Heat stress induced, ligand-independent MET and EGFR signalling in hepatocellular carcinoma

Abstract

Purpose: The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling.

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 tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining.

Results: Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and 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 signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling.

Conclusions: Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.

Keywords: AKT; EGFR; MET; heat stress; hepatocellular carcinoma; thermal ablation.

Figure 1.. Heat stress induces rapid MET and EGFR signaling in HCC cells in vitro.

(A) Western blot analysis of kinome-wide changes in protein tyrosine phosphorylation in response to heat stress in hepatocytes and HCC cells. A panel of rat hepatocyte (Clone 9) and HCC (N1S1, AS30D) cell lines and human HCC cell lines (HuH7, Hep3B, PLC/PRF/5) were heat stressed (45°C) or control (37°C) for 10 minutes, harvested immediately post-heat stress and whole-cell lysates were subjected to Western blotting using a phosphotyrosine (P-Tyr-100) Y* antibody with broad reactivity to tyrosine phosphorylated proteins and peptides. The antibody does not cross-react with phospho-serine or phospho-threonine residues. β-Actin was used as a loading control. (B) N1S1 and (C) AS30D HCC cell lines cells were heat stressed (45°C for 10 minutes), recovered up to 2-hours post heat stress and whole-cell lysates were subjected to Western blotting for phospho- and total MET and EGFR. β-actin was used as a loading control. BL= baseline, non-heat stress control; t= time post heat stress in minutes. For example, t=0 indicates immediate post-heat stress. (D) A panel of human HCC cell lines were heat stressed (45°C) or control (37°C) for 10 minutes, harvested immediately post-heat stress and whole-cell lysates were subjected to Western blotting using phospho-specific antibodies against EGFR and MET. β-actin was used as a loading control. Representative images from n ≥ 3 independent experiments.

Figure 2.. Representative phospho-MET immunohistochemical staining of the ablation zone 24-hours post-ablation in the orthotopic N1S1 HCC model.

Low power (40×; A,B) and high power (100×; C, D) photomicrographs of phospho-MET immunostained sections. (A,C) Sham ablated tumor. No evidence of MET phosphorylation in the tumor (T), background liver (L) or at the tumor-liver margin in the sham-ablated tumor. (B, D) Laser ablated tumor. Markedly increased MET phosphorylation at the tumor-ablation margin (black arrowheads) in the laser-ablated tumor (*T) with decreased MET phosphorylation further from the ablation margin toward the non-ablated tumor (T). T = non-ablated tumor; *T = ablated tumor; L = normal background liver.

Figure 3.. Effect of MET and EGFR inhibition on heat stress and/or growth factor induced MET and EGFR signaling in HCC cells.

(A) N1S1 and (B) AS30D HCC cells were treated with a dose-titration of the MET inhibitor SU11274 (0.04μM-10μM), EGFR inhibitor erlotinib (0.08μM-20μM) or vehicle control (0.1% DMSO) and 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 mean±SEM of 3 independent experiments. (C) N1S1 and (D) AS30D HCC cells pre-treated with SU11274 (1μM and 10μM), erlotinib (1μM and 10μM) or vehicle control (0.1% DMSO) for 1 hour, heat stressed (45°C) or control (37°C) for 10 minutes, harvested immediately post-heat stress and whole-cell lysates were subjected to Western blotting using phospho-specific and total antibodies against MET and EGFR. β-actin was used as a loading control. (E) N1S1 cells pre-treated for 1 hour with SU11274 (5μM) or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) ± concomitant treatment with recombinant HGF (50ng/ml) or vehicle control for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total MET. β-actin was used as a loading control. (F) AS30D cells pre-treated for 1 hour with erlotinib (5μM) or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) ± concomitant treatment with recombinant EGF (50ng/ml or vehicle control for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total EGFR. β-actin was used as a loading control.

Figure 4.. Effect of MET and EGFR inhibition on HCC clonogenic survival following heat stress.

(A) N1S1 HCC cells were pre-treated with a dose-titration of the MET inhibitor PF-042179030 (0.1μM-10μM) or vehicle control (0.1% DMSO) and (B) AS30D HCC cells were pre-treated with a dose-titration of the EGFR inhibitor erlotinib (0.1μM-10μM) or vehicle control (0.1% DMSO) for 1 hour were subjected to heat stress (45°C) or control (37°C) for 10 minutes and plated for the colony formation assay. Percent colony formation was calculated relative control to 37°C control. Data are presented as mean±SEM of 3 independent experiments and were analyzed using one-way ANOVA followed by post-hoc pairwise comparison using an unpaired t-test.. ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01. White bars = drug only; Grey bars = drug plus heat stress. (C, D) Graphs of drug plus heat stress data only from (A) and (B) for (C) N1S1 and (D) AS30D, respectively.

Figure 5.. Phosphoproteomic pathway analysis of heat stress induced MET signaling.

The protein interaction network was generated from the Ingenuity Pathway Analysis (IPA) (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) [28, 29]. Ingenuity pathway analysis of quantitative phosphoproteomic data identifies numerous signaling pathways associated with heat stress induced MET signaling (green = increased phosphorylation, red = decreased phosphorylation, grey = neutral, yellow = both increases and decreases).

Figure 6.. Heat stress induced PI3K/mTOR dependent AKT signaling is independent of MET signaling.

(A) N1S1 cells pre-treated with a dose-titration of the MET inhibitor PF-042179030 (0.1μM-10μM) or vehicle control (0.1% DMSO) for 1 hour followed by heat stress (45°C) or control (37°C) for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho-MET, AKT and ERK and total MET. β-actin was used as a loading control. (C) N1S1 cells pre-treated with the PI3K/mTOR inhibitor BEZ235 (1μM) or vehicle control (0.1% DMSO) for 1 hour followed by heat stress (45°C) or control (37°C) for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total MET, AKT, PRAS40, 4EBP1, ERK, SAPK/JNK, cJUN, ATF2 and EphA2. Vimentin was used as a loading control. See Supplemental Figure 3 for corresponding quantitative mass spectrometry (LCMS) data.