Inhibition of mTORC1 signaling sensitizes hepatocellular carcinoma cells to glycolytic stress - PubMed (original) (raw)

. 2016 Oct 1;6(10):2289-2298.

eCollection 2016.

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Inhibition of mTORC1 signaling sensitizes hepatocellular carcinoma cells to glycolytic stress

Xin Zhao et al. Am J Cancer Res. 2016.

Abstract

Reprogrammed glucose metabolism, especially glycolysis, is profoundly implicated in tumor development or metastasis. As the interconnectedness and flexibility of metabolic signaling, targeting a metabolic signaling molecule may have limited anti-tumor effects. Here, Gene set enrichment analysis (GSEA) was used to explore the accompanied effectors of glycolysis in hepatocellular carcinoma (HCC). Based on the expression of lactate dehydrogenase A (LDHA), a key enzyme in catalyzing pyruvate into lactate, the glycolytic ability of HCC was defined as low group and high group. GSEA of two independent GEO datasets showed that mTORC1 signaling was the most striking metabolic alternations in high group. Pharmacological inhibition of mTORC1 signaling with rapamycin decreased LDHA level and glycolytic capacity of six HCC cell lines. Furthermore, c-Myc was identified as a downstream target of mTORC1 signaling and mediated mTORC1-induced LDHA expression. Importantly, rapamycin sensitized HCC cells to the glycolysis inhibitor 2-deoxyglucose (2-DG) in vitro and in vivo. Meanwhile, genetic silencing several other downstream targets of mTORC1 signaling (TFEB, SREBP-1 and SKAR) failed to enhance or faintly influenced the cytotoxic effects of 2-DG. These results demonstrate that combining rapamycin with 2-DG holds significant promise as prospective clinical treatment in HCC.

Keywords: glycolysis; hepatocellular carcinoma; lactate dehydrogenase a; mTOR signaling.

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Figures

Figure 1

Figure 1

mTORC1 signaling is a key modulator of glycolysis in HCC. GSEA plot of mTORC1 signaling based on the gene expression profiles of low LDHA group versus high LDHA group in GSE6764 (A) and GSE14520 (B). NES, normalized enrichment score. (C) Six HCC cells were treated with 100 nM rapamycin or not for 12 h, then lysed and immunoblotted with the indicated antibodies. (D) The mRNA level of LDHA was detected by real-time qPCR in HCC cell lines upon rapamycin treatment. (E) The glycolytic capacity of HCC cells upon rapamycin treatment was measured by Seahorse XF Analyzers. Data shown represents the mean ± SD. *, P < 0.05; **, P < 0.01.

Figure 2

Figure 2

mTORC1 signaling regulates glycolysis through targeting c-Myc/LDHA axis. A. Gene set enrichment analysis of the two HCC dataset based on a signature of MYC target genes and LDHA expression. B. Correlation between c-Myc and LDHA was analyzed in twenty-four HCC tissues. C. Cells were cultured for further 48 h after transfection with LDHA specific siRNAs, then lysed and probed by western blotting with indicated antibodies. D. Cells were cultured for 24 h in the presence of 100 nM rapamycin, then lysed and probed by western blotting with indicated antibodies. E. Hypothesis of dampening glycolysis by combined treatment.

Figure 3

Figure 3

Rapamycin or LDHA knockdown sensitizes HCC cells to 2-deoxyglucose. (A, B) Human HCC cells were treated with 2 mM of 2-DG, 100 nM rapamycin or combined, for 48 h followed by cell viability (A) and caspase-3/7 activity (B) analysis. (C, D) The si-Ctrl or si-LDHA HCC cells were treated with 2 mM of 2-DG for 48 h followed by cell viability (C) and caspase-3/7 activity (D) analysis. Data shown represents the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4

Figure 4

Combined treatment of rapamycin and 2-DG inhibits liver tumorigenesis. A. Tumor growth curve of HepG2 cells upon indicated treatment. B. At the endpoint of animal experiment, mice were sacrificed and tumor weight was calculated. C. Immunohistochemical analysis of Ki67 in indicated treatment group. The mean positive staining cells were calculated with six random fields; Scale bar: 50 μm. *, P < 0.05; **, P < 0.01.

Figure 5

Figure 5

Silencing of TFEB, SREBP1 and SKAR fails to render HCC cells sensitive to 2-deoxyglucose. A. Effects of rapamycin on TFEB, SREBP1 and SKAR mRNA expression in LM3, Huh7 and HepG2 cells. B. Cells were cultured for further 48 h after transfection with indicated specific siRNAs, then lysed and probed by western blotting with indicated antibodies. C. The indicated HCC cells were treated with 2 mM of 2-DG for 48 h followed by cell viability analysis. Data shown represents the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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