Hepatocyte growth factor sensitizes brain tumors to c-MET kinase inhibition

Abstract

Purpose: The receptor tyrosine kinase (RTK) c-MET and its ligand hepatocyte growth factor (HGF) are deregulated and promote malignancy in cancer and brain tumors. Consequently, clinically applicable c-MET inhibitors have been developed. The purpose of this study was to investigate the not-well-known molecular determinants that predict responsiveness to c-MET inhibitors and to explore new strategies for improving inhibitor efficacy in brain tumors.

Experimental design: We investigated the molecular factors and pathway activation signatures that determine sensitivity to c-MET inhibitors in a panel of glioblastoma and medulloblastoma cells, glioblastoma stem cells, and established cell line-derived xenografts using functional assays, reverse protein microarrays, and in vivo tumor volume measurements, but validation with animal survival analyses remains to be done. We also explored new approaches for improving the efficacy of the inhibitors in vitro and in vivo.

Results: We found that HGF coexpression is a key predictor of response to c-MET inhibition among the examined factors and identified an ERK/JAK/p53 pathway activation signature that differentiates c-MET inhibition in responsive and nonresponsive cells. Surprisingly, we also found that short pretreatment of cells and tumors with exogenous HGF moderately but statistically significantly enhanced the antitumor effects of c-MET inhibition. We observed a similar ligand-induced sensitization effect to an EGF receptor small-molecule kinase inhibitor.

Conclusions: These findings allow the identification of a subset of patients that will be responsive to c-MET inhibition and propose ligand pretreatment as a potential new strategy for improving the anticancer efficacy of RTK inhibitors.

Figure 1. METi effects on c-MET activation, and molecular backgrounds of brain tumor cells

A The glioblastoma cell lines U87, A172, T98G, U373, SF-767 and U1242, primary cells GBM-10 and GBM-6, GSCs 0308 and 1228, and the medulloblastoma cell lines D425, ONS-76, DAOY and PFSK were treated with METi prior to treatment with HGF. Protein lysates were immunoblotted for phosphorylated (p-MET) or total c-MET. The results show that METi inhibits basal and HGF-induced c-MET phosphorylation in all cells. B Protein lysates from the above cells were subjected to immunoblotting for phospho-c-MET (p-MET), c-MET, HGF, PTEN, phosphor-EGFR (p-EGFR), EGFR and β-Actin using specific antibodies. C Protein levels from the blots described in (B) were quantified by densitometry on film. The data show substantial variability in the expressions of the different proteins in the different cells.

Figure 2. The effects of METi on cell death / apoptosis and proliferation vary between the different brain tumor cells

A The glioblastoma cell lines U87, A172, T98G, U373, SF-767 and U1242, primary cells GBM-10 and GBM-6, GSCs 0308 and 1228, and the medulloblastoma cell lines D425, ONS-76, DAOY and PFSK were treated with METi or control. The cells were subsequently assessed for apoptosis and cell death by AnnexinV/7AAD flow cytometry and the percentage of dead cells was determined. B The same brain tumor cells as in (A) were treated with METi or control. The cells were subsequently assessed for proliferation by cell counting over a period of 5 days and growth curves were established. All experiments were performed in triplicates and repeated three times.

Figure 3. HGF co-expression determines sensitivity to c-MET kinase inhibitor

Analysis of correlations between the levels of METi-induced inhibition of cell survival A and proliferation B with the expressions of c-MET, p-MET, HGF, PTEN, p-EGFR and EGFR using both regression and Spearman correlation analyses. The results show that METi-induced inhibition of proliferation and survival statistically significantly correlates with HGF expression but not with c-MET, p-MET, PTEN, p-EGFR or EGFR.

Figure 4. Pathway activation mapping of c-MET inhibitor effects in high HGF and low HGF expressing cell lines

Cells were treated with METi or control and phosphorylation/activation mapping by Reverse Phase Protein Microarray (RPMA) was obtained on three cell lines that comprised the top quartile of the highest HGF expression (U1242, U87, A172) and on four cell lines that comprised the bottom quartile of HGF expression (ONS-76, DAOY, GFM-10 and T98G). Statistically significant differences between the effects of c-MET inhibitor treatment (calculated as the difference between triplicate independent experiments of the treatment – control/untreated) were found for 5 biochemically interconnected ERK pathway-linked signaling proteins that are found downstream of c-MET: JAK (Y1022/Y1023) p=0.04; ERK 1,2 (T202/Y204) p=0.01; p53 (S15) p=0.06; ELK (S383) p=0.03; and RSK3 (T356/S360) p=0.05. The figure reveals the biochemically interlinked signature of differential treatment response in the context of high vs. low HGF production along with the RPMA data results as a histogram for each of the proteins.

Figure 5. The in vivo anti-tumor effects of METi are significantly greater in high HGF-expressing glioblastoma xenografts than in low-expressing xenografts

Glioblastoma cells U87 with high HGF-expression and T98G with low HGF-expression were stereotactically implanted in the striatum of immunodeficient mice (n=6 for each treatment group). METi or vehicle control was administered by daily oral gavage starting one week post-tumor implantation. The animals were euthanized 4 weeks after tumor implantation and the volumes of all tumors were measured. The results show that METi significantly inhibited the growth of U87 xenografts but not the growth of T98G xenografts. A Representative brain cross sections with implanted xenografts (arrows). B Quantification of tumor sizes. *, p<0.05.

Figure 6. Short-time exogenous HGF treatment sensitizes glioblastoma cells and GSCs to RTK inhibitor-induced apoptosis in vitro and in vivo

A Glioblastoma cell lines U87 and U373, primary cells GBM-10, and GSCs 1228 and 0308 were treated with HGF for 2–24 hours and then treated with METi. The cells were assessed for apoptosis and death by Annexin V/7AAD flow cytometry. METi induces greater apoptosis in cells pre-treated with HGF for 2–6 hr prior METi treatment. B The glioblastoma cells U373 were treated with EGF (100 ng/ml) for 3–24 hours and then treated with the EGFR inhibitor Erlotinib (2 µM). The results show that Erlotinib induces greater apoptosis in cells pre-treated with EGF for 3 hrs before Erlotinib treatment. C GSCs 1228-derived flank xenografts were pre-treated with HGF intratumoral injections 2 hr prior to treatment with METi and tumor sizes were measured after 3 weeks. The results show that HGF-pre-treated tumors were more inhibited by METi than non-pretreated tumors (n=6 for each treatment group). *, p<0.05