Arvin M. Gouw

Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression

As the result of genetic alterations and tumor hypoxia, many cancer cells avidly take up glucose and generate lactate through lactate dehydrogenase A (LDHA), which is encoded by a target gene of c-Myc and hypoxia-inducible factor (HIF-1). Previous studies with reduction of LDHA expression indicate that LDHA is involved in tumor initiation, but its role in tumor maintenance and progression has not been established. Furthermore, how reduction of LDHA expression by interference or antisense RNA inhibits tumorigenesis is not well understood. Here, we report that reduction of LDHA by siRNA or its inhibition by a small-molecule inhibitor (FX11 [3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid]) reduced ATP levels and induced significant oxidative stress and cell death that could be partially reversed by the antioxidant N-acetylcysteine. Furthermore, we document that FX11 inhibited the progression of sizable human lymphoma and pancreatic cancer xenografts. When used in combination with the NAD+ synthesis inhibitor FK866, FX11 induced lymphoma regression. Hence, inhibition of LDHA with FX11 is an achievable and tolerable treatment for LDHA-dependent tumors. Our studies document a therapeutical approach to the Warburg effect and demonstrate that oxidative stress and metabolic phenotyping of cancers are critical aspects of cancer biology to consider for the therapeutical targeting of cancer energy metabolism.

Glucose-Independent Glutamine Metabolism via TCA Cycling for Proliferation and Survival in B Cells

Because MYC plays a causal role in many human cancers, including those with hypoxic and nutrient-poor tumor microenvironments, we have determined the metabolic responses of a MYC-inducible human Burkitt lymphoma model P493 cell line to aerobic and hypoxic conditions, and to glucose deprivation, using stable isotope-resolved metabolomics. Using [U-(13)C]-glucose as the tracer, both glucose consumption and lactate production were increased by MYC expression and hypoxia. Using [U-(13)C,(15)N]-glutamine as the tracer, glutamine import and metabolism through the TCA cycle persisted under hypoxia, and glutamine contributed significantly to citrate carbons. Under glucose deprivation, glutamine-derived fumarate, malate, and citrate were significantly increased. Their (13)C-labeling patterns demonstrate an alternative energy-generating glutaminolysis pathway involving a glucose-independent TCA cycle. The essential role of glutamine metabolism in cell survival and proliferation under hypoxia and glucose deficiency makes them susceptible to the glutaminase inhibitor BPTES and hence could be targeted for cancer therapy.

Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell-autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell-autonomous dependence on GLS for cancer therapy.

MYC oncogene overexpression drives renal cell carcinoma in a mouse model through glutamine metabolism

Significance The absence of appropriate transgenic animal models of renal cell carcinomas (RCCs) has made it difficult to identify and test new therapies for this disease. We developed a new transgenic mouse model of a highly aggressive form of RCC in which tumor growth and regression is conditionally regulated by the MYC oncogene. Using desorption electrospray ionization–mass-spectrometric imaging, we found that certain glycerophosphoglycerols and metabolites of the glutaminolytic pathway were higher in abundance in RCC than in normal kidney tissue. Up-regulation of glutaminolytic genes and proteins was identified by genetic analysis and immunohistochemistry, therefore suggesting that RCC tumors are glutamine addicted. Pharmacological inhibition of glutaminase slowed tumor progression in vivo, which may represent a novel therapeutic route for RCC. The MYC oncogene is frequently mutated and overexpressed in human renal cell carcinoma (RCC). However, there have been no studies on the causative role of MYC or any other oncogene in the initiation or maintenance of kidney tumorigenesis. Here, we show through a conditional transgenic mouse model that the MYC oncogene, but not the RAS oncogene, initiates and maintains RCC. Desorption electrospray ionization–mass-spectrometric imaging was used to obtain chemical maps of metabolites and lipids in the mouse RCC samples. Gene expression analysis revealed that the mouse tumors mimicked human RCC. The data suggested that MYC-induced RCC up-regulated the glutaminolytic pathway instead of the glycolytic pathway. The pharmacologic inhibition of glutamine metabolism with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide impeded MYC-mediated RCC tumor progression. Our studies demonstrate that MYC overexpression causes RCC and points to the inhibition of glutamine metabolism as a potential therapeutic approach for the treatment of this disease.

