Anil K. Rustgi - US grants

Oral cancer is a common malignancy in the United States and worldwide. A number of studies indicate that the molecular basis of oral cancer involves the activation of oncogenes, inactivation of tumor suppressor genes, and the interaction of DNA tumor viruses. A critical genetic event in the molecular pathogenesis of oral cancer is cyclin D1 over expression. The cyclin D1 oncogene product interacts with cyclin-dependent kinases (cdks), cdk inhibitors and proliferating cell nuclear antigen (PCNA) in the G1 phase of the cell cycle. Cyclin D1 over expression results in cells traversing the G1 phase in a shorter period of time and entering the S phase more rapidly. The net result is greater proliferation and eventual transformation. Genetic models of oral carcinogenesis in animals are lacking. Transgenic mouse approaches allow the development of a model of multistage oral carcinogenesis. Ultimately, a transgenic mouse mode of oral carcinogenesis has the further advantage of developing molecular diagnostics and testing the efficacy of chemopreventive agents and novel therapeutic drugs. We have developed a novel and unique transgenic mouse model in which the cyclin D1 oncogene has been targeted to the tongue squamous epithelium by employing the Epstein-Barr virus ED-L2 promoter. Since this promoter has been shown by us to be active in basal and suprabasal cells of oral keratinocytes, we have found that cyclin overexpression in the tongue causes several dysplasia, a prominent precursor of cancer. The cyclin D1 mice also display increased cell proliferation, and acquire alterations in other genes, namely p53 mutations and epidermal growth factor receptor (EGFR) over expression. The work described in this Project is designed to focus on the role of cyclin D1 in oral carcinogenesis through a transgenic mouse model. These goals will be achieved through the following Specific Aims and the overall Program Project: (1) To study interrelated cell cycle genetic alterations in transgenic mice as a function of cyclin D1 over expression; (2) To cross-breed the cyclin D1 mice with p53 homozygous hull mice since p53 mutation is associated with dysplasia in the cyclin D1 mice; and 93) To study cyclin D1 properties in oral cell lines.

? DESCRIPTION (provided by applicant): The University of Pennsylvania Perelman School of Medicine (Penn) Gastroenterology (GI) Training Program has been instrumental in the development of academic research careers for gastroenterology trainees since 1963. During the last 15 years, there has been a tremendous expansion of biomedical research. The Penn GI Training Program has increased its base of basic science faculty coupled with impressive growth and maturation of the adult and pediatric GI divisions. Trainees have outstanding opportunities to pursue molecular and cellular biology in the field of gastroenterology, including hepatology and pancreatology. Direction: The Program Director (Dr. Anil Rustgi) and Associate Director (Dr. Gary Wu) have an administrative structure that oversees the needs of the Training Program, assisted by an Internal Advisory Board and an External Advisory Board. They are complemented by rigorous individual trainee research advisory committees. Faculty: Research faculty from the Adult and Pediatric GI divisions and basic science departments are selected based upon experience with trainees, independent and externally funded laboratories, and relevant projects in digestive, liver and pancreatic diseases. The faculty is grouped by research interests; we refer to them as affinity or thematic groups: 1) Developmental biology and genetics; 2) Immunobiology and host responses; and 3) Cell growth and differentiation. Proposed Training: The cornerstone of the Program is an intensive laboratory-based research experience (basic and translational), which entails close interaction with a training program faculty mentor and close oversight by a research advisory committee and the administrative structure. This laboratory work is supplemented by an educational curriculum that includes an introductory course in molecular and cellular biology, selected University courses, research seminars and lectures, journal clubs, written and oral research presentations, and seminars on extramural funding and the bioethics of scientific research. Candidates: Outstanding trainees with MD or MD-PhD degrees (postdoctoral fellows) or occasionally PhD degrees enter the Program from the Adult and Pediatric GI Fellowship Programs after being selected through a nationally competitive application process. Additionally, there has been growth in Penn's Physician-Scientist Residency Pathway (short-track internal medicine residents pursuing GI fellowship) and new avenues have emerged to identify future trainees in gastroenterology, including through our NIDDK R25 undergraduate training grant and our medical student T32 training grant supplements.

DESCRIPTION, OVERALL (provided by applicant): The University of Pennsylvania's Digestive Diseases Research Core Center (DDRCC), entitled Center for Molecular Studies in Digestive and Liver Diseases (CMSDLD), has been funded since 1997. This competing grant proposal continues to unite investigators from multiple disciplines for integrative digestive, live and pancreatic based research. The research base includes investigators organized in the following three interrelated scientific affinity groups: (l) Developmental biology and regenerative medicine, (2) lmmunobiology and host responses, and (3) Cell growth and differentiation. The research base consists of 58 members, with interrelated scientific programs, and represents a spectrum of Departments, Centers, Institutes, and Schools at the University of Pennsylvania and surrounding Institutions. In addition, there is a young investigator base of 11 associate members, whose own individual programs and career development are nurtured by the Center. Center members are supported by $27,301,051 in digestive-diseases related NIH research funding, of which 60% is through NIDDK. A fundamental goal of our Center is to foster interdisciplinary research that leads to a cooperative understanding of the molecular and biochemical processes that form, regulate, and operate digestive tract, pancreatic and liver organs and their organizing tissues in health and disease. In this context, our intent is to utiliz the Center as a means to develop innovative ideas by attracting and engaging established investigators into digestive, liver and pancreatic research. An equally important goal of the Center is to develop young investigators in this research. Four highly successful Scientific Core facilities are designed to provide digestive-specific services for the stimulation of collaborative research: Molecular Biology/Gene Expression, Molecular Pathology and Imaging, Transgenic and Chimeric Mouse and Cell Culture. An Administrative Core directs the fiscal and organizational aspects of the Center, including the coordination and publicity of the scientific cores, pilot and feasibility (P/F) grant program, academic enrichment program and Internal/External Advisory committees. Our DDRCC's aggregate functions maintain the digestive, liver and pancreatic programs at the forefront of biomedical science.

The identification of novel genes involved in proliferation and malignant transformation has been greatly enhanced through positional cloning after localization of chromosomal regions that are deleted (designated as loss of heterozygosity-LOH-or allelic deletion) during malignant transformation. The GI tract is no exception and indeed, identification of critical genes such as APC for adenomatous polyposis coli (chromosome 5q), p53 (chromosome 17p), DCC for deleted in colon cancer (chromosome 18q), and Smad4 (chromosome 18q). Recognizing that other chromosomal regions are frequently deleted in the progression premalignant to malignant states in the colon, we have meticulously identified a new target region of allelic loss on chromosome 22q that is involved in human colorectal carcinogenesis. Fine genetic and physical mapping with microsatellite DNA markers demonstrates that the genetic length of this interval on chromosome 22q13 is 0.57 cM which corresponds to approximately 425 kb, recognized as substantial progress in human genomics and genetics projects. In addition, we have generated clones from a BAC (bacterial artificial chromosomes) library that potentially spans this contig. We hypothesize that other genes exist whose encoded proteins harbor critical genetic, biological and biochemical properties that ultimately are important in the maintenance of intestinal epithelial cell homeostasis and linked to progression to adenomatous polyp and cancer. We are collaborating closely with Dr. James Gusella s group, renowned for their contributions in neurogenetics and chromosome 22q studies. Therefore, to achieve the identification of the gene through widely available positional cloning technologies and attain its subsequent molecular characterization, we will pursue well-integrated Specific Aims:: (1) To complete the physical and genetic definition of the chromosome 22q13 region with BAC clones. We will identify candidate genes from the BAC clones using exon trapping and when necessary, cDNA selection, followed by DNA sequencing. (2) To identify the chromosome 22q13 candidate gene from among other genes in the region, each candidate gene will be studied for tumor-specific alterations (mutations) using with PCR-SSCP followed by DNA sequencing of SSCP variants. (3) To evaluate gene expression at the RNA level, Northern blotting and in situ hybridization will be performed on human tissues. In aggregate, our novel preliminary data lay the foundation for applying timely positional cloning strategies to identify the chromosome 22q gene and ultimately, leading to mechanistic insights and translational applications.

