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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 >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

Angiosarcoma is a rare endothelial derived soft-tissue tumor with an estimated incidence of 0.2/100,000 persons/year. Current treatment regimes include surgical resection followed by chemotherapy. Even after treatment, reoccurrence is likely and usually fatal. The canine equivalent of angiosarcoma, hemangiosarcoma (HSA), is a relatively common canine endothelial neoplasm with an overall incidence of 24/100,000. In addition, canine HSA is more prevalent in specific breeds suggesting a genetic component. Similar to human angiosarcoma, HSA present as two basic forms: visceral (spleen or cardiac) and dermal. Furthermore, canine HSA occur spontaneously making canine HSA a relevant model system to study human angiosarcoma. Interestingly, MEK/MAPK signaling cascade is activated in Kaposi's Sarcoma, another human endothelial derived cancer. We hypothesize that the MEK/MAPK signaling cascade is vital for HSA and angiosarcoma growth and survival. We performed expression analysis supplemented by small molecule inhibitor studies utilizing canine primary cell isolates derived from visceral and dermal HSA. MAPK pathway expression signatures are elevated in HSA primary isolates compared to proliferating endothelial cells. Consistent with mRNA expression studies, ERK2 is constitutively phosphorylated in serum starved conditions. In addition, HSA cell viability is reduced by the MEK1/2 inhibitor CI-1040 compared to isolated canine endothelial cells. Other inhibitors targeting tyrosine kinase receptors (Sorafenib, EGFR Inhibitor, and SU11652) also decrease HSA cell viability. We conclude a network of tyrosine kinase receptors constitutively activate the MEK/MAPK signaling pathway in HSA. This work provides insights in the etiology and therapeutic potential for targeting both canine HSA and angiosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2929. doi:10.1158/1538-7445.AM2011-2929