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Supplemental Table 1. Five mice lacking intestinal SMAD4 expression ("KO") and five littermate control mice ("WT") had their colon microbiome composition compared. 16S targeted sequencing was performed on mouse stool pellets ("Pellet") and colon mucosal scrapings ("Scraping") and compared at the phylum, class, order, family, genus, and species levels. Values represent proportion of organisms detected per sample. Genotypes were split evenly between sexes. The five female mice were co-housed and the five male mice were co-housed, with bedding mixed between cages weekly., This research was funded in part by National Cancer Institute grants (F32 CA236309; T32 CA106183; R01 CA235016), the Burroughs Wellcome Fund Physician-Scientist Institutional Award #1018894/Vanderbilt Supporting Careers in Research for Interventional Physicians and Surgeons (SCRIPS), the Vanderbilt Ingram Cancer Center (VICC) grant (P30 CA068485), the Vanderbilt Digestive Diseases Research Center Grant (P30 DK058404), and the Department of Veterans Affairs Career Development Award (IK2BX004648).

Background & Aims Colonization by gut microbiota in early life confers beneficial effects on immunity throughout the host’s lifespan. We sought to elucidate the mechanisms whereby neonatal supplementation with p40, a probiotic functional factor, reprograms intestinal epithelial cells for protection against adult-onset intestinal inflammation. Methods p40 was used to treat young adult mouse colonic (YAMC) epithelial cells with and without deletion of a methyltransferase, su(var)3-9, enhancer-of-zeste and trithorax domain–containing 1β (Setd1β), and mice in early life or in adulthood. Anti–transforming growth factor β (TGFβ)-neutralizing antibodies were administered to adult mice with and without colitis induced by 2,4,6-trinitrobenzenesulfonic acid or dextran sulfate sodium. We examined Setd1b and Tgfb gene expression, TGFβ production, monomethylation and trimethylation of histone H3 on the lysine 4 residue (H3K4me1/3), H3K4me3 enrichment in Tgfb promoter, differentiation of regulatory T cells (Tregs), and the inflammatory status. Results p40 up-regulated expression of Setd1b in YAMC cells. Accordingly, p40 enhanced H3K4me1/3 in YAMC cells in a Setd1β-dependent manner. p40-regulated Setd1β mediated programming the TGFβ locus into a transcriptionally permissive chromatin state and promoting TGFβ production in YAMC. Furthermore, transient exposure to p40 during the neonatal period and in adulthood resulted in the immediate increase in Tgfb gene expression. However, only neonatal p40 supplementation induced the sustained H3K4me1/3 and Tgfb gene expression that persisted into adulthood. Interfering with TGFβ function by neutralizing antibodies diminished the long-lasting effects of neonatal p40 supplementation on differentiation of Tregs and protection against colitis in adult mice. Conclusions Exposure to p40 in early life enables an epigenetic imprint on TGFβ, leading to long-lasting production of TGFβ by intestinal epithelial cells to expand Tregs and protect the gut against inflammation., Graphical abstract

Background & AimsChronic inflammation in the colon is a predisposing factor for colon cancer. We previously reported that colon epithelial cell silencing of Smad4, the central downstream mediator of TGFβ family signaling, increased epithelial expression of inflammatory genes, including the chemokine CCL20, and increased susceptibility to colitis-associated cancer. Here, we examine the role of the chemokine/receptor pair CCL20/CCR6 in mediating colitis-associated colon carcinogenesis induced by SMAD4 loss.MethodsMice with conditional, epithelial-specific Smad4 loss with and without germline deletion of the Ccr6 gene were subjected to three rounds of dextran sodium sulfate and followed for up to 3 months. Tumors were quantified histologically, and immune cell populations were analyzed by flow cytometry and immunostaining.ResultsIn humans, SMAD4 expression inversely correlated with CCL20 expression. Smad4 loss in mouse colon epithelium led to enlarged gut-associated lymphoid tissues and recruitment of specific immune cell subsets to the mouse colon epithelium and underlying stroma, particularly Treg, Th17, and dendritic cells. Loss of CCR6 abrogated these immune responses and significantly reduced the incidence of colitis-associated tumors observed with loss of SMAD4 alone.ConclusionsRegulation of mucosal inflammation is a critical role of SMAD4 signaling within the colon epithelium and central to its tumor suppressor function in the colon. A key downstream node in this regulation is suppression of CCL20 signaling by the epithelium to CCR6 in immune cells. Loss of SMAD4 in the colon epithelium increases CCL20 expression and chemoattraction of CCR6+ immune cells, contributing to greater susceptibility to colon cancer.

BACKGROUND & AIMSAcinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression.METHODSAcinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate upon injury. Transcripts of over 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared to gastric metaplasia,KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy.KrasG12Dwas expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining.RESULTSscRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison toKrasG12D-induced ADM identifies populations associated with disease progression. Activation ofKrasG12Dexpression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype.CONCLUSIONSUnder conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse.KrasG12Dexpression is sufficient to drive neoplasia from injury-induced ADM and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

TGF-β superfamily signaling is responsible for many critical cellular functions including control of cell growth, cell proliferation, cell differentiation, and apoptosis. TGF-β appears to be critical in gastrulation, embryonic development, and morphogenesis, and it retains pleiotropic roles in many adult tissues and cell types in a highly context-dependent manner. While TGF-β signaling within leukocytes is known to have an immunosuppressive role, its immunomodulatory effects within epithelial cells and epithelial cancers is less well understood. Recent data has emerged that suggests TGF-β pathway signaling within epithelial cells may directly modulate pro-inflammatory chemokine/cytokine production and resultant leukocyte recruitment. This immunomodulation by epithelial TGF-β pathway signaling may directly impact tumorigenesis and tumor progression through modulation of the epithelial microenvironment, although causal pathways responsible for such an observation remain incompletely investigated. This review presents the published literature as it relates to the immunomodulatory effects of TGF-β family signaling within intestinal epithelial cells and carcinomas.

