Amino Acids Rather than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells

Cells must duplicate their mass in order to proliferate. Glucose and glutamine are the major nutrients consumed by proliferating mammalian cells, but the extent to which these and other nutrients contribute to cell mass is unknown. We quantified the fraction of cell mass derived from different nutrients and found that the majority of carbon mass in cells is derived from other amino acids, which are consumed at much lower rates than glucose and glutamine. While glucose carbon has diverse fates, glutamine contributes most to protein, suggesting that glutamine's ability to replenish tricarboxylic acid cycle intermediates (anaplerosis) is primarily used for amino acid biosynthesis. These findings demonstrate that rates of nutrient consumption are indirectly associated with mass accumulation and suggest that high rates of glucose and glutamine consumption support rapid cell proliferation beyond providing carbon for biosynthesis.National Institutes of Health (U.S.) (Grant U54CA143874

Environment Dictates Dependence on Mitochondrial Complex I for NAD+ and Aspartate Production and Determines Cancer Cell Sensitivity to Metformin

Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism. Keywords: cancer metabolism; metformin; biguanide; NAD+/NADH ratio; drug sensitivity; complex I; mitochondria; aspartateNational Institutes of Health (U.S.) (Grant P30CA1405141)National Institutes of Health (U.S.) (Grant GG006413)National Institutes of Health (U.S.) (Grant R01 CA168653)National Institutes of Health (U.S.) (Grant R01 CA201276

Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers

Tumor genetics guides patient selection for many new therapies, and cell culture studies have demonstrated that specific mutations can promote metabolic phenotypes. However, whether tissue context defines cancer dependence on specific metabolic pathways is unknown. Kras activation and Trp53 deletion in the pancreas or the lung result in pancreatic ductal adenocarinoma (PDAC) or non-small cell lung carcinoma (NSCLC), respectively, but despite the same initiating events, these tumors use branched-chain amino acids (BCAAs) differently. NSCLC tumors incorporate free BCAAs into tissue protein and use BCAAs as a nitrogen source, whereas PDAC tumors have decreased BCAA uptake. These differences are reflected in expression levels of BCAA catabolic enzymes in both mice and humans. Loss of Bcat1 and Bcat2, the enzymes responsible for BCAA use, impairs NSCLC tumor formation, but these enzymes are not required for PDAC tumor formation, arguing that tissue of origin is an important determinant of how cancers satisfy their metabolic requirements.National Institutes of Health (U.S.) (Grant F30CA183474)National Institutes of Health (U.S.) (Grant T32GM007753

Defining the contributors to mammalian cell mass

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.Proliferation can be thought of as the sum of many biosynthetic processes. To proliferate, a cell must not only physically divide but must also newly synthesize each of its components as it progresses through the cell cycle. Metabolism allows a cell to meet these demands. Metabolic alterations associated with proliferating cells have been characterized, and increasing research interest seeks to provide mechanistic and teleological insight into these metabolic alterations. The following dissertation provides a framework for understanding how proliferating mammalian cells use the nutrients available to them to synthesize macromolecule precursors that are ultimately used to synthesize new cell mass. Substantial research efforts have focussed on the abilities of glucose and glutamine to serve as sources of biosynthetic material for cell growth, especially since proliferating cells avidly consume these nutrients. Many other nutrients are consumed at much lower rates, and we have quantified how each contributes to biosynthesis, demonstrating that amino acids are the primary contributors to mammalian cell mass. Although glucose consumption does not directly relate to its contribution to cell mass, glycolytic flux is important to sustain cell growth, and activation of this pathway is thought to promote biosynthesis. To better understand regulation of this pathway, we have explored the biochemical properties of two glycolytic enzymes, pyruvate kinase and enolase. Although pyruvate kinase isoform M2 (PKM2) expression enables proliferation in some contexts, we demonstrate that this is not because of its putative activity as a protein kinase. We additionally characterize a novel modification of enolase by its substrate, phosphoenolpyruvate, which can covalently modify a catalytic residue and inhibit enzyme activity. These studies collectively contribute to an understanding of how metabolism can support rapid proliferation in mammalian cells, and lay the foundation for future studies to understand proliferative metabolism.by Aaron M. Hosios.Ph. D

Lack of Evidence for PKM2 Protein Kinase Activity

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.Howard Hughes Medical Institute (International Student Research Fellowship)Vertex Pharmaceuticals Incorporated. Vertex Scholars ProgramMassachusetts Institute of Technology. Department of Biology (Grant T32GM007287)David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Student Fellowship)National Cancer Institute (U.S.) (Grants R01CA168653 and P30CA14051)Burroughs Wellcome Fun

Cooperative nutrient accumulation sustains growth of mammalian cells

The coordination of metabolic processes to allow increased nutrient uptake and utilization for macromolecular synthesis is central for cell growth. Although studies of bulk cell populations have revealed important metabolic and signaling requirements that impact cell growth on long time scales, whether the same regulation influences short-term cell growth remains an open question. Here we investigate cell growth by monitoring mass accumulation of mammalian cells while rapidly depleting particular nutrients. Within minutes following the depletion of glucose or glutamine, we observe a growth reduction that is larger than the mass accumulation rate of the nutrient. This indicates that if one particular nutrient is depleted, the cell rapidly adjusts the amount that other nutrients are accumulated, which is consistent with cooperative nutrient accumulation. Population measurements of nutrient sensing pathways involving mTOR, AKT, ERK, PKA, MST1, or AMPK, or pro-survival pathways involving autophagy suggest that they do not mediate this growth reduction. Furthermore, the protein synthesis rate does not change proportionally to the mass accumulation rate over these time scales, suggesting that intracellular metabolic pools buffer the growth response. Our findings demonstrate that cell growth can be regulated over much shorter time scales than previously appreciated.National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874)National Institutes of Health (U.S.) (Contract R01GM085457)National Cancer Institute (U.S.) (Fellowship F31CA167872)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Program 5T32GM008334

Biophysical changes reduce energetic demand in growth factor–deprived lymphocytes

Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-xL expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-xL cells depleted of IL-3 and primary CD8+ T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress

PKM2 Isoform-Specific Deletion Reveals a Differential Requirement for Pyruvate Kinase in Tumor Cells

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.National Institutes of Health (U.S.) (Grant R01CA168653)National Institutes of Health (U.S.) (Grant 5P01CA117969)National Institutes of Health (U.S.) (Grant P30CA147882)National Institutes of Health (U.S.) (Grant 5P30CA14051)National Institutes of Health (U.S.) (Grant 5K08CA136983)National Institutes of Health (U.S.) (Grant DK059635)National Institutes of Health (U.S.) (Grant R01DK092606)National Institutes of Health (U.S.) (Grant R00CA131472)National Institutes of Health (U.S.) (Grant R01GM056203)American Diabetes Association (Grant 7-12-BS-09)Smith Family FoundationBurroughs Wellcome FundDamon Runyon Cancer Research FoundationStern Famil

Pyruvate Kinase Isoform Expression Alters Nucleotide Synthesis to Impact Cell Proliferation

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.United States. Dept. of Defense. Congressionally Directed Medical Research Programs (Postdoctoral Award W81XWH-12-1-0466)Smith Family FoundationBurroughs Wellcome FundDamon Runyon Cancer Research FoundationStern FamilyAmerican Association for Cancer ResearchNational Cancer Institute (U.S.) (NIH 5P30CA1405141)National Cancer Institute (U.S.) (R01CA168653