Alteration of the lipid profile in lymphomas induced by MYC overexpression

Significance Desorption electrospray ionization mass spectrometric imaging (DESI-MSI) has been shown to be particularly powerful for identifying lipids and metabolites directly from tissue sections and providing a chemical map of their distribution. We applied DESI-MSI to investigate changes in lipid profiles that occur in animal and human lymphomas associated with the overexpression of the v-myc avian myelocytomatosis viral oncogene homolog (MYC). Using statistical analysis, we found 86 lipids that were either increased or decreased in MYC-induced transgenic mouse models of lymphoma. Most of the increased lipids were glycerophosphoglycerols and cardiolipins with a higher content of monounsaturated fatty acids when compared with control tissue. The lipid profiles of MYC associated human lymphomas with overexpression of MYC resemble closely those observed in MYC-induced transgenic mouse models. Overexpression of the v-myc avian myelocytomatosis viral oncogene homolog (MYC) oncogene is one of the most commonly implicated causes of human tumorigenesis. MYC is known to regulate many aspects of cellular biology including glucose and glutamine metabolism. Little is known about the relationship between MYC and the appearance and disappearance of specific lipid species. We use desorption electrospray ionization mass spectrometry imaging (DESI-MSI), statistical analysis, and conditional transgenic animal models and cell samples to investigate changes in lipid profiles in MYC-induced lymphoma. We have detected a lipid signature distinct from that observed in normal tissue and in rat sarcoma-induced lymphoma cells. We found 104 distinct molecular ions that have an altered abundance in MYC lymphoma compared with normal control tissue by statistical analysis with a false discovery rate of less than 5%. Of these, 86 molecular ions were specifically identified as complex phospholipids. To evaluate whether the lipid signature could also be observed in human tissue, we examined 15 human lymphoma samples with varying expression levels of MYC oncoprotein. Distinct lipid profiles in lymphomas with high and low MYC expression were observed, including many of the lipid species identified as significant for MYC-induced animal lymphoma tissue. Our results suggest a relationship between the appearance of specific lipid species and the overexpression of MYC in lymphomas.

Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia–cell cycle dual reporter

Significance In this study, we report the finding that a subpopulation of hypoxic cancer cells expressed genes involved in mitochondrial function, sustained oxidative metabolism, and were fully tumorigenic. These findings indicate that, whereas the Warburg effect contributes to the metabolism of growing cancer cells, tumorigenicity does not exclusively depend on it and is not diminished by continued respiration under hypoxia. Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring (“non-Warburg”) cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic “non-Warburg” cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity.

Oncogene KRAS activates fatty acid synthase, resulting in specific ERK and lipid signatures associated with lung adenocarcinoma

Significance We studied lung tumors induced by oncogene KRAS gene mutation using transgenic mice and human lung specimens. Gene expression analyses show that KRAS induces fatty acid synthase (FASN), promoting lipogenesis. Through desorption electrospray ionization MS imaging, we found specific lipid modifications in KRAS lung adenocarcinoma. Nanoimmunoassay identified specific KRAS-associated phosphoprotein signatures. We showed that KRAS activates the ERK2 protein, whereas non-KRAS lung adenocarcinoma shows elevated ERK1. We inhibited FASN by a small molecule, cerulenin, and this inhibition blocked cellular proliferation of KRAS-driven lung cancer cells. FASN inhibitors may, thus, present promising therapeutic agents for the treatment of KRAS-associated lung adenocarcinoma. KRAS gene mutation causes lung adenocarcinoma. KRAS activation has been associated with altered glucose and glutamine metabolism. Here, we show that KRAS activates lipogenesis, and this activation results in distinct proteomic and lipid signatures. By gene expression analysis, KRAS is shown to be associated with a lipogenesis gene signature and specific induction of fatty acid synthase (FASN). Through desorption electrospray ionization MS imaging (DESI-MSI), specific changes in lipogenesis and specific lipids are identified. By the nanoimmunoassay (NIA), KRAS is found to activate the protein ERK2, whereas ERK1 activation is found in non–KRAS-associated human lung tumors. The inhibition of FASN by cerulenin, a small molecule antibiotic, blocked cellular proliferation of KRAS-associated lung cancer cells. Hence, KRAS is associated with activation of ERK2, induction of FASN, and promotion of lipogenesis. FASN may be a unique target for KRAS-associated lung adenocarcinoma remediation.