Background and Aims Activating mutation of the KRAS gene is common in some cancers, such as pancreatic cancer, but rare in other cancers. Chronic pancreatitis is a predisposing condition for pancreatic ductal adenocarcinoma (PDAC) but how it synergizes with KRAS mutation is not known. Methods We used a mouse model to express an activating mutation of Kras in conjunction with obstruction of the main pancreatic duct to recapitulate a common etiology of human chronic pancreatitis. Because the cell of origin of PDAC is not clear, Kras mutation was introduced into either duct cells or acinar cells. Results While Kras G12D expression in both cell types was protective against damage-associated cell death, chronic pancreatitis induced p53, p21, and growth arrest only in acinar-derived cells. Mutant ducts cells did not elevate p53 or p21 expression and exhibited increased proliferation driving the appearance of PDAC over time. Conclusions One mechanism by which tissues may be susceptible or resistant to KRAS G12D -initiated tumorigenesis is whether or not they undergo a p53-mediated damage response. In summary, we have uncovered a mechanism by which inflammation and intrinsic cellular programming synergize for the development of PDAC.

Background & Aims Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor β (TGFβ) family signaling through SMAD4 in colonic epithelial cells. Methods The Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFβ1 and bone morphogenetic protein 2. Results Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis–associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis–associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFβ signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions TGFβ suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082., Graphical abstract

Pancreatic ductal adenocarcinoma (PDA) initiation and progression are accompanied by an immunosuppressive inflammatory response. Here, we evaluated the immunomodulatory role of chemosensory signaling in metaplastic tuft cells (MTCs) by analyzing the role of GNAT3, a gustatory pathway G-protein expressed by MTCs, during PDA progression.Gnat3-null (Gnat3Ablation of Gnat3 in KC organoids increased release of tumor-promoting cytokines in conditioned media, including CXCL1 and CXCL2. Analysis of Gnat3Compromised gustatory sensing, achieved by Gnat3 ablation, enhanced the CXCL1/2-CXCR2 axis to alter the MDSC population and promoted the progression of metastatic PDA.

// Chandrasekhar Padmanabhan 1, 2 , Eric J. Rellinger 1, 2 , Jing Zhu 1, 2 , Hanbing An 1, 2 , Luke G. Woodbury 8 , Dai H. Chung 1, 3, 4 , Alex G. Waterson 5 , Craig W. Lindsley 6, 7 , Anna L. Means 1, 2 and R. Daniel Beauchamp 1, 2, 4, 8, 9 1 Section of Surgical Sciences, Vanderbilt University Medical Center, Nashville TN, 37232, USA 2 Department of Surgery, Vanderbilt University Medical Center, Nashville TN, 37232, USA 3 Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville TN, 37232, USA 4 Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville TN 37232, USA 5 Department of Pharmacology, Vanderbilt University School of Medicine, Nashville TN, 37232, USA 6 Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville TN, 37232, USA 7 Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville TN, 37232, USA 8 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville TN, 37232, USA 9 The Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville TN, 37232, USA Correspondence to: R. Daniel Beauchamp, email: daniel.beauchamp@vanderbilt.edu Keywords: EMT, apoptosis, cFLIP, TRAIL, isoxazole Received: April 27, 2017 Accepted: June 26, 2017 Published: July 25, 2017 ABSTRACT Epithelial cancers (carcinomas) comprise the top four causes of cancer-related deaths in the United States. While overall survival has been steadily improving, therapy-resistant disease continues to present a major therapeutic challenge. Carcinomas often exploit the normal developmental program, epithelial-to-mesenchymal transition (EMT), to gain a mesenchymal phenotype associated with increased invasiveness and resistance to apoptosis. We have previously shown that an isoxazole-based small molecule, ML327, partially reverses TGF-β-induced EMT in an immortalized mouse mammary epithelial cell line. Herein, we demonstrate that ML327 reverses much of the EMT gene expression program in cultured carcinoma cell lines. The reversal of EMT sensitizes these cancer cells to the apoptosis-inducing ligand TRAIL. This sensitization is independent of E-cadherin expression and rather relies on the downregulation of a major anti-apoptotic protein, cFLIP S . Loss of cFLIP S is sufficient to overcome resistance to TRAIL and exogenous overexpression of cFLIP S restores resistance to TRAIL-induced apoptosis despite EMT reversal with ML327. In summary, we have utilized an isoxazole-based small molecule that partially reverses EMT in carcinoma cells to demonstrate that cFLIP S critically regulates the apoptosis resistance phenotype associated with EMT.

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.

