic bacteria accelerates tumorigenesis in genetically predisposed mice whereas germ-free mice are protected from PDA. (v) Select bacterial byproducts induce recruitment of regulatory T cells, M2-polarized macrophages, and myeloid derived suppressor cells to the PDA TME. Based on these data, we postulate that pathogenic gut bacteria drive pancreatic oncogenesis in at-risk hosts via activation of specific PRRs which leads to pathogen-induced immune suppression. In Aim 1 we will define the gut and intra-pancreatic microbiome in mice developing PDA compared with WT mice, we will contrast the microbial phenotypes associated with aggressive PDA compared with slowly progressive cancer in genotypically identical hosts, and determine whether humans with PDA harbor a distinct gut and pancreatic microbiome compared with age-matched control patients. In Aim 2, we will test our hypothesis that gut microbes influence oncogenesis by examining whether directly modulating the microbiome alters the rate of tumor progression. As such, we will identify the specific endoluminal bacteria which affect the progression of pancreatic oncogenesis. In Aim 3, we will test our hypothesis that select luminal pathogens promote pancreatic tumorigenesis by inducing immune suppression via ligation of select Toll-like receptors and NOD-like receptors. Our experiments will provide critical new information on the mechanism of pancreatic oncogenesis. We will also identify novel risk factors and provide guidance for the development of innovative therapeutics aimed at PDA prevention in at-risk hosts.

Summary This is the first application for renewal of T32 CA193111 to continue funding the training of next generation of physician-scientists in gastrointestinal oncology. We believe that physician-scientists armed with rigorous research training are necessary to discover and translate new advances in GI oncology as well as to investigate critical issues facing delivery of cancer care to patients. Our trainees are drawn from NYU?s renowned clinical fellowship programs in Medical Oncology or Gastroenterology or top-notch residency programs in Radiation Oncology or Surgery. Trainees take a 2-3 year hiatus from clinical activities to engage in intensive full-time training in GI cancer research. Our program is composed of (i) a Basic/Translational Science track aimed at individuals who wish to direct their own basic science laboratory in GI oncology or individuals who want to lead translational research programs that comfortably interface between the pre-clinical laboratory and clinical investigation and (ii) a Population Sciences tract for trainees who seek careers in the diverse fields of population sciences including cancer epidemiology, comparative effectiveness, disparities, health services, or outcomes research. The program for our Basic/Translational Science track consists of two years of individualized mentored research in faculty laboratories, enhanced by a strong parallel didactic program in cancer biology. The Population Sciences track is centered on a rigorous Masters-level curriculum that teaches clinical trial design, epidemiology, comparative-effectiveness, and outcomes research methodologies along with a mentored research project based in population sciences. The two tracks are united through a weekly 2-hour core curriculum covering essential topics in GI cancer and critical lessons for cancer physicians embarking on independent investigative careers. We plan to continue to have a steady-state of 4 trainees at any given time. Our first group of graduates have been very successful in acquiring the research skills and credentials to eventually lead their own research groups. We are proud that our first graduates have obtained academic positions in oncology and submitted highly scored K grants. Our trainees are nurtured by a cadre of mentors with substantial accomplishments in cancer research and stellar training records. The aim of our training program is to continue to graduate physician- scientists armed with the intellectual capacity and tools to lead their own research programs and become leaders and role models in investigative GI oncology.