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Invasive ductal cancers (IDC) have a variety of precursor lesions and molecular subtypes. Ductal Carcinoma in Situ (DCIS) is considered an important precursor stage immediately preceding IDC and has increased in its incidence in recent times. 20-40% of DCIS form IDC however, currently we lack robust molecular signatures to predict progression prone DCIS. Performing next generation sequencing in breast cancer DCIS precursors is likely to help elucidate their progression potential. Sequencing the progression path of IDC lesions in a mouse model, and in human samples of the same molecular subtype, should help to understand the drivers of transformation. We utilized the C3-TAG genetically engineered mouse model that forms DCIS like precursor lesions, and human breast DCIS-IDC pairs for genomic analyses. Through single cell RNA-sequencing, and bulk mRNA-seq, we identified disease stage specific, and cell population specific, signatures at three stages of breast cancer tumor progression in the C3-TAG mouse model. Through integrated analysis we subset cancer cells in the DCIS and tumor stage in C3-TAG and identify genes and gene signatures relevant in the IDC stage including HIF-1-alpha pathway (Fos proto-oncogene, lactate dehydrogenase A); glycolysis pathway (phosphoglycerate mutase 1, pyruvate kinase M1/2, aldolase, fructose-bisphosphate A, enolase 1); Metastasis associated protein (MTA3) complex (tubulin alpha 1c, tubulin alpha 4a) and endocrine resistance (cyclin dependent kinase inhibitor 2A, KRAS proto-oncogene, GTPase, heparin binding EGF like growth factor, cyclin D1). We also identified microenvironment signatures and detected gene changes in the immune, endothelial and fibroblast cells as DCIS transitions to IDC, including a sharp decline of CD8 T cell genes (CD274, C-C motif chemokine ligand 5, SLAM family member 7) from DCIS to IDC, highlighting an immune dysregulation as part of the transition from DCIS to IDC. Simultaneously, we retrieved a public microarray dataset of human DCIS/IDC pairs and performed Ribo-zero RNA-sequencing of 5 human basal-like DCIS/IDC and 5 Luminal A DCIS/IDC pairs. Utilizing gene set enrichment analysis, we show that our C3-TAG DCIS stage tumor cell signature was significantly enriched in the human basal-like DCIS, but not in Luminal A DCIS. These results highlight that the C3-TAG DCIS may recapitulate the biology of basal-like DCIS and that progression in basal-like may be different than in Luminal/ER+ cancers. Further interrogation of these DCIS and IDC stage specific signatures may lead to identification of a clinical marker of progression and/or a therapeutic target of early intervention. Citation Format: Aatish Thennavan, Xiaping He, Charles M Perou. Molecular signatures of DCIS to invasive progression for basal-like breast cancers: An integrated mouse model and human tumor study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD6-9.

Purpose: To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB). Experimental Design: Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated with survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity, and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model. Results: Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of MYCN did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity, and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed. Conclusions: High expression of LDHA is independently associated with outcome of NB, and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. Clin Cancer Res; 24(22); 5772–83. ©2018 AACR.

13C MRI of hyperpolarized [1-13C]pyruvate metabolism has been used in oncology to detect disease, investigate disease progression, and monitor response to treatment with a view to guiding treatment in individual patients. This technique has translated to the clinic with initial studies in prostate cancer. Here, we use the technique to investigate its potential uses in patients with glioblastoma (GB). We assessed the metabolism of hyperpolarized [1-13C]pyruvate in an orthotopically implanted cell line model (U87) of GB and in patient-derived tumors, where these were produced by orthotopic implantation of cells derived from different patients. Lactate labeling was higher in the U87 tumor when compared with patient-derived tumors, which displayed intertumoral heterogeneity, reflecting the intra- and intertumoral heterogeneity in the patients' tumors from which they were derived. Labeling in some patient-derived tumors could be observed before their appearance in morphologic images, whereas in other tumors it was not significantly greater than the surrounding brain. Increased lactate labeling in tumors correlated with c-Myc–driven expression of hexokinase 2, lactate dehydrogenase A, and the monocarboxylate transporters and was accompanied by increased radioresistance. Because c-Myc expression correlates with glioma grade, this study demonstrates that imaging with hyperpolarized [1-13C]pyruvate could be used clinically with patients with GB to determine disease prognosis, to detect early responses to drugs that modulate c-Myc expression, and to select tumors, and regions of tumors for increased radiotherapy dose. Significance: Metabolic imaging with hyperpolarized [1-13C]pyruvate detects low levels of c-Myc–driven glycolysis in patient-derived glioblastoma models, which, when translated to the clinic, could be used to detect occult disease, determine disease prognosis, and target radiotherapy. Cancer Res; 78(18); 5408–18. ©2018 AACR.

We have shown previously that dietary administration of phenethyl isothiocyanate (PEITC), a small molecule from edible cruciferous vegetables, significantly decreases the incidence of poorly differentiated prostate cancer in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice without any side effects. In this study, we investigated the role of c-Myc–regulated glycolysis in prostate cancer chemoprevention by PEITC. Exposure of LNCaP (androgen-responsive) and 22Rv1 (castration-resistant) human prostate cancer cells to PEITC resulted in suppression of expression as well as transcriptional activity of c-Myc. Prostate cancer cell growth inhibition by PEITC was significantly attenuated by stable overexpression of c-Myc. Analysis of the RNA-Seq data from The Cancer Genome Atlas indicated a significant positive association between Myc expression and gene expression of many glycolysis-related genes, including hexokinase II and lactate dehydrogenase A. Expression of these enzyme proteins and lactate levels were decreased upon PEITC treatment in prostate cancer cells, and these effects were significantly attenuated by ectopic expression of c-Myc. A normal prostate stromal cell line (PrSC) was resistant to lactic acid suppression by PEITC treatment. Prostate cancer chemoprevention by PEITC in TRAMP mice was associated with a significant decrease in plasma lactate and pyruvate levels. However, a 1-week intervention with 10 mg PEITC (orally, 4 times/day) was not sufficient to decrease lactate levels in the serum of human subjects. These results indicated that although prostate cancer prevention by PEITC in TRAMP mice was associated with suppression of glycolysis, longer than 1-week intervention might be necessary to observe such an effect in human subjects. Cancer Prev Res; 11(6); 337–46. ©2018 AACR.

Dysregulation of metabolism is one of the cancer hallmarks. However, little is known whether long coding RNAs (lncRNAs) play a role in the regulation of metabolism in pancreatic ductal adenocarcinoma (PDAC). In the present study, we focused on lncRNA LINC00901 because a high level of LINC00901 is associated with the poor outcome of PDAC patients. Functional studies showed that LINC00901 promotes PDAC cell proliferation and invasion, whereas knockout (KO) of LINC00901 impairs these abilities of PDAC cells. Furthermore, the xenograft animal model suggested that LINC00901 promotes tumorigenesis. To determine the underlying mechanism of LINC00901-mediated tumorigenesis, we performed RNA-seq analysis in LINC00901 overexpression or KO cells. Gene Set Enrichment Analysis (GSEA) analysis showed that gene sets involved in glucose biological processes such as ‘‘MYC targets”, “Glycolysis” and “Gluconeogenesis” are significantly enriched in the LINC00901 overexpression group, whereas these genes are down-regulated in the LINC00901 KO group. The top-scoring genes recurring in the three gene sets include key glycolysis-related genes, lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and enolase 1 (ENO1), all of which were validated by qRT-PCR and western blot. Moreover, we found that LINC00901 upregulates c-Myc by stabilizing c-Myc RNA. Together, these results suggest that LINC00901 is an oncogenic lncRNA capable of regulating glucose metabolism, leading to PDAC development. Experiments are underway to determine how LINC00901 regulates c-Myc RNA stability in pancreatic cancer cells. Citation Format: Wan-Xin Peng, Liu Yang, Yin-Yuan Mo. LINC00901 regulates glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2389.

