Abstract 2963: Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development
Authors: Freeman Tanner J. , Pope Jillian , Smith Josh , Sharbel Daniel , Washington Kay , Chen Xi , Chaturvedi Rupesh , Wilson Keith , Shroyer Noah , Dhawan Punita , Means Anna , Deane Natasha G. , Beauchamp R. Daniel 
Published: May 2015

Background: Inactivation of the TGF-β signaling occurs in 50-75% of all colorectal cancer cases. Down-regulation of Smad4, the central mediator of TGF-β signaling, occurs in &gt;50% of stage III patients. How Smad4 contributes to normal intestinal homeostasis and functions as a tumor suppressor are incompletely understood. We reported that β-catenin mRNA expression is inhibited by BMP-mediated Smad4 signaling, and loss of Smad4 signaling results in biologically significant amplification of the Wnt/β-catenin signal. Here, we report on the consequences of Smad4 loss for intestinal homeostasis.

Methods: We generated mice genetically engineered to undergo inducible, tissue-specific deletion of Smad4 within intestinal epithelial cells. K19-CreERT x Smad4 flx/flx and Lrig1-CreERT x Smad4 flx/flx mice were treated with vehicle or tamoxifen to induce recombination at the Smad4 locus in the intestinal stem cell compartment. Intestines from sacrificed mice were cleansed for enteroid isolation, Ussing chamber studies, RNA-seq analysis and preservation by formalin fixation and paraffin embedding (FFPE). We validated Smad4 status via immunohistochemistry and stained FFPE sections for different cell populations within the crypt. To evaluate inflammatory cytokines, we performed Luminex cytokine array analysis on colonic mucosa from control and tamoxifen treated animals. To determine the role for Smad4 in intestinal response to injury, we also treated control and tamoxifen treated animals with dextran sodium sulfate (DSS). We utilized RNA-Seq analysis to determine an intestinal Smad4 loss gene signature.

Results: Smad4 deficient crypts exhibited an expansion of the Ki67 positive cells in the proliferative zone and fewer Carbonic anhydrase II staining cells suggesting fewer mature enterocytes. We found that loss of Smad4 in the intestinal crypt is accompanied by an accumulation of pericryptal CD3+ lymphocytes and F4/80+ macrophages and is associated with increased intestinal permeability as measured by low molecular weight FITC Dextran using the Ussing chamber. Cytokine analysis revealed Macrophage Inflammatory Protein 3a (MIP3a) to be up-regulated in Smad4 knockout mice. Guided by our RNA-Seq analysis, we observe increased levels of pErk in Smad4 deplete tissue. We also observed that mice with intestinal epithelial Smad4 depletion developed advanced mucinous adenocarcinoma after recovery from an inflammatory stimulus induced by DSS.

Conclusions: These results suggest that Smad4 loss disrupts intestinal homeostasis by enabling persistently high levels of Erk signaling to interfere with cell maturation, thereby contributing to increased inflammation. We postulate that the increased inflammation observed in Smad4 knockout mice leads to a tumorigenic environment.

Citation Format: Tanner J. Freeman, Jillian Pope, Josh Smith, Daniel Sharbel, Kay Washington, Xi Chen, Rupesh Chaturvedi, Keith Wilson, Noah Shroyer, Punita Dhawan, Anna Means, Natasha G. Deane, R. Daniel Beauchamp. Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2014-2963