(c,d) Inhibition of c-MET induces apoptosis. using tyrosine kinase inhibitors in GBM tumor cells generates a cytostatic response seen as a a cell cycle arrest, which is accompanied by a substantial change in global gene expression levels. We demonstrate that a key component of this pattern is the transcriptional activation of the MET receptor tyrosine kinase and that pharmacological inhibition of MET overcomes the resistance to EGFR inhibition in these cells. These findings provide important new insights into mechanisms of resistance to EGFR inhibition and suggest that inhibition of multiple targets will be necessary to provide therapeutic benefit for GBM patients. studies of acute and transient ligand-stimulated activation of the receptor. This pattern is disparate from the clinical setting where EGFR is chronically active in GBM as a result of autocrine and paracrine expression of EGFR and its ligands (Ekstrand model systems. Here, we describe a novel genetically engineered mouse model of EGFR-driven GBM based on co-expression of wild-type EGFR (EGFRWT) and TGF, an EGFR ligand expressed in GBM. We established that a strict spatiotemporal expression and activation of EGFRWT with loss of tumor suppressor genes p16Ink4a/p19Arf and PTEN efficiently induce gliomagenesis in adults. Using these mice, we reveal a new and distinctive mechanism of resistance to EGFR TKI treatment. EGFR inhibition causes a global change in the transcription profile of GBM tumor cells, PHA-767491 hydrochloride including expression and activation of the MET tyrosine kinase receptor. The acquired MET activity results in the persistent activation of downstream signaling pathways and pharmacological inactivation of MET reverses its resistance function. Our results demonstrate that multi target inhibition is necessary to overcome resistance in GBM. Results Sustained activation of EGFRWT and loss of tumor suppressor genes in mice form GBM tumors Ligand-receptor autocrine and paracrine loops are commonly observed between EGFR and its ligands in GBMs (Ekstrand receptor tyrosine PHA-767491 hydrochloride kinase (RTK) gene. We treated TGF-EGFRWT;Ink2/3?/?;PTENlox tumor cell cultures with gefitinib for 16 hours and harvested total RNA at different times and performed qRT-PCR to measure the relative expression levels of mRNA over time (Figure 6a). Our results demonstrate a biphasic increase in the mRNA levels upon gefitinib treatment. Within 30 min of treatment the levels of c-met mRNA doubled and stayed constant for 3.5 hours, after which the levels increased to PHA-767491 hydrochloride over 5 folds after 16 hours. This latter increase in mRNA levels corresponded to the appearance of detectable levels of activated MET receptors (increase in MET autophosphorylation sites Tyr1234/1235 levels) (Figure 6b). We also determined that this induction in MET expression upon gefitinib treatment is irrespective of PTEN status (Supplementary Figure 6). Open in a separate window Figure 6 Gefitinib treatment increases expression and activation of c-Met in PTEN deficient GBM tumor cells. (a) Representative qRT-PCR from total RNA isolated from a TGF-EGFRWT;Ink2/3?/?;PTENlox tumor culture (T5) treated with gefitinib (10 M) for the indicated time. (b) Immunoblot of total cell lysates isolated from cells as in (a) and probed using antibodies against the indicated proteins. (c) Graphical representation of luciferase reporter assay results. A 3.5 kb fragment of the LAT antibody mouse c-Met promoter was used to drive the expression of firefly luciferase. Control plasmid (pGL4.10[mRNA levels upon gefitinib treatment resulted from an enhanced transcription of the gene by using a 3.5 kilobase (kb) fragment of the promoter region (Liang promoter (Figure 6c). Finally, we validated these observations by performing IHC against MET on GBM tumor sections from mice that have been treated with erlotinib. Figure 6d demonstrates that treatment of GBM tumor-bearing mice with the EGFR TKI erlotinib resulted in the expression of MET tyrosine kinase 72 hours post treatment. Our work indicated that this gefitinib-induced increase in MET expression and activity is responsible for sustaining a pro-survival Akt-based signaling. As such, we reasoned that co-treatment of TGF-EGFRWT;Ink2/3?/?;PTENlox tumor cells with gefitinib and the MET inhibitor SU11274 may sensitize these cells to apoptosis. Treatment of TGF-EGFRWT;Ink2/3?/?;PTENlox tumor cell cultures with both gefitinib and SU11274 robustly abrogated the levels of phospho MET Tyr1234/1235, indicating a complete inhibition of MET activity (Figure 7a). Inhibition of MET activity paralleled a reduction in the activity of Akt.