Only a limited fraction of patients derives durable clinical benefit upon immune checkpoint blockade. Deepening our understanding of mechanisms of response and resistance to these therapies is thus needed to optimize their employment in rational combinations. Cellular energy metabolism reprogramming is a critical hallmark of cancer. High glucose consumption and lactate production by tumor cells restrict nutrient availability in the tumor microenvironment (TME) for effector T cells, which also rely on glycolysis to replicate and function. In addition, immune checkpoints and co-stimulatory molecules are emerging as important regulators of T cell metabolism. Exploiting the capacity of immune checkpoint blockade to perturb T cell metabolism in combination with inhibition of tumor glycolysis may thus be a rational and more effective anti-cancer approach. We investigated the link between tumor glycolysis and immune cell function using RNA sequencing data sets from patients treated with checkpoint blockade and in tumor:T cell co-culture systems. We then studied the effects of checkpoint blockade in syngeneic glycolysis-defective murine tumor models and explored mechanisms underlying anti-tumor activity. We found that expression of glycolysis-related genes is inversely correlated with infiltration of most immune cell types in melanomas from patients before CTLA-4 blockade. However, after treatment, immune-related and glycolysis-related genes were more often co-expressed, suggesting that anti-CTLA-4 may partially restore immune cell fitness in the glycolytic TME. To directly assess the effect of tumor metabolism on T cell function, we co-cultured activated T cells with the highly glycolytic murine mammary carcinoma 4T1 and observed that tumor cells, or similar non-toxic concentrations of exogenous lactate, significantly limited T cell activation and viability. We thus investigated whether inhibition of tumor glycolysis could potentiate the activity of CTLA-4 blockade. We found that neoadjuvant treatment with anti-CTLA-4 significantly prolonged survival in mice bearing glycolysis defective 4T1 tumors, where lactate dehydrogenase A (LDH-A) - the critical enzyme controlling lactate production in aerobic glycolysis - was knocked down (4T1-KD). Intriguingly, tumor protection was associated with intratumoral regulatory T cell (Treg) functional phenotypic destabilization towards IFN-gamma and TNF-alpha producing Tregs. By mimicking the LDH-A-KD and control 4T1 TME in vitro using higher and lower concentrations of glucose, respectively, we showed that CTLA-4 blockade promotes glucose uptake by Tregs and more efficiently counteracts Treg suppression and enhances CD28 co-stimulation at higher glucose concentrations. These findings indicate that rebalancing glucose utilization in favor of immune cells in the TME may facilitate the activity of CTLA-4 blockade and reveal an additional mechanism through which anti-CTLA-4 interferes with Treg function in the presence of glucose. Citation Format: Roberta Zappasodi, Inna Serganova, Ivan Cohen, Masatomo Maeda, Masahiro Shindo, Yasin Senbabaoglu, Avigdor Leftin, Rachana Maniyar, Svena Verma, Matthew Lubin, Myat Kyaw Ko, Mayuresh M. Mane, Arnab Ghosh, Ellen Ackerstaff, Jason A. Koutcher, Ping-Chih Ho, Greg M. Delgoffe, Ronald Blasberg, Jedd D. Wolchok, Taha Merghoub. CTLA-4 blockade drives loss of regulatory T cell functional stability in glycolysis defective tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3257.

Increased expression of Hypoxia-Inducible Factor 1α (HIF-1α) in breast tumours is associated with poor survival outcome. HIF-1α promotes phenotypic adaptations including metabolic reorganisation, angiogenesis and increased cell invasiveness. Notably, HIF-1α activity is increased in Triple receptor Negative Breast Cancer (TNBC) compared with other breast cancer subtypes. One reason for this elevated HIF-1α stabilisation in TNBC has been described; intracellular cysteine depletion which leads to impaired prolyl hydroxylase-domain (PHD) enzyme activity. Normally, cystine is acquired in exchange for intracellular glutamate using the xCT antiporter. However, in TNBC the excessive secretion and extracellular accumulation of glutamate impairs cystine exchange. Since the majority of glutamate produced in TNBC is derived from glutaminase-catalysed hydrolysis of glutamine, we hypothesised that glutaminase inhibition using a potent and selective inhibitor, CB-839, would improve glutamate/cystine exchange and reduce HIF-1α levels. We detected normoxic HIF-1α expression in BT549, Hs578T and SUM159PT TNBC cell cultures. In these cases HIF-1α protein was destabilised upon treatment with CB-839 and the expression of some HIF-1α-target genes was significantly decreased (e.g. CA9, BNIP3, PDK1, LDHA) but not others (e.g. ADM and VEGFA). HIF-1α destabilisation corresponded with increased PHD-dependent hydroxylation, supporting the hypothesis that glutaminase inhibition can improve PHD function. Consistently, WT but not a proline hydroxylation-null mutant HIF-1α protein was depleted by CB-839. We confirmed that CB-839 decreased glutamate secretion but, unexpectedly, HIF-1α levels were not restabilised by addition of excess extracellular glutamate. Thus glutamine metabolism drives normoxic HIF-1α stabilisation via an additional mechanism distinct from perturbation of the glutamate/cystine exchange system. To elucidate the underlying process we focused on the glutamine-derived metabolite (S)-2-Hydroxyglutarate [(S)-2HG], a competitive inhibitor of 2-Oxoglutarate (2OG)-dependent enzymes, including the PHD enzymes. Glutaminase inhibition decreased the levels of (S)-2HG. Furthermore, growing CB-839-treated cells in culture medium containing cell permeable (S)-2HG, but not (R)-2HG, could restabilise HIF-1α. In addition, increasing 2OG levels in these cells further stabilised HIF-1α, an effect that was blunted by inhibition of lactate dehydrogenase A (LDHA), a promiscuous source of (S)-2HG generated via reduction of 2OG. Conclusion: CB-839 has demonstrated encouraging clinical responses in patients with heavily pretreated and therapy-resistant metastatic TNBC. Our work demonstrates that high levels of glutamine metabolism provoke HIF-1α stabilisation through the LDHA-catalysed production of (S)-2HG. Assessment of HIF-1α activity may be a useful approach to identify glutamine-addicted tumours and predict responsiveness to glutaminase inhibitor therapy. Citation Format: Laura Cussonneau, Anne-Lise Dechaume, Pamela M. Murray, Henegama Liyanage, Tet-Woo Lee, Frederik Pruijn, Euphemia Y. Leung, Dean C. Singleton. HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3561.

Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. To uncover cell type-specific dependencies to LDH, we screened a diverse panel of 94 cancer cell lines for responsiveness to two novel LDH A/B inhibitors developed through the NCI Experimental Therapeutics Program (NExT). We found that Ewing sarcoma (EWS) cell lines were exquisitely sensitive, with IC50 values approximately ten-fold below the median IC50 of the panel. To understand the mechanism behind this sensitivity, we genetically knocked down LDHA and LDHB using siRNA, and discovered that EWS cell lines were sensitive to loss of LDHA only, which inhibited proliferation and induced apoptosis. Notably, treatment of EWS cells with the LDH inhibitors phenocopied these effects. Additionally, genetic knockdown of EWS-FLI1, the oncogenic driver of EWS, resulted in loss of LDHA, but not LDHB. Analysis of publicly available ChIP-seq data generated using shFLI1-transfected EWS cells revealed that LDHA, but not LDHB, is directly regulated by EWS-FLI1. Functional mechanistic studies of glycolytic intermediates and cellular bioenergetics in EWS cells treated with the LDH inhibitors demonstrated that loss of viability was due to impairment of glycolysis, which occurred both in vitro and in vivo, and perturbation of the NAD+/NADH ratio. The translational potential of these compounds was next evaluated using in vivo analyses of pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Intravenous administration of the LDH inhibitors resulted in diminished LDH activity, reduction of the lactate-to-pyruvate ratio, tumor cell necrosis, and a decrease in tumor growth rate in aggressive xenograft models of EWS. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, our data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as EWS that exhibit oncogene-dependent expression of LDH and increased glycolytic activity. This abstract is also being presented as Poster B33. Citation Format: Choh Yeung, Anna E. Gibson, Sameer H. Issaq, Nobu Oshima, Marielle E. Yohe, Haiyan Lei, Ganesha Rai, Daniel J. Urban, Michelle S. Johnson, Gloria A. Benevides, Giuseppe L. Squadrito, Sandy Eldridge, John Hamre III, Arnulfo Mendoza, Jack F. Shern, Lee J. Helman, Murali C. Krishna, Matthew D. Hall, Victor M. Darley-Usmar, Leonard M. Neckers, Christine M. Heske. Lactate dehydrogenase A is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR08.

