Abstract 3146: Rac1 accumulates in the nucleus at the invasive front of colorectal cancer through promoting cell motility

Abstract

Abstract [Background] Rac1 is one of the key regulators in a variety of physiological processes, including membrane trafficking, cell motility, and cancer invasiveness. Although evidence describing the function of the Rho family small GTPases in colorectal cancer has been recently accumulated, its mechanism in invasion and metastasis remains poorly explored. Here, we directly explore the interaction between Rac1 and β-catenin in colon cancer morphogenesis and metastasis. [Methods] Expressions of cell-cell junctional molecules, small GTPases and some tyrosine kinases such as IGF-1R and c-MET were evaluated by immunohistochemistry in the surgical specimens with human colon cancers. Ten colon cancer cell lines were assessed for Rac1 activation by pull-down assay. Invasion assay was performed to evaluate cells’ behavior. Cell adhesion associated proteins such as E-cadherin and β-catenin and actin cytoskeleton were evaluated with several kinase inhibitors by immunocytochemistry. [Results] First, we found that active Rac1 expression at the invasion front and distant liver metastatic site correlates with loss of E-cadherin and nuclear β-catenin accumulation in colon cancer patients. Next, rho-GTPase activation status was evaluated in various colon cancer cell lines with GST-fused Cdc42 and Rac interactive binding (CRIB) domain of PAK1 (PAK-CRIB). Expression level of active Rac1 correlates with cell invasiveness in colon cancer cell lines. The cell invasiveness was enhanced or inhibited by over expression of either constitutive active mutant of Rac (V12Rac) or dominant negative mutant of Rac (N17Rac) with Matrigel assay. The downstream signaling of Rac1-dependent pathway was inhibited by either dominant negative WAVE (WAVE-MT) or CA region of N-WASP (CA region). The finding that endogenous β-catenin and Rac1 directly were bound in nuclear fractions of cells was detected by co-immunoprecipitation assay. Rac1 inhibition by not only NSC23766, but also IGF-1R inhibitor restores the adhesion of colon cancer cells through inhibiting translocation of β-catenin to the nucleus. We confirmed that Rac1 inhibition induces cell-cell adhesion through restoring E-cadherin localization to the cell membrane and redistribution of β-catenin to the cytoplasm. [Conclusions] Taken together, our results raise the possibility that nuclear translocation of Rac1 and β-catenin at the invasive front could play a role in the invasive activity of colorectal cancer cells. Thus present study provides a concept for a new therapeutic strategy that targeting Rac1 molecul would be effective to suppress colon cancer invasive cell. Citation Format: Shinsuke Funakoshi, Hiromasa Takaishi, Ayano Niibe-Kabashima, Tomohiro Suzuki, Gen Sakai, Motoko Izumiya, Masayuki Adachi, Yasuo Hamamoto, Hajime Higuchi, Akinori Hashiguchi, Nobuhiro Tsukada, Takanori Kanai. Rac1 accumulates in the nucleus at the invasive front of colorectal cancer through promoting cell motility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3146. doi:10.1158/1538-7445.AM2014-3146