DESCRIPTION (provided by applicant): The pancreas is a complex organ with exocrine and endocrine functions that are a product of different cell lineages. It is generally believed that "precursor" cells reside in the developing pancreatic epithelium and give rise to ductal, acinar and islet cells. However, it is unclear what features distinguish such "precursor" cells and what cues trigger these cells to "transdifferentiate" into the different lineages. In addition, how these cell fate decisions are altered during pathological conditions of the pancreas, e.g. pancreatic ductal adenocarcinoma, also requires elucidation. Two important molecules that serve as "markers" of the lineages include keratin 19 (K19) for ductal epithelial cells and the PDX-1 transcription factor for beta islet cells based upon our studies and that of other groups. We have demonstrated that the K19 promoter is transcriptionaly active in pancreatic ductal epithelial cells (mediated by the GKLF/KLF4 transcription factor) but not in acinar cells. Furthermore, when the K19 promoter is fused to the lacZ reporter gene in transgenic mice, there is evidence of beta-galactosidase expression in pancreatic ductal but not acinar cells. When mice bearing a non-inducible Cre "knock-out" into the K19 locus are bred onto the Rosa26r reporter gene background, beta-galactosidase expression is largely confined to ducts. Thus, we hypothesize that K19 is associated with pancreatic ductal epithelial cell development and cell fate decisions, thereby permitting us to conduct lineage tracings in a rigorous fashion during development and adulthood. We can also dissect the conversion of the normal ductal phenotype into a premalignant/malignant phenotype. The following Specific Aims will be pursued: 1) to determine the transcriptional regulation of the K19 promoter by PDX-1 in different cell lineages and assess whether KLF4 and PDX-1 cooperate in this regulation; 2) To determine differentiation capacities of K19 expressing ductal epithelial cells in mouse pancreas during normal pancreatic development and in adult pancreas. This will be achieved by the generation and characterization of mice in which an inducible Cre recombinase is directed by the K19 promoter and bred into the Rosa26r background, allowing for temporally regulated labeling of K19-positive cells and their molecule progeny; and 3) To generate and characterize K19-mutated Ki-rats transgenic mice in order to determine the molecular basis for ductal metaplasia, a precursor to cancer. In addition, complementary approaches will be utilized to model ductal metaplasia in vitro. In summary, our interrelated studies will provide insights into cell fate decisions during pancreatic development and in adult pancreas and furnish a foundation to define the basis for premalignant stages in pancreatic ductal oncogenesis.

PROJECT SUMMARY - OVERALL Esophageal cancer is common worldwide. There are two major subtypes: esophageal squamous cell cancer (ESCC) and esophageal adenocarcinoma (EAC). Precursor lesions include esophageal squamous dysplasia and esophageal intestinal metaplasia (=Barrett's Esophagus), respectively. The molecular pathogenesis of ESCC and EAC involves genomic aberrations (e.g. cyclin D1, cyclin E, epidermal growth factor receptor or EGFR), genetic mutations or loss (e.g. TP53, p120catenin), epigenetic alterations, transcriptome derangements and interplay with environmental/lifestyle variables. As our primary overarching objective, we have made significant progress in this integrated Program Project (P01) to the identification and characterization of mechanisms underlying the molecular pathogenesis of ESCC and EAC as related to genomic and genetic ?divers? in tumor cells, novel interactions in the tumor microenvironment and approaches to tumor metastasis. As our secondary overarching objective, we are discovering common principles in biological behavior between the anatomically related ESCC with head/neck SCC and lung SCC as well as EAC with lung adenocarcinoma (LAC). For our third overarching objective, we seek to translate our novel 3D organotypic culture/3D organoid and mouse models, findings in human tissues, and preclinical therapeutic studies to clinical trials in patients. Our integrated and cohesive 3 projects and 3 core facilities, with unequivocal support from our institutions, rigorous review by internal and external advisory boards, have had significant impact upon the field of esophageal cancers and related cancers. Each Project and Core has Specific Aims and Research Strategies that are intertwined through intellectual concepts, model systems/reagents and experimental approaches that would not be possible through individual grants. Pivotal concepts and approaches in this P01 competing renewal relate to cyclin D1/CDK4 and cyclin E deregulation with new therapeutic approaches to CDK4/6 inhibition and the immune microenvironment, CDK2 inhibition (cyclin E kinase partner), and glutaminase inhibition (due to glutamine addition in the face of cyclin D1 overexpression); mutant p53 and its roles in endocytic recycling, tumor invasion and tumor metastasis; and the RANTES cytokine in the tumor microenvironment with therapeutic targeting. Our synergy has resulted in our being highly productive with visible publications, presentations at conferences, influencing the field through expansion of investigators in the field and providing leadership in task forces, which we will augment even more. In aggregate, our integrated and rigorous projects, buttressed by innovative Cores, will shape the landscape in esophageal cancer.

DESCRIPTION (provided by applicant): Ductal epithelial cells and/or centroacinar cells are believed to harbor progenitor cells and give rise to the common pancreatic adenocarcinoma. While there are experimental models to suggest that there is significant capacity for regeneration within the adult pancreatic tissue, considerable debate exists over whether these events represent true examples of "transdifferentiation" as opposed to selective expansion of undifferentiated progenitors in ductal cells or centroacinar cells. Furthermore, the molecular basis for pancreatic ductal branching morphogenesis during exocrine development and epithelial carcinogenesis has yet to be elucidated, which might provide new clues as to the events that trigger ductal differentiation, potential transdifferentiation into other cell types, and malignant transformation. The PDX-1 homeodomain transcription factor is of critical importance in endocrine cell lineage specification;however, the molecular regulation of exocrine (specifically ductal cell lineage) remains elusive. We describe in vitro and in vivo evidence that PDX-1 plays a crucial role in the regulation of ductal morphogenesis, using cytokeratin 19 as a surrogate of ductal gene expression. The overarching hypothesis of this proposal is that PDX-1 modulates pancreatic ductal branching morphogenesis, and this goes awry during malignant transformation. Using innovative three-dimensional cell culture systems (spheroid cysts) and Pdx-1 C-terminal mutant knock-in mice, this hypothesis will be pursued by the following interrelated Specific Aims: (1) To determine the molecular mechanisms underlying pancreatic ductal formation, differentiation and migration, termed branching morphogenesis or tubulogenesis, in a three-dimensional cell culture system;(2) To determine the in vivo role of PDX-1 in pancreatic ductal branching morphogenesis;and (3) To characterize terminal intercalated duct-acinar junctional cells and to characterize known factors regulating centroacinar cell proliferation, differentiation and gene expression. The insights gained from these complementary approaches will provide new insights into how pancreatic ducts develop, how they migrate and compartmentalize, and how they undergo malignant transformation. These studies will provide a platform for new molecular diagnostics and targeted therapeutics.

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) has served as a unique platform for durable insights into and advances in molecular pathogenesis, chemoprevention, diagnosis, and therapy, not only in this venue but expanded into other disease states. The elucidation of these comprehensive aspects of CRC has been facilitated through the identification of key genes and molecular pathways. It is well accepted that the majority of sporadic colorectal cancers feature the chromosomal instability pathway, involving alterations in key tumor suppressor genes (APC and p53, but also SMAD4) and oncogenes (especially Ras, but also EGFR, c-myc, B-Raf) and their downstream effectors. A subset of sporadic colon cancers undergo microsatellite instability (MSI) pathway, typically right-sided with B-Raf mutations but without Ras mutations. Recently, it has been advocated that certain colorectal cancers feature hypermethylation. We have genetically and physically mapped a region of loss of heterozygosity (LOH) on chromosome 22q13.31 as a basis to understand the role of new genes in colorectal carcinogenesis (and other cancers). We now have discovered a new telomeric region of LOH, which harbors two microRNAs, namely Let-7a3 and Let-7b, and these microRNAs are downregulated in up to 40% of colorectal cancers. MicroRNAs have become increasingly recognized for their pivotal, diverse roles in development, differentiation, proliferation, and cancer, which is achieved the degradation or interference with translation with messenger RNAs. We hypothesize that the Let-7a3 and Let-7b microRNAs are instrumental in colorectal cancer progression, and this may be achieved by targeting the Ras oncogene, and also, the c-myc oncogene. This novel hypothesis will be pursued by the following interrelated Specific Aims: (1). To identify the roles of the Let-7a3 and Let-7b microRNAs in colorectal cancer. A. To determine the relationship between these microRNAs and clinical parameters and molecular parameters;B. To determine the function(s) of Let7a and Let7b in colorectal carcinogenesis through genetic /overexpression/knockdown studies of these microRNAs with determination of effects upon cellular proliferation, apoptosis, migration and invasion, which are critical features of the colon cancer initiation and progression. (2). To determine if the Ras and c-myc oncogenes represent targets of Let-7a3 and Let- 7b. This will be achieved through examination of Ras transcriptional and protein activities in the setting of genetic approaches, namely Let-7a3 and Let-7b overexpression/knockdown. (3) To determine the functional consequences of restoration and inhibition of Let-7a3 and Let-7b in colon cancer cells in vivo. This will be assessed by innovative three-dimensional organotypic cultures, which mimic the colon cancer microenvironment, and in athymic, irradiated nude mice through bioluminescence of tumor growth. In aggregate, our studies have the means to unravel new pathways of regulation of the Ras and c-myc oncogenes by the Let-7a3 and Let-7b microRNAs in colon cancer, especially where sporadic colorectal cancers (chromosomal instability pathway) do not harbor Ras mutations. PUBLIC HEALTH RELEVANCE: Colorectal cancer (CRC) serves as a paradigm for the investigation of basic mechanisms and translation into novel chemopreventive, diagnostic and therapeutic strategies. The underlying pathogenesis of CRC may involve the Let-7a3 and Let-7b microRNAs, which in general are critical regulators of normal cellular processes to malignant transformation. Our studies hope to reveal new paradigms in CRC pathogenesis, and offer opportunities for exploiting this knowledge in novel diagnostics and therapeutics.

