Surinder K. Batra - US grants

Mucins have been implicated in the pathogenesis of benign and malignant diseases of secretory epithelial cells; however, the molecular basis of the alterations that occur in mucins during different diseases is poorly understood. We cloned a mucin gene from a cDNA library prepared from the human pancreatic tumor cell line HPAF, that showed identity to the published partial sequence of MUC4 cDNA. MUC4 showed differential and regulated expression in several pancreatic tumor cell lines but undetectable expression in normal pancreas. This leads us to propose the hypothesis that MUC4 expression is involved in the general pathogenesis and metastatic spread of pancreatic adenocarcinoma. The objectives of this proposal are to define the structure and regulation of human MUC4 apomucin gene. The recently derived cDNA fragments will be used to isolate the full-length cDNA and regulatory sequences for the MUC4 gene. Specifically, we propose to: 1) Clone and characterize the full-length MUC4 cDNA. The cDNA and gene containing MUC4 coding and regulatory sequences will be cloned and characterized. 2)Investigate the role of regulated expression of MUC4 in human pancreatic tumor cells. Pancreatic cell lines expressing regulated levels of MUC4 will be used as a model to evaluate the role of growth factor(s) in regulating MUC4 expression. The effects of MUC4 expression on the growth kinetics, metastatic properties and morphology of the MUC4 cDNA transfected cells will be investigated. 3) Generate and characterize monoclonal antibodies to the recombinant MUC4 protein and cell lines expressing transfected cloned MUC4 cDNA. Antibodies will be characterized for specificity for mucins, affinity for the antigen and reactivity against Pancreatic tumor cells. The proposed studies will determine the structure and function of the MUC4 mucin in general and may define a role for MUC4 in the pathogenesis of human pancreatic neoplasia. Characterization of this gene and its product will aid in the development of probes and antibodies that may be useful for diagnosis and treatment of pancreatic adenocarcinomas.

DESCRIPTION (provided by applicant): Pancreatic cancer (PC) is a disease of insidious progression and high lethality, with the majority of tumors having extended beyond the confines of the pancreas at the time of diagnosis. The early diagnosis of PC remains a challenging endeavor that will require multidisciplinary approaches, including imaging studies, tissue sampling, and analysis of body fluids from patients. There is no specific tumor marker available for diagnosing pancreatic cancer. The principal reason for this grim prognosis is our inability to diagnose the disease at an early, localized, and potentially curable stage. In an extensive study on the expression analysis of various mucin (MUC) genes, we demonstrated specific and differential expression of MUC4 in pancreatic adenocarcinomas as compared to the normal pancreas or chronic pancreatitis tissues. The central hypothesis of this proposal is that the MUC4 is detected in peripheral blood mononuclear cells (PBMCs) and fine needle aspirates (FNAs) of pancreatic cancer patients but not of healthy individuals or those with non- neoplastic pancreatic diseases. Three specific aims are proposed. . Aim I will compare the expression of MUC4 by real-time RT-PCR in PBMCs of patients with pancreatic cancer, pre-malignant pancreatic diseases, pancreatitis and aged-matched healthy subjects. This aim will establish the sensitivity, specificity, accuracy and diagnostic potential of MUC4 mRNA in PBMCs. Additionally, it will determine whether the detection of MUC4 mRNA in blood correlates with tumor stage, tumor progression, recurrence or response to therapy. Aim II will identify the cell types in PBMCs that are positive for MUC4. A subset of patient's PBMCs will be FACS-analyzed to investigate the nature of the cell type(s) that express MUC4 mRNA. We will investigate the possibilities that MUC4 in PBMCs is from: i) circulating pancreatic tumor cells; ii) circulating dendritic cells or macrophages that have acquired MUC4 mRNA through phagocytosis or pinocytosis of primary or metastatic tumor cells; or iii) PBMCs that have turned on the expression of MUC4. Aim III will investigate the expression of MUC4 and nanostructural cytological changes in fine needle aspirates (FNAs) from PC patients and attempt to distinguish them from chronic pancreatitis and other benign diseases using immunocytochemical and partial wave spectroscopic (PWS) analyses. MUC4 protein will be measured in FNAs and will be correlated with the sensitivity and specificity of diagnosis. In addition, partial wave spectroscopy (PWS) will be utilized to improve the ability of cytological examination on FNAs to distinguish PC cells from non-neoplastic cells. Associating PWS signatures with MUC4 expression will further assist in disease classification and planning/formulating management strategies. Taken together, these aims will establish the utility of MUC4 in the diagnosis of lethal pancreatic cancer. PUBLIC HEALTH RELEVANCE: The overall goal of this study is to develop a novel molecular diagnostic test(s) for lethal pancreatic cancer. We will investigate the diagnostic utility of MUC4 detection in peripheral blood mononuclear cells and fine needle aspirates of pancreatic cancer patients with high sensitivity and specificity. The proposed investigations are expected to improve the current diagnosis of pancreatic cancer and can easily be translated to clinics after successful multi-center evaluation.

ABSTRACT Pancreatic cancer (PC) is a disease of insidious progression and high lethality. The survival of patients with PC is less than 5% over the period of 5 years. The etiology of PC is associated with inherited gene mutations and polymorphisms (genetic susceptibility), lifestyle-related factors, such as high caloric intake, high-fat diet and smoking. Despite epidemiological evidence suggesting an association of cigarette smoking with pancreatic malignancy, the molecular consequences of cigarette smoking and nicotine (an important constituent of cigarette smoke) leading to cancerous form of the pancreas are not clear. Recently, we have observed that cigarette smoke inhalation induces a chronic pancreatic inflammatory process with fibrosis and scarring of pancreatic structures. Furthermore, we have found altered expression of genes involved in the function of the pancreas in smoke-treated rats. In parallel studies, we detected MUC4 mucin in histologically identifiable intra-ductal lesions known as Pancreatic Intraepithelial Neoplasias (PanINs), and the expression of MUC4 increased progressively with .tumorigenic and malignant phenotype. The oncogenic potential of MUC4 was also observed in the NIH3T3 mouse fibroblast cells. Interestingly, our studies have also revealed that MUC4 regulates the expression of HER2 by post-transcriptional mechanism(s). The overall objective of this research proposal is to investigate the pathological consequences of cigarette smoking toward the initiation and progression of PC and to establish the regulatory mechanism(s) underlying the cigarette smoke- induced MUC4 expression and its role in the pathogenic process. Specifically, we aim to establish the role of cigarette smoke and nicotine in the etiology of pancreatic cancer and unravel the regulatory mechanism(s) underlying nicotine-induced MUC4 expression. We hypothesize that cigarette smoking contributes to the development of pancreatic cancer, and that MUC4 is an important component in the disease process. To test our hypothesis, we propose three specific aims. In Aim 1, we will investigate the effect of cigarette-smoke/nicotine on pancreatic cancer growth by performing various functional assays in established pancreatic cancer cell lines and in an in-vitro human pancreatic cancer progression model. Aim 2 will delineate the signaling pathways implicated in mediating the effect of nicotine on MUC4 expression alone and in collaboration with other inducers of MUC4. In Aim 3, we will carry out studies in different mouse models that spontaneously develop pre-malignant and malignant pancreatic lesions to examine the effect of cigarette-smoke and nicotine in potentiating the early events of pancreatic carcinogenesis. We will also generate a mouse model in MUC4-null background to define the role of MUC4 in malignant disease initiation and progression. Taken together, these studies will establish the causal role of cigarette-smoke and nicotine in the etiology of lethal pancreatic cancer. Project Narrative The proposed research investigations are aimed at understanding the pathological consequences of cigarette smoking toward the development of pancreatic cancer and establishing the role of cigarette smoke-induced MUC4 expression in the pathogenic process. In preliminary studies, we have shown that cigarette smoke inhalation induces a chronic pancreatic inflammatory process in vivo and the treatment of pancreatic cancer cells in vitro with nicotine induces MUC4 expression, an aberrantly expressed mucin in majority of pancreatic cancer, which also possess transforming properties. The outcome of the proposed studies will establish the mechanistic role of cigarette smoke and nicotine in the etiology of lethal pancreatic cancer.

