The effect of a novel small molecule inhibitor of FAK on viability of human colon cancer cells.

Abstract

e13635 Background: Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is overexpressed of a variety of human malignancies including colon cancer. As such, FAK represents a potential target for inhibiting colon cancer growth and progression. We have recently developed a novel small molecule inhibitor of FAK (1,2,4,5-Benzenetetraamine tetrahydrochloride; Y15) that blocks the Y397 autophosphorylation site of FAK, and we therefore hypothesized that Y15 would inhibit the viability of human colon cancer cells. Methods: Two related human colon cancer cell lines were utilized: SW480 (primary tumor) and SW620 (lymph node metastasis). Cells were screened for FAK production and Y397 autophosphorylation by Western blot analysis. An MTT assay was used to assess cell viability in the presence of different concentrations of Y15 alone (1, 10, 100 mcM), and 5-fluorouracil (5-FU; 1, 5, 25 mcM) alone or in combination with Y15 (1mcM). DMSO was used as a negative control and the FAK kinase inhibitor TAE226 used as a positive control. Results: FAK protein was detected in both cell lines, however the metastatic cells (SW620) expressed more FAK than did the primary tumor cells (SW480). In addition, the metastatic cells had greater amount of phosphorylated Y397 FAK. DMSO did not affect cell viability, while TAE226 did, as expected. Y15 inhibited viability in a dose dependent manner. 5FU did not affect viability at doses < 25 mcM. Interestingly, although 1mcM Y15 had no effect on viability, the addition of 1mcM Y15 to all doses of 5FU decreased viability dose dependently, thus sensitizing cells to 5FU. FAK expression and phosphorylation of Y397 were decreased by Y15 and mirrored the viability results. Conclusions: FAK protein is expressed in SW480 and SW620 colon cancer cells, but the metastatic SW620 cell express higher levels than their primary tumor counterparts. The novel small molecule inhibitor of FAK Y397 autophosphorylation (Y15) inhibits viability of both cell lines and does so in a dose dependent manner. In addition, Y15 increases cell sensitivity to 5FU. Taken together, these data suggest that Y15 may be a useful therapeutic agent for treating patients with colon cancer. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration CureFAKtor Pharmaceuticals