DESCRIPTION (provided by applicant): Esophageal cancer is common worldwide and comprises two subtypes: esophageal squamous cell cancer (ESCC) and esophageal adenocarcinoma (EAC). Unfortunately, most patients present at late stages, thereby making survival rates low at less than 5% at 5 years. This grim prognosis mandates the utilization of innovative and unique genetically engineered mouse models developed by our research team to implement new strategies for functional genomics, prevention, imaging and therapy. The unique consortium involves long-standing collaborations and publications between recognized experts in the field of esophageal cancer from the University of Pennsylvania, Columbia University, and Massachusetts General Hospital. In particular, the Principal Investigators are Dr. Anil Rustgi (University of Pennsylvania), Dr. Timothy Wang (Columbia University) already within the NCI Tumor Microenvironment Network, and Dr. Umar Mahmood (Massachusetts General Hospital). The overarching goals of this U01 grant proposal are the following: (1) To utilize innovative models of esophageal cancer as a platform for elucidating the link between inflammation and cancer in the tumor microenvironment; and (2) To provide new approaches in the imaging of esophageal cancer for advances in translational medicine. These goals will be pursued through five interrelated Specific Aims that are trans-disciplinary and trans-institutional, and provide new We hope that the unique interrelated models developed by Drs. Rustgi and Wang may provide a new, future direction in tissue imaging of precancerous and cancerous lesions facilitated by the development of mouse upper endoscopy, non-invasive imaging technologies and molecular probes (tracking inflammatory/immune cells in the esophageal cancer microenvironment) by Dr. Umar Mahmood, which would have a great impact upon early detection of esophageal cancer in the human. In aggregate, this highly accomplished research team is poised to translate models of esophageal cancer into the eradication of human esophageal cancer, which would alter clinical approaches in the United States and worldwide. The proposed research is in concert with the recommendations by the NCI progress group report on upper Gl cancers, and the NCI Think Tank recommendations on the Tumor Microenvironment as well as Inflammation and Cancer. RELEVANCE (See instructions): Esophageal cancer comprises two main subtypes, esophageal squamous cell cancer (ESCC) and esophageal adenocarcinoma (EAC), both common in the US and worldwide. In fact, ESCC is the 5th most common cancer amongst males worldwide and afflicts African-Americans with a high predilection in the US. EAC has the fastest rate of increase of any cancer in the US. The proposed research will translate models into improving clinical outcomes of esophageal cancers.

DESCRIPTION (provided by applicant): Stem cells are defined by the capacity for long-term self-renewal and multilineage specification. Until recently, our understanding of stem cells, as well as their role in human diseases has been rather limited. Moreover, interest in harnessing the stem cell's capacity for self-renewal to promote organ and tissue regeneration traverses many medical disciplines. It is now recognized as imperative that we better understand the complex genetics and processes that support and define the stem cell. This proposal is a multidisciplinary approach to this problem, combining the scientific strengths of four investigators, as well as institutional core core facilities and support into a cohesive approach to study the intestinal stem cell. Recently, genetic studies have identified robust markers for stem cell populations in the intestine. These advances now make it possible to isolate stem cell populations for more advanced molecular investigations They also provide us an opportunity to study how disease environments impact upon stem cell viability and specification. Stem cells are highly reliant upon mechanisms to counter the numerous stresses associated with cellular replication. Defects in maintaining genome stability in the face of such challenges cause stem cell losses. If this process is unchecked, it can lead to the premature onset of age-related degenerative pathologies. Another challenge encountered by stem cells is to correctly determine their tissue identity based upon environmental cues. Errors in stem cell identity are encountered in intestinal metaplasia of the esophagus and stomach, as well as many gastrointestinal cancers. With these observations in mind, we propose to test the following hypothesis: Intestinal stem cell identity and viability can be modulated by cell-autonomous and non-cell autonomous processes. This hypothesis will be pursued by the following interrelated Specific Aims: (1) Functional characterization of the contribution by bone-marrow derived cells to the intestinal stem cell (ISC) niche in vivo. (2) The homeodomaln transcription factor Cdx2 specifies the stem cell's "intestinal" identity. (3) Develop novel strategies to identify new intestinal stem cell markers and assay stem cell functions. This proposal seeks to exploit our combined expertise in order to better understand the molecular events that support and specify intestinal stem cells. Understanding these molecular processes will greatly improve our ability to develop novel therapeutic strategies to exploit the regenerative potential of stem cells, as well as correct stem cell deficiencies that contribute to many Gl diseases. PUBLIC HEALTH RELEVANCE: Stem cells are critical for the renewal of the intestinal epithelium. Many human diseases can affect stem cell viability and capacity for self-renewal and differentiation. This proposal explores contributions to intestinal stem cell biology and survival by bone-marrow and stromal elements, as well as cell-autonomous mechanisms. Knowledge gained here will have applications for regenerative medicine, aging, and carcinogenesis.

Esophageal cancer is a common malignancy in the United States and worldwide, and yet, carries a dismal prognosis with 5-year survival of less than 30% for early stages and less than 10%for advanced stages. Progress has been hampered by the lack of robust, reproducible models. To that end, we have developed innovative models in organotypic culture and in animals that phenocopy, for the first time, human esophageal squamous cell cancer (ESCC). These models emphasize the role of critical oncogenes and tumor suppressor genes. We hypothesize that the interplay of the EGFR oncogene with the dysregulation of the p53 and p120-catenin (p120ctn) tumor suppressor genes alters the tumorigenic potential of esophageal cells, thereby resulting in increased cell migration and invasion. Furthermore, we hypothesize that changes in the mesenchymal compartment of the esophagus occur during tumorigenesis whereby tumor activated fibroblasts interact in a paracrine manner to induce increased esophageal tumor cell migration and invasion. These interdependent hypotheses will be pursued by the following interrelated Specific Aims to understand the esophageal tumor microenvironment: (1) To investigate the role of p120ctn, a functional interactor with EGFR, in fostering esophageal cancer cell migration and invasion in the tumor microenvironment. Specifically, we will assess the effects different isoforms and phosphorylation mutants of p120ctn and understand the role of p120ctn in esophageal tumorigenesis using genetically defined organotypic culture and in vivo models;(2) To investigate how EGFR overexpression and p53 mutation cooperate to induce esophageal tumorigenesis in similar models with an emphasis upon the differential biological properties of structural p53 mutants found to occur in esophageal cancer;(3) To identify critical signaling pathways in activated fibroblasts involved in paracrine signaling to tumor cells that increase tumor cell migration and 'nvasion in the microenvironment. These collective approaches provide a platform for exciting, new targeted therapeutic approaches in concert with Projects 2 and 3 that are primed to overcome resistance of esophageal cancer to current therapy. Project 1will continue to utilize the outstanding core facilities. RELEVANCE (Seeinstructions): This Project focuses upon the interplay of the EGFR oncogene with the p120ctn and p53 tumor suppressor genes in fostering esophageal squamous epithelial transformation and the interaction with stromal fibroblasts, and has unraveled new models and insights. The findings have relevance in combinatorial therapy of esophageal cancer and other squamous cell cancers.

The Administrative Core will provide comprehensive and cohesive support in administrative, fiscal and programmatic aspects of the Program Project. It will also interface with the University of Pennsylvania, the Wistar Institute, Fox Chase Cancer Center and the National Cancer Institute (NCI). The Administrative Core, in geographical proximity to the scientific core facilities and the laboratories of the project leaders, will serve many organizational and fiscal functions. These include generation and analysis of monthly budget reports, interfacing with the medical school's research services, coordinating clinical/translational programs, and completing NCI requirements. In addition, the administrative core will continue to coordinate monthly meetings with the project and core facilities leaders; weekly core facilities' meetings; regular research seminars/journal clubs/laboratory meetings; maintain a library, newsletter and website; and promote the program project within the university and medical school (especially as it relates to the internal advisory board-lAB), as well as regionally and nationally. Importantly, the Core will organize the annual conference/retreat in conjunction with the review by the external advisory board (EAB). All IAB and EAB reports will be organized by the Core that will be shared with the Dean, Cancer Center Director, Wistar Institute Director and the NCI. As additional measures, the Core will organize workshops in grant writing, manuscript writing, and career development for students, postdoctoral fellows and junior faculty in the Program Project. It will expand the human tissue banking efforts of the Morphology and Molecular Biology Cores, promote the relational databases of the Molecular Biology Core, ensure appropriate bioinformatics support through the Molecular Biology Core and integrate the new Biostatistics Core. The Administrative Core will advance the innovative and far-reaching goals articulated and envisioned by the Program Project. In aggregate, the administrative core, already in existence, will enhance the intellectual, scientific, structural and fiscal components of the Program Project. Individually and together, Drs. Rustgi and Herlyn and Ms. Hay have vast experience in administration and ensuring thematic integration between the Projects and Cores. RELEVANCE (See instructions): This Core is critical to the growth and expansion of the Program Project at Penn, regionally, nationally, and internationally. It achieves prominence of the Program Project by integrating the Projects, providing support for the other cores, nurturing new investigators-into the field, expanding the repetoire of the databases/reagents of the Program Project, and working closely with institutional leadership and the NCI.