Introduction TNBC is a heterogeneous disease and is often associated with expression of a stem cell signature (CD44+, CD24-). Recently platinum agents have been shown to improve the pathological response rates in the neo-adjuvant setting; however a significant proportion of patients with TNBC will relapse. The study objective was to evaluate the potential of targeting glycolysis, mitochondrial function and lactate metabolism in platinum resistant TNBC cell lines. Methods Stem cell signature and glycolytic phenotyping: CD44 and CD24 antibodies were used to measure the percentage of stem cells in each of the cell lines using flow cytometry(FACS). Protein expression of hexokinase, Glut1 transporter, lactate dehydrogenase A/B(LDHA and LDHB), and monocarboxylic transporter 1 and 4 (MCT1 and MCT4) was determined in MCF7(control), MDA-MB231, MDA-MB468, and HCC38 cells. Proliferation and apoptosis assays: IC50s for platinum, metformin, and MCT1 inhibitor AR-C155858, Tocris (MCT1I) were calculated by the sulforhodamine B (SRB) assay. Propidium iodide and AnnexinV staining intensity used to measure apoptosis by FACS. Metabolic analysis: The change in lactate and glucose levels in culture media post-treatment with metformin and MCT1I was quantified by 1H NMR. Intracellular lactate and ATP concentrations were determined by lactate(BioVision) and ATP determination assays(Invitrogen). Mitochondrial membrane potential and Reactive oxygen species (ROS) were measured by MitoTracker Red and MitoSOX Red. 2NBDG was used to assess cell glucose uptake. Combination index SRBs: Cells were treated with increasing concentrations of MCT1I and metformin for 72h. The combination index values were calculated using CalcuSyn® software. Results MDA-MB231 and HCC38 cells were resistant to cisplatin and associated with a stem cell signature (CD44+, CD24-) which was not detected in the other cell lines. MCT1 was highly expressed in HCC38 cells but not at all in MDA-MB231. In contrast, MCT4 was abundantly expressed MDA-MB231 but minimally in HCC38 cells. In these two cell lines, only MDA-MB231 was sensitive to metformin which reduced ATP production, induced ROS generation, and increased the percentage of apoptotic cells. HCC38 also produced ROS, but ATP production and apoptosis were unaffected. HCC38 cells were more sensitive to MCT1I than other cells, which was associated with increased intracellular lactate. The NMR data showed significant increases in lactate in culture media following treatment with metformin in MDA-MB231 and HCC38. Only HCC38 had increased 2NBDG uptake and reduced of glucose levels in culture medium by metformin. Metformin significantly enhanced the anticancer efficacy of MCT1I in HCC38 cells, as a synergistic effect of the two agents was observed across the range of different concentrations; whilst an antagonistic effect was shown in MDA-MB231 cells with almost all concentrations. Conclusions Targeting lactate metabolism selectively inhibits a platinum-resistant stem cell population of TNBC, this effect is enhanced when combined with metformin. The inhibition of glycolysis and lactate metabolism may represent a new therapeutic strategy for this population and warrants further study. Citation Format: Narumi Harada, Vidhya Varghese, Lingmin Xie, Laura M Kenny. Targeting lactate metabolism as a novel therapeutic target in platinum-resistant triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-03.

Purpose: The transcription factor Forkhead box protein M1 (FOXM1) plays critical roles in cancer development and progression. However, the regulatory role and underlying mechanisms of FOXM1 in cancer metabolism are unknown. In this study, we characterized the regulation of aerobic glycolysis by FOXM1 and its impact on pancreatic cancer metabolism. Experimental Design: The effect of altered expression of FOXM1 on expression of glycolytic enzymes and tumor development and progression was examined using animal models of pancreatic cancer. Also, the underlying mechanisms of altered pancreatic cancer glycolysis were analyzed using in vitro molecular biology. The clinical relevance of aberrant metabolism caused by dysregulated FOXM1 signaling was determined using pancreatic tumor and normal pancreatic tissue specimens. Results: We found that FOXM1 did not markedly change the expression of most glycolytic enzymes except for phosphoglycerate kinase 1 (PGK-1) and lactate dehydrogenase A (LDHA). FOXM1 and LDHA were overexpressed concomitantly in pancreatic tumors and cancer cell lines. Increased expression of FOXM1 upregulated the expression of LDHA at both the mRNA and protein level and elevated LDH activity, lactate production, and glucose utilization, whereas reduced expression of FOXM1 did the opposite. Further studies demonstrated that FOXM1 bound directly to the LDHA promoter region and regulated the expression of the LDHA gene at the transcriptional level. Also, elevated FOXM1–LDHA signaling increased the pancreatic cancer cell growth and metastasis. Conclusions: Dysregulated expression and activation of FOXM1 play important roles in aerobic glycolysis and tumorigenesis in patients with pancreatic cancer via transcriptional regulation of LDHA expression. Clin Cancer Res; 20(10); 2595–606. ©2014 AACR.

Pyruvate kinase M2 (PKM2) is crucial for aerobic glycolysis that is highly expressed in the various cancer tissues. Although high expression of PKM2 is observed in prostate tumor tissues, its functional role against affecting cancer metabolism is not clearly understood. Herein, we investigate its role in regulating autophagy and its associated pathways. In the current study, various PKM2-siRNA constructs were used to knockdown the PKM2 expression and observed its cellular pathways on autophagy. Cell viability was significantly reduced in PKM2-siRNA transfected prostate cancer cells, DU145. Acridine orange staining and immunoblotting analysis showed that downregulation of PKM2 markedly increased autophagy cell death. In addition, immunoblotting analysis exhibited the blocking the protein kinase B (Akt) and mTOR1 pathways, which subsequently down-regulated the expression of glycolytic enzymes lactate dehydrogenase A (LDHA) and Glucose transporter 1 (GLUT1). To the best of our knowledge, this is the first study that inhibition of PKM2 changes in cancer cell metabolism and induces autophagy, thus, providing new perspectives into the mechanism of its anticancer activity against prostate cancer, DU145. Citation Format: Prasanta Dey, Amit Kundu, Byung Mu Lee, Hyung Sik Kim. Knockdown of PKM2 induces autophagic cell death via Akt/mTOR pathway in human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4370.

Mitochondria evolved from free-living bacteria after being endocytosed by eukaryotic host cells millions of year ago. We hypothesized that antibiotics could cause mammalian mitochondrial damage while causing bacterial lethality. Mitochondrial toxicity of three antibiotics, azithromycin, doxycycline and ciprofloxacin, were tested in human mammary epithelia MCF-12A and fibroblast by fluorescent and transmission electron microscopy. Gene expression and DNA damage were tested by real time polymerase chain reaction (qPCR) and ELISA. We found antibiotics suppressed the mitochondrial membrane potential gradient of MCF-12A and fibroblast. Ultrastructural studies showed that antibiotics caused vacuolated swollen mitochondria with disrupted cristae in MCF-12A and fibroblast, compared to the ultrastructure of mitochondria in the cells without antibiotic treatment. Fluorescent microscopy revealed antibiotics induce mitochondrial reactive oxygen species (ROS), superoxide, after 3 hours of culture. The DNA oxidative damage product, 8-OHdG, was significantly increased in the media after MCF-12A and fibroblast were cultured in antibiotic media for 24 hours. Antibiotics upregulated gene expression of hypoxia inducible factor 1 alpha (HIF1a), glycolytic enzymes including hexokinase 2(HK2), phosphofructokinase 1 (PFKM) and pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), and glucose transporters 1 (SLC2A1) and 3 (SLC2A3) in MCF-12A and fibroblast. Lactate production was also increased in the culture media of healthy cells MCF-12A and fibroblast cells, confirming aerobic glycolysis, or “the Warburg effect”, the method to generate energy after cells are treated with antibiotics. In summary, antibiotics caused mitochondrial toxicity, ROS overproduction, DNA oxidant damage, upregulation of HIF1a gene and aerobic glycolysis in healthy mammalian cells. Over-usage of antibiotics could contribute to tumorigenesis and neurodegeneration and aggravate existing mitochondria-associated diseases. Citation Format: Robert L. Elliott, Xianpeng Jiang. Antibiotics induce mitochondrial dysfunction, DNA damage and aerobic glycolysis of human mammary epithelia and fibroblast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3795.

