Abstract 5224: hMENA isoforms impact NSCLC patient outcome through fibronectin/β1 integrin axis

Abstract

Abstract The splicing of the actin regulator hMENA generates different isoforms and we have demonstrated that the two alternatively expressed isoforms, hMENA11a and hMENAΔv6, have opposite functions in cell invasiveness. This general mechanism is of great clinical relevance in early NSCLC patients, where the pattern of hMENA isoform expression is a powerful prognostic factor. However the mechanism of action of the two isoforms have remained unclear. Herein, we evaluated whether hMENA and its isoforms influence β1 integrin expression and signaling considering the role of this integrin in cancer cell invasiveness and tumor progression. We performed hMENA silencing by siRNA and shRNA, to evaluate by QRT-PCR and biochemical approaches the expression of β1 integrin; by immunofluorescence the MRTF1 localization, by in vivo assay G-Actin/F-Actin ratio and by luciferase reporter assay the SRF activity. β1 integrin activation and signaling was evaluated by flow cytometry using an antibody specific for the β1 active conformation and by biochemical analysis of the phosphorylation of FAK, SRC and Paxillin. The secretoma of hMENA11a transfected cancer cell lines was analyzed by LC-MS/MS. Immunohistochemical analysis was performed using pan-hMENA, hMENA11a, and fibronectin antibodies in primary cancer tissues from node negative NSCLC patients. The Chi-Square or Fisher Exact tests were used to estimate associations among categorical variables and disease-free survival was calculated by the Kaplan-Meier product limit method. We show that the depletion of all hMENA isoforms inhibits the Serum Response Factor (SRF) activity, and the expression of its target gene β1 Integrin, by affecting G-Actin/F-Actin ratio, critical for the nuclear localization of the SRF co-factor myocardin related transcription factor 1 (MRTF1). Furthermore, we provide new insights into the mechanisms involved in the opposite functions of hMENA11a and hMENAΔv6 in cell invasiveness and we identify a new role of these isoforms in the β1 integrin-ECM signalling axis. Indeed, hMENAΔv6-drives cancer cell invasion by increasing β1 integrin activation and signalling, which is reduced by the anti-invasive hMENA11a isoform. Moreover, exogenous expression of hMENA11a in hMENAΔv6 positive cancer cells dramatically reduces secretion of extracellular matrix (ECM) components, including β1 integrin ligands and metalloproteinases. On the other hand overexpression of the pro-invasive hMENAΔv6 increases fibronectin production. In primary tumors high hMENA11a correlates with low stromal fibronectin and favorable clinical outcome of early node-negative non-small cell lung cancer patients. This newly discovered signature, which pays attention to the alternative splicing of hMENA and ECM components such as fibronectin in the stroma, might help fill in the gap in the still controversial clinical management of early node-negative NSCLC patients. Citation Format: Francesca Di Modugno, Sheila Spada, Belinda Palermo, Paolo Visca, Pierluigi Iapicca, Anna Di Carlo, Barbara Antoniani, Isabella Sperduti, Anna Di Benedetto, Irene Terrenato, Marcella Mottolese, Francesco Gandolfi, Francesco Facciolo, Emily Chen, Martin A. Schwartz, Angela Santoni, Mina J. Bissell, Paola Nisticò. hMENA isoforms impact NSCLC patient outcome through fibronectin/β1 integrin axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5224.