DESCRIPTION (provided by applicant): Barrett's esophagus is an increasingly prevalent, preneoplastic disorder that results primarily from gastroduodenal reflux of acid and bile. The applicant group, which includes established investigators from Columbia University, the University of Pennsylvania, and the Mayo Clinic, propose a multidisciplinary, multicenter, translational research program to study the origins and pathogenesis of the disorder. The team, many of whom have collaborated in the past, will focus on the role of chronic inflammation and bile acids in the upregulation of established and novel stem cell markers, and possible ways to target these progenitor cells. The proposal builds on extensive basic science findings, the development of novel transgenic (L2-IL-1beta) and surgical models of BE/esophageal adenocarcinoma, and preliminary analyses of human BE tissues. The groups will bring to the network a very large BE patient population, experienced Barrett's clinical investigators, and bench researchers with extensive experience with stem cells and inflammatory models of Gl cancer. Three projects are proposed. Project 1 will focus on the role of Notch signaling in models of Barrett's esophagus, and will determine the effects of Notch inhibition or Notch activation on progression to dysplasia. Project 2 will seek to characterize the cell of origin in Barrett's esophagus in our mouse models. We will use constituitive and inducible mouse models of Cre expression to lineage trace active and quiescent progenitors that are upregulated in our model and in human BE. Additionally, we will carry out a pilot clinical trial using an antagonist of a G-protein coupled receptor expressed on progenitor cells upregulated in BE. Finally, Project 3 will aim to identify novel biomarkers and gene signatures in BE, correlating data sets from animal and human models, clarifying the importance of non-goblet cell columnar epithelium and changes in the gastric cardia. We will also assemble a cohort of patients undergoing radiofrequency ablation, identifying biomarkers of response to therapy and using successfully ablated patients as a novel human model to study the development of BE. Overall, these studies aim to elucidate the earliest stages and cell types that contribute to BE pathogenesis in order to better stratify risk and develop preventive therapies.

DESCRIPTION (provided by applicant): The pancreas is an organ that has endocrine (islet lineage) and exocrine compartments (acinar and ductal lineage). The ductal tree consists of terminal, or intercalated, ducts that interface with acini or centroacinar cells, which are potentil sites of progenitor cells. Intercalated ducts merge to form intralobular ducts, and these in turn merge to form interlobular ducts, and finally, into the main duct that traverses the pancreas to the duodenum, delivering fluid laden with digestive enzymes. We have emphasized studies (in the previous cycle) on the governance of the regulation of ductal branching morphogenesis. Using this as a platform, we now wish to elucidate the transcriptional programs that regulat ductal development, but in particular regulate the emergence of ductal structures during acinar-ductal metaplasia (ADM) and the transition of normal ducts to preneoplastic ducts (termed PanIN). We hypothesize that a newly discovered homeodomain transcription factor, Prrx-1, is critical in ductal cell morphogenesis, acinar-ductal metaplasia, and re-neoplasia. This hypothesis will be pursued by the following Specific Aims through complementary in vitro and rigorous in vivo approaches: (1) To elucidate the mechanistic underpinnings of Prrx1 in ADM and PanIN. We will determine the functional roles of Prrx1+ cells in ADM through genetic lineage tracing studies in vivo. We will define the dependence of pancreatic regeneration after ADM through the pancreatic conditional knockout of Prrx1. We will determine if Prrx1+ cells are more susceptible to the effects of oncogenic KrasG12D in the development of PanIN. (2) To determine the gene targets of Prrx1 through CHiP-Seq, to validate specific gene targets identified in preliminary analysis and determine their functiona relationships. (3) To delineate the functional differences between Prrx1 isoforms in the roles in ADM and PanIN: the role of Prrx1b in self-renewal of cells and role of Prrx1a in invasion. Our novel insights and innovative approaches will for the first time help to mechanistically characterize a transcriptional factor that is critical in ductal development, ADM and PanIN, and place this gene as potentially as important as Pdx1 in the endocrine lineage and Ptf1a (p48) in the acinar lineage. These studies will help to provide opportunities for management of pancreatitis to enhance tissue regeneration and of PanIN/PDAC.

ADMINISTRATIVE LEADERSHIP AND STRUCTURE The Administrative Core has a number of critical functions: (1) to oversee the scientific, programmatic, operational and fiscal affairs of the Center for Molecular Studies in Digestive and Liver Diseases (CMSDLD); (2) to provide support for the scientific core facilities; (3) to coordinate the oversight and implementation of the pilot and feasibility grant program; (4) to furnish support to Center members; (5) to foster the career development of Center associate members; (6) to coordinate the internal and external advisory board meetings, as well as the steering committees of the Cores; (7) to promote interdisciplinary research at Penn and with other institutions in Philadelphia (Jefferson, Drexel, Temple and Fox Chase Cancer Center); (8) to interact with the NIH/NIDDK administration and other DDRCCs; and (9) to promote the CMSDLD missions in the national and regional scientific and lay communities.

¿ Pilot Projects One pilot project will be funded per year, beginning in Year 2 of the award. The purpose of these projects will be to conduct translational research in Barrett's esophagus that meets the objectives of the BETRNet, falls within one of the four designated Research Areas, and that can utilize effectively the resources available through our integrated Research Center. Cross-BETRNet Activities One Cross-BETRNet project will be funded per year, beginning in Year 2 of the award. The purpose of these projects will be to conduct translational research in Barrett's esophagus that meets the objectives of the BETRNet, falls within one of the four designated Research Areas, and that can feasibly utilize the resources available through our Research Center in combination with the other BETRNet consortia. The below outlined application details will be modified as needed based on guidelines set forth by the BETRNet Steering Committee. In order to minimize duplicated effort, much of the organization, administration, and oversight for Cross-BETRNet Projects is similar to that described above for the Pilot Projects. Additionally, we have commitment from other BETRNet Consortium applicants regarding potential future cross-BETRNet collaborative efforts (see Section 2.7. Letters of Commitment).

DESCRIPTION (provided by applicant): The RNA-binding protein LIN28B has been implicated in a variety of cancers, with their expression correlating with an aggressive phenotype, enhanced metastasis and poor prognosis. When expression is directed to the mouse intestinal epithelium we find that LIN28B causes intestinal hypertrophy, loss of Paneth cells, crypt fission and intestinal adenocarcinoma formation. LIN28B specifically targets and repress the Let-7 family of miRNAs. LIN28B inhibits Let-7 microRNA (miRNA) maturation through sequestration of pri-miRNAs n the nucleolus, which is inaccessible to microprocessor machinery. By rescuing Let-7 levels with an inducible Let- 7a transgenic mouse model, we determined that the LIN28B phenotypes noted above are due to Let-7 dependent actions of LIN28B. Furthermore, we find that LIN28B mediated downregulation of Let-7 results in the upregulation of Igf mRNA binding protein-1 (IMP1), and thus, we will investigate the role of the interplay between LIN28B and IMP-1 in intestinal homeostasis and cancer. Since Let-7-independent mechanisms of LIN28B function have been reported, we have pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (RNP CLIP-Seq) to identify mRNAs and miRNAs that are targeted directly by LIN28B.This reveals that LIN28B binds to mRNAs involved in metabolic processes, splicing, and regulation of the actin cytoskeleton. Thus, we identify a new pathway of LIN28B-Let7-IMP1 in colon carcinogenesis that adds to our knowledge of underlying molecular mechanisms, but also provides a platform in translational opportunities by using LIN28B as a biomarker for disease progression, and targeting this pathway therapeutically.

