Coactivation of Receptor Tyrosine Kinases in Malignant Mesothelioma as a Rationale for Combination Targeted Therapy

Introduction: To identify new therapeutic approaches in malignant mesothelioma (MM), we examined the expression and activation of receptor tyrosine kinases (RTKs) and the effects of specific RTK inhibitors and the mammalian target of rapamycin (mTOR) inhibitor rapamycin; the latter being of special interest in MM given the recent linkage between NF2 loss and mTOR activation. Methods: We performed a screen for mutated or activated RTKs in 14 MM cell lines and 70 primary tumors. Expression of phosphorylated RTKs was analyzed by Western blotting and a membrane-based antibody array in normal growth conditions and after treatment by specific inhibitors. MET and epidermal growth factor receptor (EGFR) mutations were screened by sequencing. MET, hepatocyte growth factor, insulin-like growth factor 1 receptor, and EGFR expression were studied by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay, and by Affymetrix expression microarrays. Results: Profiling of the phosphorylation status of 42 RTKs showed prominent coactivation of MET and EGFR in 8 of 14 (57%) MM cell lines. MET, EGFR, and insulin-like growth factor 1 receptor were the main RTKs activated after mTOR inhibition and contributed to AKT feedback activation. Knockdown of MET by RNA interference inhibited not only the phosphorylation of MET but also that of EGFR. Conversely, stimulation with hepatocyte growth factor increased both phospho-MET and phospho-EGFR. The combination of PHA-665752 and the EGFR inhibitor, erlotinib, suppressed cell growth more than either agent alone in three of six cell lines tested. Finally, combinations of rapamycin and different RTK inhibitors were more active than either drug alone in 12 of 13 cell lines. Conclusion: Combination targeting of kinase signaling pathways is more effective than single agents in most MM.