The HGF-MET axis coordinates liver cancer metabolism and autophagy for chemotherapeutic resistance

Abstract

Notwithstanding the numerous drugs available for liver cancer, emerging evidence suggests that chemotherapeutic resistance is a significant issue. HGF and its receptor MET play critical roles in liver carcinogenesis and metastasis, mainly dependent on the activity of receptor tyrosine kinase. However, for unknown reasons, all HGF-MET kinase activity-targeted drugs have failed or have been suspended in clinical trials thus far. Macroautophagy/autophagy is a protective 'self-eating' process for resisting metabolic stress by recycling obsolete components, whereas the impact of autophagy-mediated reprogrammed metabolism on therapeutic resistance is largely unclear, especially in liver cancer. In the present study, we first observed that HGF stimulus facilitated the Warburg effect and glutaminolysis to promote biogenesis in multiple liver cancer cells. We then identified the pyruvate dehydrogenase complex (PDHC) and GLS/GLS1 as crucial substrates of HGF-activated MET kinase; MET-mediated phosphorylation inhibits PDHC activity but activates GLS to promote cancer cell metabolism and biogenesis. We further found that the key residues of kinase activity in MET (Y1234/1235) also constitute a conserved LC3-interacting region motif (Y1234-Y1235-x-V1237). Therefore, on inhibiting HGF-mediated MET kinase activation, Y1234/1235-dephosphorylated MET induced autophagy to maintain biogenesis for cancer cell survival. Moreover, we verified that Y1234/1235-dephosphorylated MET correlated with autophagy in clinical liver cancer. Finally, a combination of MET inhibitor and autophagy suppressor significantly improved the therapeutic efficiency of liver cancer in vitro and in mice. Together, our findings reveal an HGF-MET axis-coordinated functional interaction between tyrosine kinase signaling and autophagy, and establish a MET-autophagy double-targeted strategy to overcome chemotherapeutic resistance in liver cancer. Abbreviations: ALDO: aldolase, fructose-bisphosphate; CQ: chloroquine; DLAT/PDCE2: dihydrolipoamide S-acetyltransferase; EMT: epithelial-mesenchymal transition; ENO: enolase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLS/GLS1: glutaminase; GLUL/GS: glutamine-ammonia ligase; GPI/PGI: glucose-6-phosphate isomerase; HCC: hepatocellular carcinoma; HGF: hepatocyte growth factor; HK: hexokinase; LDH: lactate dehydrogenase; LIHC: liver hepatocellular carcinoma; LIR: LC3-interacting region; PDH: pyruvate dehydrogenase; PDHA1: pyruvate dehydrogenase E1 alpha 1 subunit; PDHX: pyruvate dehydrogenase complex component X; PFK: phosphofructokinase; PK: pyruvate kinase; RTK: receptor tyrosine kinase; TCGA: The Cancer Genome Atlas.

Keywords: Biogenesis; Warburg effect; combined treatment; glutaminolysis; targeted therapy.

Figure 1.

HGF facilitates liver cancer metabolism and biogenesis. (a) HGF accelerates acidification of liver cancer cell culture medium. After starvation overnight, HepG2, SMMC-7721, Huh-7, MHCC-97H, Hepa1-6 and H22 liver cancer cell lines (5 × 10⁴) were individually treated with or without HGF (40 ng/ml) for 48 h, then used for photographs of the culture medium. The color of the medium indicated the degree of acidification. Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; RPMI-1640, Roswell Park Memorial Institute 1640 medium. (b) Impact of HGF on the Warburg effect in liver cancer cell lines. After starvation overnight, HepG2, SMMC-7721, Huh-7, MHCC-97H, Hepa1-6 and H22 cells (5 × 10⁴) were individually stimulated with or without HGF (40 ng/ml) for 6 h, and subsequently subjected to analysis of glucose consumption and lactate production. (c) Effect of HGF on glutaminolysis in liver cancer cell lines. After starvation overnight, HepG2, SMMC-7721, Huh-7, MHCC-97H, Hepa1-6 and H22 cells (5 × 10⁴) were individually stimulated with or without HGF (40 ng/ml) for 6 h, and subsequently subjected to analysis of glutamine consumption and glutamate production. (d) Impact of HGF on biogenesis in liver cancer cell lines. After starvation overnight, HepG2, SMMC-7721, Huh-7, MHCC-97H, Hepa1-6 and H22 cells (5 × 10⁴) were individually stimulated with or without HGF (40 ng/ml) for 24 h, and subsequently subjected to analysis of nucleotide (DNA), lipid (triglyceride) and amino acid (aspartate) synthesis. Data are presented as the means ± SD from at least 3 independent experiments. Statistically significant differences with two-tailed Student’s t-test are marked as * (p < 0.05) or ** (p < 0.01). Abbreviations: HGF, hepatocyte growth factor.