DESCRIPTION (provided by applicant): Barrett's esophagus is an increasingly prevalent, preneoplastic disorder that results primarily from gastroduodenal reflux of acid and bile. The applicant group, which includes established investigators from Columbia University, the University of Pennsylvania, and the Mayo Clinic, propose a multidisciplinary, multicenter, translational research program to study the origins and pathogenesis of the disorder. The team, many of whom have collaborated in the past, will focus on the role of chronic inflammation and bile acids in the upregulation of established and novel stem cell markers, and possible ways to target these progenitor cells. The proposal builds on extensive basic science findings, the development of novel transgenic (L2-IL-1beta) and surgical models of BE/esophageal adenocarcinoma, and preliminary analyses of human BE tissues. The groups will bring to the network a very large BE patient population, experienced Barrett's clinical investigators, and bench researchers with extensive experience with stem cells and inflammatory models of Gl cancer. Three projects are proposed. Project 1 will focus on the role of Notch signaling in models of Barrett's esophagus, and will determine the effects of Notch inhibition or Notch activation on progression to dysplasia. Project 2 will seek to characterize the cell of origin in Barrett's esophagus in our mouse models. We will use constituitive and inducible mouse models of Cre expression to lineage trace active and quiescent progenitors that are upregulated in our model and in human BE. Additionally, we will carry out a pilot clinical trial using an antagonist of a G-protein coupled receptor expressed on progenitor cells upregulated in BE. Finally, Project 3 will aim to identify novel biomarkers and gene signatures in BE, correlating data sets from animal and human models, clarifying the importance of non-goblet cell columnar epithelium and changes in the gastric cardia. We will also assemble a cohort of patients undergoing radiofrequency ablation, identifying biomarkers of response to therapy and using successfully ablated patients as a novel human model to study the development of BE. Overall, these studies aim to elucidate the earliest stages and cell types that contribute to BE pathogenesis in order to better stratify risk and develop preventive therapies.

PROJECT 1 ABSTRACT Esophageal cancer comprises two major subtypes, namely esophageal squamous cell carcinoma (ESCC), and esophageal adenocarcinoma (EAC). Esophageal cancer poses grave and pressing clinical problems in the United States and worldwide as reflected by the increasing incidence of EAC in the US, and of the worse prognoses of any cancers as evident in ESCC worldwide. We are focusing on the p120catenin (p120ctn) or CTNND1 and TP53 tumor suppressor genes. Tumor cell progression is illustrated by invasion into the extracellular matrix (ECM) or stoma. This then involves interrelated networks between tumor cells and diverse cell types in the tumor microenvironment. This cross-talk and cross-fertilization trigger a necessary cascade of events prior to dissemination of tumor cells into blood and lymphatic vessels, as well as local and distant metastasis. These include, but are not restricted to, immune cells/inflammatory cells, fibroblasts, endothelial cells, pericytes, neurons, and adipocytes. Our unified view is that p120ctn loss or mislocalization, either of which abrogates its tumor suppressor activities involved in the maintenance of the adherens junctions (complex with E-cadherin) fosters tumor initiation as revealed by our published work on the conditional loss of p120ctn in the mouse esophagus, resulting in invasive ESCC (with local metastasis to lymph nodes) accompanied by desmoplasia and the specific recruitment of myeloid derived suppressor cells (MDSCs) or immature myeloid cells. Tumor progression requires the acquisition of TP53 mutations, which conspire to drive further tumor invasion. The tumor cells interact with cancer-associated fibroblasts (CAFs) and these MDSCs in the tumor microenvironment, involving in part the IL-6 master cytokine that is pro-inflammatory and pro- tumorigenic. Thus, our overarching hypothesis is that p120ctn and TP53 proteins cooperate in tumor progression, TP53 mutation triggers an invasive gene signature that drives tumor cells to invade into the ECM and remodel the ECM, and that tumor invasion in the microenvironment involves the interactions between tumor cells, CAFs and MDSCs. This hypothesis will be pursued by the following interrelated Specific Aims. Aim 1: To evaluate CD38 induction in MDSC populations and its role in immunosuppression via iNOS activation. Aim 2: To elucidate the functional roles of IL-6 as a mediator of cross talk between tumor cells and CAFs in the ESCC microenvironment. Aim 3: To elucidate the functional interplay between p120ctn and TP53 in the esophageal tumor microenvironment. Our successful achievement of these Specific Aims is facilitated greatly by the synergy between the exceptional Projects and the exceptional support provided by the Core Facilities, apart from the broad and deep institutional support.

CORE A ABSTRACT The Administrative component of Core A will provide comprehensive and cohesive support in administrative, fiscal and programmatic aspects of the Program Project. It will also interface with the University of Pennsylvania, Dana Farber Cancer Institute, Fox Chase Cancer Center and the National Cancer Institute (NCI). The Administrative component, in geographical proximity to the scientific core facilities and the laboratories of the project leaders at Penn, will serve many organizational and fiscal functions. These include generation and analysis of monthly budget reports, interfacing with the medical school's research services, coordinating clinical/translational programs, and completing NCI requirements. In addition, the administrative component will continue to coordinate monthly meetings with the project and core facilities leaders; weekly core facilities' meetings; regular research seminars/journal clubs/laboratory meetings; maintain a library, newsletter and website; and promote the program project within the university and medical school (especially as it relates to the internal advisory board-IAB), as well as regionally and nationally. Importantly, the Core will organize the annual conference/retreat in conjunction with the review by the external advisory board (EAB). All IAB and EAB reports will be organized by the Core that will be shared with the Dean (Dr. Larry Jameson), Abramson Cancer Center Director (Dr. Chi Dang), DFCI (Dr. Griffin) and the NCI. As additional measures, the Administrative component will organize workshops in grant writing, manuscript writing, and career development for students, postdoctoral fellows and junior faculty in the Program Project. It will continue to expand the human tissue banking efforts of the Molecular Pathology/Imaging Core (MPIC) and Molecular Biology Core (MBC), promote the databases generated by the Projects and Cores, ensure appropriate the new genomics bioinformatics support through the Molecular Biology Core and integrate the Biostatistics component. There has been establishment of close communication, interaction and synergistic collaborations with Project 3 based at DFCI via direct bidirectional visits (by Project Leaders as well as lab personnel, the latter from DFCI for instruction in the MPIC and MBC), monthly P01 meetings (using current capture and video technology), weekly conference calls, and The Administrative component will advance the innovative and far- reaching goals articulated and envisioned by the Program Project. In aggregate, the administrative component will enhance the intellectual, scientific, structural and fiscal components of the Program Project. Individually and together, Dr. Rustgi and Ms. Hay have vast experience in administration and ensuring thematic integration between the Projects and Cores. The Biostatistics subcomponent will be led by Dr. Gimotty, Associate Director of Core A, who has vast cancer biostatistical experience and joint publications as evidence of the rich synergistic interactions and collaborations.Dr. Gimotty will provide her expertise and experience in cancer biostatistics in the following areas: a) Design of experimental and clinical studies. b) Statistical modeling and Statistical analysis to evaluate each project's research hypotheses. c) Interpretation of research data and collaboration with investigators to make scientifically and statistically appropriate statements, and to assist in the preparation of scientific abstracts, presentations, and manuscripts. d) Methodological investigations to solve practical problems that arise including analyses requiring non-standard methods, comparison of alternative statistical methods, and conduct of discovery studies using archived public databases relevant to the research projects.

PROJECT SUMMARY The exocrine pancreas has a remarkable ability to regenerate after injury, as illustrated in acute pancreatitis, and subsets of chronic pancreatitis. Acinar-ductal metaplasia (ADM) is critical in the ability of the exocrine pancreas to regenerate or permit progression to a preneoplastic state (pancreatic intraepithelial neoplasia or PanIN). Expression of oncogenic Kras* (=mutant Kras) in the mouse pancreas leads to formation of PanIN lesions with long latency, indicating the need for genetic and possibly epigenetic ?second hits?. Chronic pancreatitis is recognized as a strong risk factor for pancreatic ductal adenocarcinoma (PDA) in humans. In mouse models of pancreatic cancer, induction of either acute or chronic pancreatitis results in tissue-wide ADM that is followed by rapid repair (we designate this as ?Adaptive? ADM). However, in the presence of oncogenic Kras*, repair is impaired and ADM progresses to PanIN lesions (we designate this as ?Oncogenic? ADM). Currently, the mechanisms underlying the formation of ADM and how ADM progresses to PanIN in the presence of mutant Kras* remain unknown. Recently, our group performed gene expression analysis of murine ductal cells isolated from the developing pancreas, acute pancreatitis (ADM), and PanIN expressing oncogenic KrasG12D, and compared the expression profiles to that of normal pancreatic ductal cells, resulting in nearly 80 potential genes of interest. Prrx1 (paired-related homeobox 1) was the most differentially regulated transcription factor in all three processes, followed by Etv5, a member of the Ets family of transcriptional factors. Based upon compelling published and preliminary data, we hypothesize that Etv5 and Prrx1 are involved in the initiation and maintenance of ADM, respectively, following pancreatitis. Furthermore, we hypothesize that this regulation allows for subsequent transformation by oncogenic Kras*, thereby promoting progression to PanIN. This hypothesis will be tested through the following interrelated Specific Aims: (1) To determine if Prrx1 is required for ADM and PanIN following pancreatic injury; (2) To elucidate the relationship between Etv5 and Sox9 in the functional regulation of ADM; and (3) To identify and evaluate gene targets of Prrx1 and iKras* (inducible mutant Kras) in the development of ?Oncogenic? ADM (to PanIN). This aim will identify effectors of Prrx1 and iKras*. Our innovative and integrated research will define the transcriptional regulation of ADM and provide a basis for new perspectives in the therapy of pancreatitis and PanIN.