Metabolic vulnerabilities of MYC-induced cancer

All highly proliferating cells have two absolute requirements: abundant sources of energy and building blocks such as proteins, carbohydrates, and lipids. Specific gene products that coordinate massive induction of both energy and these chemical products are essential for normal organismal development and when constitutively activated would be anticipated to elicit uncontrolled growth or cancer. MYC is candidate gene product that can coordinate the transcription of genes that regulate energy and intermediary metabolism through its canonical targets through specific and more general mechanisms of gene binding, induction, and suppression [1, 2, 3]. Previous reports have noted MYCs role in the regulation of glucose and glutamine pathways to drive energy production. Further, MYC regulates nearly all of the central metabolic pathways in the cells, such as glycolysis, glutaminolysis, and nucleotide biosynthesis [1, 2, 3].

Correspondence: Oncogenic MYC persistently upregulates the molecular clock component REV-ERBα

Correspondence: Oncogenic MYC persistently upregulates the molecular clock component REV-ERBα

Abstract 4616: Oncogenic c- and N-Myc disrupt circadian rhythm.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Circadian rhythms are regulated by feedback loops comprising a network of factors that regulate Clock-associated genes. Chronotherapy seeks to take advantage of altered circadian rhythms in some cancers to better time administration of treatments to increase efficacy and reduce toxicity. Taking advantage of cancers that have substantially different circadian rhythms, or are ‘out of phase’, with normal tissues, could open a wide therapeutic window to make them vulnerable to chemotherapy or targeted drugs at different times than normal tissue. However, there is currently no basis to identify which cancers have disrupted circadian rhythms and would be amenable to chronotherapy. c- and N-Myc are oncogenic transcription factors translocated or amplified in many cancers. While the role of Myc in circadian rhythm is currently unknown, it may affect circadian rhythm by binding to the same E-box promoter regions used by the central regulators of circadian rhythm, Clock/Bmal1. Additionally, Myc increases NAD+ levels through upregulation of NAMPT, and NAD+ is a crucial cofactor in the activity of the circadian regulator Sirt1. Thus, we hypothesized that Myc may disrupt circadian rhythm through two mechanisms: inappropriate engagement of E-box promoters and also upregulation of NAMPT leading to dysregulated Sirt1 activity. Here we show in neuroblastoma, osteosarcoma, and hepatocellular carcinoma cells that overexpressed Myc specifically upregulated the negative circadian regulator Rev-erbα, which in turn decreased expression of Bmal1. Inhibition of NAMPT downstream of Myc upregulation also led to major perturbations in circadian gene expression, suggesting a role for NAD modulation downstream of Myc in disruption of circadian rhythm. Importantly, My-expressing cells showed dramatically disrupted circadian oscillations, which could be partially rescued by inhibiting expression of Rev-erbα. Together, these data suggest that Myc-driven cancers have altered circadian oscillation due to upregulation of Rev-erbα and NAMPT, and that cancers driven by Myc may thus be good candidates for chronotherapy. We thank the following funding sources: NIH R01CA051497, R01CA57341, LLS 636311 Citation Format: Brian J. Altman, Annie Hsieh, Arvin Gouw, Anand Venkataraman, Bo Li, David Bellovin, M. Celeste Simon, Dean Felsher, John Hogenesch, Chi V. Dang. Oncogenic c- and N-Myc disrupt circadian rhythm. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4616. doi:10.1158/1538-7445.AM2013-4616