Mitochondria evolved from free-living bacteria via endocytosis within a eukaryotic host cell millions of year ago. We hypothesized that antibiotics might damage mitochondria of human cancer cells and lead to oxidative damage of cancer cells. We examined three classes of antibiotics, azithromycin, doxycycline and ciprofloxacin in the human breast cancer cell lines MCF-7 and MDA-MB-231. The combination of antibiotics and the anti-glycolytic agent, 2-Deoxy-D-glucose (2-DG) was also tested in the cells. Cell growth was measured by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay. Mitochondrial membrane potential and reactive oxygen species were tested by fluorescent microscopy. Gene expression and DNA damage were measured by real time polymerase chain reaction (qPCR) and ELISA. All antibiotics suppressed mitochondrial membrane potential and growth of MCF-7 and MDA-MB-231 cells in a dose dependent pattern. The IC50of all three antibiotics were less than clinical patient’s maximum serum concentration (Cmax) or concentrations in tissues. The IC50 of ciprofloxacin in MCF-7 (1.25-2.5µM) and MDA-MB-231 (2.5-3.5µM) are much lower than the Cmax 7.7µM while patients take 500mg every 12 hours orally in clinical practice. The DNA oxidative damage metabolite, 8-OHdG, in the media was significantly increased after cancer cells were cultured in the media containing antibiotics for 24 hours. Three antibiotics upregulated gene expression of glycolytic enzymes including hexokinase 2(HK2), phosphofructokinase 1 (PFKM) and pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), and glucose transporters 1 (SLC2A1) and 3 (SLC2A3). The antibiotics increased uptake of 3[H]-2-DG of MCF-7 and MDA-MB-231. These results suggest that cancer cells increase aerobic glycolysis to produce ATP after antibiotic treatment. Thus, we combined 2-DG and antibiotics to treat the breast cancer cell lines. The antibiotics and 2-DG synergistically inhibited growth of the breast cancer cells. MDA-MB-231, a chemo-drug resistant cell line, has similar sensitivity to antibiotics and 2-DG as the MCF-7, a drug sensitive cell line. In summary, azithromycin, doxycycline and ciprofloxacin suppress the growth of breast cancer cells and augment the cytotoxicity of 2-DG to breast cancer cells. These findings suggest we can treat cancer with antibiotics similar to treating an infection. Citation Format: Xianpeng Jiang, Amanda Benoit, Toby Jiang, Robert L. Elliott. Antibiotics suppress growth of breast cancer cells and synergize cytotoxicity of 2-Deoxy-D-glucose: Treating cancer like an infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3600.

Objective: Determine whether LDH-A knockdown enhances CAR T-cell tumor-targeting and treatment response.Background: It has been shown that T-cells are restricted from entering many human and murine solid tumors, including both murine and human prostate tumors. CD3(+) T-cells are restricted from Myc-CaP murine prostate tumors and localize around the periphery of the tumor nodules (1). Human prostate specific membrane antigen (hPSMA) targeted CAR T-cells are also restricted from hPSMA(+) Myc-CaP tumors and this is only partially reversed by anti-PD1 treatment (1). This report describes the benefit of LDH-A depletion and a greater response of CAR T-cells targeting Myc-CaP tumors with low expression of LDH-A. Methods: LDH-A depletion in hPSMA(+) Myc-CaP cells bearing a bioluminescence reporter (Renilla Luciferase), was achieved by shRNA knockdown (KD) (1). A scrambled IgG shRNA was used as a control (NC). LDH-A expression was quantified by digital droplet PCR (ddPCR), Western blotting and LDH enzyme activity. The glycolytic activity of KD and NC cells was measured using Seahorse XF96 and XFp analyzers. Intratumoral lactate levels were monitored by magnetic resonance spectroscopy (MRS). The preparation and characterization of “second generation” hPSMA-directed CAR T-cells and the hPSMA(+) Myc-CaP tumor models in SCID mice have been described (1). To monitor CAR T-cell trafficking, T-cells were transduced with a Click Beetle Red luciferase reporter to enable efficient visualization by bioluminescence imaging (BLI) of CAR T-cell trafficking, persistence and viability within the hPSMA(+)Myc-CaP tumor mass. Tumor volume was calculated from caliper measurements. CD31 and PD-L1 expression was quantified with immunofluorescent staining and Metamorph Offline image analysis. Results and Discussion: These studies demonstrated that LDH-A KD was the dominant factor in reducing tumor growth. The difference in tumor doubling time (DT) between KD and NC tumors in the presence of CAR T-cell therapy was significant (p Citation Format: Mayuresh M. Mane, Khalid Shalaby, Ivan Cohen, Avi Albeg, Jenny Ijoma, Myat Ko, Masatomo Maeda, Kiranmayi Vemuri, Jaya Satagopan, Anna Moroz, Juan Zurita, Larissa Shenker, Ellen Ackerstaff, Masahiro Shindo, Ekaterina Moroz, Maxim A. Moroz, Inna Serganova, Jason Koutcher, Vladimir Ponomarev, Ronald G. Blasberg. The effect of lactate dehydrogenase-A (LDH-A) knockdown and human prostate-specific membrane antigen (hPSMA) directed CAR T-cell treatment on hPSMA(+) Myc-CaP tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A089.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a hereditary cancer syndrome in which affected individuals are at risk for development of cutaneous and uterine leiomyomas and an aggressive form of type II papillary kidney cancer. HLRCC is characterized by germline mutation of the tricarboxylic acid (TCA) cycle enzyme, fumarate hydratase (FH). FH-deficient kidney cancer is characterized by impaired oxidative phosphorylation and a metabolic shift to aerobic glycolysis, a form of metabolic reprogramming referred to as the Warburg effect. Increased glycolysis generates ATP needed for increased cell proliferation. In FH-deficient kidney cancer, levels of AMP-activated protein kinase (AMPK), a cellular energy sensor, are decreased resulting in diminished p53 levels, decreased expression of the iron importer, DMT1, leading to low cellular iron levels, and to enhanced fatty acid synthesis by diminishing phosphorylation of acetyl CoA carboxylase, a rate-limiting step for fatty acid synthesis. Increased fumarate and decreased iron levels in FH-deficient kidney cancer cells inactivate prolyl hydroxylases, leading to stabilization of hypoxia-inducible factor (HIF)-1α and increased expression of genes such as VEGF and glucose transporter 1 (GLUT1) to provide fuel needed for rapid growth demands. Several therapeutic approaches for targeting the metabolic basis of FH-deficient kidney cancer are under development or are being evaluated in clinical trials, including the use of agents such as metformin, which would reverse the inactivation of AMPK, approaches to inhibit glucose transport, lactate dehydrogenase A (LDHA), the antioxidant response pathway, the heme oxygenase pathway, and approaches to target the tumor vasculature and glucose transport with agents such as bevacizumab and erlotinib. These same types of metabolic shifts, to aerobic glycolysis with decreased oxidative phosphorylation, have been found in a wide variety of other cancer types. Targeting the metabolic basis of a rare cancer such as FH-deficient kidney cancer will hopefully provide insights into the development of effective forms of therapies for other, more common forms of cancer. Clin Cancer Res; 19(13); 3345–52. ©2013 AACR.