Project Summary The Tumor Biology Program aims to catalyze research within the ACC to advance understanding of the molecular mechanisms underlying cancer pathogenesis and to translate this knowledge to identify new and more effective preventive, diagnostic, prognostic and therapeutic approaches. This Program, which was founded in the early 1970s, continues to progressively respond to advances in cancer research. Its overarching scientific aims are to: 1) Elucidate the molecular and cellular basis of carcinogenesis, and 2) Translate these findings into durable clinical applications. To that end, the Program is organized around three central goals or themes: 1) Structural biology of molecules relevant to cancer, 2) The molecular and cellular basis of cancer, and 3) The use of model organisms to study cancer in vivo. Interactive, intra-Programmatic scientific affinity groups foster collaboration within and across themes, including: 1) Tthe tumor microenvironment, 2) RNA biology, 3) DNA repair and genomic stability, 4) Cancer cell metabolism (this spawned a new ACC Basic Science Center of Excellence), and 5) Organ-specific cancers, such as pancreatic (this has been integrated into the new ACC Pancreatic Translational Center of Excellence). The Program continues under the strong leadership of Anil Rustgi, MD, who fosters deep and impactful intra- and inter-Programmatic collaborative relationships. Program Leadership is also instrumental in recruiting new members (e.g., Drs. Berger, Feldser, Garcia, and Puré), mentoring junior faculty, establishing scientific affinity groups to leverage and further develop common research interests among faculty members, and organizing conferences and symposia. Program members are extremely actively involved in the training and mentorship of PhD students, MD/PhD students, and MD or PhD postdoctoral fellows by virtue of their leadership roles in the Penn Biomedical Graduate Studies, MD/PhD (MSTP) program and NIH T32 training grants. Program Leadership was highly successful in harnessing Institutional support to catalyze a tumor biology translational research initiative and increased translational research has been pursued in both thematic and organ-specific contexts. Its success is evident in the emergence of new transdisciplinary, disease-specific collaborations that join Program members with colleagues in Clinical Research and Population Science to study cancers, such as pancreatic and esophageal, in which there is an unmet need for improved diagnosis and treatment. Currently, the Program has 50 members from 16 departments and four different schools with total funding of $22M (annual direct costs) of which $18.7M is peer-reviewed and $5.6M is NCI-funded. Since 2010, members published 466 cancer-related papers, of which 15% were intra-Programmatic, 32% were inter-Programmatic, and 30% were multi-institutional.

PROJECT SUMMARY (PILOT & FEASIBILITY PROGRAM) The goal of this program is to provide funding for innovative projects compatible with the scientific focus of the Center for Molecular Studies in Digestive and Liver Diseases (CMSDLD). We have had high success in collaborations with other Penn Centers/Institutes/Children's Hospital of Philadelphia for joint RFAs that have served to increase funding for awardees in a significant fashion. Furthermore, we strive to support new investigators, including both young scientists and those who have not previously pursued research in digestive or liver diseases. The desired outcomes of the Pilot and Feasibility Grant Program are new R01 grant proposals (and equivalent, e.g. VA Merit grant), new interdisciplinary grant proposals (e.g. NIH U01/U54, P01, P50 mechanisms) and new significant private foundation/national society grant proposals. Indeed, our pilot and feasibility grant program has had an impressive return of investment in new grants, exceeding 10:1. This is complemented by a vast array of support through scientific core facilities, structured mentorship programs, and deep conviction to augment the campus wide and national careers of recipients, and facilitate the academic promotions of recipients. This program is structured to be inclusive of scientists throughout the University of Pennsylvania. Importantly, we have a robust track record of identifying young scientists who are women and represent diversity and inclusion, consistent with our Center's philosophy and vision.

PROJECT SUMMARY (ENRICHMENT PROGRAM) The University of Pennsylvania's DDRCC is the Center for Molecular Studies in Digestive and Liver Diseases (CMSDLD). The CMSDLD's vision and missions revolve around interdisciplinary, innovative and impactful scientific research that is translated into understanding human health and ameliorating human diseases and disorders. The CMSDLD catalyzes advances in scientific research through a comprehensive and vibrant library of integrated educational and training programs. These enrichment programs bring together Center members to foster new research directions, promote the professional, academic and scientific development of Center associate members, host weekly research seminars/joint lab meetings/journal clubs/Advances in Technology series, host Visiting Professorships (monthly), build upon a history of growing Endowed Lectureships, and highlight a didactic series of academic skills workshops (for Center associate members and trainees) as well as a new Introduction to Molecular Biology course (for Center lab personnel that are new to science). In addition, our CMSDLD is at the vanguard of training programs spanning undergraduate students (NIDDK R25 training grant in GI basic sciences and an NIH R25 training grant in GI clinical sciences under review), medical students (MD year out programs, as well as the largest MD PhD program in the country), graduate students, and postdoctoral fellows (two NIH T32 training grants). Our CMSDLD members hold various leadership positions in all these endeavors, thereby ensuring the development and maturation of a ?pipeline? of young scientists for future engagement in digestive, liver and pancreatic diseases research. Finally, our CMSDLD emphasizes gender, diversity and inclusion in all its pathways, and is committed to successful outcomes in this context as well.

OVERALL PROJECT SUMMARY Barrett?s esophagus is an increasingly prevalent, preneoplastic disorder resulting from acid/bile reflux and chronic inflammation at the GE junction. This application is a renewal of a long-standing multicenter, translational research program from Columbia University, the University of Pennsylvania and the Mayo Clinic. The team, which has been highly productive, will now focus on the role of microbiota and the tumor microenvironment in the development and progression of Barrett?s esophagus and esophageal adenocarcinoma. The group has added additional collaborative sites at MIT, the Dana Farber Cancer Institute and Munich Technical University, and will utilize heavily a new Microbiome and Metabolomics Core at Penn- CHOP. Thus, the team comprises broad and unique expertise in mouse models, genomics, microbiology and clinical research. The application is built around the hypothesis that the inflammation-dependent tumor microenvironment, modulated by the GE junction microbiome, is critical for early progression of esophageal carcinogenesis. The proposal will utilize both the novel transgenic (L2-IL-1?) and innovative 3D organoid models, along with a cross-sectional study of 100 BE patients. Project 1 will study the role of microbiota and myeloid cells in the L2-IL-1? mouse model of Barrett?s esophagus. This project will incorporate germ-free housing, antibiotic eradication, colonization with defined flora, myeloid cell ablation and correlative human studies. Project 2 is focused on the characterization of microenvironment drivers in BE, and will include FACS/IHC analysis of CAFs and immune cells (MDSCs/Tregs) in BE patients, along with 3D organoids in culture. The role of IL-6 in response to epithelial TP53 mutations and immune cell activation will be defined. Finally, Project 3 will seek to identify novel biomarkers and gene signatures related to the microbiome and microenvironment. The study will analyze bile acids, a product of microbes, and minimally invasive tests such as saliva/breath test/tethered capsule sponge to analyze microbes to develop screening/surveillance strategies. Overall, these projects will advance the science of the microbiome and microenvironment in BE that will hopefully lead to translational applications.