DESCRIPTION (provided by applicant): The proposed application represents a comprehensive plan of three projects to improve the capacity of existing tests for pancreatic cancer, and to validate novel biomarkers discovered as part of collaborative efforts during the previous phase of the EDRN. The first project further develops a broad based diagnostic test for pancreatic cancer that enhances the CA19-9 test and incorporates elements of early detection of cancer by detecting autoantibodies to tumor specific glycopeptide structures found on mucins. The earliest premalignant and malignant lesions in the pancreas express MUCI, MUC4, MUC5ac, MUC16, and MUC17 with truncated O-linked structures attached (Tn, sialyl Tn, T) but may not produce a sufficient amount to be detected in serum. Instead, autoantibodies to these specific structures are produced at sufficiently high concentrations to be detected. As lesions progress mucins are detected in circulation, initially as immune complexes with the autoantibodies, and in later stages in the absence of immune complexes. This overall hypothesis will be tested by developing a set of three integrated tests for serum: a test that quantifies autoantibodies, a test that quantifies immune complexes, and a test that quantifies circulating mucins for both oligosaccharide and core protein (on the same molecule). A complementary study in collaboration with Dr. Sam Hanash will identify non-mucin proteins that carry Tn and sialyl Tn antigens and evaluate autoantibodies to these proteins. The second project led by Dr. Surinder Batra proposes to further develop a novel analytical test that shows enhanced sensitivity for detection mucin type glycoprotein (which can be difficult to detect using conventional techniques) using surface enhanced Raman spectroscopy (SERS). This technique will be used to develop an assay for multiple biomarkers in serum. The third project, led by Dr. Schmittgen and in collaboration with Dr. Hollingsworth, will undertake discovery of potential miRNA biomarkers in plasma and urine of patients with different stages of pancreatic cancer and evaluate the prognostic utility for patients with resected pancreatic cancer of evaluating the tissue expression of a set of microRNAs that are associated with early metastasis of pancreatic cancer.

ABSTRACT Prostate cancer (PC) is among the most commonly diagnosed malignancies and the second leading cause of cancer-related deaths in men. Although significant progress in the development of early detection tests has led to improved management of patients diagnosed with organ-confined PCs, the progression to locally invasive and metastatic hormone-refractory PCs (HRPCs) typically leads to treatment resistance, disease relapse and the death of patients. Therefore, a better understanding of etiological causes responsible for PC initiation and progression and treatment resistance is needed to identify novel molecular therapeutic targets. Recent lines of evidence have revealed that the malignant transformation of adult prostatic stem/progenitor cells into highly tumorigenic PC-initiating cells may provide critical functions in PC progression, metastases at distant tissues, treatment resistance and disease relapse. In considering these advances, the central hypothesis of this proposal is that the activation of specific oncogenic products induced in PC-initiating cells and their progenies during PC etiology and progression, including an up-regulation of EGFR and hedgehog signaling elements, may cooperate for their sustained growth, survival, invasion, metastases, treatment resistance and disease relapse. Based on this hypothesis, the long-term objective is to evaluate the potential benefit to simultaneously target EGFR and hedgehog cascades for eradicating PC- and metastasis-initiating cells and their progenies and improving the current clinical anti-androgenic treatments and docetaxel-based chemotherapies against locally invasive, androgen independent and metastatic PCs. For this, we will use in vitro and in vivo human PC cell models, and a large panel of patient's prostatic tissues relevant to prostate carcinogenesis. Three specific aims are proposed. AIM I will establish the specific functions and molecular mechanisms of EGFR and hedgehog pathways in the malignant transformation of PC stem/progenitor cells and their progenies during PC initiation and progression. AIM II will identify the drug resistance-associated molecules modulated through the activation of EGF-EGFR and sonic hedgehog cascades in PC stem/progenitor cells versus their progenies. AIM III will establish the therapeutic benefit of co-targeting EGFR and sonic hedgehog pathways for eradicating PC- and metastasis-initiating cells and their progenies and improving current clinical therapies. Altogether, these studies should delineate the specific functions provided by EGFR and hedgehog pathways in the acquisition of a more malignant phenotype and resistance of PC stem/progenitor cells and their progenies to current clinical therapies. The therapeutic interest of co-targeting EGFR and hedgehog cascades to eradicate the total PC cell mass and improve current anti-androgenic treatments and docetaxel-based chemotherapies for treating PC patients and thereby prevent disease relapse and the death of patients will be established.

Competitive Revision Project 3: Project Leaders: Surinder K. Batra, Ph.D.; Aaron Sasson, M.D Title: Blomarkers for the diagnosis of pancreatic cancer Introduction. Impact of the revised competitive application.The revision of the application contains parallel new Alms on the global analysis of surrogate markers in peripheral blood nuclear cells (PBMCs) for the early diagnosis of pancreatic cancer. The proposed funding will support a new graduate student, Michael Blaine. (MD, Ph.D. Program, joining from June), Brigid O'Hollerman (medical summer student-working during summer) and a post-doc (TBA) and will use the human samples obtained in the parent grant application. Other proposed members will be partly supported for FTEs for the overall retention at UNMC. The proposal will heavily Core Microarray facility and will help towards the retention of people in that facility.

DESCRIPTION (provided by applicant): This U54 application submitted in response to RFA CA10-021 Tumor Microenvironment (TMEN) is from the University of Nebraska Medical Center. The overall goal of this application is to define the role of interactions between pancreatic tumor cells and the tumor microenvironment during the development and progression of pancreatic cancer. A hallmark of pancreatic cancer is an extreme fibrotic response, and it is our collective hypothesis that fibrosis promotes signaling to the tumor cells, which promotes tumor growth, invasion and metastasis. Specifically, we will investigate interactions, regulation and contributio of secreted and cell surface molecules expressed by stromal cells, premalignant epithelial cells, and malignant cells. This project brings together investigators with experience in the biology of pancreatic cancer. The Pancreatic Tumor Microenvironment Network (TMEN) will include 4 research projects and 3 shared resources. Project 1: Interplay of tumor microenvironment and MUC4 in pancreatic cancer. Surinder K. Batra, Ph. D. Project 2: Lymphangiogeneis and metastasis during pancreatic cancer. Michael A. Hollingsworth, Ph. D Project 3: Role of N-cadherin in pancreatic tumor microenvironment. Keith Johnson, Ph.D. Project 4: CXCR2-dependent pancreatic cancer progression and metastasis. Rakesh K. Singh. Shared resource 1: Administrative Core;Shared resource 2: Rapid Autopsy Program (RAP) Core Shared resource 3: Genetically engineered Model (GEM) Core, Kay Wagner, Ph.D. The four research projects will investigate the role of microenvironment in the early stages of tumor development (Project 1), tumor progression (Projects 3 and 4) and angiogenesis and metastasis (Project 2). Together the group of investigators will exploit the powerful resources comprising of clinical samples, in vitro cell models and genetically engineered animal models of spontaneous tumorigenesis that exist at UNMC, to unravel the complex interplay between the components of tumor microenvironment and tumor cells in pancreatic cancer initiation and progression. With the expertise of the involved investigators in TME, we seek to improve our understanding of the underappreciated role of tumor microenvironment in pancreatic cancer and establish potential therapeutic relevance.