OBJECTIVES: Since the cancer stem-like cells (CSCs) play an important role in the resistance against radiation and/or chemotherapy due to its different metabolic profile, to investigate the specific metabolic characteristics of CSCs is valuable. Dynamic nuclear polarization (DNP) technique using 13C-labeled substrates enables magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors. In this study, DNP-MRI along with hyperpolarized [1-13C] pyruvate was conducted to evaluate the difference in glycolytic profile between a glioblastoma cell line and a CSC-derived glioma using orthotopic xenograft mouse model. The imaging findings were compared to those in immunohistology and cell cultures. METHODS: A 3T MRI scanner along with a custom-made head coil was used for imaging. To obtain hyperpolarized [1-13C] pyruvate, 30 μL of [1-13C] pyruvic acid containing 15 mM of OX063 (trityl radical compound) and 2.5 mM of the gadolinium chelate were polarized at 3.35 T and 1.4 K in the Hypersense DNP polarizer. The hyperpolarized sample dissolved in a buffered medium was injected via the tail vein followed by a 13C-two-dimensional spectroscopic imaging. During the acquisition, the body temperature was kept in the range of 35.0 to 36.9 Celsius degrees. Orthotopic brain tumor model was developed in nude mice by intracranial implantation of glioblastoma cell lines, U251 and glioma stem-like cells, NSC11. DNP-MRI was performed when the tumor size ranged 50 + 10 mm3 on T2-weighted MRI. Independently, immunofluorescent analyses of monocarboxylate transporter 1(MCT1) and MCT4 was performed using paraffin-embedded tumor tissue. Expressed protein level of lactate dehydrogenase A (LDHA), LDHB, MCT1 and MCT4 under the normal cell culture conditions were evaluated by Western blotting. RESULTS: 13C-MRI showed an increase in lactate-to-pyruvate ratio (Lac/Pyr) both in U251 and NSC11 compared to the contralateral normal brain tissue, suggested elevated anaerobic glycolysis (P CONCLUSION: The quality of 13C-DNP-MRI using [1-13C] pyruvate was validated and considered to be feasible to apply to the brain tumors. The result was reasonable in that malignant tumors enhance anaerobic glycolytic pathway even in the aerobic environment. 13C-DNP-MRI also demonstrated a difference in the extent of Lac/Pyr in relation to the normal brain between the two tumor models probably related to expression levels of glycolysis-related proteins. Citation Format: Tatsuya Kawai, Murali C. Krishna, Jeffery R. Brender, Jennifer Lee, Tamalee Kramp, Kazu Yamamoto, Shun Kishimoto, Tomohiro Seki, Kevin A. Camphausen. Metabolic imaging of glioblastoma using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4110.

Increased conversion of pyruvate to lactate by Lactate Dehydrogenase A (LDHA) is a feature of many neoplasms. Therefore, LDHA inhibition is considered a promising approach toward developing a new therapeutic strategy against cancers expressing the Warburg phenotype. To develop this strategy for clinical use, a feasible and sensitive noninvasive imaging approach that can dynamically evaluate LDHA activity in vivo would be highly beneficial since in vitro metabolic profiling dose not always predict in vivo cancer metabolism. Hyperpolarized (HP) 13C Magnetic Resonance Imaging (MRI) can be used to perform dynamic 13C metabolic flux analysis in vivo. In particular, use of [1-13C]pyruvate (13C-pyr) with 13C MRI permits real-time monitoring of intratumoral LDHA activity through dynamic observation of conversion of 13C-pyr to [1-13C]lactate (13C-lac). This study aimed to apply 13C MRI technology in support of a therapeutic strategy to explore efficacy of a newly developed and highly potent LDH inhibitor (LDHi) in a glycolytic tumor model using MiaPaCa2 xenografts in mice. In vitro analysis showed that the LDHi dose-dependently inhibited human LDH activity and suppressed in vitro cell growth in MiaPaCa2 cells. By ex vivo assay, LDH activity in MiaPaCa2 xenografts was significantly suppressed (82.2 ± 5.6 % decrease compared to vehicle controls) following a single intravenous(IV) injection of 50 mg/kg LDHi. Next, 13C MRI imaging of HP 13C-pyr metabolism was performed before and after a single LDHi IV injection to assess inhibitor impact on intratumoral metabolic flux in vivo. 13C MR spectroscopy confirmed that LDHi suppressed intratumor LDHA activity dose- and time-dependently. The maximum effective dose was 50 mg/kg and inhibitor impact in the tumor persisted for 10-12 hrs after a single injection. In addition, lactate production in the tumor was suppressed 30 minutes after IV administration of LDHi, as was the 13C-lac to 13C-pyr ratio decreased by 83.3 ± 4.4 % compared to vehicle controls. Importantly, the close correlation of these data with the results of the ex vivo LDH activity assay, suggests that 13C MRI can reliably monitor in vivo on target effects of LDHi without need for tissue sampling. Based on the data using 13C MRI, we developed a therapeutic strategy for using LDHi in an efficacy study of MiaPaCa2 xenografts. Intermittent IV administration of LDHi significantly suppressed tumor growth in this glycolytic model. In conclusion, intratumoral inhibition of LDHA in vivo upon IV administration of a novel LDHi was readily visualized by HP 13C MRI, confirming the utility of this noninvasive method. This methodology can be of great value in developing new therapeutic strategies using LDHi and perhaps other metabolic inhibitors to treat cancers characterized by the Warburg phenotype. Further, HP 13C MRI should allow for selection of those patients likely to respond to such treatments. Citation Format: Nobu Oshima, Shun Kishimoto, Kristin Beebe, Dan Crooks, Michael Moses, Kazutoshi Yamamoto, Jeffry R. Brender, Anastasia Sowers, Ganesha Rai, Daniel Urban, Goria Benavides, Giuseppe Squadrito, Victor Darley-Usmar, Matthew Hall, James B. Mitchell, Murali C. Krishna, Leonard M. Neckers. Evaluation of a novel LDH inhibitor efficacy in vivo in a glycolytic cancer model using hyperpolarized 13C magnetic resonance imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4104.

Although cancers have altered glucose metabolism, termed the Warburg effect, which describes the increased uptake and conversion of glucose to lactate by cancer cells under adequate oxygen tension, changes in the metabolism of glutamine and fatty acid have also been documented. The MYC oncogene, which contributes to the genesis of many human cancers, encodes a transcription factor c-Myc, which links altered cellular metabolism to tumorigenesis. c-Myc regulates genes involved in the biogenesis of ribosomes and mitochondria, and regulation of glucose and glutamine metabolism. With E2F1, c-Myc induces genes involved in nucleotide metabolism and DNA replication, and microRNAs that homeostatically attenuate E2F1 expression. With the hypoxia inducible transcription factor HIF-1, ectopic c-Myc cooperatively induces a transcriptional program for hypoxic adaptation. Myc regulates gene expression either directly, such as glycolytic genes including lactate dehydrogenase A (LDHA), or indirectly, such as repression of microRNAs miR-23a/b to increase glutaminase (GLS) protein expression and glutamine metabolism. Ectopic MYC expression in cancers, therefore, could concurrently drive aerobic glycolysis and/or oxidative phosphorylation to provide sufficient energy and anabolic substrates for cell growth and proliferation in the context of the tumor microenvironment. Collectively, these studies indicate that Myc-mediated altered cancer cell energy metabolism could be translated for the development of new anticancer therapies. (Clin Cancer Res 2009;15(21):6479–83)

