Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth

Abstract

Although aerobic glycolysis (the Warburg effect) is a hallmark of cancer, key questions, including when, how, and why cancer cells become highly glycolytic, remain less clear. For a largely unknown regulatory mechanism, a rate-limiting glycolytic enzyme pyruvate kinase M2 (PKM2) isoform is exclusively expressed in embryonic, proliferating, and tumor cells, and plays an essential role in tumor metabolism and growth. Because the receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR) signaling cascade is a frequently altered pathway in cancer, we explored its potential role in cancer metabolism. We identified mTOR as a central activator of the Warburg effect by inducing PKM2 and other glycolytic enzymes under normoxic conditions. PKM2 level was augmented in mouse kidney tumors due to deficiency of tuberous sclerosis complex 2 and consequent mTOR activation, and was reduced in human cancer cells by mTOR suppression. mTOR up-regulation of PKM2 expression was through hypoxia-inducible factor 1α (HIF1α)-mediated transcription activation, and c-Myc-heterogeneous nuclear ribonucleoproteins (hnRNPs)-dependent regulation of PKM2 gene splicing. Disruption of PKM2 suppressed oncogenic mTOR-mediated tumorigenesis. Unlike normal cells, mTOR hyperactive cells were more sensitive to inhibition of mTOR or glycolysis. Dual suppression of mTOR and glycolysis synergistically blunted the proliferation and tumor development of mTOR hyperactive cells. Even though aerobic glycolysis is not required for breach of senescence for immortalization and transformation, the frequently deregulated mTOR signaling during multistep oncogenic processes could contribute to the development of the Warburg effect in many cancers. Components of the mTOR/HIF1α/Myc-hnRNPs/PKM2 glycolysis signaling network could be targeted for the treatment of cancer caused by an aberrant RTK/PI3K/AKT/mTOR signaling pathway.

Fig. 1.

Hyperactive mTOR promotes aerobic glycolysis. (A) Cultures of primary Tsc1^+/+, Tsc1^+/−, and Tsc1^−/− MEF isolated from a Tsc1^+/− interbreeding. Tsc1^−/− (B), Tsc2^−/− (C), or Pten^−/− (D) and WT MEFs were treated with or without 5 nM rapamycin (R) for 48 h. The cultured media were collected for the measurement of glucose and lactate. Data represent mean ± SEM. *P < 0.05. RU, relative unit.

Fig. 2.

mTOR is a positive regulator of Pkm2 expression. Total RNA and protein lysates were extracted from WT, Tsc2^−/− (A), or Pten^−/− (B) MEFs treated with or without 10 nM rapamycin (R) for 24 h for RT-qPCR and immunoblotting, respectively. *P < 0.05. (C) Age-matched kidneys from two normal mice and kidney tumors from two Tsc2^del3/+ mice were immunoblotted. (D) Human pancreatic (PANC-1), prostate (PC3), and liver (HepG2) cancer cell lines were treated with or without 10 nM rapamycin for 24 h and then subjected to immunoblotting.

Fig. 3.

Pkm2 is critical for aerobic glycolysis of cells with activated mTOR. Lysates of Tsc2^−/− (A) or Pten^−/− (B) MEFs stably expressing the shRNA for Pkm2 were subjected to immunoblotting, and the conditioned media from the cultures of these MEFs were examined for glucose and lactate. Data represent mean ± SEM. *P < 0.05. RU, relative unit.

Fig. 4.

mTOR stimulates Pkm2 expression through induction of HIF1α. RT-qPCR (A) and immunoblotting (B) analysis for WT and Tsc2^−/− MEFs treated with or without 10 nM rapamycin (R) for 24 h. *P < 0.05. RU, relative unit. (C) Tsc2^−/− MEFs were transfected with shHIF1α or scramble shRNA (shV) in pSilencer2.1 and then subjected to immunoblotting. (D) Schematic representation of the promoter regions of mouse PKM gene. E1 and E2 indicate the location of PKM exons 1 and 2. Dark rectangles indicate predicted HIF1α binding regions; two-way arrows indicate fragment amplified in ChIP real-time PCR analysis. The transcription start site is indicated by an arrow above the gene. PBR, predicted binding region; NBR, nonspecific binding region. (E) Tsc2^−/− MEFs treated with or without 10 nM rapamycin (R) for 24 h. HIF1α antibody-immunoprecipitated DNA was PCR amplified for regions indicated in D. The data are plotted as the ratio of immunoprecipitated DNA subtracting nonspecific binding to IgG vs. total input DNA. Representative data from two independent experiments are shown. Data represent mean ± SEM of replicate real-time PCR. *P < 0.05.

Fig. 5.

mTOR up-regulates Pkm2 expression through the c-Myc–hnRNPs axis. (A) WT and Tsc2^−/− MEFs treated with or without 10 nM rapamycin for 24 h were subjected to immunoblotting. (B) Tsc2^−/− MEFs were transfected with siRNA of c-Myc or scramble siRNA and then subjected to immunoblotting.

Fig. 6.

Inhibition of mTOR, glycolysis, and PKM2 suppresses cell proliferation and tumorigenesis. WT and Tsc2^−/− MEF cells were treated with 3-BrPA (A) and rapamycin (B) for 48 h at the indicated concentration. Cell viability was determined with MTT assay. P < 0.05. (C) Cells were treated with or without 0.5 nM rapamycin (R) for 18 h before the addition of 25 μM 3-BrPA or solvent control for 48 h. Cell viability was detected with MTT assay. Combination index < 1; *P < 0.05. (D) Nude mice bearing xenografted s.c. tumor from PC3 cells were treated with a single drug or the combination of 3-BrPA and rapamycin. The relative tumor sizes were plotted. Data represent mean ± SEM. *P < 0.05. (E) PC3 cells were transduced with the shPKM2 (shPKM2) or scramble shRNA lentiviruses (shV) and then inoculated s.c. into nude mice. (Upper) Immunoblotting. (Lower) Kaplan–Meier survival analysis of the mice with xenografting tumors. P < 0.05. (F) Schematic illustration of the RTK/PI3K/AKT/mTOR pathway regulated glycolysis and tumorigenesis through the HIF1α/c-Myc–hnRNPs/PKM2 network. Active mTOR switches on PKM2 production through up-regulation of HIF1α-mediated transcriptional activation, and the c-Myc–hnRNPs regulated alternative splicing. The consequent activation of glycolysis promotes tumorigenesis.