131 - Disrupted regulation of Nox1 by protein disulfide isomerase-A1 in colon cancer cells with KRas overactivity

Abstract

Tumor cells, e.g., colorectal cancer cells (CRC) often upregulate Nox NADPH oxidases, but underlying mechanisms are unclear. Thiol isomerases such as protein disulfide isomerase-A1 (PDIA1) are thioredoxin superfamily redox chaperones overexpressed in cancer and associated with tumoral progression. We previously showed an essential requirement of PDIA1 for agonist-triggered Nox1 activation in vascular smooth muscle . We thus investigated PDIA1/Nox1 crosstalk in CRC, known to highly express Nox1 and mutated overactive KRas. Our analysis of RNAseq databanks and direct validation in CRC cell lines indicated significant positive correlation between PDIA1 expression and Ras activity. We questioned whether Ras activity might influence PDIA1/Nox1 crosstalk and studied CRC with constitutive high (HCT116) vs. moderate (HKE3) or basal (CaCo2) Ras activity. In Caco2 and HKE3, PDIA1 siRNA silencing promoted decreased superoxide generation (HPLC/2OH-ethidium). Unexpectedly, in HCT116, PDIA1 silencing promoted increased superoxide output. Effects of NoxA1ds peptide inhibitor confirmed the Nox1 source. Thus, analogous to vascular cells, PDIA1 supports Nox1 in CRC with basal or moderate Ras activity, while PDIA1 silencing with overactive Ras unleashes Nox1 activity. Since vascular PDIA1 effects involve Rac1 RhoGTPase, a known Nox1 subunit, we hypothesized that direct Ras-mediated Rac1 activation could bypass PDIA1 regulation. Indeed, in HCT116 treated with Rac1 inhibitor, PDIA1 became supportive of superoxide production; conversely, Rac1-G12V active mutant transfection into HKE3 induced PDIA1 to restrict Nox1-dependent superoxide. PDIA1 silencing impaired cell proliferation and migration in HKE3, an effect lost in HCT116. Also, E-cadherin expression enhanced in HKE3 and decreased in HCT116 upon PDIA1 silencing, consistent with PDIA1 supporting epithelial-mesenchymal transition. Thus, PDIA1 may be a servomechanism of Nox1 regulation, supporting it within its operational range but capping its overactivation. Ras upregulation, unleash Nox1 activation possibly via Rac1, inducing PDIA1 capping mechanism switch. PDIA1 may crucially regulate redox-dependent adaptive processes related to cancer progression.