Background & Aims Identification and validation of new functionally relevant and pharmacologically actionable targets for pancreatic ductal adenocarcinoma (PDAC) remains a great challenge. Premalignant acinar cell reprogramming (acinar-to-ductal metaplasia [ADM]) is a precursor of pancreatic intraepithelial neoplasia (PanIN) lesions that can progress to PDAC. This study investigated the role of proline-rich tyrosine kinase 2 (PYK2) in mutant Kras-induced and pancreatitis-associated ADM and PanIN formation, as well as in PDAC maintenance. Methods Genetically engineered mouse models of mutant Kras (glycine 12 to aspartic acid) and Pyk2 deletion were used for investigating the role of PYK2 in PDAC genesis in mice. In vitro ADM assays were conducted using primary pancreatic acinar cells isolated from mice. Immunohistochemistry, immunofluorescence, and a series of biochemical experiments were used to investigate upstream regulators/downstream targets of PYK2 in pancreatic carcinogenesis. PDAC cell line xenograft experiments were performed to study the role of PYK2 and its downstream target in PDAC maintenance. Results PYK2 was increased substantially in ADM lesions induced by mutant Kras or inflammatory injury. Pyk2 deletion remarkably suppressed ADM and PanIN formation in a mutant Kras-driven and pancreatitis-associated PDAC model, whereas PYK2 knockdown substantially inhibited PDAC cell growth in vitro and in nude mice. This study uncovered a novel yes-associated protein 1/transcriptional co-activator with PDZ binding motif/signal transducer and activator of transcription 3/PYK2/β-catenin regulation axis in PDAC. Our results suggest that PYK2 contributes to PDAC genesis and maintenance by activating the Wnt/β-catenin pathway through directly phosphorylating β-cateninY654. Conclusions The current study uncovers PYK2 as a novel downstream effector of mutant KRAS signaling, a previously unrecognized mediator of pancreatitis-induced ADM and a novel intervention target for PDAC., Graphical abstract

Objectives: CD24 has been considered a normal and cancer stem cell marker. Potential intestinal stem cells weakly express CD24. In the pancreas, CD24 is a possible cancer stem cell marker for ductal adenocarcinoma. Methods: Expression of CD24 in intestinal and pancreatic neuroendocrine tumors (NETs) was examined. Immunohistochemistry was performed on benign duodenum, ileum mucosa, and pancreas, as well as primary duodenal, primary and metastatic ileal, and pancreatic NETs. Results: Scattered CD24-positive cells were noted in the duodenal and ileal crypts, most of which showed a strong subnuclear labeling pattern. Similar expression was observed in 41 (95%) of 43 primary ileal NETs but in only four (15%) of 26 duodenal NETs ( P < .01). In addition, metastatic ileal NETs retained CD24 expression. Pancreatic islets did not express CD24, and only rare cells had subnuclear labeling of CD24 in the pancreatic ducts. Unlike ileal NETs, only five (5%) of 92 pancreatic NETs expressed CD24 in the subnuclear compartment ( P < .01). All five NETs showed a unique morphology with prominent stromal fibrosis. Conclusions: CD24 expression was frequent in primary and metastatic midgut NETs but rare in pancreatic and duodenal NETs. Expression of CD24 in ileal NETs may have future diagnostic and therapeutic implications.

Cancer-associated fibroblasts (CAFs) in the tumor stroma play a key role in tumor progression. Erdogan et al. show that CAF-mediated alignment of the fibronectin matrix is a key factor promoting directional cancer cell migration., Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF–cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α–mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration.

Normal pancreatic epithelium progresses through various stages of pancreatic intraepithelial neoplasms (PanINs) in the development of pancreatic ductal adenocarcinoma (PDAC). Transcriptional regulation of this progression is poorly understood. In mouse, the hepatic nuclear factor 6 (Hnf6) transcription factor is expressed in ductal cells and at lower levels in acinar cells of the adult pancreas, but not in mature endocrine cells. Hnf6 is critical for terminal differentiation of the ductal epithelium during embryonic development and for pancreatic endocrine cell specification. We previously showed that, in mice, loss of Hnf6 from the pancreatic epithelium during organogenesis results in increased duct proliferation and altered duct architecture, increased periductal fibrosis and acinar-to-ductal metaplasia. Here we show that decreased expression of HNF6 is strongly correlated with increased severity of PanIN lesions in samples of human pancreata and is absent from >90% of PDAC. Mouse models in which cancer progression can be analyzed from the earliest stages that are seldom accessible in humans support a role for Hnf6 loss in progression from early- to late-stage PanIN and PDAC. In addition, gene expression analyses of human pancreatic cancer reveal decreased expression of HNF6 and its direct and indirect target genes compared with normal tissue and upregulation of genes that act in opposition to HNF6 and its targets. The negative correlation between HNF6 expression and pancreatic cancer progression suggests that HNF6 maintains pancreatic epithelial homeostasis in humans, and that its loss contributes to the progression from PanIN to ductal adenocarcinoma. Insight on the role of HNF6 in pancreatic cancer development could lead to its use as a biomarker for early detection and prognosis.

Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.

