(A) Sham ablated tumor. inhibitor SU11274 (0.04M-10M) or vehicle control (0.1% DMSO). Cells were assessed for viability at 72 hours using WST-1 assay. Data were normalized to vehicle control and the IC50 estimated using non-linear regression curve fitting. Data are presented as meanSEM of 3 independent experiments NIHMS1507456-supplement-Supp2.tiff (7.2M) GUID:?DFDCE6D7-7926-43F6-A555-75BECBC3F428 Supp3: Supplemental Figure 3: Quantitative mass spectrometry. N1S1 cells pre-treated with the PI3K/mTOR inhibitor BEZ235 (1M) or vehicle control (0.1% DMSO) for 1 hour followed by heat stress (45C) or control (37C) for 10 minutes were harvested immediately post-heat stress. Following peptide enrichment cell lysates were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data are presented as relative mean abundance. NIHMS1507456-supplement-Supp3.pdf (202K) GUID:?8402560E-9ADF-47D9-9554-3D3DE6C21AD8 Supp4. NIHMS1507456-supplement-Supp4.docx (112K) GUID:?3146BC88-F160-40C0-97B9-1D4F6DF46AC9 Abstract Purpose. The aims of the present study were two-fold: first, to test the hypothesis that AZ-960 heat stress induces MET and Smoc1 EGFR signaling in hepatocellular carcinoma (HCC) cells and inhibition of this signaling decreases HCC clonogenic survival; and second, to identify signaling pathways associated with heat-stress induced MET signaling. Materials and Methods: MET+ and EGFR+ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumors and liver/tumor assessed for phospho-MET and phospho-EGFR immunostaining. Results: Heat stress induced rapid AZ-960 MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation AZ-960 at the HCC tumor ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and AZ-960 EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signaling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signaling did not block AKT signaling Conclusions: Heat stress induced MET and EGFR signaling is distinct from growth factor mediated signaling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism. heat stress conditions (45C for 10 minutes) that induce a significant reduction in cell survival but still result in colony formation whereas 50C for 10 minutes resulted in complete cell killing with no colony formation and 37C for 10 minutes had no effect on cell survival or colony formation [11]. This model recapitulates the thermal injury experienced at the margin of the ablation zone where both neoplastic and parenchymal cells experience heat stress that may or may not induce complete cell killing. As such, 45C for 10 minutes was used as the thermal dose based on the incomplete HCC cell killing. For all experiments using a small molecule inhibitor, cells were pre-treated with the designated small molecule inhibitor (SU11274, PF-04217903, Erlotinib or BEZ235) or vehicle control (0.1% DMSO) for one hour prior to heat stress. For experiments utilizing recombinant growth factors, HGF or EGF was administered concurrently at the time of heat stress for 10 minutes after the one-hour pre-treatment with small molecule inhibitor or vehicle control. Cells were harvested immediately after the 10-minute heat stress, rinsed in PBS and snap frozen in liquid N2 for Western blot or mass spectrometry analysis or analyzed using a colony formation assay. For the dose titration experiments in Figure 3A and AZ-960 3B, N1S1 and AS30D HCC cells were treated with a dose-titration of the MET inhibitor SU11274 (0.04M-10M), EGFR inhibitor erlotinib (0.08M-20M) or vehicle control (0.1% DMSO) and assessed for viability at 72 hours using a WST-1 assay. For the combination small molecule inhibitor plus heat stress experiments in Figure 3C and 3D, N1S1 and AS30D HCC were cells pre-treated with SU11274 (1M and 10M), erlotinib (1M and 10M) or vehicle control (0.1% DMSO) for 1 hour, heat stressed (45C) or control (37C) for 10 minutes, harvested immediately post-heat stress and whole-cell lysates were subjected to Western blotting. For the combination.
