Contribution of the epithelial-mesenchymal (EMT) phenotype to the sensitivity of colorectal cancer cell lines to the p21-activated kinase inhibitor, PF-3758309.

Abstract

438 Background: Increased expression of p21–activated kinase (PAK) family proteins has been observed in a range of malignancies including those of breast, ovarian, colorectal (CRC), and pancreatic origin. PAK (PAK 1-6) proteins are critical mediators of cell proliferation, motility, transcription, and translation. In initial studies, we demonstrated that the PAK4 inhibitor, PF-3758309, exhibited robust single agent efficacy against selected CRC cell lines, with IC50s of less than 0.015 uM, whereas some cell lines were relatively resistant to the agent, with IC50s of greater than 1 uM. In subsequent gene array analyses, CRC cell lines sensitive or resistant to PF-3758309 exhibited overexpression of core genes associated with a mesenchymal or epithelial phenotype, respectively. Thus, the goal of this study was to assess the functional consequences of altering the expression of EMT-associated genes and to seek rational combination partners in CRC. Methods: CRC cell lines that were sensitive to PF-3758309 (IC50=0.015 uM) were transfected with selected short hairpin- (sh)RNAs or micro-(mi)RNAs that are known to regulate EMT. Semi-quantitative RT-PCR and immunoblotting were performed to confirm target knockdown. The transfected cell lines were then exposed to increasing concentrations of PF-3758309 to determine the functional role of these genes in conferring responsiveness to PF-3758309. Results: Sensitive CRC cell lines were transfected with shRNAs to Zeb1, vimentin, and caldesmon, genes that are associated with a mesenchymal phenotype. Interestingly, when the cell lines were exposed to increasing concentrations of PF- 3758309 they demonstrated a ′right shift′ towards a more resistant phenotype. Likewise, transfection with miRNA 200c, a known suppressor of Zeb1, resulted in a similar shift towards resistance. Conclusions: These data suggest that the EMT phenotype may play a functional role in determining CRC sensitivity to the PAK4 inhibitor PF-3758309, and in addition, a rational combination targeting the epithelial phenotype with epidermal growth factor receptor inhibitors, may be warranted. [Table: see text]