P2-02-05: Molecular Mechanism for Src Homology Phosphotyrosyl Phosphatase 2 Regulation of Cell Motility and Migration.

Abstract

Abstract Background: The Src homology phosphotyrosyl phosphatase 2 (SHP2) acts as a transducer of mitogenic and cell survival signaling downstream of receptor tyrosine kinases, cytokine receptors and integrins. As such, SHP2 promotes cell growth, transformation, survival, and motility, among other processes. Recent studies suggest that SHP2 is overexpressed in breast cancer and is essential for the maintenance of the transformed phenotype both in the HER2−positive and the basal-type/triple-negative breast cancer (BTBC) cells. Materials and Methods: The expression of SHP2 in BTBC cells was suppressed constitutively by lenti virus-mediated transduction of specific shRNA. Impact of SHP2 inhibition on cell migration was determined by the monolayer wound healing assay. The SHP2 substrate-trapping mutant was used to identify FAK as an SHP2 substrate. Site-directed mutagenesis, binding studies and phosphatase assays were employed to further characterize FAK as a bona fide SHP2 substrate. Immunofluorescence microscopy was conducted to visualize the amounts of phosphorylated FAK and its location in the cell after adhesion. Results: We demonstrate that SHP2 is essential for the migration of BTBC cells as evidenced by loss of the enhanced migratory behavior upon its inhibition. Using the SHP2 substrate-trapping mutant, FAK was identified as a biological substrate of SHP2. Site-directed mutagenesis, binding studies and phosphatase assays confirmed that SHP2 indeed associates with and dephosphorylates FAK at the pY397 site, a site known to promote the focal adhesion activity of FAK. Immunofluorescence of breast cancer cells with SHP2 inhibition were found to have higher levels of FAK pY397 further confirming that FAK is an SHP2 substrate. The increase in pY397 level correlated with loss of cell polarity, enhanced focal adhesion formation, and acquisition of non-transformed morphology. Discussion: These results suggest that dephosphorylation of pY397 of FAK by SHP2 is the major mechanism by which it is able to modulate cancer cell migration. Together with previous findings, the current studies show that SHP2 is a suitable target for therapeutic intervention to combat breast cancer metastasis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-05.