Mechanisms underlying the loss of protein disulfide isomerase-dependent regulation of NADPH oxidase in colon carcinoma cells with Ras overactivation

Abstract

Protein Disulfide Isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone mainly from endoplasmic reticulum (ER), where it assists redox protein folding. Recent works indicate PDI involvement in redox cell signaling, bearing a close functional and physical interaction with the NADPH oxidase complex, the main cellular source of signaling oxidants. In Vascular smooth muscle cells (VSMC), PDI is required for Nox1 expression and related superoxide generation, cell migration and RhoGTPase activation. PDI also converges with phagocyte NADPH oxidase. However the mechanisms by which PDI interacts with Noxes is unclear. To investigate such mechanisms, we studied HT29-D4 colon cancer cells bearing high constitutive Nox1 expression and overactivated Ras/Raf/Erk pathway (due to a mutation of Ras effector B-Raf). Intriguingly, in this model, PDI silencing by siRNA did not affect Nox1 activity. In this BEPE project, we focus in tumor cells, investigating whether Ras overactivation could be a potential mechanism for disrupted protein disulfide isomerase/Nox interaction. This BEPE will be performed in the laboratory of our collaborator at Aix-Marseille University and is likely to enrich the ongling candidate's project. The specific aims are: 1) To investigate the functional effect of PDIA1 silencing in cells with constitutively active Ras pathway, focusing on cell migration, which is an important process for tumor progression; 2) To assess the production of distinct ROS and NOX activity in this model; 3) To address possible mechanisms underlying the effects of Ras on PDI/NOX convergence. In line with data already obtained in this project, as well as other works from our group, together with the main expertise of the french group, we propose to focus our efforts on the role of RhoGTPases and their regulator RhoGDI. We will investigate whether Ras overactivation promotes changes in RhoGTPase activity and their interaction with RhoGDI± or PDI, and how PDI silencing affects such variables. This BEPE project is likely to contribute to our project and to provide substantial academic gains that will be further valuable for our group in Brazil.