Figure 2.

HGF-MET signaling promotes liver cancer metabolism and biogenesis via PDHC and GLS. (a) Screening for critical enzymes under HGF-MET regulation in cancer metabolism. After starvation overnight, HepG2-derived CRISPR-Cas9 system-mediated vehicle control (MET WT) or MET knockout (MET KO) cells (5 × 10⁴) were treated with or without HGF (40 ng/ml) for 2 h, and subsequently subjected to activity analysis for the indicated enzymes. (b) Identification for interaction targets of MET from important enzymes and transporters in cancer metabolism. HepG2 cell lysates (5 × 10⁵) were subjected to co-immunoprecipitation with anti-MET antibody, and then analyzed by western blot with the indicated antibodies. (c and d) Effect of MET on PDHC and GLS activity in liver cancer cell lines. SMMC-7721, Huh-7, MHCC-97H, Hepa1-6 and H22 cells (2 × 10⁴) were individually transfected with siRNAs to knock down MET (siMET) or not (siCtrl). Seventy-two h after transfection, cells were starved overnight, then treated with or without HGF (40 ng/ml) for 2 h, and subsequently subjected to analyze activity of PDHC (c) and GLS (d). (e) Contribution of PDHC and GLS to the HGF-MET signaling-mediated Warburg effect. After pre-incubation with PDHC inhibitor (CPI-613, 100 μM) or GLS inhibitor (BPTES, 100 nM) overnight under starvation, HepG2 MET WT or KO cells (5 × 10⁴) were treated with or without HGF (40 ng/ml) for 6 h, and subsequently subjected to analysis of glucose consumption and lactate production. (f) Contribution of PDHC and GLS to HGF-MET signaling-mediated glutaminolysis. HepG2 MET WT or KO cells (5 × 10⁴) were treated as mentioned above, and subsequently subjected to analysis of glutamine consumption and glutamate production. (g) Contribution of PDHC and GLS to HGF-MET signaling-mediated biogenesis. After pre- and co-incubation with PDHC inhibitor (CPI-613, 100 μM) and GLS inhibitor (BPTES, 100 nM) overnight under starvation, HepG2 MET WT or KO cells (5 × 10⁴) were treated with or without HGF (40 ng/ml) for 24 h, and subsequently subjected to analysis of DNA, triglyceride and aspartate contents. Data are presented as the means ± SD from at least 3 independent experiments. Statistically significant differences with two-tailed Student’s t-test are marked as * (p < 0.05) or ** (p < 0.01). Abbreviations: WT, wild type; KO, knockout; PDHC, pyruvate dehydrogenase complex; GLS, glutaminase; PDHi, PDHC inhibitor; GLSi, GLS inhibitor.

Figure 3.

MET phosphorylates PDHC and GLS in vivo and in vitro. (a) Stimulation of HGF-MET signaling on phosphorylation of PDHC and GLS in vivo. WT and MET KO HepG2 cells (5 × 10⁵) were individually treated with or without HGF (40 ng/ml) for 2 h, then subjected to immunoprecipitation with anti-PDHA1, anti-DLAT/PDCE2, anti-PDHX and anti-GLS antibodies, and subsequently analyzed by western blot using anti-phospho-tyrosine or the other indicated antobodies. (b–e) Direct phosphorylation effects of MET on PDHC and GLS in vitro. Recombinant HIS-PDHA1 (1 μg) (b), HIS-DLAT/PDCE2 (1 μg) (c), HIS-PDHX (1 μg) (d) or HIS-GLS (1 μg) (e) were individually incubated with FLAG-MET (400 ng) in kinase reaction buffer at 37°C for 45 min, with or without ATP (10 mM) and MET kinase inhibitor (JNJ-38877605, 50 nM). After the reaction, the mixtures were subjected to western blot analysis with anti-phospho-tyrosine or the other indicated antobodies. Abbreviations: PDHA1, pyruvate dehydrogenase E1 alpha 1 subunit; DLAT/PDCE2, dihydrolipoamide S-acetyltransferase; PDHX, pyruvate dehydrogenase complex component X; GLS, glutaminase.

Figure 4.