Interplay of tumor microenvironment and MUC4 in pancreatic cancer. Clinically, MUC4 mucin is aberrantly overexpressed in pancreatic cancer (PC) tissues and its expression is associated with poor prognosis (1). Studies from our laboratory have conclusively established that MUC4 is involved in enhanced motility, invasiveness and drug resistance of tumor cells in vitro and promotes tumorigenicity and metastasis in vivo (2). MUC4 upregulation is one of the early events in pancreatic cancer as indicated by its upregulation in precursor lesions (3). Our previous studies have demonstrated that MUC4 expression in pancreatic cancer cells in part is regulated by cytokines like IFN- y which are potentially secreted by cells of tumor/inflamed pancreas microenvironment. Inflammation is regarded as a precursor to cancer and in case of pancreatic cancer the increasing evidence supporting the association of pancreatitis to pancreatic cancer risk/development underscores the importance of inflammation in this lethal malignancy. It is thus not surprising that like high MUC4 levels, high levels of pro-inflammatory cytokines (IL-1 & IL-12) in tumor correlates with poor prognosis (4). Given the importance of inflammation and involvement of MUC4 in pancreatic cancer development, and regulation of MUC4 by inflammatory cytokines like IFN- y, the overall objective of this proposal is to understand the modulation of MUC4 expression by tumor microenvironment Another aspect of MUC4 and tumor microenvironment is the ability of MUC4 to interact with and possibly modulate the components of extracellular matrix via the unique domain (NIDO). However, to keep the proposal focused and avoid overlap with our ongoing studies the emphasis of the current application is on the regulation of MUC4 by tumor microenvironment (TME).

DESCRIPTION (provided by applicant): Pancreatic cancer (PC) is the fourth leading cause of cancer deaths in the U.S. and has a five-year survival rate of only 5%. The current standard of care for advanced PC, gemcitabine, prolongs survival by only a few weeks, and only 25% of patients respond to this treatment. Resistance to gemcitabine is a major problem in the treatment of pancreatic cancer. We have recently shown that MUC4 contributes to the resistance of PC cells to gemcitabine-induced apoptosis. MUC4 mucin is a large glycoprotein aberrantly expressed by PC cells. We have previously shown that MUC4 downregulation induces apoptosis, inhibits proliferation, blocks invasion and metastasis and sensitizes PC cells to gemcitabine. These results suggest that MUC4 could be an extremely relevant therapeutic target in PC. However, there are currently no therapeutic strategies to downregulate MUC4 expression in vivo. Fluticasone propionate (FP), a potent anti-inflammatory glucocorticoid used clinically in treating bronchial asthma, has previously been reported to repress the expression of MUC4 mRNA in cultured nasal polyp cells. However, its role in regulating MUC4 expression in PC cells and on PC cell behavior has never been examined. The central hypothesis of the proposal is that Pharmacological downregulation of MUC4 with glucocorticoids would enhance the sensitivity of PC cells to chemotherapeutic agents and a combination of GCs will synergistically enhance the therapeutic efficacy of chemotherapy. The preliminary studies have indicated that FP downregulates MUC4 expression at the transcript level via the glucocorticoid receptor (GR). To investigate further the mechanism by which FP affects MUC4 expression and to assess its potential therapeutic relevance in PC, we propose three specific aims. In aim 1 we will investigate the mechanisms underlying the observed downregulation of MUC4 by FP in PC cells using GR specific shRNAs, promoter-reporter assays and chromatin immunoprecipitation. In aim 2 we will examine the effect of FP treatment on PC cell function in vitro, compare FP with dexamethasone (a glucocorticoid currently used in co-treatment with cancer chemotherapy) in affecting the sensitivity of PC cells to gemcitabine, and examine the effect of FP treatment on PC tumor growth and chemosensitivity in vivo in an orthotopic xenograft model in nude mice. In aim 3, we will examine the effect of FP treatment on PC tumor growth and chemosensitivity in vivo in a clinically relevant spontaneous PC mouse model. Taken together, the proposed studies will investigate the therapeutic potential of this novel MUC4 repressing agent for possible application as an adjuvant to existing PC chemotherapy regimens.

The administrative core will provide support for personnel involved in the administration, scientific oversight and coordination, communication, budgetary oversight, and clerical functions associated with the overall research program proposed in this Tumor Microenvironment Network (TMEN) application.

Overview of the Resource. Given the inherent difficulties in obtaining large quantities of tissues from pancreatic cancer patients, we have devised and instituted a rapid autopsy program that is designed to harvest primary tumor and organs containing all metastatic deposits of tumor from individuals who die of pancreatic cancer. The process we have instituted allows us to harvest and rapidly freeze these tissues in a process that is completed within 5 hours of death. This process is unique and highly innovative as a tissue resource in that it allows us to capture the entire history disease progression for pancreatic cancer patients (from intact primary tumor and remaining precursor lesions through to distant metastases at all locations). For the purposes of this application, the resource will provide high quality frozen samples, slides for frozen sections, formalin fixed tissues, and associated body fluids (blood, serum, ascites) for all projects requiring tissues. The costs of acquiring these tissues in the rapid autopsy program are supported in part by our Cancer Center Support Grant and our Pancreatic Cancer SPORE Grant. The costs requested here are supplemental to that support and are required for the requisite sample processing and pathology support that is associated with supporting the projects proposed in this application.

DESCRIPTION (provided by applicant): Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the U.S. Only a subset of patients initially responds to the current chemotherapeutic agent gemcitabine. Traditional cytotoxic agents are ineffective at controlling the signaling pathways that ultimately drive the disease progression. Hence, there is an urgent need to target signaling pathways specifically amplified during disease progression. An important theme that has emerged from our preliminary studies is that afatinib, an irreversible inhibitor of all ErbB family members, would be an alternative strategy for the combined approach of targeting all EGFR family members. Through this proposal we seek to develop an effective therapy for PC by combining the afatinib and the cytotoxic agent gemcitabine to provide broad spectrum molecular (EGFR family proteins and also mucins) targeted therapeutic protocol with enhanced antitumor efficacy and control of chemoresistance. Based on previous studies and our preliminary observations we hypothesize that afatinib, a pan-EGFR family inhibitor will enhance the efficacy of gemcitabine by modulating/downregulation EGFR family proteins along with mucins, augmenting tumor microenvironment and targeting cancer stem cells. We have proposed three specific aims to test this hypothesis. The first aim is to evaluate the functional effect of afatinib, alone and in combination with gemcitabine in PC cells, functiona significance and intrinsic resistance by using the combinational approach in PC cells. In this aim we will explore the mechanism of action of afatinib in augmenting mucin-mediated therapeutic resistance in PC cells. We will also predict the sensitivity of the combination of gemcitabine with afatinib in overcoming intrinsic resistance in vitro in PC cells. The second aim is focused at exploring the efficacy of the combination of gemcitabine with afatinib in genetically engineered mouse model of PC. The third aim is to investigate the potential impact of pan-EGFR Inhibitor in overcoming intrinsic resistance imposed by pancreatic cancer stem cells (PCSC) and tumor microenvironment. In this aim, we will analyze the effect of combination therapy on pancreatic cancer stem cells and tumor microenvironment to overcome the chemotherapeutic resistance. The significance of the present proposal is to understand the central mechanism(s) of the afatinib-induced enhanced chemotherapeutic efficacy of gemcitabine in lethal PC. Altogether, the overall results of this proposal would be enormously promising for initiating clinical trials consisting of a combination of EGFR family member specific inhibitor afatinib with gemcitabine as a therapeutic tool in PC patients. The outcome of the proposed studies will have high impact on thousands of pancreatic cancer patients currently receiving gemcitabine as a first line chemotherapy.

