HGF/SF promotes an aggressive phenotype in c-Met-overexpressing fulvestrant-resistant MCF-7 cells - Evidence for MMP-9 and PI3k involvement [Abstract]

Introduction: Acquired resistance to anti-hormonal agents in breast cancer is frequently associated with altered growth factor expression and the development of an aggressive cell phenotype in vitro. Chronic exposure of MCF-7 cells to the pure antioestrogen Fulvestrant results in acquired Fulvestrant resistance. Significantly, these resistant cells also overexpress the c-Met receptor, a protein that clinically has been linked to invasive breast carcinoma and metastasis. Subsequent exposure of Fulvestrant-resistant (Fas-R) cells to the c-Met ligand, hepatocyte growth factor/scatter factor (HGF/SF), results in a profound increase in their motile and invasive capacities. In other cancer cells, HGF/SF is known to induce invasive behaviour by matrix metalloproteinase (MMP) overproduction via the phosphoinositide-3 kinase (PI3K)/AKT pathway. In order to investigate whether a similar mechanism might operate in our resistant cell line, we first established whether HGF/SF stimulated MMP expression and subsequently determined the effects of PI3K pathway inhibition on both MMP expression and the aggressive phenotype. Methods: MCF-7 and Fas-R cells were exposed to HGF/SF (20ng/ml). MMP mRNA and protein expression were assessed using RT-PCR and zymography respectively. In vitro migratory and invasive capacities of both cell types were measured by migration and invasion assays. Pharmacological inhibition of the PI3K/AKT pathway was achieved using LY294002; changes in PI3K signalling pathway activity were assessed by Western Blotting and phosphospecific antibodies. Results: HGF/SF significantly promoted the migration and invasion of Fas-R cells whilst having only a modest effect in MCF-7 cells. Moreover, in Fas-R cells HGF/SF induced MMP-1, -9 and -10 expression and increased the level of AKT phosphorylation. Inclusion of LY294002 inhibited HGF/SF-enhanced Fas-R cell migration and invasion. AKT inhibition furthermore downregulated MMP-9 mRNA levels and abolished MMP-9 protein expression. Conclusion: These data suggest that HGF/SF-induced stimulation of c-Met, overexpressed in Fulvestrant-resistant breast cancer cells, promotes an aggressive, invasive phenotype through a mechanism involving PI3K/AKT-mediated MMP-9 expression