In humans, tissue homeostasis has to be maintained for decades without significant loss of functionality. Epithelia such as the oral mucosa achieve this through the self-renewing capabilities of stem cells. These life-long residents of the oral mucosa maintain homeostasis of the epithelium and at the same time have to be able to protect themselves from the main threats to longevity, i.e., stem cell exhaustion and malignant conversion and carcinogenesis. A key pathway in mediating cell cycle arrest in oral keratinocytes is mediated by TGFB superfamily signaling. There is good evidence implicating TGFB signaling as a tumor suppressor pathway in oral cancer. We have previously shown that disruption of TGFB signaling and cell adhesion can lead to oral squamous cell carcinoma. In vitro using oral keratinocytes, we found that TGFB regulates the stem cell compartment, inducing gene expression of quiescent stem cell markers MECP2, XPC, ITGB1 and PDPN. Using multiplex immunofluorescence, we have stained a tissue microarray containing 28 cases of squamous cell carcinoma, 4 adenocarcinoma, 4 metastatic carcinoma, 6 hyperplasia, and 5 adjacent inflammatory as well as normal adjacent and normal tissues. The antibodies selected were MECP2, XPC, ITGB1 and K15 in combination with the proliferation marker ki67 to determine if TGFB signaling regulates markers of stem cell quiescence. This quiescent gene signature highlighted a basal niche for slow-cycling cells, reserve stem cells. During oral disease progression, marker expression was abandoned in favor of increased proliferation. In vivo, a mouse model in which KrasG12D expression was induced and the Smad4 gene deleted specifically in the adult epithelium showed invasive cancers in the oral cavity. These Smad4-negative tumors express Sox9, a marker of human OSCCs and a TGFB target, which we have also observed to be upregulated in human premalignant lesions. Sox9-positive areas in the mouse tumors are associated with a downregulation of quiescent oral stem cell markers Krt15 and Mecp2, same as we observed in immunostainings of human tissues. In conclusion, TGFB and SMAD4 regulate quiescent stem cells and loss of this pathways allows invasive cancers to develop in the setting of oncogene expression. Furthermore, we could show that MECP2 in combination with other markers identifies quiescent basal cells in the human oral mucosa and is lost as TGFB signaling is disrupted in tumorigenesis. Citation Format: Claudia D. Andl, Anna L. Means, James S. Lewis, Alexandra Rothaus, Thomas Andl. TGFB signaling disruption in the formation of oral pre-neoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4682.

Pancreatic cancer remains as one of the most deadly cancers with few treatment options at late stages and little information about how it develops through earlier stages. Activating mutation of the Kras gene has been implicated in, but is not sufficient for, tumorigenesis. In mouse models of pancreatic cancer, loss of tumor suppressor genes in conjunction with Kras mutation leads to gradual stochastic acquisition of neoplastic precursors and carcinomas, whereas many cells remain phenotypically unaltered in younger mice. Here, we demonstrate that two oncogenic events, mutation of Kras and production of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF), are sufficient for rapid and complete neoplastic transformation of the exocrine pancreas. We found that macrophages are the major source of HB-EGF production in pancreatic cancer tissue samples, and that macrophages are present in high density and in close association with human pancreatic cancer lesions. In a mouse model, high macrophage density was observed at the earliest stages of neoplastic transformation. The consequence of elevated HB-EGF signaling was investigated without the confounding effects of other macrophage-produced factors via transgenic overexpression of the active form of HB-EGF. In this model, HB-EGF was sufficient to promote Kras-initiated tumorigenesis, inducing rapid and complete neoplastic transformation of the entire exocrine pancreas shortly after birth. HB-EGF overexpression and Kras(G12D) together, but neither alone, increased proliferation with increased cyclinD1 and decreased Cdkn2a/2d (p16/p19(Ink4A/Arf)). These findings establish the importance of oncogenic synergy in cancer initiation and promotion, and establish a molecular link between inflammation and the earliest stages of tumor induction.

Background & Aims Pancreatitis is the most important risk factor for pancreatic ductal adenocarcinoma (PDAC). Pancreatitis predisposes to PDAC because it induces a process of acinar cell reprogramming known as acinar-to-ductal metaplasia (ADM)—a precursor of pancreatic intraepithelial neoplasia lesions that can progress to PDAC. Mutations in KRAS are found at the earliest stages of pancreatic tumorigenesis, and it appears to be a gatekeeper to cancer progression. We investigated how mutations in KRAS cooperate with pancreatitis to promote pancreatic cancer progression in mice. Methods We generated mice carrying conditional alleles of Yap1 and Taz and disrupted Yap1 and Taz using a Cre-lox recombination strategy in adult mouse pancreatic acinar cells (Yap1fl/fl;Tazfl/fl;Ela1-CreERT2). We crossed these mice with LSL-KrasG12D mice, which express a constitutively active form of KRAS after Cre recombination. Pancreatic tumor initiation and progression were analyzed after chemically induced pancreatitis. We analyzed pancreatic tissues from patients with pancreatitis or PDAC by immunohistochemistry. Results Oncogenic activation of KRAS in normal, untransformed acinar cells in the pancreatic tissues of mice resulted in increased levels of pancreatitis-induced ADM. Expression of the constitutive active form of KRAS in this system led to activation of the transcriptional regulators YAP1 and TAZ; their function was required for pancreatitis-induced ADM in mice. The JAK–STAT3 pathway was a downstream effector of KRAS signaling via YAP1 and TAZ. YAP1 and TAZ directly mediated transcriptional activation of several genes in the JAK–STAT3 signaling pathway; this could be a mechanism by which acinar cells that express activated KRAS become susceptible to inflammation. Conclusions We identified a mechanism by which oncogenic KRAS facilitates ADM and thereby generates the cells that initiate neoplastic progression. This process involves activation of YAP1 and TAZ in acinar cells, which up-regulate JAK–STAT3 signaling to promote development of PDAC in mice.