An atypical cancer biogenesis is actuated in HGF-MET-targeted drug-resistant cells. (a) Analysis of MET kinase activity in MET-targeted inhibitor- or antibody-insensitive cells. After 3 rounds of selection with the MET-specific small molecule inhibitor JNJ-38877605 (50 nM) and anti-MET monoclonal antibody 5D5 (40 μg/ml), MET-targeted inhibitor- or antibody-insensitive HepG2 cells (5 × 10⁴) were harvested for western blot with the indicated antibodies. WT and MET KO HepG2 cells were used as controls. (b) Assessment of the Warburg effect and glutaminolysis in MET-targeted inhibitor- or antibody-insensitive cells. MET-targeted inhibitor- or antibody-insensitive HepG2 cells (5 × 10⁴) were subjected to analysis of glucose consumption, lactate production, glutamine consumption and glutamate production. WT and MET KO HepG2 cells were used as controls. (c) Assessment of nucleotide, lipid and amino acid synthesis in MET-targeted inhibitor- or antibody-insensitive cells. MET-targeted inhibitor- or antibody-insensitive HepG2 cells (5 × 10⁴) were subjected to analysis of DNA, triglyceride and aspartate contents. WT and MET KO HepG2 cells were used as controls. (d) Contribution of glucose and glutamine to biogenesis in HGF-MET-targeted drug-insensitive cells. After being pre-cultured in normal medium or medium without glucose and glutamine for 12 h, HepG2 MET WT or KO and MET-targeted inhibitor- or antibody-insensitive cells (5 × 10⁴) were individually subjected to analysis of DNA, triglyceride and aspartate contents. Data are presented as the means ± SD from at least 3 independent experiments. Statistically significant differences with two-tailed Student’s t-test are marked as * (p < 0.05) or ** (p < 0.01). Abbreviations: p-Y, p-MET Y; Y1234/5, Y1234/1235; In-insen, inhibitor-insensative; Ab-insen, antibody-insensitive; Ctrl, normal medium; No Glu&Gln, both glucose- and glutamine-deleted medium.

Figure 5.

HGF-MET-targeted drugs induce autophagy to reset biogenesis in liver cancer. (a) Assessment of autophagy state in MET-targeted inhibitor- or antibody-insensitive cells. Cell lysates from MET-targeted inhibitor- or antibody-insensitive HepG2 cells (5 × 10⁴) were analyzed by western blot with the indicated antibodies. WT and MET KO HepG2 cells were used as controls. Quantification of LC3 turnover and SQSTM1 degradation are shown on the right. (b) Contribution of autophagy to HGF-MET-targeted drugs-reprogrammed biogenesis. WT and ATG5 KD HepG2 cells (5 × 10⁴) were individually treated with vehicle control, inhibitor of MET (JNJ-38877605, 50 nM) or anti-MET antibody (5D5, 40 μg/ml) for 24 h, and subsequently subjected to analysis of DNA, triglyceride and aspartate contents. (c) Impact of MET-targeted inhibitor or antibody on LC3 turnover in liver cancer cell lines. HepG2, SMMC-7721, Huh-7, MHCC-97H, Hepa1-6 and H22 cells (5 × 10⁴) were individually treated with vehicle control, an inhibitor of MET (JNJ-38877605, 50 nM) or anti-MET antibody (5D5, 40 μg/ml) for 8 h, and subsequently subjected to western blot analysis with the indicated antibodies. (d) Effect of MET-targeted inhibitor or antibody on aggregation of GFP-LC3 puncta. HepG2 cells stably expressing GFP-LC3 (1 × 10⁴) were individually treated with vehicle control, inhibitor of MET (JNJ-38877605, 50 nM) or anti-MET antibody (5D5, 40 μg/ml) for 6 h. Representative images and quantification of MET-targeted inhibitor or antibody-induced GFP-LC3 punctate cells were shown as indicated by confocal fluorescence microscopy. (e) Effect of MET inhibition on autophagic flux. After pre-incubation with or without BAFA1 (50 nM) for 2 h, HepG2, SMMC-7721 and Huh-7 cells (5 × 10⁴) were individually treated with inhibitor of MET (JNJ-38877605, 50 nM) for 6 h or not treated, and subsequently subjected to western blot analysis with the indicated antibodies. (f) Analysis of autophagic flux in MET-targeted inhibitor- or antibody-insensitive cells. MET-targeted inhibitor- or antibody-insensitive cells (5 × 10⁴) were individually treated with or without BAFA1 (50 nM) for 4 h, and subsequently subjected to western blot analysis with the indicated antibodies. Data are presented as the means ± SD from at least 3 independent experiments. Statistically significant differences with two-tailed Student’s t-test are marked as * (p < 0.05) or ** (p < 0.01). Abbreviations: MET-in, MET-targeted inhibitor; MET-ab, MET-targeted antibody; ATG5, autophagy related 5; KD, knockdown; BAFA1, bafilomycin A₁; R.U., relative units.

