Abstract A36: Interruption of K-Ras/PAK1/Crk pathway has a dramatic anti-tumor effect in NSCLC

Abstract

Abstract Purposes of the study: The key downstream effector(s) of mutant K-Ras involved in lung cancer metastasis are not known. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic K-Ras signal in non-small cell lung cancer (NSCLC). Experimental procedures: We have carried out three types of experiments. Exp.1: NSCLC clinical specimens were used to examine correlation between K-Ras mutation, activation of PAK1/Crk axis and expression of adhesion molecules. K-Ras genotype was determined by sequencing K-Ras on exon 2 and the activation of PAK1/Crk axis was determined by quantifying PAK1 phosphorylation (Thr 423) and Crk phosphorylation (Ser 41) by immunohistochemical staining of the surgically resected, paraffin embedded NSCLC specimens. Exp.2: In order to assess the role of proto-oncogene c-Crk as a downstream effector of K-Ras, we inhibited K-Ras in NSCLC cells and examined Crk phosphorylation. Specifically, we inhibited K-Ras in NSCLC cells by exposing cells to a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and examined p-Crk-II (Ser41) by western blotting. Exp.3: We examined the functional role of K-Ras/PAK1/Crk axis in controlling malignant behavior of NSCLC cells. For this purpose, we interrupted the K-Ras/PAK1/Crk axis in NSCLC cells by combination of K-Ras inhibition (FTI + GGTI) and PAK1 inhibition and examined the motility and morphology of the cells in a wound healing assay. Findings: Exp.1: Immunohistochemical analysis of the specimens demonstrated a complete and inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. PAK1/Crk phosphorylation was also associated with disease stage at presentation (Pearson's R = 0.57). Furthermore, we observed a positive correlation between K-Ras mutation on codons 12 or 13 and PAK1/Crk phosphorylation (Pearson's R = 0.67) even in a small number of analyzed specimens. Exp.2: K-Ras inhibition by combination of FTI and GGTI completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change in cells exposed to individual inhibitors or inhibitor diluents. Exp.3: Combination of K-Ras inhibition (FTI + GGTI) and PAK1 inhibition resulted in a dramatic morphological change in H157 cells. H157 cells which typically harbor a mesenchymal morphology, obtained a tightly conjoined epithelial phenotype with a smooth border when exposed to K-Ras and PAK1 inhibitors. Cells exposed to K-Ras inhibitors and PAK1 inhibitor almost completely lost their motility as well. These dramatic effects were not seen in cells exposed to either K-Ras inhibitor combination, PAK1 inhibitor or inhibitor diluents. Conclusion: Our data provide strong evidence that proto-oncogene c-Crk is operative downstream of K-Ras in NSCLC. Considering that Crk as an oncogene has cell transforming ability, it seems that the oncogenic K-Ras signal is transduced through Crk. Our previous work demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of K-Ras signaling, bring forward the important role of K-Ras/PAK1/Crk axis as a key pathway in transduction of the oncogenic K-Ras signal in lung cancer metastasis. The very significant anti-tumor effects that we observed as a result of K-Ras/PAK1/Crk pathway inhibition, is further supportive of the prominent role of this pathway in transduction of the oncogenic K-Ras signal in NSCLC. Citation Format: Fariborz (Fred) Mortazavi, Jie Lu, Aljilani Amir, Ryan Phan, Fuyuhiko Tamanoi. Interruption of K-Ras/PAK1/Crk pathway has a dramatic anti-tumor effect in NSCLC. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A36. doi: 10.1158/1557-3125.RASONC14-A36