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/β-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/β-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Background: The assembly of distinct proteins into tight junctions results in the formation of a continuous barrier that regulates the paracellular flux of water, ions, and small molecules across epithelia. The claudin protein family encompasses numerous major structural components of tight junctions. These proteins specify the permeability characteristics of tight junctions and consequently, some of the physiological properties of epithelia. Furthermore, defective claudin expression has been found to correlate with some diseases, tumor progression, and defective morphogenesis. Investigating the pattern of claudin expression during embryogenesis or in certain pathological conditions is necessary to begin disclosing the role of these proteins in health and disease. Results: This study analyzed the expression of several claudins during mouse pancreas organogenesis and in pancreatic intraepithelial neoplasias of mouse and human origin. Conclusions: Our results underscored a distinctive, dynamic distribution of certain claudins in both the developing pancreas and the pancreatic epithelium undergoing neoplastic transformation. Developmental Dynamics 241:583–594, 2012. © 2012 Wiley Periodicals Inc.

Type 1 and type 2 diabetes result from an absolute or relative reduction in functional β-cell mass. One approach to replacing lost β-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying factors that enhance β-cell differentiation and proliferation in vivo or in vitro. Connective tissue growth factor (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and embryonic β cells that we previously showed is required for β-cell proliferation, differentiation, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or β cells results in decreased embryonic β-cell proliferation, making CTGF unique as an identified β cell-derived factor that regulates embryonic β-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in β-cell proliferation. Furthermore, CTGF overexpression in β cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature β cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature β cells in directed differentiation or regeneration protocols.

Studies in both humans and rodents have found that insulin(+) cells appear within or near ducts of the adult pancreas, particularly following damage or disease, suggesting that these insulin(+) cells arise de novo from ductal epithelium. We have found that insulin(+) cells are continuous with duct cells in the epithelium that makes up the hyperplastic ducts of both chronic pancreatitis and pancreatic cancer in humans. Therefore, we tested the hypothesis that both hyperplastic ductal cells and their associated insulin(+) cells arise from the same cell of origin. Using a mouse model that develops insulin(+) cell-containing hyperplastic ducts in response to the growth factor TGFalpha, we performed genetic lineage tracing experiments to determine which cells gave rise to both hyperplastic ductal cells and duct-associated insulin(+) cells. We found that hyperplastic ductal cells arose largely from acinar cells that changed their cell fate, or transdifferentiated, into ductal cells. However, insulin(+) cells adjacent to acinar-derived ductal cells arose from pre-existing insulin(+) cells, suggesting that islet endocrine cells can intercalate into hyperplastic ducts as they develop. We conclude that apparent pancreatic plasticity can result both from the ability of acinar cells to change fate and of endocrine cells to reorganize in association with duct structures.

Cre/LoxP-mediated DNA recombination allows for gene function and cell lineage analyses during embryonic development and tissue regeneration. Here, we describe the derivation of a K19(CreERT) mouse line in which the tamoxifen-activable CreER(T) was knocked into the endogenous cytokeratin 19 locus. In the absence of tamoxifen, leaky Cre activity could be detected only in less than 1% of stomach and intestinal epithelial cells, but not in pancreatic or hepatic epithelial tissues. Tamoxifen administration in postnatal animals induced widespread DNA recombination in epithelial cells of pancreatic ducts, hepatic ducts, stomach, and intestine in a dose-dependent manner. Significantly, we found that Cre activity could be induced in the putative gut stem/progenitor cells that sustained long-term gut epithelial expression of a Cre reporter. This mouse line should therefore provide a valuable reagent for manipulating gene activity and for cell lineage marking in multiorgans during normal tissue homeostasis and regeneration.

Background & Aims: Increase of intramucosal transforming growth factor α (TGFα) levels in the gastric fundus leads to oxyntic atrophy and massive foveolar hyperplasia in both metallothionein (MT)-TGFα mice and patients with Menetrier's disease. We have evaluated the hypothesis that increased levels of TGFα in the fundus induces an antral pattern of cell differentiation in fundic glands by studying Pdx1, a transcription factor whose expression normally is confined to the gastric antrum. Methods: Induction of Pdx1 expression was evaluated in Pdx1 lacZ/+ /MT-TGFα bigenic mice treated with zinc. The distribution of Pdx1 in MT-TGFα mice and Menetrier's disease patients was evaluated with anti-Pdx1 antibodies. Transcript levels were evaluated by quantitative polymerase chain reaction in mouse and human tissues and AGS cells. Results: In Pdx1 lacZ/+ mice, Pdx1 was expressed in antral mucosal cells including gastrin cells and TFF2-expressing deep glandular mucous cells. Zinc treatment for 2 to 8 weeks in Pdx1 lacZ/+ /MT-TGFα transgenic mice resulted in expression of Pdx1 throughout the fundus. No ectopic fundic Pdx1 expression was observed in either H felis -infected or DMP777-treated mice. In MT-TGFα mice, 8 weeks of zinc treatment elicited nuclear Pdx1 staining throughout the fundic mucosa. TGFα treatment in AGS cells led to increases in Pdx1 and gastrin messenger RNA expression. Fundic sections from Menetrier's disease patients showed nuclear Pdx1 staining throughout the fundic glands. Treatment of a Menetrier's disease patient with an anti-epidermal growth factor receptor monoclonal antibody reduced fundic expression of both Pdx1 and gastrin. Conclusions: Overexpression of TGFα in MT-TGFα mice and Menetrier's disease patients elicits ectopic expression in the fundus of Pdx1, consistent with the phenotype of antralization.

