Brandon Faubert

Signaling Kinase AMPK Activates Stress-Promoted Transcription via Histone H2B Phosphorylation

Regulation of Energy Homeostasis The mammalian AMP-activated protein kinase (AMPK) is a serine/threonine kinase complex that regulates cellular energy homeostasis. However, the mechanisms by which AMPK mediates transcriptional responses to metabolic perturbations has been unclear. Bungard et al. (p. 1201; published online 17 August; see the Perspective by Hardie) have found that AMPK activated transcription directly on chromatin, combined with phosphorylation of histone H2B at Serine-36. Both signals colocalized at genes regulated in the pathway, and both the enzyme and phosphorylation were required for the direct transcription of stress-responsive genes. The energy sensor AMPK facilitates gene transcription by localizing to chromatin and phosphorylating histone H2B. The mammalian adenosine monophosphate–activated protein kinase (AMPK) is a serine-threonine kinase protein complex that is a central regulator of cellular energy homeostasis. However, the mechanisms by which AMPK mediates cellular responses to metabolic stress remain unclear. We found that AMPK activates transcription through direct association with chromatin and phosphorylation of histone H2B at serine 36. AMPK recruitment and H2B Ser36 phosphorylation colocalized within genes activated by AMPK-dependent pathways, both in promoters and in transcribed regions. Ectopic expression of H2B in which Ser36 was substituted by alanine reduced transcription and RNA polymerase II association to AMPK-dependent genes, and lowered cell survival in response to stress. Our results place AMPK-dependent H2B Ser36 phosphorylation in a direct transcriptional and chromatin regulatory pathway leading to cellular adaptation to stress.

Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress

Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

Metabolic Heterogeneity in Human Lung Tumors

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability

A characteristic of memory T (TM) cells is their ability to mount faster and stronger responses to reinfection than naïve T (TN) cells do in response to an initial infection. However, the mechanisms that allow this rapid recall are not completely understood. We found that CD8 TM cells have more mitochondrial mass than CD8 TN cells and, that upon activation, the resulting secondary effector T (TE) cells proliferate more quickly, produce more cytokines, and maintain greater ATP levels than primary effector T cells. We also found that after activation, TM cells increase oxidative phosphorylation and aerobic glycolysis and sustain this increase to a greater extent than TN cells, suggesting that greater mitochondrial mass in TM cells not only promotes oxidative capacity, but also glycolytic capacity. We show that mitochondrial ATP is essential for the rapid induction of glycolysis in response to activation and the initiation of proliferation of both TN and TM cells. We also found that fatty acid oxidation is needed for TM cells to rapidly respond upon restimulation. Finally, we show that dissociation of the glycolysis enzyme hexokinase from mitochondria impairs proliferation and blocks the rapid induction of glycolysis upon T-cell receptor stimulation in TM cells. Our results demonstrate that greater mitochondrial mass endows TM cells with a bioenergetic advantage that underlies their ability to rapidly recall in response to reinfection.

Loss of the tumor suppressor LKB1 promotes metabolic reprogramming of cancer cells via HIF-1α

Significance Liver kinase B1 (LKB1) is a serine/threonine kinase often inactivated in human cancer. We demonstrate here that loss of LKB1 expression in cancer cells promotes a progrowth metabolic profile that enables increased cell growth and proliferation. Loss of LKB1 promotes increased tumor cell metabolism through mammalian target of rapamycin complex 1- and reactive oxygen species-dependent increases in hypoxia-inducible factor-1α (HIF-1α). LKB1-null cells are dependent on HIF-1α to maintain cellular ATP and viability under poor nutrient conditions, raising the possibility of targeting HIF-1α for synthetic lethality in LKB1-deficient tumors. Together, our data reveal that regulation of cellular metabolism is a key function of LKB1 that may contribute to its tumor-suppressor function in human cancer. One of the major metabolic changes associated with cellular transformation is enhanced nutrient utilization, which supports tumor progression by fueling both energy production and providing biosynthetic intermediates for growth. The liver kinase B1 (LKB1) is a serine/threonine kinase and tumor suppressor that couples bioenergetics to cell-growth control through regulation of mammalian target of rapamycin (mTOR) activity; however, the influence of LKB1 on tumor metabolism is not well defined. Here, we show that loss of LKB1 induces a progrowth metabolic program in proliferating cells. Cells lacking LKB1 display increased glucose and glutamine uptake and utilization, which support both cellular ATP levels and increased macromolecular biosynthesis. This LKB1-dependent reprogramming of cell metabolism is dependent on the hypoxia-inducible factor-1α (HIF-1α), which accumulates under normoxia in LKB1-deficient cells and is antagonized by inhibition of mTOR complex I signaling. Silencing HIF-1α reverses the metabolic advantages conferred by reduced LKB1 signaling and impairs the growth and survival of LKB1-deficient tumor cells under low-nutrient conditions. Together, our data implicate the tumor suppressor LKB1 as a central regulator of tumor metabolism and growth control through the regulation of HIF-1α–dependent metabolic reprogramming.

