Abstract 2367: RhoA GTPase but not cortactin is required for tumor cell migration of human epidermoid carcinoma cells in vivo

Abstract

Abstract Metastasis contributes to over 90% of cancer mortalities. Metastatic disease is a dynamic, multi-step process that requires invasion and migration of cancer cells from the primary tumour site and leads to colonization at a distant location. Migration events mediated by the family of Rho GTPases are facilitated by acto-myosin contraction at the cell rear. In vitro studies have demonstrated that knockdown of RhoA causes insufficient actomyosin contraction and decreased cell detachment. Migration of cancer cells in vivo does not only require detachment from the primary tumor, but also relies on invasion of the cell into the surrounding stromal tissue. Formation of functional invadopodia drives invasion and a key component of invadopodia is cortactin. The objective of this study is to combine inhibitory short hairpin RNA (shRNA) technology with an in vivo metastasis model to identify mechanisms of cancer cell migration that have not previously been studied in an animal model. I hypothesize that inhibition of mediators required for cellular rear detachment and invadopodia formation, such as RhoA and cortactin respectively, will prevent migration of human fibrosarcoma (HT1080) and human epidermoid carcinoma (Hep3) cell lines in vivo. Inhibition of RhoA and cortactin was performed by lentiviral infection of Hep3 and HT1080 cell lines with individual pLOK.1-puro vectors containing a 21-mir sequence to target and decrease mRNA expression. The migratory ability of both cell lines following RNA inhibition was analyzed in vitro using a scratch wound assay and in vivo using a migration and metastasis model in the shell-less chicken embryo, whereby transduced HT1080 and Hep3 cells are injected intravenously and subsequently arrest in the chorioallantoic membrane (CAM). We used a microscope mounted incubator and spinning disc confocal microscopy to visualize cells in real time and quantify their migration parameters. Results obtained from in vitro migration assays indicate that inhibition of cortactin does not affect the migration of Hep3 and HT1080 cell lines across a rigid substratum. Diminished levels of cortactin also had no significant effect on the migration of these cells in vivo. Importantly, inhibition of RhoA caused a significant decrease in cell migration in vitro for both cell lines and in vivo for Hep3 cells (p<0.05). This data suggests that RhoA, a key mediator of cellular rear detachment, is required for in vivo migration of human epidermoid carcinoma cells while cortactin, implicated in invadopodia structures, is not required. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2011-2367