The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice ...with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.
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•PKM2 is not required by all tumor cells•The need for pyruvate kinase is most apparent in nonproliferating tumor cells•PKM2 expression is variable in human cancers•Recurrent point mutations disrupting pyruvate kinase are found in human cancers
Loss of PKM2, an isoform of pyruvate kinase that was previously associated with a number of cancers, is in fact found to exacerbate tumor growth. Proliferating tumor cells do not seem to require M1 isoform either, suggesting that it is the inactive state of PKM2 that these cells seem to prefer.
Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the ...electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.
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•Use of 2H stable isotopes to trace NAD(P)H metabolism in cells•Quantification of pentose phosphate pathway contribution to cytosolic NADPH•Reporter system to distinguish cytosolic and mitochondrial NADPH•Resolve direction of otherwise identical compartmentalized redox reactions
NAD(P)H cofactors are not transported across cell membranes, with metabolic shuttles required for transport of reducing equivalents between the mitochondria and cytosol. Lewis et al. develop an approach to resolve NADPH-dependent pathways in these distinct metabolic compartments and show that directionality of serine/glycine interconversions can be determined within intact cells.
The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial. A unique function of PKM2 proposed to be important for the proliferation of some cancer cells involves the direct ...activity of this enzyme as a protein kinase; however, a detailed biochemical characterization of this activity is lacking. Using 32P-phosphoenolpyruvate (PEP) we examine the direct substrates of PKM2 using recombinant enzyme and in vitro systems where PKM2 is genetically deleted. Labeling of some protein species from 32P-PEP can be observed; however, most were dependent on the presence of ADP, and none were dependent on the presence of PKM2. In addition, we also failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein. These findings argue against a role for PKM2 as a protein kinase.
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•PEP-dependent phosphorylation was assayed in cell lysates using radioactive PEP•Trace adenine nucleotides can confound PEP-dependent phosphorylation assay results•PEP-dependent phosphorylation is rare and is not dependent on PKM2•We are unable to detect PKM2 protein kinase activity in a variety of contexts
Hosios et al. use radioactive assays to investigate PEP-dependent phosphorylation. While some proteins can be labeled by phosphate from PEP, this activity is not dependent upon PKM2. They did not observe protein kinase activity involving this metabolic enzyme, and reports of PKM2 protein kinase activity may be partially explained by ATP regeneration by pyruvate kinase.
The
serine synthesis pathway is upregulated in many cancers. However, even cancer cells with increased serine synthesis take up large amounts of serine from the environment1 and we confirm that ...exogenous serine is needed for maximal proliferation of these cells. Here we show that even when enzymes in the serine synthesis pathway are genetically upregulated, the demand for oxidized NAD+ constrains serine synthesis, rendering serine-deprived cells sensitive to conditions that decrease the cellular NAD+/NADH ratio. Further, purine depletion is a major consequence of reduced intracellular serine availability, particularly when NAD+ regeneration is impaired. Thus, cells rely on exogenous serine consumption to maintain purine biosynthesis. In support of this explanation, providing exogenous purine nucleobases, or increasing NAD+ availability to facilitate
serine and purine synthesis, both rescue maximal proliferation even in the absence of extracellular serine. Together, these data indicate that NAD+ is an endogenous limitation for cancer cells to synthesize the serine needed for purine production to support rapid proliferation.
Most patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with advanced disease and survive less than 12 months. PDAC has been linked with obesity and glucose intolerance, but whether ...changes in circulating metabolites are associated with early cancer progression is unknown. To better understand metabolic derangements associated with early disease, we profiled metabolites in prediagnostic plasma from individuals with pancreatic cancer (cases) and matched controls from four prospective cohort studies. We find that elevated plasma levels of branched-chain amino acids (BCAAs) are associated with a greater than twofold increased risk of future pancreatic cancer diagnosis. This elevated risk was independent of known predisposing factors, with the strongest association observed among subjects with samples collected 2 to 5 years before diagnosis, when occult disease is probably present. We show that plasma BCAAs are also elevated in mice with early-stage pancreatic cancers driven by mutant Kras expression but not in mice with Kras-driven tumors in other tissues, and that breakdown of tissue protein accounts for the increase in plasma BCAAs that accompanies early-stage disease. Together, these findings suggest that increased whole-body protein breakdown is an early event in development of PDAC.
Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour ...microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.
Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, ...Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in ...cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.
Copy number gain of the D-3-phosphoglycerate dehydrogenase (PHGDH) gene, which encodes the first enzyme in serine biosynthesis, is found in some human cancers including a subset of melanomas.
In ...order to study the effect of increased PHGDH expression in tissues in vivo, we generated mice harboring a PHGDH
allele that allows tissue-specific, doxycycline-inducible PHGDH expression, and we analyzed the phenotype of mice with a ubiquitous increase in PHGDH expression.
Tissues and cells derived from PHGDH
mice exhibit increased serine biosynthesis. Histological examination of skin tissue from PHGDH
mice reveals the presence of melanin granules in early anagen hair follicles, despite the fact that melanin synthesis is closely coupled to the hair follicle cycle and does not normally begin until later in the cycle. This phenotype occurs in the absence of any global change in hair follicle cycle timing. The aberrant presence of melanin early in the hair follicle cycle following PHGDH expression is also accompanied by increased melanocyte abundance in early anagen skin.
These data suggest increased PHGDH expression impacts normal melanocyte biology, but PHGDH expression alone is not sufficient to cause cancer.
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