The MYC oncogene belongs to the family of MYC genes, including MYCN (N-MYC) and MYCL (L-MYC), which are linked to human cancers such as Burkitt's lymphoma, neuroblastoma and lung cancer, respectively. MYC proteins belong to the MAX-MLX network of heterodimeric transcription factors that bind ‘E-boxes’ (5’-CACGTG-3’) to regulate genes involved in cell proliferation, differentiation and metabolism. MYC is downstream of many signal transduction pathways in normal cells; when stimulated through pathways such as tyrosine receptor kinases, MYC is induced to produce the MYC protein that dimerizes with MAX to bind DNA and regulate a transcriptional program of metabolism, cell growth and proliferation (1). MYC is under the control of growth factor signaling as well as nutrient availability, such that nutrient deprivation could result in diminished normal MYC expression and cell growth arrest. By contrast, deregulation of MYC in normal cells results in MYC binding not only to high affinity physiological binding sites but also invading into low-affinity binding sites and enhancers, resulting in an imbalance amplification of gene expression that triggers stress and cell death through activation of checkpoints such as p53. Elimination of p53 during tumorigenesis, however, can unleash MYC's transcriptional power to drive deregulated cell growth that renders cells addicted to nutrients, such that deprivation of glucose or glutamine results in cell death. The conceptual framework has been exploited to target metabolism for therapy against MYC-driven cancers, particularly aiming at glycolysis and glutaminolysis - metabolic processes that are increased by MYC. While lactate dehydrogenase A (LDHA) inhibition could curb MYC-mediated tumorigenesis (2), glutaminolysis remains an alternative survival pathway that could be targeted through inhibition of glutaminase (3-9). Glutaminase is normally expressed from two different genes: GLS that is normally expressed in kidney and brain, and GLS2 that is normally expressed in liver. Indeed, we have shown that knockdown of glutaminase (GLS) or inhibition with a small molecule (BPTES) diminishes the progression of a MYC-inducible human lymphoma xenograft model (3-7). We further documented that glutaminase (Gls) is induced by MYC and is required for early tumor development in a MYC-inducible model of mouse liver cancer (8), which displayed a decrease in the expression of normal liver Gls2. In this regard, the isoform switch from Gls2 to Gls1 in mouse (and also human) liver cancer renders tumors vulnerable to loss of one copy of Gls, which delayed tumorigenesis. We further showed that treatment with BPTES as a single agent was sufficient to prolong survival of mice bearing these MYC-induced liver cancers, providing proof-of-concept that targeting a single enzyme, in this case GLS, could change the course of the disease (8). Targeting GLS, however, has limitations, since the oncogenotypes of cancers result in different re-wiring of metabolism that is best illustrated by metabolomics studies of different mouse models of cancer (10). The MET oncogene-driven liver cancer model largely relies on glucose, whereas MYC oncogene-driven liver and lung cancers rely on both glutamine and glucose. As such, glutaminase inhibition should be considered in the context of the oncogenotype and metabolic profile of specific cancer types. Further, targeting metabolism could be constrained by the circadian regulation of cellular metabolism, rendering proliferating normal cells more vulnerable to inhibition of metabolism at specific times of the day. In this regard, we tested the hypothesis that high oncogenic MYC would ectopically invade the circadian Clock regulated genes that are also driven by E-boxes (11). The cell intrinsic clock machinery comprises of the central Clock-Bmal1 transcription factor, which induces Rev-erb’s, Cry’s, and Per's that in turn negatively regulated Bmal1 expression or Clock-Bmal1 levels, resulting in a circadian oscillation of Clock-Bmal1 function. Oscillation of the central clock transcription, which drives metabolic genes with E-boxes, results in oscillatory metabolism. Indeed, using an inducible MYC-ER system, we demonstrate that MYC could activate the negative clock regulators PER, CRY, and REV-ERBs and documented that the suppression of Bmal1 expression by MYC is mediated transcriptionally through REV-ERBs. Metabolic profiling in time-series experiments reveals oscillation of glucose and glutamine metabolic in the MYC-OFF state in U2OS cells that have very low basal endogenous MYC expression. In the MYC-ON state, intracellular glucose levels cease to oscillate and are barely detectable by NMR. Additional evidence suggests that it is converted toward lactate and biomass for cell growth. MYC, hence, can invade and disrupt the molecular circadian clock as well as metabolism, in favor of an anabolic growth program. These observations have key implications for targeting metabolic enzymes that have been documented as being induced by Clock-Bmal1 and MYC. For example, ornithine decarboxylase (ODC) and nicotinamide phosphoribosyltransferase (NAMPT) are common targets of MYC and Clock-Bmal1. Hence, we hypothesize that the dose-limiting toxicity of NAMPT inhibition, being thrombocytopenia (12), could be curbed in a lymphoma xenograft model by applying chronotherapy. In this regard, our preliminary studies illustrate that while NAMPT inhibition at two different times of the day resulted in similar efficacy in reducing lymphoma xenograft growth, one of administration time resulted in thrombocytopenia while the other time of drug administration was indistinguishable from the control treated animals (unpublished). These observations indicate that the combination of identifying metabolic vulnerabilities of MYC-driven cancer along with understanding circadian could strategically guide the use of metabolism-targeted drugs in the clinic through oncogenotyping and potentially chronotherapy. 1.Stine ZE, et al. MYC, Metabolism, and Cancer. Cancer Discov. 2015 Oct;5(10):1024-39. 2. Le A, et al. Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2037-42. 3. Gao P, et al. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature. 2009 Apr 9;458(7239):762-5. 4. Wang JB, et al. Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer Cell. 2010 Sep 14;18(3):207-19. 5. Le A, et al. Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell Metab. 2012 Jan 4;15(1):110-21. 6. Shukla K, et al. Design, synthesis, and pharmacological evaluation of bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) analogs as glutaminase inhibitors. J Med Chem. 2012 Dec 13;55(23):10551-63. 7. Dutta P, et al. Evaluation of LDH-A and glutaminase inhibition in vivo by hyperpolarized 13C-pyruvate magnetic resonance spectroscopy of tumors. Cancer Res. 2013 Jul 15;73(14):4190-5. 8. Xiang Y, et al. Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis. J Clin Invest. 2015 Jun;125(6):2293-306. 9. Shroff EH, et al. MYC oncogene overexpression drives renal cell carcinoma in a mouse model through glutamine metabolism. Proc Natl Acad Sci U S A. 2015 May 26;112(21):6539-44. 10. Yuneva MO, et al. The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type. Cell Metab. 2012 Feb 8;15(2):157-70. 11. Altman BJ, et al. MYC Disrupts the Circadian Clock and Metabolism in Cancer Cells. Cell Metab. 2015 Sep 16. pii: S1550-4131(15)00460-X. doi: 10.1016/j.cmet.2015.09.003. 12. von Heideman A, et al. Safety and efficacy of NAD depleting cancer drugs: results of a phase I clinical trial of CHS 828 and overview of published data. Cancer Chemother Pharmacol. 2010 May;65(6):1165-72. Citation Format: Chi Van Dang. MYC-mediated metabolic vulnerabilities and the circadian clock. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY12-03.

Neuroblastoma (NB) is a common pediatric malignancy that often carries a poor prognosis. Aerobic glycolysis, i.e. the Warburg effect, is a hallmark of cancers, including NB. Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate. It is considered a pivotal component of the LDH tetramer, which is central in the Warburg effect. LDHA is overexpressed in many cancers. Little is known about the role of LDHA in NB. We therefore determined the expression of LDHA mRNA and protein in 651 and 112 clinically annotated patient samples, respectively. We show that enhanced mRNA expression and predominant cytoplasmic protein expression of LDHA, as detected by immunohistochemistry, is markedly associated with increased tumor aggressiveness and decreased patient survival. Surprisingly, despite the proposed pivotal role of LDHA in the Warburg effect, LDHA was dispensable for aerobic glycolysis in NB cells. Complete depletion of LDHA by CRISPR/Cas9-mediated knock-out in several NB cell lines did not decrease glucose consumption and production of pyruvate and ATP. In accordance, LDH activity was maintained despite loss of LDHA, as LDHB and LDHC were still expressed, most likely compensating for the loss of LDHA. In line with LDHA being dispensable for aerobic glycolysis in NB cells, LDHA depletion did not decrease malignant characteristics of NB cells. Thus, clonogenic growth, migration and tumorigenicity remained unaffected. Extending these findings from manifest to developing NB, we show that in the TH-MYCN NB mouse model the development of NB from normal sympathetic ganglia is not associated with increased LDHA expression. Taken together, this work establishes LDHA as a novel predictive marker in NB and calls into question the pivotal role in aerobic glycolysis attributed to LDHA. Citation Format: Carmen Dorneburg, Lisa Christner, Thomas F. Barth, Wolfgang Mueller-Klieser, Matthias Fischer, Hero Barbara, Anneleen Beckers, Frank Speleman, Klaus-Michael Debatin, Christian Beltinger. High LDHA expression predicts decreased survival in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-051. doi:10.1158/1538-7445.AM2017-LB-051

