There is a fundamental gap in understanding how identified mutations interact with one another to promote carcinogenesis and progression to metastatic disease in colorectal cancer. Since >90% of epithelial cancer related deaths are due to metastatic disease, the continued existence of this gap represents a hindrance in properly treating the cause of the vast majority of cancer-related mortality. The long-term goal is to bette understand the mechanisms by which altered signaling pathways contribute to the development of metastatic disease, particularly in colorectal cancer. The objective in this particular applicaton is to elucidate the role of the transcription factor Smad4 in maintaining intestinal homeostasis and how Smad4 loss contributes to colorectal cancer progression through regulation of b-catenin, the central mediator of Wnt signaling. The central hypothesis is that Smad4 signaling promotes intestinal epithelial homeostasis by repressing Wnt signaling. The rationale for the proposed research is that there is increased expression of b-catenin mRNA in colon cancer cell lines in which Smad4 expression is experimentally repressed. Upon restoration of Smad4 expression to SW480 cells, a Smad4-null colon cancer cell line, Wnt signaling and b-catenin mRNA levels are decreased. In our tissue-specific, inducible Smad4 knockout mouse (K19CreERT2Smad4lox/lox), there is an extension of the proliferative zone where Smad4 is depleted. When crossing the Smad4 knockout mouse with the Apc1638 mouse, there is increased polyp burden and increased nuclear localization of b-catenin in polyps, with Smad4 depletion. Upon knowing how Smad4 regulates Wnt signaling, therapies can be better targeted to prevent progression to metastatic disease. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine the cis-elements necessary for b-catenin mRNA suppression;and 2) Determine the effect of Smad4 depletion on Wnt target gene expression in intestinal epithelial cells. Under the Aim 1, we have sub-cloned the ctnnb1 (b-catenin gene) promoter into the pGL2 luciferase based reporter vector to identify regions that are important for Smad4 regulation by measuring luciferase activity.
In Aim 2, Smad4 knockout mice, Wnt reporter mice, laser capture microdissection and intestinal epithelial cells in culture will be used to determine activation of Wnt signaling upon Smad4 inhibition. Using tissues from our murine models, differentiation (intestinal alkaline phosphatase, muc2, chromogranin A, lysozyme) and stem cell marker expression (Lgr5, Lrig1, Ascl2, Olfm4, DcamKL1, Mshi1) will be determined by qPCR and localized by IF and/or in situ hybridization. We will confirm loss of Smad4 expression by immunoblotting, IF and qPCR. This approach is innovative because it utilizes a tissue-specific, inducible recombination system to knockout Smad4 within the murine intestine permitting precise time and space control. By knocking out Smad4 on the background of well-characterized mouse models with Apc mutations, this model closely recapitulates the pathogenesis of colorectal cancer.

Public Health Relevance

The proposed research is relevant to public health because it will elucidate the role of the transcription factor Smad4 in maintaining intestinal epithelial homeostasis through regulation of canonical Wnt signaling, the signaling pathway which is thought to drive the progression of colorectal carcinoma. We have recently shown that Smad4 loss results in increased mRNA expression of the central mediator of Wnt signaling - b-catenin, yet the precise mechanism of how Smad4 represses b-catenin, and thus, Wnt signaling is unknown. This research is relevant to the part of NIH's mission that pertains to understanding human disease as this research will contribute models which closely recapitulate the pathogenesis of colorectal cancer and provide a system to study Smad4 loss within the intestinal tract.

National Institute of Health (NIH)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZRG1)
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Damico, Mark W
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Barrett, Caitlyn W; Reddy, Vishruth K; Short, Sarah P et al. (2015) Selenoprotein P influences colitis-induced tumorigenesis by mediating stemness and oxidative damage. J Clin Invest 125:2646-60
Hansen, Amanda G; Freeman, Tanner J; Arnold, Shanna A et al. (2013) Elevated ALCAM shedding in colorectal cancer correlates with poor patient outcome. Cancer Res 73:2955-64