Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth
Materials and Methods
For descriptions of reagents, cell culture, cell proliferation assay, immunoblotting, measurements of glucose and lactate, siRNA gene knockdown, generation of stable gene knockdown cell lines, mRNA expression profiling analysis, RT-qPCR, real-time PCR analysis of ChIP DNA, mouse kidney tumor assessment, xenografting tumorigenesis and treatment, and statistics, see SI Materials and Methods.
Author contributions: Q.S., X.C., H. You, and H.Z. designed research; Q.S., X.C., J.M., H.P., F.W., X.Z., Y.W., Y.J., H. Yang, R.C., L.C., Y.Z., and J.G. performed research; H.O., T.C., H. You, and D.K. contributed new reagents/analytic tools; Q.S., X.C., M.-R.W., Y.L., H. You, D.K., and H.Z. analyzed data; and Q.S., X.C., H. You, and H.Z. wrote the paper.
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.
Unlike in normal cells, glycolysis is induced by hypoxia, and cancer cells preferentially metabolize glucose by glycolysis, even in an aerobic environment (1–3). Increased glucose consumption and an elevated rate of lactate production by cancer cells are characteristics of glycolysis, first described by Otto Warburg in the 1920s and thereafter known as the Warburg effect (4). Because this altered metabolism can occur even in the presence of oxygen, glycolysis presumably confers a selective advantage for the survival and proliferation of cancer cells. This catabolic process is, however, inefficient for energy production in that it generates only 2 mol of ATP, instead of an additional 36 mol through the tricarboxylic acid (TCA) cycle, in the presence of oxygen by using 1 mol of glucose (2, 3, 5).
Although the Warburg effect is a well-recognized hallmark of cancer metabolism, its regulatory mechanism is still largely obscure. Critical issues, including how and when cancer cells acquire this highly glycolytic phenotype, and its causal relationship with cancer progression, remain to be addressed. In addition, a cellular adaptation model could not explain the constitutively high rate of glycolysis in cancer cells in tissue culture conditions with 20% oxygen in vitro or in pseudohypoxic tumors in vivo. Nevertheless, changes of rate-limiting glycolytic enzymes are observed during tumor formation. Among these enzymes is pyruvate kinase, which catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate and yields one molecule of ATP independent of oxygen supply during glycolysis (6). There are four pyruvate kinase isoenzymes: PKL and PKR are encoded by the PKLR gene but under the control of different promoters; and PKM1 and PKM2 are two different splicing forms of the same mRNA transcribed from the PKM gene. The ratio of PKM1 to PKM2 is dictated by heterogeneous nuclear ribonucleoproteins (hnRNPs) (7, 8). PKL, PKR, and PKM1 are tissue-specific isoenzymes, whereas PKM2 is considered an embryonic and cancer cell-specific isoform. By still-unknown regulatory mechanisms, PKM2 is gradually replaced by the respective tissue-specific isoenzyme during embryogenesis. By contrast, tissue-specific isoenzymes are switched to PKM2 during tumorigenesis, and PKM2 plays a critical role in tumor metabolism and growth (6, 9). Because PKM2 is an inactive form of pyruvate kinase for the last step of glycolysis, the buildup of phosphoenolpyruvate is then shunted to an alternative glycolytic pathway for anabolic synthesis and cell growth (10).
The receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR) signaling pathway plays a crucial role in regulating cell growth, survival, and metabolism (11, 12). Various alterations of the proto-oncogenes and tumor suppressors along this pathway mark this network as one of the most frequently dysregulated signaling cascades in cancers (13–16). A major activated effector of this pathway is mTOR, a serine/threonine protein kinase. mTOR integrates a broad spectrum of input, ranging from growth factor signaling to cellular nutrient status to energy supply, for regulation of protein synthesis and cell growth (14, 17). Even though the downstream events of oncogenic mTOR leading to cancer development are still largely unknown, among numerous mTOR effectors, the proto-oncogene Myc family and hypoxia-inducible factors (HIFs) are often activated in various cancers and have been considered an “axis of evil” in cancer development (5, 18). Although the function of normal c-Myc is inhibited by physiological HIF1α signaling, oncogenic Myc and HIFs collaborate with each other (though the molecular detail of this interaction is less certain) to confer metabolic advantages to cancer cells by induction of the Warburg effect through transcriptional activation of glycolytic enzymes (19).
We initially noticed prominent glycolysis in cells with constitutively active mTOR while characterizing primary and immortalized tuberous sclerosis 1 (Tsc1)- or tuberous sclerosis 2 (Tsc2)-null MEFs. Subsequently we found that mTOR was a major positive regulator of the Warburg effect, not only in cancer cells but also, surprisingly, in benign tumor cells and even in premature senescent primary cells with activated mTOR signaling, under normoxic conditions. We then identified PKM2 as a critical glycolytic enzyme in oncogenic mTOR-induced Warburg effect. Next, we determined that HIF1α and c-Myc–hnRNPs cascades were the transducers of mTOR regulation of PKM2. Moreover, disruption of mTOR signaling, PKM2, and/or glycolysis suppressed the cell proliferation and tumorigenesis caused by oncogenic mTOR signaling. We suggest that PKM2-stimulated glycolysis contributes to the development of tumors caused by hyperactive mTOR, and therefore this cascade may be targeted for the treatment of these cancers.Click here to view.
This study was supported by National Natural Science Foundation of China Grant 30788004, the National Basic Research Program of China 973 Program Grants 2009CB522202 and 2009CB522203, and Ministry of Education of China 111 Project B08007.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1014769108/-/DCSupplemental.
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