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

PGC-1α supports glutamine metabolism in breast cancer

BackgroundGlutamine metabolism is a central metabolic pathway in cancer. Recently, reductive carboxylation of glutamine for lipogenesis has been shown to constitute a key anabolic route in cancer cells. However, little is known regarding central regulators of the various glutamine metabolic pathways in cancer cells.MethodsThe impact of PGC-1α and ERRα on glutamine enzyme expression was assessed in ERBB2+ breast cancer cell lines with quantitative RT-PCR, chromatin immunoprecipitation, and immunoblotting experiments. Glutamine flux was quantified using 13C-labeled glutamine and GC/MS analyses. Functional assays for lipogenesis were performed using 14C-labeled glutamine. The expression of glutamine metabolism genes in breast cancer patients was determined by bioinformatics analyses using The Cancer Genome Atlas.ResultsWe show that the transcriptional coactivator PGC-1α, along with the transcription factor ERRα, is a positive regulator of the expression of glutamine metabolism genes in ERBB2+ breast cancer. Indeed, ERBB2+ breast cancer cells with increased expression of PGC-1α display elevated expression of glutamine metabolism genes. Furthermore, ERBB2+ breast cancer cells with reduced expression of PGC-1α or when treated with C29, a pharmacological inhibitor of ERRα, exhibit diminished expression of glutamine metabolism genes. The biological relevance of the control of glutamine metabolism genes by the PGC-1α/ERRα axis is demonstrated by consequent regulation of glutamine flux through the citric acid cycle. PGC-1α and ERRα regulate both the canonical citric acid cycle (forward) and the reductive carboxylation (reverse) fluxes; the latter can be used to support de novo lipogenesis reactions, most notably in hypoxic conditions. Importantly, murine and human ERBB2+ cells lines display a significant dependence on glutamine availability for their growth. Finally, we show that PGC-1α expression is positively correlated with that of the glutamine pathway in ERBB2+ breast cancer patients, and high expression of this pathway is associated with reduced patient survival.ConclusionsThese data reveal that the PGC-1α/ERRα axis is a central regulator of glutamine metabolism in ERBB2+ breast cancer. This novel regulatory link, as well as the marked reduction in patient survival time associated with increased glutamine pathway gene expression, suggests that targeting glutamine metabolism may have therapeutic potential in the treatment of ERBB2+ breast cancer.

Metformin Antagonizes Cancer Cell Proliferation by Suppressing Mitochondrial-Dependent Biosynthesis

Metformin is a biguanide widely prescribed to treat Type II diabetes that has gained interest as an antineoplastic agent. Recent work suggests that metformin directly antagonizes cancer cell growth through its actions on complex I of the mitochondrial electron transport chain (ETC). However, the mechanisms by which metformin arrests cancer cell proliferation remain poorly defined. Here we demonstrate that the metabolic checkpoint kinases AMP-activated protein kinase (AMPK) and LKB1 are not required for the antiproliferative effects of metformin. Rather, metformin inhibits cancer cell proliferation by suppressing mitochondrial-dependent biosynthetic activity. We show that in vitro metformin decreases the flow of glucose- and glutamine-derived metabolic intermediates into the Tricarboxylic Acid (TCA) cycle, leading to reduced citrate production and de novo lipid biosynthesis. Tumor cells lacking functional mitochondria maintain lipid biosynthesis in the presence of metformin via glutamine-dependent reductive carboxylation, and display reduced sensitivity to metformin-induced proliferative arrest. Our data indicate that metformin inhibits cancer cell proliferation by suppressing the production of mitochondrial-dependent metabolic intermediates required for cell growth, and that metabolic adaptations that bypass mitochondrial-dependent biosynthesis may provide a mechanism of tumor cell resistance to biguanide activity.

Oncogenic Myc Induces Expression of Glutamine Synthetase through Promoter Demethylation

c-Myc is known to promote glutamine usage by upregulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis and suggest a molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.

Depletion of the novel p53-target gene carnitine palmitoyltransferase 1C delays tumor growth in the neurofibromatosis type I tumor model

Despite the prominent pro-apoptotic role of p53, this protein has also been shown to promote cell survival in response to metabolic stress. However, the specific mechanism by which p53 protects cells from metabolic stress-induced death is unknown. Earlier we reported that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific member of a family of mitochondria-associated enzymes that have a central role in fatty acid metabolism promotes cell survival and tumor growth. Unlike other members of the CPT family, the subcellular localization of CPT1C and its cellular function remains elusive. Here, we report that CPT1C is a novel p53-target gene with a bona fide p53-responsive element within the first intron. CPT1C is upregulated in vitro and in vivo in a p53-dependent manner. Interestingly, expression of CPT1C is induced by metabolic stress factors such as hypoxia and glucose deprivation in a p53 and AMP activated kinase-dependent manner. Furthermore, in a murine tumor model, depletion of Cpt1c leads to delayed tumor development and a striking increase in survival. Taken together, our results indicate that p53 protects cells from metabolic stress via induction of CPT1C and that CPT1C may have a crucial role in carcinogenesis. CPT1C may therefore represent an exciting new therapeutic target for the treatment of hypoxic and otherwise treatment-resistant tumors.