PROJECT SUMMARY (OVERVIEW) The NIDDK P30 Digestive Diseases Research Core Center at the University of Pennsylvania is called the Center for Molecular Studies in Digestive and Liver Diseases (CMSDLD). Constituted in 1997 and funded continuously since then (with highly successful competitive renewals in 2002, 2007 and 2012), the CMSDLD provides an exceptional platform for basic and translational research in digestive, liver and pancreatic diseases with a vision of understanding human health and ameliorating the public health burden associated with these diseases. This overarching vision is executed through the interrelated missions or Specific Aims of the CMSDLD: (1) to support impactful interdisciplinary and collaborative digestive, liver and pancreatic research through its Members/Associate Members, who span 4 Schools at the University of Pennsylvania, multiple Departments/Centers/Institutes, importantly, Children's Hospital of Philadelphia, as well as institutions within and surrounding Philadelphia; (2) to foster the academic and professional development of its Associate Members; (3) to provide state-of-the art services and technologies through its scientific core facilities (with quality and cost-effectiveness), which in turn support the Members and Associate Members; (4) to oversee innovative enrichment (inclusive of education and mentorship) programs and a highly successful pilot and feasibility grant program; (5) to promote gender, diversity and inclusion as part of our CREED (Clinical care, Research, Education, Encouragement and Diversity) and (6) to collaborate with other Penn Centers/Institutes as well as national universities, academic medical centers, other DDRCCs and the NIH/NIDDK, and conduct outreach with the lay public. Rigor, reproducibility and transparency are also critical aspects of the CMSDLD. The CMSDLD has an administrative structure, or Core, which is overseen by the Dean, and guidance from highly interactive Penn/CHOP leaders, and internal and external advisory boards. The CMSDLD may be viewed as a stem cell, which self-renews and concurrently gives rise to differentiated entities on campus that in turn continue to interact with the CMSDLD: (A) Members who apply for and receive new interdisciplinary grants spawned by the CMSDLD; (B) Penn-CHOP Joint Center in Transitional Medicine (adolescence to adulthood) in digestive, liver and pancreatic medicine; (C) Penn-CHOP Microbiome Program. The Perelman School of Medicine and Children's Hospital of Philadelphia have invested heavily in the CMSDLD and are committed to continue to do so in the next funding cycle. The CMSDLD is positioned to build upon its successes and be a vanguard of research, education and translational clinical care in the future.

PROJECT SUMMARY The intestinal epithelium is in a dynamic equilibrium of proliferation, differentiation and apoptosis along the crypt-villus gradient. Normal intestinal homeostasis is disturbed during states of infection, inflammation and malignant transformation (adenomatous polyps, colorectal cancer). The pathogenesis of sporadic colorectal cancer involves distinct pathways with characteristic genomic and genetic alterations. Despite significant advances in leveraging our understanding of these pathways for diagnostic, prognostic, and therapeutic strategies, colorectal cancer (CRC) remains a leading cause of cancer-related mortality. This underscores a specific need to identify and understand novel, therapeutically tractable pathways in intestinal homeostasis that may drive CRC. Our work has introduced and elucidated the novel role of mRNA binding proteins in intestinal/colonic epithelial homeostasis, as well as aberrations including hyperproliferation, altered metabolism and transformation. LIN28B, an mRNA binding protein, also critical in embryonic stem cells, post- transcriptionally regulates the let-7 microRNA family and results in suppression of differentiation. In turn, Let-7 microRNAs have diverse mRNA targets, including IMP1 (Igf2 mRNA binding protein-1), another mRNA binding protein. We have demonstrated that LIN28B and IMP1 separately drive tumor-initiating cell phenotypes associated with their roles in regulating proliferation and differentiation during normal homeostasis; however, it remains unclear if LIN28B-mediated hyperproliferation, altered differentiation, and associated tumorigenesis requires IMP1. In addition, it remains unknown if the tumor promoting or metastatic roles of IMP1 depend entirely on its regulation by Let-7 downstream of LIN28B. Our overarching hypothesis of this proposal is that LIN28B and IMP1 comprise cooperative roles in controlling post-transcriptionally the pathways (some known) associated with epithelial cell fate, which may favor malignant transformation. We will pursue this hypothesis through the following interrelated Specific Aims: Aim 1: To evaluate the interdependence of LIN28B and IMP1 in proliferation, differentiation, and malignant transformation in vivo. Aim 2: To determine and validate common and divergent pathways mediated by LIN28B and IMP1 via RNA-Sequencing (?transcriptome?) and ribosome profiling (?translatome?) in vivo. Aim 3: To evaluate Let-7 dependent regulation of IMP1 to promote hyperproliferation and metastasis.

PROJECT SUMMARY Barrett?s esophagus (BE) is the replacement of the normal squamous esophageal epithelium with an incompletely intestinalized columnar epithelium. It occurs in response to chronic acid and bile reflux injury to the esophagus and is the most substantial risk factor for esophageal adenocarcinoma (EAC), a disease whose incidence has risen at an alarming rate. Therefore, improving our understanding of the pathogenesis of BE and its progression to EAC is a critical research imperative. A key lesson of recent genomic studies led in part by this team has demonstrated that many potentially pathogenic somatic mutations are present in the BE epithelium of those who do not progress to dysplasia or cancer, highlighting the important role of processes other than genomic mutations for promoting disease onset and progression. Here, we focus on the pivotal role of the tumor microenvironment in the pathogenesis of BE and EAC pathogenesis through aims that are integrated and complementary. We provide new and compelling data implicating a subset of infiltrating activated fibroblasts and immune cells in an interconnected web of mutually reinforcing signaling pathways with BE epithelial cells that enhances carcinogenesis. We hypothesize that these tumor-promoting cell populations in the BE microenvironment are key to understanding how and why BE progresses to EAC and that characterization of these cells and regulatory pathways will serve as a foundation for developing new approaches for screening and therapeutics. This hypothesis will be pursued through the following inter-related Specific Aims: 1) To determine the nature and function of the immune cells and fibroblasts present in human BE and EAC patients. 2) To demonstrate how IL-6 secreted by cancer associated fibroblasts and TP53 mutant epithelium promotes BE pathogenesis and progression to EAC. 3) To examine the effect of Myeloid-Derived Suppressor Cells (MDSC) and regulatory T-cells (TReg) on the progression of BE to EAC. Summary and

PROJECT SUMMARY ? PROJECT 1 Esophageal cancers comprise two major subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Herein, we focus upon the pathogenesis and translational therapeutics of ESCC and draw comparisons to EAC when possible. ESCC is the deadliest of all human squamous cell carcinomas. Its common genetic lesions include the p53 tumor suppressor and epidermal growth factor receptor (EGFR) as revealed in Project 3. Mutant p53 impairs cell-cycle arrest or apoptosis in response to cellular stress. EGFR activates mitogen-activated protein kinase (MAPK). Additionally, EGFR and mutant p53 facilitate epithelial-mesenchymal transition (EMT). EMT fosters intra-tumoral heterogeneity and tumor stroma remodeling in ESCC. The malignant properties of ESCC may stem from a subset of cells characterized by high CD44 expression (=CD44H) that in turn promote tumor invasion in the stroma, tumor metastasis (e.g. lung) and conventional therapeutic resistance. The overall objective in Project 1 is to elucidate the molecular mechanisms by which mutant p53 and the tumor microenvironmental cues cooperate to drive ESCC invasion and metastasis, and as a result, unravel new therapeutic targets in synergy with Projects 2 and 3. Utilizing cell lineage-traceable mice treated with the esophageal carcinogen 4-nitroquinoline 1-oxide (4NQO), three- dimensional (3D) culture systems that recapitulate human pathology, and patient-derived xenograft (PDX) tumors with clinically available pharmacological inhibitors, our published and unpublished preliminary data indicate that the interplay between ESCC cells (harboring mutant p53) and cancer-associated fibroblasts (CAFs) and ESCC cells triggers EMT to induce CD44H cells. CD44H cells produce cytokines RANTES and interleukin (IL)-6 to drive ESCC invasion. CD44H cells also display downregulation of the Rab GTPase- coupling protein (RCP)-mediated endocytosis recycling pathway to stabilize cell surface proteins, including EGFR and the RANTES receptor CCR5. Moreover, mutant p53 (p53R172H) facilitates ESCC EMT and lung metastasis. Our central hypothesis is that mutant p53 and CAFs foster the emergence of CD44H ESCC cells with increased malignant properties and possible metastasis. We will test our hypothesis by pursuing the following interrelated Specific Aims: (1) To elucidate the role of RANTES as a mediator of the crosstalk between ESCC cells and CAFs; (2) To delineate how mutant p53 facilitates metastasis via CD44H cells; and (3) To determine how mutant p53 impacts upon the endocytic recycling pathway in ESCC invasion. We will work closely with Projects 2 and 3, with support from the Core Facilities, to translate our findings into new therapeutics that disable the protumorigenic ESCC microenvironment and subsequent proclivity for metastasis.

PROJECT SUMMARY ? CORE A The Administrative and Biostatistics Core (referred to as Core A). Core A has two interrelated components: Administrative and Biostatistics, which serve to advance the missions of the P01 that are not able to be served by other entities. The overriding objectives or Specific Aims of the Administrative component, under the direction of Dr. Anil Rustgi (also overall Director of Core A), is to coordinate an integrated research program between the Projects and with strong support from the scientific Cores. This Administrative component also facilitates interactions with Penn's Perelman's School of Medicine, its NCI designated Abramson Cancer Center, external institutions, enrichment programs, career development pathways, catalyzes new directions for our P01, and ensures that the P01 has the unequivocal and wholehearted institutional support. The Biostatistics component, under the direction of Dr. Qi Long, Professor of Biostatistics, is located in the Penn Center for Clinical Epidemiology and Biostatistics (CCEB). Its objectives or Specific Aims are to provide comprehensive support for the Projects in experimental design, statistical modeling/analysis, data interpretation and methodological investigations. In aggregate, this Core enables innovative basic science research with translation of robust preclinical models into new perspectives in therapeutics of esophageal cancer, which is a major gap in the field.

