Sulindac Treatment and Fas Deletion: Effects on a Colon Cancer Mouse Model

The ApcMin/+ mouse, a genetic model of human familial adenomatous polyposis (FAP), was used in this study to determine effects of chemical treatments and genetic alterations on colon cancer. Non-steroidal anti-inflammatory drugs (NSAIDs) have shown potential as chemo-preventive agents against cancer formation, especially colorectal cancers. However, the mechanisms by which these drugs act are not fully understood. In this study, ApcMin/+ mice were treated with sulindac, a NSAID that functions as a cyclooxygenase (COX) inhibitor. These mice demonstrated a tumor reduction of over 80%, consistent with previous reports. Gene microarray analyses of RNA from adenoma-derived dysplastic epithelial cells revealed that collagen genes, viz., Col1a2, Col5a2, Col6a2, and Col6a3, were upregulated, and matrilysin/metalloproteinase 7 (Mmp7) was downregulated, in sulindac-treated mice. Validation of the gene expression profile of the Col6a2 subunit of collagen VI and of Mmp7 was confirmed by RT-PCR. Confocal microscopy and immunofluorescence measurements showed that collagen VI was present in low amounts within the tumors of nontreated mice, but was enhanced within the tumors of sulindac-treated mice. Collagens I and V demonstrated similar patterns, but were not as prominent as collagen VI. Mmp7 was found in 'hot spot' areas within the tumors of ApcMin/+ mice treated with the vehicle, but was greatly diminished in those treated with sulindac. Studies with ApcMin/+/Mmp7-/- double-deficient mice demonstrated a reciprocal relationship of Mmp7 expression and the levels of these three collagens in vivo. Gene expression analysis of the human colon cancer cell line, HT-29, demonstrated that MMP7 and HMGB1 were downregulated after sulindac treatment, resembling similar gene alterations observed in mice. Enzymatic assays demonstrated that sulindac treatment altered gelatinase activity. Activity-based protein profiling (ABPP) targeted to serine hydrolases and metallohydrolases revealed a sulindac effect on many members of these proteases. Fas, a member of the tumor necrosis family, is responsible for initiating the apoptotic pathway when bound to its ligand, Fas-L. Defects in the Fas-mediated apoptotic pathway have been reported in colorectal cancer. In the present study, a variant of the ApcMin/+ mouse was generated with an additional deficiency of Fas (ApcMin/+/Faslpr) by multiple cross-breedings of ApcMin/+ mice with Fas deficient (Faslpr) mice. One of the main limitations of the ApcMin/+ mouse model is that it only develops benign polyps. However, ApcMin/+/Faslpr mice presented with dramatic increases in tumor burden relative to ApcMin/+ mice, with invasive lesions at advanced ages. Proliferation and apoptosis markers revealed an increase in cellular proliferation, but negligible changes in apoptosis, while p53 was enhanced at early ages. Fas-L was lower in ApcMin/+/Faslpr mice relative to ApcMin/+ cohorts. The results of this study demonstrated that sulindac is effective in increasing the expression of different collagens and decreasing the expression of Mmp7 and Hmgb1. These effects are also seen in the human colon cancer cell line HT-29 and may contribute to altered tumor burden in cancer patients undergoing NSAIDS treatments. It was also demonstrated that imposition of a Fas deletion in an ApcMin/+ background resulted in a more aggressive phenotype of the ApcMin/+ mouse model characterized by more rapid development of invasive intestinal tumors and a decrease in Fas-L levels.