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.

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.

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.

Fibrogenesis in pancreatic cancer is a dynamic process regulated by macrophage-stellate cell interaction.

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.