hMENA splicing program in tumor progression: functional role of isoforms in pancreatic cancer cell adhesion and migration

hMENA, a member of the Ena/VASP proteins involved in the cytoskeletal regulation during cell motility and adhesion, is up-regulated in invasive tumor cells and plays a crucial role in metastatic progression. We show that hMENA alternative splicing is related to EMT process and this is in line with recent data demonstrating that hMENA belongs to a cluster of genes whose alternative splicing is related to EMT. Our group has previously characterized two hMENA isoforms, one 88-kDa isoform defined as “classic” and one splice variants, named hMENA11a (90 kDa). Here we report a novel splice variant of human MENA, named hMENAΔv6 (80 kDa). hMENA11a characterizes epithelial cancer cell lines expressing E-Cadherin, whereas hMENAΔv6 is expressed in breast and cervix tumor cell lines displaying the phenotypic features of EMT with migratory behaviour. The analysis of pancreatic cell lines indicates that hMENA11a is expressed in the normal human pancreatic ductal epithelial cell line (HPDE) besides epithelial pancreatic tumor cell lines (CFPAC, T3M4, and PACA44), whereas hMENAΔv6 is expressed in PANC1 cells that lack hMENA11a and express Vimentin. We observed that the Ser157 of VASP is phosphorylated either in normal or pancreatic cancer cell lines with an epithelial phenotype and expressing hMENA11a, whereas the hMENA11a negative/hMENAΔv6 positive PANC1 cell line did not show appreciate level of P-Ser157 VASP. By gain and loss of function, we demonstrate that hMENA11a espression is able to induce VASP phosphorylation at Ser157 and that hMENA11a transduction in PANC1 cells resembles the pattern of VASP phosphorylation observed in HPDE cells. We analyzed the effects of hMENA11a mutants in PANC1 cells by the substitution of the two putative serine sites of phosphorylation present in 11a peptide to alanine. Biochemical results evidenced that only wild type hMENA11a transduction is able to induce VASP phosphorylation at Ser157, whereas the two mutants did not affect VASP phosphorylation. Epithelial cells express one or more of the Ena/VASP proteins, and these in turn localize to the leading edges of lamellipodia, the tips of filopodia, focal adhesions, cell–cell junctions. Our results demonstrate that P-Ser157 VASP interacts with hMENA11a and both proteins localize within focal adhesions in HPDE cells. PKA activation increases the pool of P-Ser157 VASP and hMENA11a at the cell periphery, whereas both proteins disappear from the cell periphery when PKA is inhibited. hMENA11a transduction in PANC1 resembles P-Ser157 VASP and hMENA11a localization at the cell periphery. At functional level, we analyze whether the hMENA splicing program differently affect the ability of PANC1 cells to adhere and invade ECM. hMENA11a or ESRP1 transduction increases P-Ser157 level in cells plated on collagen type I or fibronectin, together with an increase of cell-matrix adhesion. Furthermore, hMENA11a or ESRP1 transduction determines a reduction of cancer cell invasion in a BM-coated Boyden chamber. Differently from the breast model, where hMENA is not expressed in normal epithelium but overexpressed following transformation and tumor progression, hMENA and hMENA11a are co-expressed in normal pancreas whereas primary tumors lack the expression of the epithelial hMENA11a isoform, suggesting that hMENA splicing may be altered in pancreatic tumor progression. Altogheter, our results demonstrate that hMENA splicing is a critical step in cancer and the lost of epithelial hMENA11a isoform strongly contributes to the aggressivness of this incurable neoplasia.