Targeting c-Met receptor overcomes TRAIL-resistance in brain tumors

Abstract

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis specifically in tumor cells. However, with approximately half of all known tumor lines being resistant to TRAIL, the identification of TRAIL sensitizers and their mechanism of action become critical to broadly use TRAIL as a therapeutic agent. In this study, we explored whether c-Met protein contributes to TRAIL sensitivity. We found a direct correlation between the c-Met expression level and TRAIL resistance. We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5. This interruption greatly induces the formation of death-inducing signaling complex (DISC) and subsequent downstream apoptosis signaling. Using intracranially implanted brain tumor cells and stem cell (SC) lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that SC expressing a potent and secretable TRAIL (S-TRAIL) have a significant anti-tumor effect in mice bearing c-Met knock down of TRAIL-resistant brain tumors. To our best knowledge, this is the first study that demonstrates c-Met contributes to TRAIL sensitivity of brain tumor cells and has implications for developing effective therapies for brain tumor patients.

Figure 1. Knock down of c-Met protein sensitizes resistant medulloblastoma (MB) cells to S-TRAIL treatment.

(A) MB cell viability analysis showing the effect of varying concentrations of S-TRAIL treatment (24 h) on different MB cell lines. (B) Left, Western blot analysis of c-Met and tubulin levels in indicated MB cell lines. Right, plot showing the statistic results of c-Met protein levels in the MB lines. (C) MB lines UW473 and DAOY were pre-incubated with c-Met inhibitor (PHA665752) before treatment with HGF (25 ng/ml). Protein lysates were then immunoblotted for phosphorylated (p-Met) or total c-Met. (D) Cell viability analysis showing the effect of S-TRAIL treatment on both DAOY and UW473 cells with (+) or without (−) the presence of PHA665752 (PHA). (E) Western blot analysis c-Met and tubulin levels in UW473 cells stable-infected with LV-scrambled (UW473scr) or LV-shMet virus (UW473shMet). (F) Cell viability analysis showing the effect of S-TRAIL treatment (24 h) on UW473, UW473scr, or UW473shMet respectively. * denotes P<0.05.

Figure 2. Knock down of c-Met protein sensitizes resistant glioma cells to S-TRAIL treatment.

(A) Western blot analysis of c-Met and tubulin levels in indicated glioma lines. (B) Plot showing the statistic results of c-Met protein levels in both TRAIL-resistant and TRAIL-sensitive glioma lines. (C) LN229 cell line viability analysis showing the effect of S-TRAIL treatment (24 h and 72 h) on indicated groups. * P<0.05.

Figure 3. c-Met interacts with DR5 and affects DISC formation upon S-TRAIL stimulation.

(A) Western blot analysis of caspase 8, cleaved (cl)-caspase 3, cleaved (cl)-PARP, and tubulin levels in UW473scr and UW473shMet cells upon S-TRAIL stimulation for indicated time periods. (B) Western blot analysis showing DR5, DR4, caspase 8, FLIP, and tubulin levels in UW473, UW473scr and UW473shMet cells. (C) Upper panel, Western blot analysis showing the DR5 levels in the immunoprecipitates of IgG, anti-c-Met, and anti-DR5 (5% of each input was also blotted for total DR5 as control); Lower panel, Western blot analysis showing the c-Met levels in the immunoprecipitates of IgG, anti-DR5 and anti-c-Met (5% of each input was also blotted for total c-Met as control). (D) Left panel, Western blot analysis showing DR5 levels in the immunoprecipitates of anti-caspase 8 from UW473scr or UW473shMet cells with (+) or without (−) the presence of S-TRAIL stimulation (5% of each input was also blotted for total DR5 as control). Right panel, plot showing the quantitative analysis of the immunoblots on the left. * denotes P<0.05.

Figure 4. Knock down of c-Met sensitizes resistant MB cells to stem cell-delivered S-TRAIL both in vitro and in vivo.

(A) Cell viability measured by Fluc signal showing the effects of MSC-derived S-TRAIL in the co-cultures of MSC or MSC-S-TRAIL with UW473scr-Fmc (UW473scr), or UW473shMet-Fmc (UW473shMet) cells at various culturing ratios. (B) Left panel: Fluc bioluminescence imaging (BLI) on days 1, 3 and 7 of mice intraparenchymally implanted with UW473scr or UW473shMet cells admixed with MSC-S-TRAIL. Representative images of mice for each group are shown. Right panel, statistical analysis of Fluc bioluminescence activity observed in respective groups of mice represented on the left. (C) Plot showing Fluc bioluminescence intensities (representative of in vivo tumor growth). (D) Left, the brain sections from the indicated groups (UW473scr+MSC-S-TRAIL or UW473shMet+MSC-S-TRAIL) stained with cl-caspase 3 (blue) and imaged together with mCherry (red, represents tumor cells) and GFP (green, represents MSC secreting S-TRAIL). Right panel, plot representing the number of calculated activated caspase 3 positive cells in the corresponding samples on the left panel (scale bar  =  20 µm). ** denotes P<0.01.