Despite being a staple therapy for pancreatic cancer (PC), Radiation Therapy (RT) provides limited objective clinical response due to inherently high radioresistance (RR) of PC. As the risk of radiation-induced toxicity for PC patients far outweighs the therapeutic benefits attained, effective methods to improve the radiosensitivity of PC are urgently needed. The overall objective of this project is to identify and characterize pathway(s) contributing to RR in PC that can be explored as novel targets for radiosensitization (RS). Preliminary global gene expression analysis suggested novel involvement of cholesterol biosynthesis pathway in the RR in PC cells. Inhibition of cholesterol biosynthesis (CBS) by Zoledronic acid (Zometa) resulted in RS of panel of RR murine and human PC cells. Further, this RS was recapitulated by the inhibition of the small GTPase Rac1, whose activity is controlled by the CBS pathway. Therefore, we seek to delineate mechanisms of RR mediated by cholesterol biosynthesis pathway, and evaluate the potential of Zometa as a radiosensitizer (RST) in preclinical and clinical studies. We propose to exploit the strength of stereotactic radiation therapy and genetically engineered mouse models to comprehensively test the hypothesis that ¿cholesterol biosynthesis pathway contributes to radioresistance in PC and Zometa inhibits specific pathways consistently implicated in RR and its use will radiosensitize PC both in vitro and in vivo¿. To achieve our goal, three specific aims are proposed. Aim 1 will elucidate the mechanisms of radioresistance in PC and validation of FDPS/Rac1 inhibitors (Zometa and NSC23766) as RSTs. The functional role of critical genes identified will be determined by knockdown and overexpression studies, use of specific inhibitors, and immunohistochemistry on clinical samples. Aim 2 will determine the efficacy of Zometa as RST in mouse models (xenograft and autochthonous). A novel strategy of stereotactic irradiation for murine models will be developed. In Aim 3, a Phase-I/II study will be undertaken to assess the radiosensitizing potential of Zometa in human subjects and determine if Zometa is well tolerated in PC patients undergoing RT.

? DESCRIPTION: Successful treatment of pancreatic adenocarcinoma (PDAC) remains a challenge due to the desmoplastic microenvironment that promotes both tumor growth and metastasis and forms a barrier to chemotherapy. Hedgehog (Hh) signaling plays a crucial role in PDAC progression and contributes to desmoplasia. Hh inhibitor GDC-0449 can overcome desmoplastic reaction. While Hh levels are increased in pancreatic cancer stem cells (CSCs), tumor suppressor miR-let7b targets several genes involved in PDAC pathogenesis. Therefore, inhibition of Hh pathway and restoration of miR-let7b could effectively treat PDAC. In our preliminary studies, miR-let7b was downregulated in pancreatic cancer cell lines, human patient's cancer tissues and during progression of spontaneous pancreatic cancer in genetically engineered KPC mice. Our in-silico analysis and in vitro experiments indicate that miR-let7b targets several genes like MUC4, NCOA3, Kras, HMGA2 and TGFßR1, which are upregulated in PDAC. miR-let7b and GDC-0449 could inhibit the proliferation of human pancreatic cancer cells (Capan-1, HPAFII and T3M4) and there was synergistic effect when miR-let7b and GDC- 0449 were co-formulated into micelles using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl- propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) copolymer. This copolymer self-assembles into micelles and encapsulates hydrophobic GDC-0449 into its core and allows complex formation between miR-let7b and cationic pendant chains. This combination therapy effectively inhibited tumor growth when injected to ectopic tumor bearing mice compared to micelles carrying GDC-0449 or miR-let7b alone. Cetuximab, a monoclonal antibody for EGFR receptors, was conjugated to the nanomedicine to achieve active targeting to PDAC. Therefore, we hypothesize that combination therapy of miR-let7b with GDC-0449 using cetuximab conjugated nanomedicines can effectively chemosensitize PDAC cells by reducing desmoplasia and treat PDAC by reducing their tumorigenicity. Our overall objective is to develop novel combination therapy for PDAC using nanoformulations targeting multiple signaling pathways by miR-let7b and Hh pathway by GDC-0449. Our specific aims are to: i) determine the efficacy and molecular mechanisms of miR-let7b and GDC-0449 combination therapy in PC cells; ii) co-formulate miR-let7b and GDC- 0449 in cetuximab conjugated polymeric micelles and assess their synergistic effect on the inhibition of pancreatic cancer cells, iii) determine the therapeutic efficacy of micelles encapsulating miR-let7b and GDC-0449 in orthotopic NSG and genetically engineered KPC mouse models of PDAC. Long- term significance is to develop nanomedicines of miR-let7b and GDC-0449 that increase the local drug concentrations within the fibrotic stroma of these tumors and bypass the resistance mechanisms that allow tumor growth and inhibit the efficacy of current standard chemotherapies.

? DESCRIPTION (provided by applicant): Pancreatic cancer (PC) is a highly metastatic and therapy-resistant malignancy with patients presenting with local and distant metastases at the time of diagnosis. The metastatic lesions while still undetectable contribute to the lethality of te disease and hence should be treated effectively. Antibodies directed against tumor antigens are ideal vehicles for delivering cytotoxic agents including therapeutic radionuclides to the known and occult metastatic cancer cells and can be a promising therapeutic option for treating PC. However, pancreatic tumors present a complex and highly obstructive microenvironment that is characterized by insufficient and heterogeneous tumor blood flow and extensive desmoplasia. Cell surface mucin, MUC4, is promising target for PC therapy due to its specific overexpression in tumor that correlates positively with disease progression and negatively with patient survival. Monoclonal antibody MAb 8G7, due to its reactivity to a repetitive epitope on MUC4, can potentially serve as a highly efficient targeting agent for PC. The central hypothesis of this proposal is: MUC4 is a novel molecular target for targeted radionuclide therapy (TRT) of lethal pancreatic cancer and selective modulation of tumor microenvironment can improve the delivery and enhance the therapeutic efficacy of MUC4-targeted radiopharmaceuticals. The overall objective of the proposed studies is to determine the utility of targeting pancreatic cancer with novel radiolabeled anti-MUC4 antibodies in combination with rationally-selected specific modulators of tumor microenvironment (TME). Tumor vasculature is characterized by structural and functional anomalies as compared to normal vasculature and these differences result in differential responses when the normal and tumor vasculature are exposed to various vasoactive agents. Modulation of tumor stromal compartment can be achieved by selectively targeting the signaling pathways that regulate various components of tumor stroma. Three specific aims are proposed: 1) Evaluation of MUC4 as a target for TRT of PC; 2) Selective Modulation of tumor microenvironment for improved delivery of radiopharmaceuticals; and 3) Determine the therapeutic efficacy of MUC4 and TME targeted combination therapy. Aim 1 will provide information about the efficiency of anti-MUC4 MAb for targeting PC in vivo and characterize the MAb in terms of biodistribution and pharmacokinetics. Further, the studies will allow us to determine which therapeutic radionuclide is compatible with anti-MUC4 MAb. Aim 2 will evaluate a combination of tumor-selective agents for their ability to improve TME in xenograft and autochthonous tumors for enhanced delivery of radiopharmaceuticals. Aim 3 will allow us to determine if the improved delivery, distribution and retention of radiolabeled antibodies translates to improved therapeutic efficacy. The proposed studies represent the first comprehensive effort to overcome physiological barriers for macromolecular radiopharmaceuticals and utilize clinically relevant transgenic mouse models of PC. The preclinical results obtained from the proposed study will form the basis of clinical trial in PC patients.