Figure 6.

MET conceals a conserved LIR motif in Y1234/1235 to recruit LC3 and drive autophagy under kinase inhibition. (a) Schema of potential LIR motifs hidden in key tyrosine-phosphorylated modification sites of the MET kinase center. Underlined and capital highlighted letters indicate LIR constitution in MET. (b and c) Endogenous immunoprecipitation assay between MET and LC3. Cell lysates of HepG2, SMMC-7721 and Huh-7 cells (5 × 10⁵) were individually immunoprecipitated with anti-MET antobody (b) or anti-LC3 antibody (c), and subsequently subjected to western blot analysis with the indicated antibodies. (d) Construction of MET dephosphorylated mutants. Schematic diagrams of MET and its dephosphorylated mutants are depicted as indicated. (e) Identification of Y1234/1235 as key points for MET-LC3 interaction. HepG2 MET KO cells (5 × 10⁵) were individually co-transfected with vehicle control (Flag), or plasmids encoding WT or dephosphorylated mutants of MET and GFP-LC3. 24 h after transfection, cell lysates were subjected to immunoprecipitation and western blot analysis with the indicated antibodies. (f) Conservative analysis of Y1234, Y1235 and V1237-constituted LIR motif (YYxV) in MET. Alignment of amino acid sequences in evolutionarily different species is shown as indicated, and the YYxV motif is highlighted with red and green letters. (g) Effects of MET dephosphorylated mutants on autophagy. HepG2 MET KO cells (5 × 10⁴) were individually transfected with vehicle control, or plasmids encoding WT or dephosphorylated mutants of MET. Twenty-four h after transfection, cell lysates were analyzed by western blot with the indicated antibodies. Abbreviations: LIR, LC3-interacting region; WT, wild-type; Y, tyrosine residue; Y1234/5F, both Y1234 and Y1235 mutated to F; Y-full-F, all Y sites mutated to F; V, valine residues.

Figure 7.

MTOR signaling is invovled in MET inhibition-induced autophagy. (a) Inhibitory effect of rapamycin on MET inhibition-induced autophagy. After pre-incubation with or without rapamycin (50 nM) for 4 h, HepG2 cells (5 × 10⁴) were individually treated with an inhibitor of MET (JNJ-38877605, 50 nM) or vehicle control (DMSO) for 8 h, and subsequently subjected to western blot analysis with the indicated antibodies. (b) Rescue effect of MTOR activation on MET inhibition-induced autophagy. HepG2 cells (5 × 10⁴) were individually transfected with or without a plasmid encoding the constitutively active RRAGB^Q99L mutant (RRAGB^GTP) for 48 h, and then treated with inhibitor of MET or vehicle control as above. Cell lysates were subjected to immunoblot with the indicated antibodies. (c) Impact of the HGF-MET axis on MTOR signaling. After stavation overnight, HepG2 MET WT or KO cells (5 × 10⁴) were individually treated with or without HGF (40 ng/ml) for 2 h, and subsequently subjected to western blot analysis with the indicated antibodies. (d) Contribution of the HGF-MET axis to redox homeostasis through PDHC and GLS. HepG2 MET WT or KO cells (5 × 10⁴) were individually transfected with GLS-specific siRNA (siGLS), PDHC-specific siRNA (siPDHA1) or non-targeting siRNA control (siCtrl). Seventy-two h after transfection, cells were starved overnight and treated with or without HGF (40 ng/ml) for 6 h, and then subjected to analysis of GSSG:GSH, NADP:NADPH and NAD:NADH ratios. Data are presented as the means ± SD from at least 3 independent experiments. Statistically significant differences with two-tailed Student’s t-test are marked as * (p < 0.05) or ** (p < 0.01). Abbreviations: GSSG:GSH, oxidized glutathione:reduced glutathione; NADP:NADPH, nicotinamide adenine dinucleotide phosphate:reduced NADP; NAD:NADH, nicotinamide adenine dinucleotide:reduced NAD.

Figure 8.

