Oncogenic GNAS drives a gastric pylorus program in intraductal papillary mucinous neoplasms of the pancreas.

OBJECTIVE: Intraductal Papillary Mucinous Neoplasms (IPMNs) are cystic lesions and bona fide precursors for pancreatic ductal adenocarcinoma (PDAC). Recently, we showed that acinar to ductal metaplasia, an injury repair program, is characterized by a transcriptomic program similar to gastric spasmolytic polypeptide expressing metaplasia (SPEM), suggesting common mechanisms of reprogramming between the stomach and pancreas. The aims of this study were to assay IPMN for pyloric markers and to identify molecular drivers of this program. DESIGN: We analyzed RNA-seq studies of IPMN for pyloric markers, which were validated by immunostaining in patient samples. Cell lines expressing Kras G12D +/- GNAS R201C were manipulated to identify distinct and overlapping transcriptomic programs driven by each oncogene. A PyScenic-based regulon analysis was performed to identify molecular drivers in the pancreas. Expression of candidate drivers was evaluated by RNA-seq and immunostaining. RESULTS: Pyloric markers were identified in human IPMN. GNAS R201C drove expression of these markers in cell lines and siRNA targeting of GNAS R201C or Kras G12D demonstrates that GNAS R201C amplifies a mucinous, pyloric phenotype. Regulon analysis identified a role for transcription factors SPDEF, CREB3L1, and CREB3L4, which are expressed in patient samples. siRNA-targeting of Spdef inhibited mucin production. CONCLUSION: De novo expression of a SPEM phenotype has been identified in pancreatitis and a pyloric phenotype in Kras G12D -driven PanIN and Kras G12D ;GNAS R201C -driven IPMN, suggesting common mechanisms of reprogramming between these lesions and the stomach. A transition from a SPEM to pyloric phenotype may reflect disease progression and/or oncogenic mutation. IPMN-specific GNAS R201C amplifies a mucinous phenotype, in part, through SPDEF.

Differential spatial distribution of HNF4α isoforms during dysplastic progression of intraductal papillary mucinous neoplasms of the pancreas.

Hepatocyte Nuclear Factor 4-alpha (HNF4α) comprises a nuclear receptor superfamily of ligand-dependent transcription factors that yields twelve isoforms in humans, classified into promoters P1 or P2-associated groups with specific functions. Alterations in HNF4α isoforms have been associated with tumorigenesis. However, the distribution of its isoforms during progression from dysplasia to malignancy has not been studied, nor has it yet been studied in intraductal papillary mucinous neoplasms, where both malignant and pre-malignant forms are routinely clinically identified. We examined the expression patterns of pan-promoter, P1-specific, and P2-specific isoform groups in normal pancreatic components and IPMNs. Pan-promoter, P1 and P2 nuclear expression were weakly positive in normal pancreatic components. Nuclear expression for all isoform groups was increased in low-grade IPMN, high-grade IPMN, and well-differentiated invasive adenocarcinoma. Poorly differentiated invasive components in IPMNs showed loss of all forms of HNF4α. Pan-promoter, and P1-specific HNF4α expression showed shifts in subnuclear and sub-anatomical distribution in IPMN, whereas P2 expression was consistently nuclear. Tumor cells with high-grade dysplasia at the basal interface with the stroma showed reduced expression of P1, while P2 was equally expressed in both components. Additional functional studies are warranted to further explore the mechanisms underlying the spatial and differential distribution of HNF4α isoforms in IPMNs.

SMAD4 Suppresses Colitis-associated Carcinoma Through Inhibition of CCL20/CCR6-mediated Inflammation.

BACKGROUND & AIMS: We previously reported that colon epithelial cell silencing of Smad4 increased epithelial expression of inflammatory genes, including the chemokine c-c motif chemokine ligand 20 (CCL20), and increased susceptibility to colitis-associated cancer. Here, we examine the role of the chemokine/receptor pair CCL20/c-c motif chemokine receptor 6 (CCR6) in mediating colitis-associated colon carcinogenesis induced by SMAD4 loss. METHODS: In silico analysis of SMAD4, CCL20, and CCR6 messenger RNA expression was performed on published transcriptomic data from human ulcerative colitis (UC), and colon and rectal cancer samples. Immunohistochemistry for CCL20 and CCR6 was performed on human tissue microarrays comprising human UC-associated cancer specimens, Mice with conditional, epithelial-specific Smad4 loss with and without germline deletion of the Ccr6 gene were subjected to colitis and followed for up to 3 months. Tumors were quantified histologically, and immune cell populations were analyzed by flow cytometry and immunostaining. RESULTS: In human UC-associated cancers, loss of epithelial SMAD4 was associated with increased CCL20 expression and CCR6+ cells. SMAD4 loss in mouse colon epithelium led to enlarged gut-associated lymphoid tissues and recruitment of immune cells to the mouse colon epithelium and stroma, particularly T regulatory, 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. CONCLUSIONS: Regulation of mucosal inflammation is central to SMAD4 tumor suppressor function in the colon. A key downstream node in this regulation is suppression of epithelial CCL20 signaling 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.

Single-Cell Transcriptomics Reveals a Conserved Metaplasia Program in Pancreatic Injury.

BACKGROUND & AIMS: Acinar 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. METHODS: Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate post-injury. Transcripts of more than 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared with gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. RESULTS: scRNA-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 with KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression 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. CONCLUSIONS: Under 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. KrasG12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

Colon epithelial cell TGFß signaling modulates the expression of tight junction proteins and barrier function in mice.

Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Although TGFß signaling defects have been associated with IBD and CAC, few studies have examined the relationship between TGFß and intestinal barrier function. Here, we examine the role of TGFß signaling via SMAD4 in modulation of colon barrier function. The Smad4 gene was conditionally deleted in the intestines of adult mice and intestinal permeability assessed using an in vivo 4 kDa FITC-Dextran (FD4) permeability assay. Mouse colon was isolated for gene expression (RNA-sequencing), Western blot, and immunofluorescence analysis. In vitro colon organoid culture was utilized to assess junction-related gene expression by qPCR and transepithelial resistance (TER). In silico analyses of human IBD and colon cancer databases were performed. Mice lacking intestinal expression of Smad4 demonstrate increased colonic permeability to FD4 without gross mucosal damage. mRNA/protein expression analyses demonstrate significant increases in Cldn2/Claudin 2 and Cldn8/Claudin 8, and decreases in Cldn3, Cldn4, and Cldn7/Claudin 7 with intestinal SMAD4 loss in vivo without changes in Claudin protein localization. TGFß1/BMP2 treatment of polarized SMAD4+ colonoids increases TER. Cldn2, Cldn4, Cldn7, and Cldn8 are regulated by canonical TGFß signaling, and TGFß-dependent regulation of these genes is dependent on nascent RNA transcription (Cldn2, Cldn4, Cldn8) but not nascent protein translation (Cldn4, Cldn8). Human IBD/colon cancer specimens demonstrate decreased SMAD4, CLDN4, CLDN7, and CLDN8 and increased CLDN2 compared with healthy controls. Canonical TGFß signaling modulates the expression of tight junction proteins and barrier function in mouse colon.NEW & NOTEWORTHY We demonstrate that canonical TGFß family signaling modulates the expression of critical tight junction proteins in colon epithelial cells, and that expression of these tight junction proteins is associated with maintenance of colon epithelial barrier function in mice.

Exposure to p40 in Early Life Prevents Intestinal Inflammation in Adulthood Through Inducing a Long-Lasting Epigenetic Imprint on TGFß.

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 1ß (Setd1ß), and mice in early life or in adulthood. Anti-transforming growth factor ß (TGFß)-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, TGFß 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 Setd1ß-dependent manner. p40-regulated Setd1ß mediated programming the TGFß locus into a transcriptionally permissive chromatin state and promoting TGFß 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 TGFß 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 TGFß, leading to long-lasting production of TGFß by intestinal epithelial cells to expand Tregs and protect the gut against inflammation.

Pancreatic islet beta cell-specific deletion of G6pc2 reduces fasting blood glucose.

The G6PC1, G6PC2 and G6PC3 genes encode distinct glucose-6-phosphatase catalytic subunit (G6PC) isoforms. In mice, germline deletion of G6pc2 lowers fasting blood glucose (FBG) without affecting fasting plasma insulin (FPI) while, in isolated islets, glucose-6-phosphatase activity and glucose cycling are abolished and glucose-stimulated insulin secretion (GSIS) is enhanced at submaximal but not high glucose. These observations are all consistent with a model in which G6PC2 regulates the sensitivity of GSIS to glucose by opposing the action of glucokinase. G6PC2 is highly expressed in human and mouse islet beta cells however, various studies have shown trace G6PC2 expression in multiple tissues raising the possibility that G6PC2 also affects FBG through non-islet cell actions. Using real-time PCR we show here that expression of G6pc1 and/or G6pc3 are much greater than G6pc2 in peripheral tissues, whereas G6pc2 expression is much higher than G6pc3 in both pancreas and islets with G6pc1 expression not detected. In adult mice, beta cell-specific deletion of G6pc2 was sufficient to reduce FBG without changing FPI. In addition, electronic health record-derived phenotype analyses showed no association between G6PC2 expression and phenotypes clearly unrelated to islet function in humans. Finally, we show that germline G6pc2 deletion enhances glycolysis in mouse islets and that glucose cycling can also be detected in human islets. These observations are all consistent with a mechanism by which G6PC2 action in islets is sufficient to regulate the sensitivity of GSIS to glucose and hence influence FBG without affecting FPI.

Differential Cell Susceptibilities to KrasG12D in the Setting of Obstructive Chronic Pancreatitis.

BACKGROUND & 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: Although KrasG12D 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 duct 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 KRASG12D-initiated tumorigenesis is whether 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.

Immunomodulatory Effects of TGF-ß Family Signaling within Intestinal Epithelial Cells and Carcinomas.

TGF-ß superfamily signaling is responsible for many critical cellular functions including control of cell growth, cell proliferation, cell differentiation, and apoptosis. TGF-ß 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-ß 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-ß pathway signaling within epithelial cells may directly modulate pro-inflammatory chemokine/cytokine production and resultant leukocyte recruitment. This immunomodulation by epithelial TGF-ß 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-ß family signaling within intestinal epithelial cells and carcinomas.

Epithelial Smad4 Deletion Up-Regulates Inflammation and Promotes Inflammation-Associated Cancer.

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 ß (TGFß) 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 TGFß1 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, TGFß signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions: TGFß 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.

cFLIP critically modulates apoptotic resistance in epithelial-to-mesenchymal transition.

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-ß-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, cFLIPS. Loss of cFLIPS is sufficient to overcome resistance to TRAIL and exogenous overexpression of cFLIPS 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 cFLIPS critically regulates the apoptosis resistance phenotype associated with EMT.

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin.

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 α5ß1 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.

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction.

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/ß-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/ß-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.

Prox1-Heterozygosis Sensitizes the Pancreas to Oncogenic Kras-Induced Neoplastic Transformation.

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.