Abstract 4064: Rac1 activation promotes invasive phenotype of side population in colon cancer cells by destabilizing the E cadherin-β-catenin ligation.
Authors: Funakoshi Shinsuke , Azuma Toshifumi , Higuchi Hajime , Hamamoto Yasuo , Adachi Masayuki , Kabashima-Niibe Ayano , Hashiguchi Akinori , Takaishi Hiromasa , Hibi Toshifumi 
Published: August 2014

Abstract
[Background] Side population (SP) cells are small fraction of cancer cells that have tumorigenic activity, chemotherapy resistance and metastatic potential. However, it remains to be clarified what characteristics SP cells possess. Here we report SP cells possess more invasive property than non-SP cells, occurring coincident with enhanced activation of a member of Rho-GTPase, Rac1. [Methods] Thirteen colon cancer cell lines were assessed for Rac1 activation by pull-down assay and the percentage of SP cell fraction within total number of cells by the flow cytometry-based side population (SP) technique to get invasive phenotype. Invasion assay was performed to evaluate SP cells’ behavior. Cell adhesion associated proteins such as E-cadherin and β-catenin and actin cytoskeleton were evaluated by immunocytochemistry [Results] We identified positive correlation between active Rac1 expression and SP cell frequency. In addition, Enhanced Rac1 activation and SP cell frequency were associated with poorly differentiated histology. Isolated SP cells expressed more active Rac1 as compared to non-SP cells, being associated with increased invasive activity in SP cells. Mesenchymal and amoeboid types of migration are based on reorganization of the actin cytoskeleton, but their requirements for Rho and Rac signaling differ. With respect to mesenchymal migration, membrane protrusions at the leading edge are formed in a Rac-dependent manner. Expression of a dominant-negative form of Rac in elongated cells inhibits the formation of such protrusive membrane structures, causing decreased cell migration. In contrast, Rho signaling is not essential for mesenchymal migration. With respect to amoeboid migration, the actin cytoskeleton is reorganized along the plasma membrane, causing dynamic membrane blebbing along the cell surface. Suppression of Rac1 activity by either dominant-negative Rac1 or dominant negative WAVE, a downstream mediator of Rac1 signaling reduced SP cell invasion activity. Over expression of constitutively active mutant of Rac1 enhanced SP cell motility. Furthermore, the E-cadherin attenuation and β-catenin enhancement were confirmed by immunohistocytochemistry after Rac1 inhibition. [Conclusions] We identified that Rac1 would appear to be a key molecule to mediate SP cells’ motility. We also implicate that suppression of Rac1 activity leads to reduction in number of invasive SP cells through restored E-cadherin function. Thus present study provides a concept for a new therapeutic strategy that targeting Rac1 molecule would be effective to suppress colon cancer invasive cell.
Citation Format: Shinsuke Funakoshi, Toshifumi Azuma, Hajime Higuchi, Yasuo Hamamoto, Masayuki Adachi, Ayano Kabashima-Niibe, Akinori Hashiguchi, Hiromasa Takaishi, Toshifumi Hibi. Rac1 activation promotes invasive phenotype of side population in colon cancer cells by destabilizing the E cadherin-β-catenin ligation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4064. doi:10.1158/1538-7445.AM2013-4064