? DESCRIPTION (provided by applicant): Prostate cancer (PCa) is the most common non-cutaneous malignancy in the United States males and is the second leading cause of cancer deaths in men. In this regard, epidemiologic data have also provided evidence of racial disparity, including younger age, higher incidence and aggressiveness of PCa at diagnosis and inferior survival rates of African Americans (AA) relative to Caucasian patients in the Unites States. The explanation for these differences is still unknown. On the other hand, our recent results combined with prior studies have revealed that MIC-1 and its functional partners EGFR and CXCR4 were more frequent in AA compared to Caucasian PCa patients. Importantly, recent reports have revealed that EGFR and CXCR4 mediate Docetaxel resistance in PCa cells. Therefore, the central hypothesis of this proposal is that the up- regulation of MIC-1 signaling cascade may cooperate with EGFR and CXCR4 oncogenic pathways for the acquisition of a more aggressive behavior, survival and therapeutic resistance of PCa cells and contribute to the racial disparity between AA versus Caucasian patients. The overall objective is to establish the signaling elements modulated by MIC-1/transforming growth factor-ß receptors (TGF-ßRs) and their functional cross- talks with EGFR and CXCR4 cascades in tumor and tumor-associated stromal cells during prostate carcinogenesis and bone metastases that contribute to the ethnic disparity in AA vs. Caucasian patients. The therapeutic benefit to targeting MIC-1 cascade and its functional partners for reversing the chemoresistance of PC cells will also be investigated using PCa cell models and human PCa tissues from AA and Caucasian men. To test this hypothesis, three specific aims were proposed. Aim I will establish the involvement of MIC-1, EGFR and CXCR4 in racial disparity by performing immunohistochemical analyses of their expression on a large panel of benign and malignant tissues and DNA microarrays with PC cell lines relevant to AA and Caucasian men. Aim II will delineate molecular mechanisms of MIC-1 and its signaling cross-talks with EGFR and CXCR4 cascades involved in the growth, invasion and alterations of metabolic pathways using in vitro PCa cell models relevant to AA and Caucasian PCa patients. Aim III will determine the therapeutic benefit to target MIC-1 cascade for improving the anti-carcinogenic effects of docetaxel using in vitro and in vivo PCa cell models. Finally, we also demonstrate the molecular mechanism associated with Docetaxel resistance and its relationship with MIC-1 in PCa cells relevant to AA and Caucasian patients. Taken together, these studies will delineate the functional interplay between MIC-1 and its interacting partners EGFR and CXCR4 and their potential role in disease aggressiveness and therapy resistance to better understand their involvement in the racial disparity between AA and Caucasian patients. The overall outcome of this application will reveal the clinical interest to use combination of MIC-1, EGFR and CXCR4 as molecular biomarkers for an earlier and accurate diagnosis, response to therapy and impact on therapeutic intervention of AA and Caucasian patients.

? DESCRIPTION (provided by applicant): Activating mutations in the K-Ras gene are the earliest and most common genetic alterations detected in human pancreatic cancer (PC) specimens and are associate with progression and metastasis. In genetically engineered mouse models (GEMMs) of PC, oncogenic K-Ras drives the formation of pancreatic intraepithelial neoplasia (PanIN lesions) and their progression to invasive PC. Oncogenic K-Ras has, at least, four effector pathways that play distinct roles in malignant transformation: MAPK pathway (mitogen-activated protein kinase), PI3K pathway (phosphoinositide 3-kinase), Ral-GDS (Ral guanine nucleotide dissociation stimulator), and Rac1 GTPase (Ras-related C3 botulinum toxin substrate 1). Among these effectors, the least well understood is the Rac1 pathway. Yet, this pathway has recently been shown to be critical for Ras-driven PC development. In transformation assays, Rac1 is required for transformation by the Ras oncogenes, and activated mutants of Rac1 can cooperate with the MAPK pathway to elicit malignant transformation. However, the specific mechanisms by which Rac1 promotes anchorage-independent growth and tumorigenicity are still unclear, so are the mechanisms responsible for its cooperation with the MAPK pathway. The objective of this proposal is to elucidate these mechanisms. Our central hypothesis is that the Rac1 and MAPK pathways act conjointly to control the expression/activity of proteins that play critical roles in integrating signals from cell-cell and ell-matrix interactions, most notably the YAP protein (Yes-associated protein 1). This hypothesis is based on preliminary data indicating that the inhibition of either Rac1 or MAPK pathway causes the degradation of the YAP protein. YAP is a transcriptional co-activator that controls genes involves in proliferation. The stability, location, and activity of YAP is controlled by signaling pathways involved in sensing cell-cell and cell-matrix interactions, such as the Hippo and Wnt pathways. In GEMMs of Ras-driven PC, YAP is required for the formation of PanIN lesions and their progression to invasive PC. In our telomerase-immortalized human pancreatic ductal cells, overexpressed YAP alone is sufficient to allow anchorage-independent growth. To test our central hypothesis and define the role of the Rac1 pathway in the initiation and progression of PC and its cooperation with the MAPK pathway, we propose three Specific Aims: Aim 1 will elucidate the mechanisms responsible for the transforming activity of Rac1 and its cooperation with the MAPK pathway; Aim 2 will evaluate Rac1 activity and YAP levels in human PC specimens and assess their pathobiological significance; and Aim 3 will delineate the cooperation and relative contributions of Rac1 and MAPK pathways in the initiation and progression of PC in GEMMs. Altogether, the proposed work will shed light on the mechanisms underlying Ras-driven carcinogenesis and reveal novel therapeutic targets, which could potentially be exploited for the treatment of pancreatic cancers and other Ras-driven malignancies.

