An epitope tag alters phosphoglycerate dehydrogenase structure and impairs ability to support cell proliferation

Background The gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region of focal genomic copy number gain in human cancers. Cells with PHGDH amplification are dependent on enzyme expression for proliferation. However, dependence on increased PHGDH expression extends beyond production of serine alone, and further studies of PHGDH function are necessary to elucidate its role in cancer cells. These studies will require a physiologically relevant form of the enzyme for experiments using engineered cell lines and recombinant protein. Results The addition of an N-terminal epitope tag to PHGDH abolished the ability to support proliferation of PHGDH-amplified cells despite retention of some activity to convert 3-PG to PHP. Introducing an R236E mutation into PHGDH eliminates enzyme activity, and this catalytically inactive enzyme cannot support proliferation of PHGDH-dependent cells, arguing that canonical enzyme activity is required. Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation. To enable studies of PHGDH function, we report a method to purify recombinant PHGDH and found that untagged enzyme activity was greater than N-terminally tagged enzyme. Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure. Conclusions Purification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies. Furthermore, while tagged PHGDH retains some ability to convert 3PG to PHP, the structural alterations caused by including an epitope tag disrupts the ability of PHGDH to sustain cancer cell proliferation.National Science Foundation (U.S.). Graduate Research Fellowship (DGE-1122374)T32GM007287National Cancer Institute (U.S.) (R01CA168653)National Cancer Institute (U.S.) (P30CA14051)Burroughs Wellcome FundAmerican Association for Cancer Researc

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

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.National Institutes of Health (U.S.) (NIH grant R01 GM56203)National Institutes of Health (U.S.) (grant NIH 5P01CA120964)Dana-Farber/Harvard Cancer Center (NIH 5P30CA006516)National Institutes of Health (U.S.) (NIH grant R03MH085679)National Human Genome Research Institute (U.S.) (Intramural Research Program)National Institutes of Health (U.S.) (Molecular Libraries Initiative of the NIH Roadmap for Medical Research

The role of phosphoglycerate dehydrogenase in cell proliferation and tumor progression

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis. Vita.Includes bibliographical references.The metabolic needs of proliferating cells are distinct from those of quiescent, terminally differentiated cells. Cancer is a disease of inappropriate cell proliferation, and tumor cells require a unique metabolic program to support proliferation. Phosphoglycerate dehydrogenase (PHGDH) catalyzes the first step in the serine biosynthesis pathway, and the gene encoding this enzyme is amplified in human cancer. PHGDH-amplified cells depend on PHGDH expression to proliferate, and upon PHGDH knockdown, supplying exogenous serine cannot restore proliferation. We hypothesized that PHGDH is an unappreciated source of the reductive currency NADPH for biosynthesis and/or redox maintenance. PHGDH can utilize NADP+ as a cofactor in vitro, and tritiated glucose tracing shows that PHGDH produces exclusively NADPH, not NADH, in cells. We therefore designed and validated mutant PHGDH enzymes with altered selectivity for NAD(P) (H) that can be used to test whether an ability of PHGDH to produce NADPH is required for cell proliferation. In the course of these experiments, we found that N-terminally tagged PHGDH exhibits structural alterations compared to the wildtype enzyme and is incapable of supporting proliferation. To enable future studies of a physiologically relevant form of PHGDH, we developed a method to purify untagged recombinant PHGDH. In order to model PHGDH-amplified cancer in vivo, we developed a doxycycline-inducible PHGDH transgenic mouse. When PHGDH expression is increased ubiquitously in mouse tissues, melanin granules are present in hair follicles at an inappropriate stage of the hair follicle cycle. This melanocyte-related abnormality is provocative, as PHGDH amplification occurs in human melanoma. When PHGDH is overexpressed in a model of melanoma driven by mutant Braf and Pten loss, tumor growth is accelerated. Mice with mutant Braf normally develop growth arrested melanocytic nevi, and when PHGDH is overexpressed in this context an increased in nevi is observed and malignant melanoma arises with incomplete penetrance. These data identify PHGDH as the first metabolic enzyme that can be overexpressed in its wild type form and promote cancer initiation and/or progression to a malignant state. We conclude that PHGDH metabolic activity is important for cancer cell proliferation and that PHGDH amplification or overexpression can promote tumorigenesis at multiple stages.by Katherine R. Mattaini.Ph. D

Human Phosphoglycerate Dehydrogenase Produces the Oncometabolite d-2-Hydroxyglutarate

