M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth - PubMed (original) (raw)

M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth

Marta Cortés-Cros et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Many cancer cells have increased rates of aerobic glycolysis, a phenomenon termed the Warburg effect. In addition, in tumors there is a predominance of expression of the M2 isoform of pyruvate kinase (PKM2). M2 expression was previously shown to be necessary for aerobic glycolysis and to provide a growth advantage to tumors. We report that knockdown of pyruvate kinase in tumor cells leads to a decrease in the levels of pyruvate kinase activity and an increase in the pyruvate kinase substrate phosphoenolpyruvate. However, lactate production from glucose, although reduced, was not fully inhibited. Furthermore, we are unique in reporting increased serine and glycine biosynthesis from both glucose and glutamine following pyruvate kinase knockdown. Although pyruvate kinase knockdown results in modest impairment of proliferation in vitro, in vivo growth of established xenograft tumors is unaffected by PKM2 absence. Our findings indicate that PKM2 is dispensable for tumor maintenance and growth in vivo, suggesting that other metabolic pathways bypass its function.

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Conflict of interest statement

Conflict of interest statement: All of the authors are employees and shareholders of Novartis Pharma AG (Switzerland) or Novartis Institutes for BioMedical Research, Inc.

Figures

Fig. 1.

Fig. 1.

Knockdown of PKM2 leads to decreased PK activity, accumulation of phosphoenolpyruvate, decreased glycolytic rates, and increased oxygen consumption in HCT116 cancer cells. (A) Schematic representation of PKM1 and PKM2 with the alternatively spliced exons 9 and 10. The location of the shRNAs is specified by a star. sh566 and sh1021 knockdown PKM1 and PKM2. sh1493 is specific to PKM1 and sh1408 and sh1411 are specific for PKM2. (B) qRT-PCR quantification of the levels of PKM2 (Upper) and PKM1 (Lower) mRNA 7 d after shRNA knockdown. Specificity of the shRNAs is shown at the top of the panel. sh566 and sh1021 knock down both PKM1 and PKM2, sh1493 only PKM1, and sh1408 and sh1411 PKM2. (C) Immunoblotting shows shRNA-mediated stable knockdown of endogenous PKM2 protein 4 and 7 d after infection. (D) PK catalytic activity from cell lysates generated from HCT116 cells expressing the different shRNAs targeting PK. Asterisks indicate P = 0.0022 (sh1411 vs. shAlk), P < 0.0001 (sh1408 vs. shNSQ), and P = 0.0023 (sh566 vs. sh1408). (E) PEP levels from cellular lysates. PEP levels are obtained by IC-MS/MS and values depicted in an arbitrary unit that reflects the integrated peak area of a MS signal. Data were normalized by total protein content in the cells. Error bars represent SD of the mean from triplicate. Asterisks indicate P = 0.0392 (sh566 vs. sh1411), P = 0.0029 (sh1021 vs. sh1408), P = 0.0021 (sh1021 vs. sh1411), and P = 0.0192 (sh1408 vs. shAlk).

Fig. 2.

Fig. 2.

Decreased glucose uptake and lactate production and accumulation of metabolic intermediates in HCT116 colon carcinoma cells upon PKM2 knockdown. (A, Left) Immunoblotting shows doxycycline- (Dox) inducible, shRNA-mediated knockdown of endogenous PKM2 protein in HCT116 cells 7 d after infection with lentivirus containing shRNAs as indicated. (Right) PK catalytic activity from cell lysates from same experiment. Closed bars, no doxycycline, open bars, with doxycycline. (B) Consumed 13C6-glucose (mM, Left) and released 13C3-lactate (mM, Right) in media from PKM1/2 knockdown HCT116 cultures incubated with 13C6-glucose for 8 h, as determined by NMR. Approximately 6% of glucose in the naive media was unlabeled. This value remained constant for all samples at the 8-h time point. (C) Intracellular metabolites derived from 13C6-glucose; (Left) 13C3-PEP levels obtained by IC-MS/MS and values depicted in an arbitrary units. 13C3-alanine (Center), and 13C3-serine (Right); values were determined by LC-MS/MS and are expressed as a percent of the total cellular pool for each metabolite. (D) Consumed 13C5-glutamine (mM, Left) and released 13C3-lactate (mM, Right) in media from PKM1/2 knockdown HCT116 cultures incubated with 13C5-glutamine for 8 h as determined by NMR. (E) 13C3-serine as percent of the total cellular serine pool from cellular lysates of cells cultured for 8 h with 13C5-glutamine. Approximetly 8% of glutamine in the naive media was unlabeled. Measurements were made by NMR (media) or LC-MS/MS (cell lysates). Error bars represent SE of the mean from triplicate wells. P values depicted. Closed bars indicate cells without doxycycline, and open bars cells treated with doxycycline in all graphs.

Fig. 3.

Fig. 3.

Knockdown of PKM2 leads to impaired in vitro cellular proliferation in HCT116 colon carcinoma cells but no effect on in vivo tumor xenograft growth. (A) Clonogenic survival assays of HCT116 and (B) monolayer proliferation of HCT116 cells upon knockdown of PKM1 and PKM2. Proliferation was measured at 4 and 7 d after lentiviral infection and selection. Error bars represent SD of the mean from octoplicate wells. (C) PEP levels from tumor extracts from D. Each point represents a separate animal. Asterisks indicate P = 0.0057 (WT veh vs. sh1021 Dox), P = 0.0325 (WT Dox vs. sh1021 Dox), and P = 0.0317 (WT Dox vs. sh1408 Dox). (D) Immunoblotting revealing PKM2 levels in HCT116 tumor xenografts harvested from mice at 28–32 d postimplantation. (E) PK catalytic activity from HCT116 tumor xenografts expressing the different shRNAs targeting PK. (F) Mean tumor volume (TVol) from HCT116 xenografts expressing shRNAs to PKM1/2 as indicated. Xenografts were allowed to grow for 14 d, then dosed with doxycycline or vehicle daily for 14 d. Each point represents the mean tumor volume of eight mice per group ± SEM.

Fig. 4.

Fig. 4.

Knockdown of PKM1/2 leads to impaired in vitro cellular proliferation in RKO colon carcinoma cells but no effect on in vivo tumor xenograft growth in nude mice. (A) Immunoblotting shows doxycycline-inducible, shRNA-mediated knockdown of endogenous PKM2 protein in RKO cells at 4 and 7 d after infection with lentivirus containing shRNAs. (B) Cellular proliferation and (C) colony formation capacity of the RKO cells upon doxycycline induction. Proliferation was measured at 4 and 7 d after lentiviral infection as described above. Error bars represent SD of the mean from octoplicate wells. (D) Immunoblotting revealing PKM2 levels in RKO tumor xenografts harvested from mice dosed with doxycycline or vehicle daily for 14 d. PKM2 is the band indicated by the arrow. Each lane is a separate animal. (E) PEP levels from tumor extracts from D. Each point represents a separate animal. *P = 0.0008 (sh1021 Veh vs. sh1021 Dox). (F) Mean tumor volume (TVol) from RKO xenografts expressing shRNAs to PKM1/2 as indicated. Each point represents the mean tumor volume of eight mice per group ± SEM.

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