? DESCRIPTION (provided by applicant): A majority of pancreatic cancer (PC) patients (> 80%) present with an unresectable primary tumor with distant metastasis at the time of diagnosis due to its asymptomatic nature and lack of methods for early detection. While the overall 5-year survival rate of PC is dismal, signi?cantly better outcomes have been reported for early stage smaller tumors. PC is believed to progress through a series of histological changes and recent estimates indicate that these changes require 15-20 years to develop into invasive metastatic disease, hence providing a window of opportunity to intervene. While early diagnosis is an obvious strategy to improve the survival of PC patients, lack of non-invasive biomarkers for risk prediction of precursor lesions or early stage invasive disease impedes our ability to diagnose PC during this window. Premalignant cystic lesions of the pancreas offer unique opportunity for early diagnosis. To meet the EDRN stated goal of performing a validation study for early cancer detection and risk assessment likely to yield meaningful results within 5 years, we propose an innovative approach that could lead to the development of a clinically useful biomarker in this time frame. Additionally, we propose to study cystic neoplasms since IPMN and MCN offer a unique opportunity to identify pancreatic premalignant lesions and serve as a target for early detection strategies. The candidate biomarkers studies in our application have been identified and evaluated over the past 5 years in EDRN-funded biomarker developmental laboratories (BDLs). Studies from the laboratories of participating investigators have conclusively established that mucin overexpression is a hallmark of pancreatic cancer and have identified biomarkers (MUC5AC and its 2 glycoforms, MUC4 and a glycoform of endorepellin) that can effectively distinguish a) patients with resectable PC patients from patients with benign pancreatic diseases; b) mucinous and non- mucinous cysts; c) high-grade from moderate- and low-grade mucinous cysts and d). high-risk intestinal type IPMNs and low risk gastric type IPMNs. The overall objective of this CVC proposal is to demonstrate the ability of the aforementioned biomarkers to distinguish pancreatic adenocarcinoma (PC) patients from healthy controls, patients with benign biliary obstruction and chronic pancreatitis (CP), and to identify those cysts with high malignant potential. We have a large number of high quality, well-characterized specimens from patients with pancreatic disease and diseased controls that were collected and processed following protocols developed through the EDRN. Two specific aims are proposed. Aim 1 will use available samples from our biospecimen repository to evaluate MUC5AC and MUC4 as biomarkers to distinguish pancreatic adenocarcinoma (PC) patients from healthy controls, and from patients with benign biliary obstruction and chronic pancreatitis (CP). Aim 2 will determine if our biomarkers can identify those cysts with high lethal potential from those with a low risk for malignant transformation.

A majority of pancreatic cancer (PC) patients (> 80%) present with an unresectable primary tumor with distant metastasis at the time of diagnosis due to its asymptomatic nature and lack of methods for early detection. While the overall 5-year survival rate of PC is dismal, significantly better outcomes have been reported for early stage smaller tumors. PC is believed to progress through a series of histological changes and recent estimates indicate that these changes require 15-20 years to develop into invasive metastatic disease, hence providing a window of opportunity to intervene. While early diagnosis is an obvious strategy to improve the survival of PC patients, lack of non-invasive biomarkers for risk prediction of precursor lesions or early stage invasive disease impedes our ability to diagnose PC during this ?window. Premalignant cystic lesions of the pancreas offer unique opportunity for early diagnosis. To meet the EDRN stated goal of performing a validation study for early cancer detection and risk assessment likely to yield meaningful results within 5 years, we propose an innovative approach that could lead to the development of a clinically useful biomarker in this time frame. Additionally, we propose to study cystic neoplasms since IPMN and MCN offer a unique opportunity to identify pancreatic premalignant lesions and serve as a target for early detection strategies. The candidate biomarkers studies in our application have been identified and evaluated over the past 5 years in EDRN-funded biomarker developmental laboratories (BDLs). Studies from the laboratories of participating investigators have conclusively established that mucin overexpression is a hallmark of pancreatic cancer and have identified biomarkers (MUC5AC and its 2 glycoforms, MUC4 and a glycoform of endorepellin) that can effectively distinguish a) patients with resectable PC patients from patients with benign pancreatic diseases; b) mucinous and nonmucinous cysts; c) high-grade from moderate- and low-grade mucinous cysts and d). high-risk intestinal type IPMNs and low risk gastric type IPMNs. The overall objective of this CVC proposal is to demonstrate the ability of the aforementioned biomarkers to distinguish pancreatic adenocarcinoma (PC) patients from healthy controls, patients with benign biliary obstruction and chronic pancreatitis (CP), and to identify those cysts with high malignant potential. We have a large number of high quality, well-characterized specimens from patients with pancreatic disease and diseased controls that were collected and processed following protocols developed through the EDRN. Two specific aims are proposed. Aim 1 will use available samples from our biospecimen repository to evaluate MUC5AC and MUC4 as biomarkers to distinguish pancreatic adenocarcinoma (PC) patients from healthy controls, and from patients with benign biliary obstruction and chronic pancreatitis (CP). Aim 2 will determine if our biomarkers can identify those cysts with high lethal potential from those with a low risk for malignant transformation.

Recent evidence has provided an understanding of the molecular mechanisms and genetic changes underlying pancreatic ductal adenocarcinoma (PDAC) pathogenesis; however, limited information is available on progression in the disease. Studies in several genetically engineered mouse models for PDAC suggest that acinar cells, centroacinar cells, and/or postulated stem cells can be responsible for the development of PDAC. This is believed to occur via a process termed ?acinar-to-ductal metaplasia? (ADM), during which a differentiated cell type (acinar) is reversibly replaced with another mature, differentiated cell type, a condition also visible during inflammation, thereby underlining the predisposition of chronic pancreatitis patients to PDAC. Pancreatic differentiation 2 (PD2), also known as polymerase associated factor-1 (Paf1), has been found to be overexpressed in PDAC and to exhibit oncogenic potential. Our previous studies have further defined the role of PD2/Paf1 in cell cycle regulation and in inducing chromatin structure remodeling in PDAC cells. We have also demonstrated that PD2/Paf1 plays a major role in the multi-lineage differentiation of mouse embryonic stem cells and maintains pancreatic cancer stem cells (PCSCs). Our preliminary efforts to investigate the expression of PD2/Paf1 in KrasG12D;Pdx1Cre mouse model of PDAC showed that it is differentially overexpressed in neoplastic pancreatic ducts during murine PDAC progression, as compared to its strictly acinar expression in normal pancreas. PD2/Paf1 was specifically expressed in `intermediate structures' expressing both acinar and ductal specific markers, representing `transitional cells' during pancreatic acinar to ductal metaplasia. Further, we found that PD2/Paf1 is overexpressed along with PCSCs markers in PDAC progression in mouse tissues and isolated CSCs. The multi-potent property of these CSCs allows them to differentiate into several cell types. Therefore, the overall goal of this study is to define the role of PD2/Paf1 in trans-differentiation of acinar cells to ductal cells during PDAC progression, through lineage- differentiation of pancreatic CSC population. Based on these observations our central hypothesis is that ?PD2/Paf1 plays a significant role in the process of acinar-to-ductal metaplasia, thereby contributing to PDAC progression, and its overexpression contributes to the ductal lineage-differentiation of pancreatic CSCs.? To test this hypothesis, Aim 1 will focus on investigating the potential link of PD2/Paf1 in acinar-to-ductal metaplasia using cerulein-induced mouse model of PDAC progression. Aim 2 will elucidate the mechanism(s) of PD2/Paf1 in acinar-to-ductal metaplasia using acinar cells, organoid 3D-cultures, and PD2-/- animals. In Aim 3, we will understand the functional mechanism of PD2/Paf1 in ductal lineage-differentiation of pancreatic CSCs in PDAC progression. Taken together, understanding of novel roles of PD2/Paf1 in ADM progression through stem-like cell differentiation will lead to critical information for the long-term goal of developing novel, targeted therapy against PDAC.