Y1234/1235 unphosphorylated MET correlates with autophagy in clinical liver cancer patients. (a) Clinical liver tissue microarray assay for correlation between Y1234/1235 unphosphorylated MET and the autophagic marker SQSTM1. A total of 208 cases of clinical liver tissue samples, including normal liver tissue (8 cases), cancer adjacent normal liver tissue (8 cases), hepatocellular carcinoma (152 cases), bile duct adenocarcinoma (12 cases) and metastatic adenocarcinoma (28 cases), were analyzed by immunohistochemistry (IHC) with the indicated antibodies and H&E staining. Full scans of tissue microarray are shown as indicated. (b) Representative images of Y1234/1235 unphosphorylated MET and SQSTM1 staining under high power field (HPF) from clinical liver tissue microarray. Scale bar: 100 µm. (c) Qualitative analysis for expression pattern and stained grade of Y1234/1235 unphosphorylated MET and SQSTM1 in clinical liver tissue microarray. Statistics in cancer, normal tissue or total samples were individually presented as indicated. Abbreviations: H&E staining, hematoxylin-eosin staining; ‘-’, negative; ‘+’, positive or weak; ‘++’, moderate; ‘+++’, strong.

Figure 9.

Autophagy blockage improves HGF-MET-targeted drug efficiency in liver cancer. (a) Synergistic effect of CQ and MET-targeted drugs on proliferation capacity of liver cancer cells. HepG2 cells (1 × 10⁴) were individually treated with vehicle control, an inhibitor of MET (JNJ-38877605, 50 nM), anti-MET antibody (5D5, 40 μg/ml) or/and CQ (20 μM), and subsequently subjected to analysis of cell proliferation capacity at the indicated times. (b) Synergistic effect of CQ and MET-targeted drugs on viability of liver cancer cells. HepG2 cells (2.5 × 10³) were individually treated with vehicle control, an inhibitor of MET (JNJ-38877605, 50 nM), anti-MET antibody (5D5, 40 μg/ml) or/and CQ (20 μM) for 8 h, and then subjected to analysis of cell viability. (c and d) Synergistic effect of CQ and MET-targeted drugs on colony-formation capacity of liver cancer cells. HepG2 cells (0.5 × 10³) were individually treated with a single dose of vehicle control, an inhibitor of MET (JNJ-38877605, 50 nM), anti-MET antibody (5D5, 40 μg/ml) or/and CQ (20 μM) for 2 wk, and subsequently subjected to analysis of colony-formation capacity of cells (c). Representative images of clones are shown as indicated (d). (e) Synergistic impact of CQ and MET-targeted drugs on biogenesis of liver cancer cells. HepG2 cells (5 × 10⁴) were individually treated with vehicle control, an inhibitor of MET (JNJ-38877605, 50 nM), anti-MET antibody (5D5, 40 μg/ml) or/and CQ (20 μM) for 24 h, and then subjected to analysis of DNA, triglyceride and aspartate contents. Data are presented as the means ± SD from at least 3 independent experiments. (f and g) Synergistic effect of autophagy blockage and HGF-MET inhibition on tumor growth in xenograft-bearing mice. SMMC-7721 and Huh-7 cells (1 × 10⁶) were individually inoculated subcutaneously into the flanks of athymic nu/nu mice. Once palpable, the tumors were individually treated with PBS (vehicle control, 100 μl), MET inhibitor (20 mg/kg in 100 μl of PBS) or/and the autophagic blocker CQ (10 mg/kg in 100 μl of PBS) every 5 d. Tumor growth was recorded every 5 d during treatment, and reported as the mean tumor surface size ± SD with n = 10 animals per group (f). Tumor weight was detected after sacrifice and shown on the right (g). (h) Synergistic impact of autophagy blockage and HGF-MET inhibition on cancer biogenesis in xenograft tumors. Individual samples (n = 3) from each treated group in SMMC-7721 and Huh-7 xenograft tumors were randomly selected and subjected to analysis of DNA, triglyceride and aspartate contents. Statistically significant differences with two-tailed Student’s t-test are marked as * (p < 0.05) or ** (p < 0.01). Abbreviations: MET-in, MET inhibitor; MET-ab, MET antibody; CQ, chloroquine.

Figure 10.

Schematic diagram for HGF-MET-coordinated metabolic modes in liver cancer therapeutic resistance. HGF activates MET to facilitate the Warburg effect and glutaminolysis for liver cancer biogenesis and growth. Upon treatment with HGF-MET-targeted drugs, dephosphorylated MET promotes autophagy to maintain biogenesis homeostasis for survival. Double inhibition of HGF-MET and autophagy overcomes liver cancer therapeutic resistance by disrupting biogenesis.