Rates of obesity are increasing and 640 million adults were considered obese in 2014 an increase of 27.5% over the past three decades. Obesity is strongly linked with increased cancer risk and particularly so for liver cancer in men. Although virus infection had been the leading cause of hepatocellular carcinoma (HCC) in the past, the incidence of obesity induced Non-Alcoholic Fatty Liver Disease (NAFLD) a precursor to HCC is increasing and the number of new HCC cases increased 38% and HCC deaths 56% in the last decade. In this study, we conducted meta-analysis of gene expression profiles obtained from livers of high fat diet fed and chemically induced liver cancers of male C57BL/6 mice in order to elucidate the characteristics of fatty liver and HCC. Three separate studies fed mice high fat diets with 60% kcal% fat (from lard and soy bean oil) diet starting at 7 or 8 week old, and continued for 7-10 weeks (7 weeks, GSE40638; 10 weeks, GSE52333; and 8 weeks, GSE79434). We compared these three studies with a liver cancer study (GSE51188) which induced cancers following a single dose of 25 mg/kg diethyl nitrosamine. Affymetrix Mouse Gene 1.0 ST Array was used for high fat diet studies, and Illumina MouseRef-8 x2.0 BeadChips was used for cancer study. We analyzed each study data with GEO2R (comparison against respective normal liver) and performed meta-analysis of the four data sets by Advaita Pathway Guide software. The analysis identified 118 genes ≥0.2 log2 fold-change (Log2FC) and 0.05 for 1/3 high fat diet studies to increase the candidate genes, but the direction of changes is the same with other two studies). Among these 118 genes, 22 genes were up-regulated and another 17 genes were down-regulated in all four data sets. 29 genes were elevated in tumor, but lowered in high fat diet. On the other hand, 50 genes were lowered in tumor and elevated in high fat diet. We identified Annexin A2 (Anxa2) among the 22 genes which were up-regulated in all data sets, Anxa2 has consistently been reported to be increased in liver tumors and is associated with poor prognosis. The Anxa2 finding suggests our methodology is robust. Besides Anxa2, Nucleoporin 62 (Nup62), Lactate Dehydrogenase A (Ldha), Oxysterol Binding Protein-Like 3 (Osbpl3) were also up-regulated in all studies (Log2FC of liver tumor: Anxa2=2.75, Nup62=0.35, Ldha=0.53, Osbpl3=1.82). Osbpl3 had higher Log2FC value following Anxa2, and its expression was found to be upregulated in some cancers. Importantly, all these were significantly associated with lower overall survival rates in male HCC patients of The Cancer Genome Atlas (TCGA Genes; ANXA2, p=0.00032; NUP62, p=0.000003; LDHA, p=0.0154; OSBPL3, p=0.0255). This data analysis identified several new genes that may be involved in developing cancer from fatty liver, and they might be useful cancer biomarkers based on their association with poor survival. Citation Format: Kumiko Kato-Shoji, Noriyuki Miyoshi, Miki Igarashi, John I. Risinger, Yutaka Shoji. Meta analysis of gene transcripts from fatty livers and liver cancers of C57BL6 mice identifies potential drivers of HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-281. doi:10.1158/1538-7445.AM2017-LB-281

[aim] Increased lactate production is a feature of many neoplasms, and Lactate Dehydrogenase A (LDH-A) plays a key role in conversion of pyruvate to lactate. LDHA inhibition, therefore, is considered to be a promising approach toward developing a new therapeutic strategy for cancer treatment focused on targeting cancer metabolism. Non-invasive imaging approaches able to monitor metabolic fluxes in vivo will be useful for this purpose. Hyperpolarized 13C Magnetic Resonance Imaging (MRI) has been well known as a valuable technology to investigate metabolic processes in tumor xenografts, allowing us to perform dynamic 13C-metabolic flux analysis in vivo. Use of [1-13C]pyruvate with this technology provides the ability to monitor LDHA activity in real time through dynamic observation of conversion of [1-13C]pyruvate to [1-13C]lactate. This study aimed to monitor drug efficacy of a newly developed LDH inhibitor (LDHI, obtained from National Cancer Institute Experimental Therapeutics Program, NExT) in a xenograft tumor model using 13C MRI technology with hyperpolarized 13C-labeled pyruvate. [Results] Hyperpolarized [1-13C]pyruvate MR studies were performed before and after LDHI administration to assess the impact on metabolic flux in vivo. Using hyperpolarized [1-13C]pyruvate MR Spectroscopy (MRS), we found that lactate production was significantly suppressed by LDHI administration in MiaPaca (a glycolytic pancreatic cancer cell line) tumors, as was the [1-13C]lactate to [1-13C]pyruvate ratio ([1-13C]-Lac/Pyr), which was calculated from the areas under the curves (AUC) using time-intensity data. This ratio decreased from 1.08 to 0.128 (88.1% decrease) 30 minutes after intravenous administration of the LDHI. In addition, hyperpolarized [1-13C]pyruvate MRS revealed that LDHI significantly suppressed lactate production in a dose dependent manner. Furthermore, Chemical Shift Imaging with 13C MRI demonstrated that the [1-13C]lactate signal in each voxel clearly decreased, compared to that before LDHI administration. The sum of [1-13C]lactate signals in the tumor region decreased after LDHI administration, resulting in a significant decrease in the tumor-specific [1-13C] Lac/Pyr ratio (1.463±0.31 before LDHI to 0.134±0.036 30 minutes after LDHI administration, a 90.67±2.56% decrease, n=3, p [Conclusions] These results indicate that hyperpolarized 13C-MRI is a useful method to evaluate on-target efficacy of novel LDH inhibitors in vivo, and this technique can be used to determine optimum dose and exposure time of the LDHI in the tumor region. The current method can be of great value in providing an in vivo pharmacodynamic biomarker for this novel anti-cancer therapeutic targeting deregulated tumor metabolism. Citation Format: Nobu Oshima, Shun Kishimoto, Kristin Beebe, Keita Saito, Kazutoshi Yamamoto, Jeffery Brender, Anastasia Sowers, Ganesha Rai, Bryan T. Mott, David J. Maloney, James B. Mitchell, Murali K. Cherukuri, Leonard M. Neckers. Monitoring the impact on metabolic flux in vivo of a newly developed LDH inhibitor using hyperpolarized 13C magnetic resonance spectroscopic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2017-2852

Introduction and objectives: It is well known as Warburg effect that anaerobic glycolytic pathway is activated in various type of advanced cancers including prostate cancer (PC). Lactate dehydrogenase-A (LDH-A) controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. Since LDH-A pathways have been implicated in chemoresistance in various cancers, we investigated whether inhibition of LDH-A pathway could mediate the sensitivity to docetaxel (DOC) in human PC cells. Materials and Method: Four PC cell lines (PC3, DU145, LNCaP, LN-CSS ) were used. LN-CSS is one of the LNCaP-derived castration-resistant PC (CRPC) cell lines established in our laboratories. Sodium oxamate (SO) was used as a specific LDH-A inhibitor. The protein expression was detected by western blot analysis using specific antibodies. Cell growth and survival were evaluated by WST-1 assays. Cell cycle progression and apoptotic inducibility were evaluated by flow cytometry using propidium iodide and Annexin V. The cytotoxicity of SO/DOC combination on PC cells was evaluated using the Chou-Talalay combination index (CI) method which offers quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations. Result: Western blot analysis showed that LDH-A protein was highly expressed in LN-CSS cells compared with other PC cell lines including the parental LNCaP. WST-1 assays showed that treatment with SO (50 mM) for 72 hours in PC cells resulted in growth inhibition (PC3; ~30%, DU145; ~55%, LNCaP; ~20%,LN-CSS; ~55% ), while SO has little growth inhibitory effects on normal lymphocytes in the concentrations between 1-100 mM. IC50 to DOC in PC cells were showed to be 4 nM, 1 nM, 1nM, and 4.5 nM in PC3, DU145, LNCaP and LN-CSS, respectively, suggesting that both PC3 and LN-CSS were relatively resistant to DOC compared with DU145 and LNCaP. Synergistic cytotoxicity was observed after the combination therapy with DOC and SO in LN-CSS (CI: 0.5) but not in PC3 (CI: 1.9), DU145 (CI: 2.0), or LNCaP (CI: 6.5). Cell cycle analyses revealed that the combination with DOC and SO for 72 hours resulted in the accumulation of cells in G2-M phase followed by sub-G1 accumulation in LN-CSS cells. Annexin V assays showed that 43% apoptosis was induced by the combination therapy in LN-CSS cells, while only 12% by DOC only in LN-CSS cells. Conclusion: Our results strongly suggest that LDH-A plays an important role in DOC resistance in advanced PC cells and inhibition of LDH-A promotes DOC-sensitivity especially in CRPC cells. Our study may provide valuable information for the future development of targeted therapies in patients with CRPC. Citation Format: Hiroyuki Muramatsu, Makoto Sumitomo, Shingo Morinaga, Hiroshi Saiki, Ikuo Kobayashi, Keishi Kajikawa, Genya Nishikawa, Yoshiharu Kato, Masahito Watanabe, Kent Kanao, Kogenta Nakamura, kazuhiro Yoshikawa. Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4400. doi:10.1158/1538-7445.AM2017-4400