Background & Aims: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in both normal pancreatic islets and in pancreatic cancers, but its role in pancreatic physiology and disease is not known. This report examines the effects of HB-EGF overexpression in mouse pancreas. Methods: Transgenic mice were established using a tissue-specific promoter to express an HB-EGF complementary DNA in pancreatic β cells, effectively elevating HB-EGF protein 3-fold over endogenous levels. Results: Mice overexpressing HB-EGF in pancreatic islets showed both endocrine and exocrine pancreatic defects. Initially, islets from transgenic mice failed to segregate α, β, δ, and PP cells appropriately within islets, and had impaired separation from ducts and acini. Increased stroma was detected within transgenic islets, expanding with age to cause fibrosis of both endocrine and exocrine compartments. In addition to these structural abnormalities, subsets of transgenic mice developed profound hyperglycemia and/or proliferation of metaplastic ductal epithelium. Both conditions were associated with severe stromal expansion, suggesting a role for islet/stromal interaction in the onset of the pancreatic disease initiated by HB-EGF. Supporting this conclusion, primary mouse fibroblasts adhered to transgenic islets when the 2 tissues were cocultured in vitro, but did not interact with nontransgenic islets. Conclusions: An elevation in HB-EGF protein in pancreatic islets led to altered interactions among islet cells and among islets, stromal tissues, and ductal epithelium. Many of the observed phenotypes appeared to involve altered cell adhesion. These data support a role for islet factors in the development of both endocrine and exocrine disease.

Pancreatic duodenal homeobox factor-1, PDX-1, is required for pancreas development, islet cell differentiation, and the maintenance of beta cell function. Selective expression in the pancreas appears to be principally regulated by Area II, one of four conserved regulatory sequence domains found within the 5'-flanking region of the pdx-1 gene. Detailed mutagenesis studies have identified potential sites of interaction for both positive- and negative-acting factors within the conserved sequence blocks of Area II. The islet beta cell-enriched RIPE3b1 transcription factor, the activator of insulin C1 element-driven expression, was shown here to also stimulate Area II by binding to sequence blocks 4 and 5 (termed B4/5). Accordingly, B4/5 DNA-binding protein's molecular mass (i.e. 46 kDa), binding specificity, and islet beta cell-enriched distribution were identical to RIPE3b1. Area II-mediated activation was also unaffected upon replacing B4/5 with the insulin C1/RIPE3b1 binding site. In addition, the chromatin immunoprecipitation assay showed that the Area II region of the endogenous pdx-1 gene was precipitated by an antiserum that recognizes the large Maf protein that comprises the RIPE3b1 transcription factor. These results strongly suggest that RIPE3b1/Maf has an important role in generating and maintaining physiologically functional beta cells.

Inflammation regulates many aspects of gut homeostasis but is also a key component of colon cancer progression. While TGFβ signaling is known to regulate inflammatory responses within immune cells, we have uncovered a novel regulatory pathway in which TGFβ and BMP signaling suppress responses to inflammatory stimuli within the colonic epithelium. Using mice with conditional deletion of Smad4 in intestinal epithelium, we found that CCL20 expression was increased with Smad4 loss. Similarly, in murine immortalized colonocytes and human colon cancer cell lines, blocking TGFβ and/or BMP receptors increased CCL20 expression. CCL20 is upregulated in response to inflammatory signals such as TNF and IL-1β. CCL20 is also upregulated in colon cancer but the mechanism is not understood. We found that pre-treatment of colonocytes or colon cancer cells with TGFβ1 and BMP2 completely suppressed TNF- or IL-1β-induced CCL20 expression at the level of gene transcription. By chromatin immunoprecipitation, we found that TGFβ1/BMP2 treatment impaired binding of NFκB and phospho-STAT3 to the CCL20 promoter. To understand the significance of this regulation in chronic inflammation, we subjected Smad4 deleted and control mice to three rounds of dextran sodium sulfate (DSS)-mediated damage to the distal colon. We found that loss of Smad4 in mouse colonic epithelium was sufficient to induce tumorigenesis following damage-induced inflammation. Following DSS-mediated damage, Smad4-null epithelium developed invasive colorectal adenocarcinoma within two months of DSS treatment while Smad4+ control mice never develop tumors following DSS exposure. The Smad4 null tumors were histologically similar to those of human colitis-associated colon cancers. Prior to tumor formation, we saw an increase in CD8+ cells in Smad4-deleted colons, suggesting that tumor progression involves bidirectional crosstalk between the epithelium and immune cells and that this crosstalk is regulated in part by Smad4-mediated signaling within the epithelium. SMAD4, TGFβ receptors, or BMP receptors are often mutated in colon cancer. This loss of TGFβ and/or BMP signaling likely facilitates epithelial-immune cell crosstalk in colitis-associated colon cancers. Citation Format: Anna L. Means, Tanner J. Freeman, Connie J. Weaver, Chanjuan Shi, Mary K. Washington, Bronson C. Wessinger, Tasia Brown, David K. Flaherty, Kevin P. Weller, Robert J. Coffey, Keith T. Wilson, Robert D. Beauchamp. Smad4 pathways modulate induction of the chemokine Ccl20 and repress inflammation-induced carcinogenesis in mouse colon. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A16.

