Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with the worst survival rate among solid cancers. The pressing needs for extending life expectancy of patients are the identification of early prognostic markers and novel druggable pathways. PDAC arises generally from pancreatic intraepithelial neoplasia (PanIN) and a dynamic interactions between tumor, stromal cells and autocrine and paracrine signaling lead to epithelial to mesenchymal transition (EMT), an early process in the natural history of pancreatic cancer. Cytoskeletal reorganization, extracellular matrix (ECM) remodeling, and matrix metalloproteinases (MMPs) contribute to PDAC aggressiveness in cooperation with soluble growth factors or cytokines, with TGF-β1 as crucial player. hMENA is an actin regulatory protein whose splicing program, mediated by the epithelial splicing regulatory proteins (ESRPs), has been associated with the EMT process. Our previous studies indicated that alternative splicing of hMENA, generates hMENA11a and hMENAΔv6 isoforms with opposite roles in cell proliferation and invasion in breast and lung cancers. Alternative splicing is known to play a prominent role in tumor progression and tumorigenesis and the derived isoforms may represent powerful diagnostic and prognostic factors as we have recently shown for hMENA alternative splicing in early stage non-small cell lung cancer (NSCLC). The aim of this study is to investigate the role of TGF-β1 on the expression and function of hMENA isoforms in PDAC, and verify whether the expression pattern of hMENA isoforms may impact patient outcome.
Methods: We analyzed the expression pattern of hMENA isoforms by immunohistochemistry, using anti-pan hMENA and specific anti-hMENA11a antibodies, in 285 PDACs, 15 PanINs, 10 pancreatitis, and normal pancreas, evaluating the patient outcome. The functional role of hMENA isoforms were analyzed by loss and gain of function experiments in untreated and TGF-β1-treated PDAC cell lines.
Results: In a panel of pancreatic cancer cell lines, hMENA11a expression correlates with an epithelial phenotype, while hMENAΔv6 expression with a mesechymal phenotype, with low E-cadherin and high vimentin expression. hMENA11a knock-down in PDAC cell lines affected cell-cell adhesion but not cell invasion. TGF-β1 cooperated with β-catenin signalling to up-regulate hMENA and hMENAΔv6 expression but not hMENA11a. The hMENA/hMENAΔv6 up-regulation play a crucial role in cell invasiveness and in TGF-β1-induced EMT. After TGF-β1 treatment, hMENA/hMENAΔv6 were mobilized from focal adhesion to actin stress fibers, and the silencing of these isoforms significantly inhibited the TGF-β1-induced EMT in PANC-1. Functionally, in the absence of hMENA11a, the hMENA/hMENAΔv6 up-regulationis crucial for SMAD2-mediated TGF-β1 signalling, migration, invasion and MMPs activities. Pan hMENA immunostaining, absent in normal pancreas and low-grade PanINs, was weak in PanIN-3 and had higher levels in virtually all PDACs with 64% of cases showing strong staining. Conversely, the anti-invasive hMENA11a isoform only showed strong staining in 26% of PDAC. The absence of hMENA11a in a subset (34%) of pan-hMENA-positive tumors significantly correlated with poor outcome, in agreement with experimental results.
Conclusions: hMENA isoforms are regulated differently by TGF-β1, and the pattern of expression of hMENA isoforms is crucial in TGF-β1-dependent EMT and cell invasion. The pattern of expression of hMENA isoforms correlates with PDAC patient outcome and it could be used in specific clinical settings for the choice of the most effective treatment of PDAC patients. Our data provide new insights into molecular pathways involved in PDAC biology and suggest that hMENA-related pathways are promising targets for the development of new prognostic and therapeutic tools in PDAC.
Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Isabella Sperduti, Matteo Fassan, Ivana Cataldo, Borislav C. Rusev, Rita T. Lawlor, Maria Grazia Diodoro, Michele Milella, Gian Luca Grazi, Mina J. Bissell, Aldo Scarpa, Paola Nisticò. The pattern of hMENA isoforms is regulated by TGF-β1 in pancreatic cancer and may predict patient outcome [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A113.
- ©2016 American Association for Cancer Research.