The adhesion molecule NCAM promotes ovarian cancer progression via FGFR signalling.
Bottom Line: NCAM is absent from normal ovarian epithelium but becomes highly expressed in a subset of human EOC, in which NCAM expression is associated with high tumour grade, suggesting a causal role in cancer aggressiveness.This pro-malignant function of NCAM is mediated by its interaction with fibroblast growth factor receptor (FGFR).Our results point to NCAM-mediated stimulation of FGFR as a novel mechanism underlying EOC malignancy and indicate that this interplay may represent a valuable therapeutic target.
Affiliation: IFOM - The FIRC Institute of Molecular Oncology, Milano, Italy.Show MeSH
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Mentions: Following the observation that NCAM/FGFR interplay is necessary for EOC cell migration and invasion, we asked whether it is also sufficient. To address this question, we selected two human EOC cell lines, SKOV3 and OVCA-433, which express no endogenous NCAM (Figs S4 and S5 of Supporting Information, panels A and B). Both cell lines express various FGFR family members (our unpublished observations; Chandler et al, 1999; Valve et al, 2000), thus providing a suitable experimental system to investigate the impact of ectopically expressed NCAM on FGFR activity. SKOV3 and OVCA-433 cells were stably transfected with full-length NCAM (Figs S4 and S5 of Supporting Information, panels A and B). SKOV3 cells were also retrovirally transduced with a GFP-encoding vector (Fig S4A and B of Supporting Information) for xenotransplantation purposes (see below). In agreement with the data on NCAM silencing in MOVCAR cells, ectopic expression of NCAM in human EOC cells had no effect on cell proliferation (Fig S4C of Supporting Information and data not shown). Rather, NCAM-expressing SKOV3 and OVCA-433 cells exhibited a remarkable increase in their migratory activity as compared to mock-transfected cells (Fig 3A and Fig S5C of Supporting Information). Previous studies have established that the interaction with FGFR involves the two FNIII repeats of NCAM (referred to as FN1 and FN2; Kiselyov et al, 2003). Therefore, to determine the relative contribution of FGFR binding in NCAM-induced EOC cell migration, SKOV3 and OVCA-433 cells were transfected with a mutant version of NCAM lacking FN2 (ΔFN2), in which the interaction with FGFR is disrupted (Francavilla et al, 2007). Mutant NCAM was expressed at a level comparable to full-length NCAM and retained the localization at the cell surface (Figs S4 and S5 of Supporting Information, panels A and B), as well as the ability to induce cell–cell adhesion (not shown), which is mediated by distal Ig domains (Soroka et al, 2003). Furthermore, deletion of the FN2 domain did not alter folding, localization or the adhesive properties of NCAM. The ability of NCAM to stimulate FGFR signalling has been mostly characterized on FGFR1 (Francavilla et al, 2009; Kiselyov et al, 2003), a member of the FGFR family that is prominently expressed in both SKOV3 and OVCA433 cells (Fig S6B of Supporting Information and data not shown). Therefore, we focused on FGFR1 to define the impact of NCAM expression on FGFR activation. First, we confirmed that full-length NCAM, but not ΔFN2, forms a complex with FGFR1 in EOC cells (Fig S6A of Supporting Information). Accordingly, only full-length NCAM was able to induce autophosphorylation of FGFR1 in SKOV3 cells (Fig S6B of Supporting Information), confirming that also in EOC cells NCAM stimulates FGFR activation through its FN domains. In both SKOV3 and OVCA-433 cells, NCAM-ΔFN2 failed to promote cell migration (Fig 3A and Fig S5C of Supporting Information), thus implicating the interaction with FGFR as a pre-requisite for NCAM-induced migration of EOC cells. To further confirm this notion, SKOV3 cells expressing NCAM or a control vector were either treated with the FGFR inhibitor PD173074 or transfected with a dominant-negative version of FGFR1. In both cases, FGFR inhibition resulted in the abrogation of NCAM-dependent migration (Fig 3A) supporting the results obtained with NCAM-ΔFN2. By analogy to MOVCAR cells (see above), FGF-2 showed no pro-migratory activity on SKOV3 cells, a situation that was not changed by the ectopic expression of NCAM (not shown). This suggests that FGF does not cooperate with NCAM in the stimulation of FGFR-mediated EOC cell migration.
Affiliation: IFOM - The FIRC Institute of Molecular Oncology, Milano, Italy.