Abstract 280: Met (the HGF receptor) localizes to the nucleus in wound healing

Abstract

Abstract Background: The hepatocyte growth factor (HGF)/Met signaling pathway has been shown to be important for stimulating cell proliferation, motility, invasion and metastasis. Recent work from our lab has also demonstrated patients with high levels of nuclear Met have a worse prognosis even in lymph node negative breast cancer and has also identified a 60 kDa fragment from the carboxy-terminal domain of Met that localizes to the nucleus. Methods: We developed a method for quantification of in vitro wounding of epithelial cellular monolayers that we have utilized to further characterize nuclear Met. Nuclear Met was identified by immunofluorescence with the carboxy-terminal Met specific antibody CVD13 (Invitrogen) in H1650, A431 and Caco2 cells. The Met ligand (HGF) was used to stimulate wound healing and the Pfizer Met kinase inhibitor PHA-665752 (PHA) was used to determine the dependence of activated Met for nuclear localization. Results: As previously observed, stimulating cells with HGF (50 ng/mL) increases overall wound healing and inhibition of Met kinase activity with PHA reduces overall wound healing. Using immunofluorescence we have determined nuclear Met is present at the wound edge of lung (H1650), colon (CaCo2), and skin (A431) cancer cell lines. Additionally, nuclear localization of Met is also observed in H1650 cells by immunofluorescence with a phospho-Met antibody (CST). Nuclear localization of Met is inhibited when cells are pretreated with PHA even in the presence of HGF. However, HGF stimulation alone is not sufficient to induce nuclear Met in the A549 lung cancer cell line. Finally, the 60 kDa Met fragment is absent by Western blot when H1650 cells are grown in serum-starved conditions. Conclusion: Nuclear Met is observed during the wound healing of cellular monolayers and can be inhibited with Met specific kinase inhibitors. Nuclear Met, as defined by the presence of the 60 kDa fragment, is lost in serum-starved growth conditions. Since, several pharmaceutical companies are currently developing Met-based therapies, the role of Met the nucleus may be important in future targeted cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 280.