Helicobacter pyloriinduces cancer cell motility independent of the c-Met receptor

Abstract

Background:The hepatocyte growth factor (HGF) receptor, c-Met, is strongly implicated in late-stage cancer progression and poor patient prognosis. The stomach pathogen, Helicobacter pylori (H. pylori), was recently proposed to stimulate c-Met phosphorylation dependent upon interaction of c-Met with the bacterial CagA protein required for H. pylori-induced cancer cell motility and invasion.Materials and Methods:In this report, we employed short hairpin RNA (shRNA), western blot analysis using antibodies recognizing phosphorylation at discrete c-Met residues, and immunofluorescence microscopy to investigate the CagA-c-Met interaction.Results:The data showed that shRNA-mediated c-Met knockdown did not reduce H. pylori-induced cell motility, suggesting that c-Met was not required for motility. Surprisingly, c-Met knockdown did not reduce the level of an H. pylori-induced protein recognized by a phospho-c-Met antibody. This 125 kD protein was 10 kD smaller than c-Met, suggesting that H. pylori did not phosphorylate c-Met but cross-reacted with another protein. This hypothesis was confirmed when c-Met phosphorylation inhibitors did not lower the levels of the bacteria-induced 125 kD protein, and c-Met immunoprecipitation (IP) did not detect this 125 kD protein from H. pylori-treated lysates. This protein was identified as a product of antibody cross reactivity with phosphorylated CagA. We also confirmed that CagA interacts with c-Met, but this interaction may have caused previous authors to misinterpret phosphorylated CagA as c-Met phosphorylation. Finally, pretreatment with the proteasomal inhibitor, lactacystin, caused prolonged HGF-induced c-Met phosphorylation and facilitated a CagA-negative H. pylori to stimulate AGS cell motility, suggesting that sustained c-Met phosphorylation compensates for the loss of CagA-dependent signaling.Conclusions:These data demonstrate that H. pylori stimulates cancer cell motility independent of the c-Met receptor. We further hypothesize that although H. pylori does not target c-Met, the bacteria may still utilize c-Met effector signaling to stimulate CagA-independent cancer cell motility, which may provide a further mechanism of H. pylori-dependent gastric cancer progression.