Abstract Pancreatic cancer (PC) is a highly metastatic and therapy-resistant malignancy with patients presenting with local and distant metastases at the time of diagnosis. Like other epithelial cancers, PC progression is characterized with aberrant mucin overexpression. Our previous study indicated that while MUC16 was undetectable in the normal pancreas, there was a progressive increase in MUC16 expression with the increase in grade of PanIN lesions, tumor and metastasis. We also demonstrated that MUC16 and MUC16-Cter play critical roles in metastasis of pancreatic cancer. However, functional and mechanistic involvement of MUC16 in pancreatic cancer metastasis remains poorly understood. MUC16 is a multi-domain protein that can potentially play multifaceted role in metastasis of PC. In our preliminary studies, silencing of MUC16 in PC cells resulted in reduced cell growth and migration along with alterations in EMT markers. We thus hypothesize that MUC16 is associated with PC metastasis, which, in part, is mediated by MUC16-Cter domain. To test the hypothesis and achieve the aforementioned objectives, three specific aims are proposed. First aim will evaluate the role of MUC16 in mediating cell-to-cell interactions during metastatic spread of PC cells. In this aim, we will analyze the cellular and molecular functions of MUC16 by analyzing cell-cell and cell-extracellular matrix interactions. These studies will provide insights into the role of MUC16 in facilitating the interaction of tumor cells with the endothelial cells, platelets and leukocytes during metastasis. Studies in Aim 2 will delineate the molecular mechanism(s) of MUC16-Cter-mediated PC metastasis and identify MUC16 interacting partners. Since MUC16-Cter is enriched in the chromatin bound fraction in the nucleus, its effect on global gene expression will be evaluated using ChIP- Seq analyses. Further, the effect of various point mutations affecting N-glycosylation, ubiquitylation and phosphorylation will be addressed in the light of its functional consequences. Third aim will investigate the cooperative action of MUC16 with other defined oncogenic mutations in the metastasis of PC using Muc16?/?and MUC16Cter transgenic mice. In this context, it is important to determine the role of MUC16, alone and in combination with oncogenic K-ras and p53, during PC development. To validate our in vitro findings of the role of MUC16 in PC, we will generate MUC16Cter transgenic with pancreas specific expression. Further, depending on the phenotypes of MUC16-Cter N-glycosylation, ubiquitylation and/or phosphorylation mutants, we will generate mutant specific transgenic to understand the in vivo relevance of such mutations. Overall the proposed studies will allow us to define the molecular mechanisms by which MUC16 and its Cter facilitate metastasis and contribute to the aggressiveness of PC. These novel insights into the underlying mechanisms of PC metastasis combined with the new data, reagents and models will provide novel avenues for targeting metastatic PC in future.

ABSTRACT The overall goal of this P01 proposal is to define the mechanistic role of MUC16 in facilitating pancreatic cancer (PC) metastasis. It is well known pancreatic cancer is one of the lethal cancers and also a diffuse metastatic disease with approximately 45% to 55% of patients diagnosed after the cancer has spread. The molecular mechanisms of metastasis are poorly understood. In cancer cells depolarized mucin expression contributes to altered cell-cell adhesion, aberrant potentiation of growth factor receptor signaling, epithelial-to- mesenchymal transition, and drug resistance. In our recent study we have observed that MUC16/CA125 is a membrane bound mucin, and we have observed its aberrant overexpression during PC progression while no expression was observed in the normal pancreas. Similarly our collaborative study demonstrates that the prognosis of patients with MUC16 cytoplasmic expression was significantly poorer in pancreatic cancer patients. Based on these studies and our preliminary results our general hypothesis for the program project is that MUC16-Cter-mediated signaling, biological effects resulting from mutations of MUC16, and MUC16- induced metabolic reprograming contribute to PC metastasis. To test this hypothesis we are proposing three highly integrated projects investigating the mechanisms by which a multi-domain, muti-functional mucin MUC16 promotes metastasis. First project will focus on the role and mechanism(s) of MUC16 and its C- terminal domain in the metastatic progression of PC. Project 1 will be led by Batra, who has 26 years of experience in the field of pancreatic cancer and mucin biology. Second project will explore the roles of mutated forms of membrane bound and circulating MUC16 and its glycosylation in PC metastasis. This project will be led by Hollingsworth, who has 27 years of experience in the field of pancreatic cancer and mucin biology. Third project will focus on MUC16-mediated metabolic reprograming that induces PC metastasis. This project will be led by Singh, who has 14 years of experience in the field of pancreatic cancer biology. The projects are supported by two scientific cores to facilitate animal studies (Core B), evaluate therapeutic strategies and support clinical and animal tissue collection and analysis (Core C). Additionally, an Administrative and Bioinformatics Core (A) is proposed to integrate the different projects, cores and investigators, provide a coordinated management structure and bioinformatics and statistical data analysis. Overall this P01 program builds on the ongoing strengths in pancreatic cancer research that exist at UNMC and has resulted from a long and productive history of collaboration of participating investigators. We hope to define novel mechanisms of MUC16 mediated PC metastasis which will pave a way for better management of this lethal disease.

Abstract Pancreatic cancer (PC) is the fourth leading causes of cancer-related deaths in both men and women and is one of the most lethal malignancies with a 5-year survival rate of ~8% and median survival duration of fewer than six months. Our long-term goal is that an understanding of the cellular and molecular mechanism(s) of the metastatic phenotype and pro-inflammatory/pro-angiogenic tumor milieu and its judicious manipulation can improve the treatment outcomes for PC patients. PC is frequently associated with oncogenic K-ras mutations (90-95%). Our preliminary data and published reports suggest that CXCR2 and its ligands regulate the behavior of tumor cells and recruitment of leukocytes, and endothelial (ECs) precursor cells. Limited literature exists on the role of Kras-driven CXCR2-dependent signaling in the de novo pathogenesis of PC. Thus, molecules driving a tumor-associated pro-inflammatory and pro-angiogenic phenotype have considerable potential as therapeutic targets, yet this area remains relatively under-explored in PC. To our knowledge, very little is known about the role of K-ras-driven CXCR2-dependent de novo PC development and impact of CXCR2 targeting in pancreatic cancer progression and metastasis. Our central hypothesis is that oncogenic Kras-induced expression of CXCR2 ligands plays a critical pro-tumorigenic role in inflammation-driven spontaneous tumorigenesis, angiogenesis and metastasis. A corollary to this hypothesis is that CXCR2 and its ligands are potential novel anti-tumorigenic and anti-metastatic therapeutic targets in PC. We will explore the role of CXCR2 and its ligands using xenogenic transplant models and autochthonous animal models (K- ras-driven) in PC pathology and develop a novel therapeutic strategy. To test our hypothesis, we will: 1) Define the mechanism by which K-ras-induced CXCR2 ligands expression promotes tumorigenesis and recruit pro- inflammatory and angiogenesis promoting precursor cells; and 2) Assess the therapeutic efficacy of targeting CXCR2-mediated mechanisms in pancreatic cancer treatment. Together, these studies will provide a mechanism for K-ras-dependent expression of CXCR2 ligands, delineate their role in PC pathobiology and assess their potential as therapeutic targets. With pharmacologic inhibitors of CXCR2 already in clinical trials for the treatment of inflammatory disorders, the rapid development of innovative cancer therapies targeting CXCR2 in combination with conventional anti-tumor regimens is a highly attractive option for making inroads in lethal PC. 1

