Oxidative-stress-induced FAK activation contributes to uterine serous carcinoma aggressiveness

Abstract

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer (EC), characterized by its high propensity for metastases. In fact, while endometrioid endometrial carcinoma (EEC), which accounts for 85% of EC, presents a good prognosis, USC is the most frequently fatal. Herein, we used for the first time a peptide-based tyrosine-kinase-activity profiling approach to quantify the changes in tyrosine kinase activation between USC and EEC. Among the tyrosine kinases highly activated in USC, we identified focal adhesion kinase (FAK). We conducted mechanistic studies using cellular models. In a USC cell line, targeting FAK either by inhibitors PF-573228 and defactinib (VS-6063) or by gene silencing limits 3D cell growth and reduces cell migration. Moreover, results from our studies suggest that oxidative stress is increased in USC tumors compared to EEC ones. Reactive oxygen species (ROS) induce tyrosine phosphorylation of FAK and a concomitant tyrosine phosphorylation of paxillin, a mediator of FAK signal transduction. Mechanistically, by tracking hundreds of individual cells per condition, we show that ROS increased cell distance and migration velocity, highlighting the role of ROS-FAK-PAX signaling in cell migration. Both defactinib and ROS scavenger N-acetylcysteine (NAC) revert this effect, pointing toward ROS as potential culprits for the increase in USC cell motility. A proof of concept of the role of FAK in controlling cell growth was obtained in in vivo experiments using cancer-tissue-originated spheroids (CTOS) and a patient-derived orthotopic xenograft model (orthoxenograft/PDOX). Defactinib reduces cell proliferation and protein oxidation, supporting a pro-tumoral antioxidant role of FAK, whereas antioxidant NAC reverts FAK inhibitor effects. Overall, our data points to ROS-mediated FAK activation in USC as being responsible for the poor prognosis of this tumor type and emphasize the potential of FAK inhibition for USC treatment.

This study has been funded by the Asociacion Espanola contra el Cáncer (AECC) through the projects ‘targeting the most aggressive type of endometrial cancer’ with a code GCTRA 18014MATI. Scientific Foundation (grupos estables 5690), and (project LABAE19004LLOB), the Instituto de Salud Carlos III (ISCIII) through the projects CP17/00063 and PI18/ 00795 (Cofounded by European Regional Development Fund (ERDF) ‘a way to build Europe’) and the Xarxa de Bancs de Tumors de Catalunya and sponsored by the Pla Director d’Oncologia de Catalunya (XBTC), IRBLleida Biobank (B.00000682) and PLATAFORMA BIOBANCOS PT20/00021. DLN is recipient of a Miguel Servet scheme (MS17/00063) (Cofounded by the European Social Fund (ESF), ‘investing in your future’) Ministerio de Ciencia Innovacion y Universidades, Gobierno de España. We also thank the CERCA program/Generalitat de Catalunya for institutional support. ICLM is supported by fellowships from SNSF (Ambizione PZ00P3_168077 and Prima PR00P3_193166). We acknowledge Alba Garcia and the Universitat de Lleida-IRBLleida Microscopy Facility for support in image acquisition and analysis. Figure S6 was created with Biorender.com