Understanding the progression of human pancreatic cancer is difficult due to the late stage of diagnosis of this deadly disease. We must therefore rely upon robust model systems to understand how pancreatic cancer arises, how its acute and chronic phases of progression are regulated, and how best to identify and treat it, hopefully still in the early stage. Models of pancreatic ductal adenocarcinoma (PDAC) that accurately reflect human disease are still being developed. To mimic human disease, PDAC must arise from mutations occurring in adult animals and with etiologies relevant to humans. We developed a new mouse model that incorporates obstructive chronic pancreatitis with tissue-specific, adult-onset expression of mutant Kras. We found that KrasG12D expression in duct cells but not in acinar cells led to progression of metaplastic ducts to eventual dysplasia and cancer. In early-stage disease, Kras mutation in acinar cells led to increased acinar-to-ductal metaplasia but did not downregulate p53, thus leading to reduced cell survival. Ducts naturally express much lower p53 levels, however, and there was increased survival of KrasG12D-mediated, duct-derived metaplastic cells. Furthermore, acinar cells upregulated Pdx1 during acinar-to-ductal metaplasia while duct cells did not do so during their metaplastic transition. Without chronic pancreatitis, Kras mutation in acinar cells causes abundant Pancreatic Intraepithelial Neoplasm (PanIN)-like lesions. However, overexpression of Pdx1 in acinar cells in this context permitted acinar-to-ductal metaplasia but prevented PanIN-like lesion formation, suggesting that Pdx1 can repress neoplastic progression. In summary, in the setting of obstructive chronic pancreatitis, ducts are the principal source of cancer development via reduced Pdx1 and p53 levels and increased cell survival. Citation Format: Fong C. Pan, Jessica N. Kim, Chanjuan Shi, Mary K. Washington, Maike Sander, Maureen Gannon, Robert D. Beauchamp, Christopher V. Wright, Anna L. Means.{Authors}. Kras mutation imparts neoplastic potential on duct cells but not acinar cells in a mouse model of obstructive chronic pancreatitis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B19.

Exocrine pancreatic cell types comprise greater than 90% of parenchymal cell mass in the adult pancreas. However, the factors regulating differentiation of acinar and ductal epithelial cells remain incompletely characterized. Like pancreatic islet cells, acinar and ductal cells arise from pluripotent precursors within embryonic pancreatic epithelium. Recent studies have suggested that a common pool of pluripotent stem cells is responsible for generating both endocrine and exocrine cell types, and that specific signaling pathways regulate a critical balance between endocrine and exocrine lineage commitment.

The CRABP I gene is expressed in a spatiotemporal pattern in neural and mesenchymal tissues at the onset of organogenesis. The neural pattern of CRABP I expression includes specific rhombomeres of the hindbrain, neural crest cells and their derivatives the optic stalk, and the central area of the neural retina. We have created transgenic mouse lines with CRABP I 5' and transcribed regions fused to the lacZ structural gene that recapitulate much of this neural pattern of expression. Sequences 5' of the transcription initiation site between -7.8 and -3.2 kb confer beta-galactosidase expression to specific rhombomeres, migrating neural crest cells, trigeminal ganglion, the optic stalk, and the neural retina. We have also defined a region located between exon 1 and exon 8 that confers a portion of this expression pattern, including the mesencephalic projections of the trigeminal ganglion, the inner layer of the neural retina, and the peripheral layer of the posterior hindbrain. CRABP I expression in mesenchyme appears to require sequences in addition to or outside of those examined here.

TGFβ and BMP signaling through receptor-mediated activation of SMAD proteins is crucial for normal homeostasis in intestinal epithelium. Alterations in this pathway contribute to the development of colorectal cancers. To understand the role of these pathways in homeostasis of the colon we conditionally deleted loxP-flanked alleles of Smad4 in adult mouse colon. We used Lrig1CreERT allele to achieve greater than 80% SMAD4 gene deletion and a CK19CreERT allele for mosaic deletion in colonic epithelium. Our data indicates that SMAD4 activity regulates multiple interactions between intestinal epithelium cells and surrounding stroma and that deletion of SMAD4 in adult mouse colonic epithelial cells alters expression of cytokines, increases immune cell infiltration and predisposes mice to tumorigenesis. Increases in the chemokine CCL20 have been reported in CRC tumors beginning at the adenoma stage and it may regulate both Tregs and Th-17 cells in an active immune response. Luminex cytokine/chemokine array analysis revealed increased levels of CCL20 protein in colonic mucosa and RNAseq data revealed that when SMAD4 is knocked out in the epithelium there is an upregulation of CCL20 and CCR6 (receptor) mRNA in the epithelium and stroma, respectively. To further explore the regulation of CCL20, we used immortalized mouse colonocytes (IMC cells) to further determine how the TGFβ and BMP signaling pathways may be involved in CCL20 expression. We found that CCL20 mRNA expression was suppressed by TGFβ1 and BMP2 and increased by inhibitors of these pathways. The inhibition of CCL20 by the TGFβ and BMP2 pathways occurs even in the presence of a known strong inducer of CCL20, such as TNFα. We have further observed in mice induced to undergo deletion of SMAD4 in the intestinal epithelium that treatment with DSS results in 100% of mice developing mucinous adenocarcinomas in the distal colon. We conclude that ligand-dependent Smad4-mediated signaling provides important negative homeostatic regulation of CCL20 expression in colonic epithelium. Loss of Smad4 results in increased CCL20 expression and we are currently testing the hypothesis that CCL20 overexpression contributes to the inflammation induced carcinogenesis in this mouse model. Citation Format: Tasia D. Brown, Anna Means, Tanner Freeman, Connie Weaver, Keith Wilson, R. Daniel Beauchamp. CCL20 regulation via TGFB /BMP signaling pathway in murine immortalized colonocytes and colonic epithelium. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4429.

Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1(+) colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5(+) colonic stem cells; genes upregulated in the Lrig1(+) population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1(+) cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.

Notch signaling and hepatocyte nuclear factor-6 (HNF-6) are two genetic factors known to affect lineage commitment in the bipotential hepatoblast progenitor cell (BHPC) population. A genetic interaction involving Notch signaling and HNF-6 in mice has been inferred through separate experiments showing that both affect BHPC specification and bile duct morphogenesis. To define the genetic interaction between HNF-6 and Notch signaling in an in vivo mouse model, we examined the effects of BHPC-specific loss of HNF-6 alone and within the background of BHPC-specific loss of recombination signal binding protein immunoglobulin kappa J (RBP-J), the common DNA-binding partner of all Notch receptors. Isolated loss of HNF-6 in this mouse model fails to demonstrate a phenotypic variance in bile duct development compared to control. However, when HNF-6 loss is combined with RBP-J loss, a phenotype consisting of cholestasis, hepatic necrosis, and fibrosis is observed that is more severe than the phenotype seen with Notch signaling loss alone. This phenotype is associated with significant intrahepatic biliary system abnormalities, including an early decrease in biliary epithelial cells, evolving to ductular proliferation and a decrease in the density of communicating peripheral bile duct branches. In this in vivo model, simultaneous loss of both HNF-6 and RBP-J results in down-regulation of both HNF-1β and Sox9 (sex determining region Y–related HMG box transcription factor 9). Conclusion: HNF-6 and Notch signaling interact in vivo to control expression of downstream mediators essential to the normal development of the intrahepatic biliary system. This study provides a model to investigate genetic interactions of factors important to intrahepatic bile duct development and their effect on cholestatic liver disease phenotypes. (HEPATOLOGY 2012;55:232–242)

Activating mutations in the Kras gene are commonly found in some but not all epithelial cancers. In order to understand the susceptibility of different epithelial tissues to Kras-induced tumorigenesis, we introduced one of the most common Kras mutations, Kras(G12D), broadly in epithelial tissues. We used a mouse model in which the G12D mutation is placed in the endogenous Kras locus controlled by inducible, Cre-mediated recombination in tissues expressing cytokeratin 19 including the oral cavity, GI tract, lungs, and ducts of the liver, kidney, and the pancreas. Introduction of the Kras(G12D) mutation in adult mouse tissues led to neoplastic changes in some but not all of these tissues. Notably, many hyperplasias, metaplasias and adenomas were observed in the oral cavity, stomach, colon and lungs, suggesting that exposure to products of the outside environment promotes Kras(G12D)-initiated tumorigenesis. However, environmental exposure did not consistently correlate with tumor formation, such as in the small intestine, suggesting that there are also intrinsic differences in susceptibility to Kras activation. The pancreas developed small numbers of mucinous metaplasias with characteristics of early stage pancreatic intraepithelial neoplasms (PanINs), supporting the hypothesis that pancreatic ducts have the potential to give rise pancreatic cancer.

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary.

Within the developing pancreas Hepatic Nuclear Factor 6 (HNF6) directly activates the pro-endocrine transcription factor, Ngn3. HNF6 and Ngn3 are each essential for endocrine differentiation and HNF6 is also required for embryonic duct development. Most HNF6(-/-) animals die as neonates, making it difficult to study later aspects of HNF6 function. Here, we describe, using conditional gene inactivation, that HNF6 has specific functions at different developmental stages in different pancreatic lineages. Loss of HNF6 from Ngn3-expressing cells (HNF6(Delta endo)) resulted in fewer multipotent progenitor cells entering the endocrine lineage, but had no effect on beta cell terminal differentiation. Early, pancreas-wide HNF6 inactivation (HNF6(Delta panc)) resulted in endocrine and ductal defects similar to those described for HNF6 global inactivation. However, all HNF6(Delta panc) animals survived to adulthood. HNF6(Delta panc) pancreata displayed increased ductal cell proliferation and metaplasia, as well as characteristics of pancreatitis, including up-regulation of CTGF, MMP7, and p8/Nupr1. Pancreatitis was most likely caused by defects in ductal primary cilia. In addition, expression of Prox1, a known regulator of pancreas development, was decreased in HNF6(Delta panc) pancreata. These data confirm that HNF6 has both early and late functions in the developing pancreas and is essential for maintenance of Ngn3 expression and proper pancreatic duct morphology.