ABSTRACT Dr. Surinder Batra will lead the Administrative and Bioinformatics Core (ABC) as a Program Director. Dr. Batra will provide oversight of the scientific and administrative activities of the program and chair the P01 Executive Committee which includes all research project leaders and shared core resource directors (Drs. Hollingsworth, Singh, Jain, and Thayer). Together, the team will foster communication and collaboration between the research projects and shared resource cores and monitor and assess progress. Dr. Batra will meet weekly with research project leaders and shared core resource directors, and he and his staff will serve as the editorial review/clearing house for all publications resulting from POI activities. The core will plan and coordinate regular seminars, which will include the project leaders, core directors, and investigators. These seminars will be a part of the monthly Pancreatic Cancer Program organized through other network programs in pancreatic cancer (SPORE, EDRN CVC, and TMEN). Core A will encourage, facilitate and coordinate the participation of PPG investigators in national and international workshops, conferences, and meetings on pancreatic cancer research. Also, Dr. Batra will coordinate all the activities of the Program including Internal and External Advisory Committees and will serve as the Program liaison with other appropriate academic units within University of Nebraska Medical Center and with outside entities. Core support is requested for a Staff Specialist who will be responsible for scheduling all internal and external meetings, budgetary monitoring, and general administrative assistance. Core A will provide administrative support for all three research projects and the two shared resource cores. Dr. Jane Meza will provide supervision for the overall statistical analysis and study design of the program and will lead a team of biostatisticians that have been assigned to individual projects and cores. Besides, Dr. Babu Guda will provide bioinformatics support including data mining and comparisons. This is particularly important because some of the studies proposed in project 1 and 2 will yield a large amount of data from RNA sequencing analysis which will require the employment of powerful bioinformatics tools to integrate the data and derive meaningful basic and clinical inferences. Altogether, the scientific and administrative coordination is needed for the success of the proposed Program where data will come from Research Projects and Cores, and the ABC core is well equipped to fulfill the administrative and scientific requirements databases through effective management and state-or-art Bioinformatics approaches.

ABSTRACT Resistance to therapeutic agents is the predominant cause of high lethality of pancreatic cancer (PC). Inadequate and heterogeneous blood flow and obstructive desmoplastic stromal compartment are the major impediments to the delivery and intratumoral distribution of therapeutic agents and are the extrinsic determinants of therapy resistance in PC. Intrinsic chemoresistance of PC cells is dictated by a unique population of drug resistant cancer stem cells. Hence selective modulation of blood flow and extracellular matrix can lead to improved delivery of therapeutic agents into the tumors, while elimination of CSCs can improve the sensitivity of tumor cells to chemotherapy. Elevated endothelin (ET)-1 levels and overexpression of the two ET receptors (ETAR and ETBR) are observed in tumors. Importantly, ET-1 is a strong vasomodulator and induces vasoconstriction via ETAR is believed to be an important contributor to the tumor blood flow heterogeneity in tumors. Our preliminary studies indicate that the components of ET-axis are expressed in pancreatic tumors in various components of TME including tumor cells, blood vessel and stromal cells and CSCs. Further, targeting of ETAR with a specific inhibitor BQ123 selectively enhanced perfusion and reduced hypoxia in xenograft PC tumors, while prolonged inhibition of ET-axis in autochthonous tumors (in KPC mouse model of PC) with dual specificity inhibitor (Bosentan) resulted in marked inhibition of desmoplasia. We also observed that ET- 1 exerts pro-fibrogenic effects on murine pancreatic stellate cells via ETBR. We hypothesize that: ?Sequential inhibition of ETBR and ETAR can modulate stroma and perfusion for enhanced delivery, distribution and efficacy of therapeutic agents and clinically, combination of abraxane and gemcitabine will be more efficacious with the modulation of ET axis?. Three specific aims are proposed. Studies in Aim 1 will determine the effect of selective ETAR and ETBR antagonists BQ123 and BQ788, on the delivery and distribution of macromolecule (ABX) and small molecule-based (GEM) therapeutic agents. Biodistribution of radiolabeled ABX and GEM will be studied for quantitative estimation of tumor uptake. Further, we will decipher the mechanistic role of ET-axis-mediated cross-talk between cancer and stromal cells in PC in the context of desmoplasia. Studies proposed in Aim 2 will determine the impact of ET-axis targeting on the efficacy of ABX+GEM both in vitro (using unique cancer cell- stellate cell co-cultures and tumor organoid cultures) and in vivo using genetically engineered mouse (KPC) model of PC. Finally, Aim 3 is designed to clinically evaluate the safety and perform initial screening for efficacy of combining the ET-axis antagonist Bosentan with FDA-approved combination therapy Gemcitabine (GEM) plus Abrexane (ABX) in PC patients. Altogether, the proposed studies will demonstrate the preclinical feasibility of eliminating both extrinsic and intrinsic determinants of therapy resistance by targeting a single signaling axis and clinically determine the safety of combining ET-axis antagonism with approved chemotherapeutic regimens of PC. The data generated from the three aims will form the basis of future Phase 1/II clinical trials in lethal PC.

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, primarily due to most cases being diagnosed at an advanced, incurable stage. While 5-year survival of metastatic PDAC is <5%, outcomes dramatically improve for localized PDAC. Poor prognosis is due to a lack of biomarkers for diagnosing PDAC at an early, asymptomatic stage when cure is possible. Effective diagnosis of early stage PDAC depends on identification of accurate, non-invasive biomarkers in combination with a strategy for screening increased risk populations. Our primary objective is to identify non-invasive protein biomarkers in serum, urine, and exosomes that accurately distinguish between patients with and without early stage resectable PDAC that is amenable to curative surgery. Novel diagnostics would also improve discrimination between PDAC and benign pancreatic pathologies. The goal of the proposed research is to develop clinically translatable noninvasive biomarkers- based tests for screening (in high risk groups) and differential diagnosis of PDAC. Our central hypothesis is that combinations of urinary, serum, and exosome derived biomarkers could be synergistic offering a superior classification power. We have used urine and serum samples from retrospective and prospective cohort studies to identify a range of strong candidate combinatorial multimarker algorithms for early detection and diagnosis of PDAC. In Aim 1, we will optimize the performance of a PDAC differential diagnosis algorithm and will validate the optimized algorithm in samples collected prior to clinical diagnosis in the Pancreatic Adenocarcinoma Gene Environment Risk (PAGER) study. In Aim 2, we will optimize the performance of an early detection algorithm for resectable PDAC in pre-diagnostic samples from three prospectively collected cohorts and validate the optimized EDA in blinded parallel serum/urine samples from the Southern Community Cohort Study (SCCS). If successful, our project will yield novel, validated algorithms for risk assessment and early detection and for differential diagnosis of PDAC. These algorithms when combined will result in a new pioneering screening paradigm for PDAC allowing for timely live-saving interventions. Our strong preliminary data, powerful and synergistic investigative team, and the availability of parallel urine and serum samples from unique prospective cohorts contribute to the high probability of successful accomplishing the proposed studies.