Abstract 4301: Shear stress induces Rac1/Rab5 circular ruffling/endosomes at the apical surface of metastatic esophageal tumor cells enhancing spreading and motility responses

Abstract

Abstract A coordinated mode of motility is required for dissemination and invasion of esophageal metastases through the vasculature. This motility is largely driven by the activation of GTPases Rac1 and Rab5 which regulate 2 and 3-dimensional motility respectively; however, little is known about the localization and activation of these GTPases under hydrodynamic flow. In this study, the effect of Rac1 activation on tumor motility was investigated in a controlled microfluidic chip that simulates venous flow conditions. A metastatic esophageal cell line derived from the ascites of a male patient with squamous cell cancer of the esophagus (OC-1), was transiently transfected with cDNA encoding active (L61), dominant negative (N17) and wild type (WT) Rac1 fused to GFP to enable visual localization of Rac1 in real time under static and fluid flow conditions. Transfected OC-1 cells were allowed to adhere to fibronectin (Fn) for 30 minutes under static conditions or allowed to adhere to Fn for 5 minutes followed by exposure to a continuous venous shear rate of 200 s-1 for 25 minutes. Cell motility and Rac1 localization was monitored over a 30-minute period followed by cell fixation and co-staining with a polyclonal antibody directed against Rab5 to visualize endosomal trafficking within the cells. The results of our study show that the over-expression of constitutively active Rac1 enhances circular ruffling, pseudopodia formation and motility in OC-1 cells under static conditions when compared to N17 and WT Rac1. Moreover, this activation response in L61-transfected cells is significantly increased in the presence of venous shear rates, (p<0.0013) and completely inhibited in cells expressing the N17 active mutant which display rounded quiescent morphology states. Co-localization studies confirmed that both Rac1 and Rab5 GTPases are recruited to distinct populations of endosomes containing actin at the apical membrane of OC-1 cells under shear, measuring between 0.6 and 1.5 μm. These results suggest that Rab5/Rac1 circular ruffling/endosomal recruitment at the apical surface is an important adaptive response to enhance motility under fluid flow conditions. Moreover, this study may offer insight into mechano-receptor signaling and how metastatic cells adapt and disseminate in circulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4301. doi:1538-7445.AM2012-4301