Human d-3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in the serine biosynthetic pathway, is genomically amplified in tumors including breast cancer and melanoma. In PHGDH-amplified cancer cells, knockdown of PHGDH is not fully rescued by exogenous serine, suggesting possible additional growth-promoting roles for the enzyme. Here we show that, in addition to catalyzing oxidation of 3-phosphoglycerate, PHGDH catalyzes NADH-dependent reduction of α-ketoglutarate (AKG) to the oncometabolite d-2-hydroxyglutarate (d-2HG). Knockdown of PHGDH decreased cellular 2HG by approximately 50% in the PHGDH-amplified breast cancer cell lines MDA-MB-468 (normal concentration 93 μM) and BT-20 (normal concentration 35 μM) and overexpression of PHGDH increased cellular 2HG by over 2-fold in non-PHGDH-amplified MDA-MB-231 breast cancer cells, which normally display very low PHGDH expression. The reduced 2HG level in PHGDH knockdown cell lines can be rescued by PHGDH re-expression, but not by a catalytically inactive PHGDH mutant. The initial connection between cancer and d-2HG involved production of high levels of d-2HG by mutant isocitrate dehydrogenase. More recently, however, elevated d-2HG has been observed in breast cancer tumors without isocitrate dehydrogenase mutation. Our results suggest that PHGDH is one source of this d-2HG.National Institutes of Health (U.S.) (Grants 1R01CA163591, P50GM07150 and P30CA14051)American Association for Cancer ResearchNational Science Foundation (U.S.)Burroughs Wellcome Fun

Increased Serine Synthesis Provides an Advantage for Tumors Arising in Tissues Where Serine Levels Are Limiting

Tumors exhibit altered metabolism compared to normal tissues. Many cancers upregulate expression of serine synthesis pathway enzymes, and some tumors exhibit copy-number gain of the gene encoding the first enzyme in the pathway, phosphoglycerate dehydrogenase (PHGDH). However, whether increased serine synthesis promotes tumor growth and how serine synthesis benefits tumors is controversial. Here, we demonstrate that increased PHGDH expression promotes tumor progression in mouse models of melanoma and breast cancer, human tumor types that exhibit PHGDH copy-number gain. We measure circulating serine levels and find that PHGDH expression is necessary to support cell proliferation at lower physiological serine concentrations. Increased dietary serine or high PHGDH expression is sufficient to increase intracellular serine levels and support faster tumor growth. Together, these data suggest that physiological serine availability restrains tumor growth and argue that tumors arising in serine-limited environments acquire a fitness advantage by upregulating serine synthesis pathway enzymes. Nutrient availability can constrain tumor growth. Sullivan et al. demonstrate that in some cancers, physiological levels of the amino acid serine are insufficient to support maximal tumor growth and that melanoma and breast tumors derive a growth advantage by upregulating serine biosynthesis.National Science Foundation (Grant DGE-1122374)National Science Foundation (Grant T32-GM007287)National Science Foundation (Grant F32CA213810)National Science Foundation (Grant R21CA198028)National Science Foundation (Grant R01CA168653

Metabolic Pathway Alterations that Support Cell Proliferation

Proliferating cells adapt metabolism to support the conversion of available nutrients into biomass. How cell metabolism is regulated to balance the production of ATP, metabolite building blocks, and reducing equivalents remains uncertain. Proliferative metabolism often involves an increased rate of glycolysis. A key regulated step in glycolysis is catalyzed by pyruvate kinase to convert phosphoenolpyruvate (PEP) to pyruvate. Surprisingly, there is strong selection for expression of the less active M2 isoform of pyruvate kinase (PKM2) in tumors and other proliferative tissues. Cell growth signals further decrease PKM2 activity, and cells with less active PKM2 use another pathway with separate regulatory properties to convert PEP to pyruvate. One consequence of using this alternative pathway is an accumulation of 3-phosphoglycerate (3PG) that leads to the diversion of 3PG into the serine biosynthesis pathway. In fact, in some cancers a substantial portion of the total glucose flux is directed toward serine synthesis, and genetic evidence suggests that glucose flux into this pathway can promote cell transformation. Environmental conditions can also influence the pathways that cells use to generate biomass with the source of carbon for lipid synthesis changing based on oxygen availability. Together, these findings argue that distinct metabolic phenotypes exist among proliferating cells, and both genetic and environmental factors influence how metabolism is regulated to support cell growth.Burroughs Wellcome FundDamon Runyon Cancer Research FoundationSmith Family FoundationStarr Cancer ConsortiumNational Institutes of Health (U.S.