Introduction: MET overexpression has been shown to correlate with clinical outcomes in gastric cancer. The objective of this study was to evaluate MET expression and its downstream signaling mediators, STAT3, ERK, and AKT, as potential therapeutic targets in human gastric cancer. We evaluated cabozantinib, a multi-targeted receptor tyrosine kinase inhibitor with potent activity against MET and VEGFR2, in MET-expressing (MET(+)) gastric cancer cells.
Methods: Surgical gastric cancer specimens were evaluated for expression of MET, phospho-ERK and phospho-STAT3 by immunohistochemistry (IHC). Cabozantinib was tested alone and in combination with a MEK inhibitor (U0126) and a JAK1/2 inhibitor (ruxolitinib). Half maximal inhibitory concentrations (IC50) for each drug were determined by MTS assay to determine appropriate treatment concentrations in two MET(+) human gastric cancer cell lines (Hs746T and SNU5). Protein phosphorylation levels of MET, STAT3, ERK, and AKT were determined by Western blot to assess downstream activation before and after drug treatment.
Results: MET protein expression by IHC was detected in 18/47 (38.3%) gastric cancer specimens. One-half of MET(+) specimens stained with moderate or strong intensity, and 15/18 (83.3%) showed ≥ 25% membrane staining. In the MET(+) specimens, 9/18 (50.0%) samples expressed phospho-ERK and 3/18 (16.7%) expressed phospho-STAT3. Next, gastric cancer cell lines were examined by Western blot showing elevated levels of baseline phosphorylation of MET, STAT3 and ERK in Hs746T and SNU5 cells. Treatment of SNU5 (IC50 0.37 μM) and Hs746T (IC50 2.3 μM) with cabozantinib (0.1 μM) completely blocked downstream phosphorylation of MET, STAT3, AKT and ERK. Addition of MET's ligand hepatocyte growth factor (HGF, 25 ng/ml) overcame this observed inhibition at a lower cabozantinib concentration (0.01 μM), as evidenced by phosphorylation of STAT3 and ERK. Combining U0126 or ruxolitinib with cabozantinib (0.01 μM) effectively blocked ERK and STAT3 phosphorylation, respectively, even in the presence of HGF. This demonstrates that downstream signaling in MET(+) lines can be effectively and synergistically blocked by two targeted drug therapies.
Conclusion: MET, ERK and STAT3 signaling are activated in gastric cancer cell lines, suggesting that ERK and STAT3 may serve as additional points of therapeutic intervention in MET(+) gastric cancer. Combining cabozantinib with a MEK or STAT3 inhibitor was necessary to inhibit phosphorylation of their respective targets in the presence of HGF, suggesting that drug synergy may be effective in blocking multiple activated downstream pathways.
Citation Format: Audrey H. Choi, Jianming Lu, Sangjun Lee, Peiguo Chu, Vincent Chung, Ren-Jang Lin, Joseph Kim, Joseph Chao. Multi-targeted tyrosine kinase inhibitor Cabozantinib as a therapeutic agent in MET-overexpressing gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 780. doi:10.1158/1538-7445.AM2015-780