DESCRIPTION (provided by applicant): The Herbert Irving Comprehensive Cancer Center (HICCC) was designated as an NCI Cancer Center in 1972 and gained comprehensive status in 1979. The HICCC is a component of Columbia University Medical Center is associated with New York-Presbyterian Hospital (NYPH). During the period 2008-2013, CUMC and NYPH committed over $350 million for i) new research initiatives in basic, clinical and population science; ii) new and expanded facilities for laboratory research and clinical activities; iii) recruitment and program restructuring; and iv) support of the Center's administrative office. A new Director, Stephen G. Emerson M.D., Ph.D. was appointed in 2012 and given: i) authority over new facilities, including the Irving Cancer Research Center (ICRC), a 10-story building dedicated to cancer research, with state-of-the-art laboratories and clinical space in the Herbert Irving Pavilion (HIP); ii) a broad-based recruitment plan, which has already attracted 40 new faculty members to the HICCC; and iii) restructuring and expansion of the shared resources. The structure of the HICCC has been organized to increase cancer focus and interdisciplinary collaboration, and now includes 245 members from 22 Departments and 6 Schools, assigned to two Basic Research programs (Cancer Regulatory Networks and Cancer Genetics & Epigenetics), four Disease-Specific programs (Breast Cancer, Prostate Cancer, Neuro-Oncology and Lymphoid Development & Malignancy), and two Population Science programs (Cancer Epidemiology and Prevention, Control & Disparities). The HICCC administers and supports a total of 15 Shared Resources. During the period 2008-2013, the HICCC research activities have been documented in a total of 3057 publications of which 17% are inter-programmatic and 15% are intra-programmatic. The current NCI funding base is $26M (total direct costs), a 13% increase over the 2007 NCI funding base of $23M (total direct costs). The Center is requesting CCSG support of $2,607,495 (total direct costs) for the initial budget period.

PROJECT SUMMARY The SARS-CoV-2 pandemic has led to massive morbidity and mortality worldwide due to COVID-19, with the United States particularly affected. Risk factors for COVID-19 include male sex, older age, and obesity, amongst others, which are also key risk factors for Barrett?s esophagus (BE) and esophageal adenocarcinoma (EAC). Thus, patients with BE will likely be infected with SARS-CoV-2 at markedly higher rates compared to the general population. Thus, patients with BE will likely be infected with SARS-CoV-2 at markedly higher rates compared to the general population. SARS-CoV-2 infects cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor and subsequent viral spike protein cleaving by transmembrane serine protease 2 (TMPRSS2). Infection induces key pathways and downstream effectors, resulting in pronounced inflammation. ACE2 is highly expressed in esophageal tissue, ACE inhibitors reduce NF-?B protein expression in BE, and ACE inhibitor use is associated with reduced risk of EAC. It is therefore plausible that SARS-CoV-2 infection in BE patients can result in direct effects on BE tissues that accelerate neoplasia. We published a retrospective cohort study of >1,600 hospitalized COVID-19 patients and found that use of the histamine-2 receptor antagonist famotidine was associated with a >2-fold reduction in the risk of death. While potential mechanisms underlying this observation remain poorly understood, famotidine may not only improve short-term clinical outcomes in these patients but also ameliorate the pro-neoplastic effects of SARS-CoV-2 in BE. Proton pump inhibitors (PPIs) were associated with worse outcomes in the cohort study, and we previously showed that PPI administration leads to increases in the renin-angiotensin pathway in the gut microbiome. Thus, we hypothesize the following: 1) BE patients with a history of COVID-19 are at increased risk for progression to EAC; and 2) famotidine may be indicated instead of PPIs for BE patients with a documented history of COVID- 19. In this proposal we will address the following specific aims: Aim 1) To define the functional role of ACE2 and TMPRSS2 in BE and EAC cells; Aim 2) To determine whether famotidine influences SARS-CoV-2 infection in BE and EAC cells; Aim 3) To assess the relationship between TMPRSS2 and ACE2 expression and immune cell populations in BE. A history of COVID-19 may represent a novel marker for risk for progression to EAC among BE patients, and this may need to be incorporated into clinical profiles aimed at identifying appropriate high-risk patients for screening and surveillance. Furthermore, treatment with famotidine and not PPIs may be more appropriate for BE patients with mild reflux symptoms and a history of documented COVID-19.

LEADERSHIP, PLANNING, AND EVALUATION: PROJECT SUMMARY The vision of the Herbert Irving Comprehensive Cancer Center (HICCC) is that discoveries here will end cancer everywhere. This is achieved through a dynamic, multi-pronged mission: (1) foster discovery science, (2) translate discovery science into innovative clinical trials and interdisciplinary clinical care, and (3) promote bidirectional community outreach and engagement, all of which is promulgated through a Strategic Plan. The goals of Leadership, Planning, and Evaluation (LPE) are to create and sustain this vision by nurturing an innovative and nimble leadership team, buttressed by internal and external advisory groups, and inclusion of the HICCC membership for the identification and prioritization of new opportunities. The HICCC furthermore charges its leadership and advisory bodies to develop and maintain the Strategic Plan, which interdigitates with the institutional Strategic Plan. Leading the HICCC?s leadership, planning and evaluation activities is the Director, Anil K. Rustgi, MD, a nationally known translational researcher who was recruited last year from the University of Pennsylvania?s Abramson Cancer Center. Rustgi is supported by a highly experienced and cohesive team of Senior Leadership that includes a Deputy Director and six Associate Directors. Together, this team comprises the Senior Leadership Team (SLT). The SLT actively engages Program Leaders and a core of internal advisors in strategy and decision making through Cancer Center Leadership Team (CCLT) meetings, where strategic and operational matters are discussed and recommendations made to the Center Director. A highly productive committee structure, which includes an Internal Advisory Board (IAB), External Advisory Board (EAB), Community Advisory Board (CAB), and Cancer Research Career Enhancement (CRCE) Committee, complements HICCC leadership and allows for diverse perspectives to help shape and guide the future of the HICCC. Under Rustgi, the HICCC has set forth a new vision, which will help realize the Cancer Center?s full potential over the next project period and beyond. Concurrently, care was taken to ensure that the HICCC continuously responds to the CCSG Summary Statement, changing expectations for NCI-designated Comprehensive Cancer Centers, and ongoing feedback and guidance from the IAB, EAB, CAB, and others. All contributors have enthusiastically endorsed the Strategic Plan, along with the initiatives that have been identified for the years ahead. The current LPE, which is hallmarked by active listening, inclusion, and an unyielding commitment to serving the community, has resulted in a time of unprecedented growth. This is evidenced throughout the application in the accomplishments and plans for Research Programs, COE, Shared Resources, and CRCE, including noteworthy accomplishments in translation by bringing HICCC discoveries into the clinic.

DEVELOPMENTAL FUNDS: PROJECT SUMMARY Developmental Funds are critical to promote synergistic, collaborative research that is aligned with the Herbert Irving Comprehensive Cancer Center (HICCC) strategic plan and fueled by our Catchment Area. The HICCC continues to utilize Developmental Funds in a strategic manner, facilitating high-priority research with high probability of external grant funding, fostering the research careers of junior investigators, facilitating team science, increasing translational and clinical research, and promoting new research areas relevant to the Catchment Area. During the project period, HICCC invested a total of $4.4M in 39 pilot projects. Of this total, $856,000 in Cancer Center Support Grant (CCSG) Developmental Funds funded 14 pilot projects. These projects have led to $15.3M in external grant funding, a nearly 18-fold return on investment. From 2017 to 2019, HICCC also funded 15 pilots using $2.1M non-CCSG funds made possible through philanthropy and industry funding. A new funding partnership in 2019, with the Emerson Collective Cancer Research Fund, provided an additional $1.44M which funded nine high-risk, high-reward projects in its first year. Lastly, $17M in non-CCSG Developmental Funds, derived from a combination of institutional, philanthropic, and industry funds, were used for Shared Resources. In this application, the HICCC requests $300,000 per year in Developmental Funds, an increase of $123,000 per year and 7% of the total CCSG budget request (direct costs). These funds will be utilized to meet the following interrelated Specific Aims: (1) Award pilot projects to HICCC investigators to promote team science, high risk/high reward ideas, junior investigators, and pre-clinical and clinical concepts; (2) Invest in Shared Resources in order to support leading-edge research and priorities set forth in the 2019-2025 strategic plan; and (3) Support the planning of multi-investigator grants (e.g. P01, U01/U54, SPOREs) that aim to foster transdisciplinary collaboration.