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

Cell Mol Gastroenterol Hepatol. 2018 May 24;6(3):257-276. doi: 10.1016/j.jcmgh.2018.05.006. eCollection 2018.


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.

Keywords: AOM, azoxymethane; APC, adenomatous polyposis coli; BMP, bone morphogenetic protein; CAC, colitis-associated carcinoma; CCL20, Chemokine (C-C motif) ligand 20; CRC, colorectal cancer; CRISPR/Cas9, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9; Colitis-Associated Carcinoma; DMEM, Dulbecco's modified Eagle medium; DSS, dextran sodium sulfate; FBS, fetal bovine serum; FDR, false discovery rate; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; IBD, inflammatory bowel disease; IL, interleukin; IMCS4fl/fl, immortalized mouse colonoctye cell line with loxP-flanked Smad4 alleles; IMCS4null, immortalized mouse colonocyte cell line with deletion of the Smad4 alleles; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; PE, phycoerythrin; R-SMAD, Receptor-SMAD; SFG, retroviral vector; STAT3, signal transducer and activator of transcription 3; TGFβ; TGFβ, transforming growth factor β; TNF, tumor necrosis factor; Tumor Necrosis Factor; UC, ulcerative colitis; WNT, wingless-type mouse mammary tumor virus integration site; YAMC, young adult mouse colon epithelial cells; mRNA, messenger RNA; sgRNA, single-guide RNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Bone Morphogenetic Protein 2 / genetics
  • Bone Morphogenetic Protein 2 / metabolism
  • Carcinoma / etiology
  • Carcinoma / immunology*
  • Carcinoma / pathology
  • Cell Line
  • Cell Line, Tumor
  • Colitis / chemically induced
  • Colitis / complications
  • Colitis / immunology*
  • Colorectal Neoplasms / etiology
  • Colorectal Neoplasms / immunology*
  • Colorectal Neoplasms / pathology
  • Dextran Sulfate / pharmacology
  • Humans
  • Inflammation / chemically induced
  • Inflammation / complications
  • Inflammation / immunology*
  • Intestinal Mucosa / drug effects
  • Intestinal Mucosa / immunology
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Smad4 Protein / genetics
  • Smad4 Protein / immunology*
  • Transforming Growth Factor beta1 / genetics
  • Transforming Growth Factor beta1 / metabolism


  • Bmp2 protein, mouse
  • Bone Morphogenetic Protein 2
  • Smad4 Protein
  • Smad4 protein, mouse
  • Transforming Growth Factor beta1
  • Dextran Sulfate