Background: Breast cancer (BC) has historically been considered immunologically silent; however, several observations indicate that potentiation of immune functions can benefit BC patients. Intratumor T-cell infiltration has prognostic significance in patients with BC across different molecular and histological categories. In addition, PD-L1 can be overexpressed in BC, in particular in the highly aggressive triple negative BC (TNBC) subtype. This is associated with poor prognosis specifically in patients with luminal B and basal-like phenotypes, thus making these subtypes rational targets of PD-1/PD-L1 axis blockade treatments. However, initial results from early-phase clinical trials show a modest activity of immune checkpoint blockade monotherapy against BC, with 19% TNBC and 3-12% hormone receptor-positive patients achieving a clinical response to anti-PD-1/PD-L1 therapies. Lack of inflammation and T-cell infiltration at the tumor site are characteristic features of tumors that do not respond to checkpoint blockade. Since BC is typically poorly infiltrated by T cells, having strategies that reverse this immune exclusion as well as non-invasive modalities to predict this effect are crucial to improve the clinical efficacy of immune checkpoint blockade. Experimental Design: Our working model is that tumor glycolysis (lactate) and T-cell infiltration are mechanistically interdependent, since we reasoned that a highly glycolytic tumor microenvironment (due to lactate production) could hamper survival, expansion and effector functions of T cells in BC lesions, thus explaining T-cell exclusion. Therefore, we tested whether genetic and/or pharmacologic inhibition of lactate production/consumption could restore intratumor T-cell infiltration and immune function in BC models, thus favoring tumor responsiveness to checkpoint blockade. Results: By interrogating a compendium of 4 BC patient gene expression datasets, we found that patients harboring tumors with high expression of lactate dehydrogenase A (LDH-A) or the lactate transporters MCT-1/4 have a significantly higher risk to develop metastases (p Citation Format: Roberta Zappasodi, Arnab Ghosh, Inna Serganova, Ivan Cohen, Yasin Senbabaoglu, Masahiro Shindo, Mayuresh M. Mane, Avigdor Leftin, Ellen Ackerstaff, Jason A. Koutcher, Jedd D. Wolchok, Ronald G. Blasberg, Taha Merghoub. Overcoming intratumor T-cell exclusion by modulation of lactate metabolism to improve immune checkpoint therapies in aggressive breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR06.

Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the KRAS mutation status, share one common parameter which is increased reactive oxygen species (ROS) when compared to their parental cells counterpart. Importantly, we found that CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism (OXMET) for energy and biosynthesis. Thus, alteration in mitochondria which resulted in metabolic changes and higher basal levels of ROS may be one of the major factors contributing to cisplatin resistance. We have investigated mitochondrial morphology in parental vs. CR cells using transmission electron microscopy (TEM). Our data clearly showed that CR cells possessed significantly higher number of mitochondria per total cell area (n = 4; p = 0.0006). CR cells also consumed 2-4 fold more oxygen when compared to their parental cells. To further confirm that oxidative phosphorylation was utilized more in CR cells, we assayed mitochondrial membrane potential (MMP) using TMRE staining. MMP was significantly higher in CR cells suggesting that CR cells possess highly active mitochondria. Key glycolytic enzymes hexokinase II (HKII) and lactate dehydrogenase A (LDHA), as well as lactate production were decreased in all CR cell lines which corresponded to the resistance to glycolytic inhibitor, 2DG (n = 8; p Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Sumedh Shah, Shu-Mei Chen, Vy Dinh, Macus T. Kuo, Lynn G. Feun, Niramol Savaraj. Exploiting ROS and metabolic differences to selectively killing cisplatin resistant lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1038.

Metabolic reprogramming is a hallmark of cancer. Elevated glycolysis in cancer cells switches the cellular metabolic flux to produce more biological building blocks, thereby facilitating cellular adaptation to the adverse environment and sustaining rapid proliferation. Recently, new evidence has emerged that abnormal metabolic reprogramming may occur at the early-stages of neoplasia and critically contribute to early transformation and cancer initiation. Previously, we reported that 14-3-3ζ overexpression is an early event in human breast cancer, taking place as early as in the atypical ductal hyperplasia (ADH) stage. We therefore hypothesized that 14-3-3ζ overexpression in breast epithelial cells may trigger metabolic reprogramming by elevating glycolysis that contributes to cancer initiation. In the present study, our bioinfomatics analysis of microarray profiling data from the early-stages breast neoplastic lesions revealed that 14-3-3ζ expression is strongly correlated with the expression of genes of the canonical glycolytic pathway, particularly lactate dehydrogenase A (LDHA). Experimentally, increasing 14-3-3ζ expression in human non-transformed mammary epithelial cells (hMECs), MCF10A and MCF12A, transcriptionally up-regulated LDHA expression, elevated glycolytic activity, and promoted early transformation of hMECs. Conversely, knockdown of LDHA in the 14-3-3ζ-overexpressing hMECs significantly reduced glycolytic activity and inhibited transformation. Together, our data demonstrate that overexpression of 14-3-3ζ in early stage pre-cancerous breast epithelial cells may trigger an elevated glycolysis by activating the MEK-ERK-CREB signaling axis and transcriptionally up-regulating LDHA, thereby critically contributes to human breast cancer initiation. Citation Format: Chia-Chi Chang, Chenyu Zhang, Ozgur Sahin, Qingling Zhang, Hai Wang, Jian Zhang, Sumaiyah Rehman, Ping Li, Dihua Yu. Upregulation of lactate dehydrogenase A by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-313.

Somatic mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) contribute to the pathogenesis of cancer via production of the ‘oncometabolite’ D-2-hydroxyglutarate (D-2HG). Elevated D-2HG can block differentiation of malignant cells by functioning as a competitive inhibitor of alpha-ketoglutarate (alpha-KG) dependent enzymes, including Jumonji family histone lysine demethylases. 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although cancer-associated IDH1/2 mutations exclusively produce D-2HG, biochemical studies have demonstrated that L-2HG functions as a more potent inhibitor of alpha-KG-dependent enzymes. Here we report that under conditions of oxygen limitation, mammalian cells selectively produce L-2HG via enzymatic reduction of alpha-KG. Hypoxia-induced L-2HG is not mediated by IDH1 or IDH2, but instead results from promiscuous substrate usage by lactate dehydrogenase A (LDHA). During hypoxia, the resulting increase in L-2HG is necessary and sufficient for the induction of increased methylation of histone repressive marks, including histone 3 lysine 9 (H3K9me3). Thus, L-2HG appears to function as a metabolic signaling intermediate, translating information about oxygen availability into epigenetic modifications that can influence gene expression and cellular differentiation. Citation Format: Craig B. Thompson, Andrew M. Intlekofer, Raymond G. Dematteo, Sriram Venneti, Lydia W. S. Finley, Chao Lu, Ariën S. Rustenburg, Patrick B. Grinaway, John D. Chodera, Justin R. Cross, Alexander R. Judkins. Stereochemistry matters: L-2HG as a tumor response to hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY43-01. doi:10.1158/1538-7445.AM2015-SY43-01

Introduction: Radiation therapy (RT) is an important treatment option for prostate cancer (CaP) patients at both early and advanced-stage disease. Many CaP patients suffer from relapse and metastasis due to radio-resistance. In this study, we aimed to develop a radio-resistant CaP (CaP-RR) xenograft model, to identify potential protein markers associated with radio-resistance using proteomic approach, and to validate the identified biomarkers in CaP cell lines and animal xenografts. Methods: Subcutaneous (s.c) CaP animal model was established using PC3-RR and PC3-control CaP cells in NODSCID mice. Tumor xenografts were harvested 5 weeks post cell inoculation and the proteins were extracted. Using LC-MS/MS expression protein profiles from the two groups were created. Statistical analysis was performed using one-way ANOVA for all features. Mascot was employed for peptide identification. Differential protein expressions were identified by re-importing the Mascot data to Progenesis software. The protein pathways and the top KEGG terms were searched against KEGG database using ingenuity software. Selected pathway markers or modulators were validated in CaP-RR cell lines and xenograft tumors using western blotting and immunohistochemistry, respectively. Lactate dehydrogenase A (LDHA) was selected for functional studies including radio-sensitivity, apoptosis and autophagy assays. Results: We found for the first time that higher levels of vascularity, proliferation, and proteins from the glycolysis pathway are associated with CaP-RR xenograft tumors and that more than two hundred proteins were differentially expressed between CaP-RR and CaP naïve tumor xenografts. In addition, we demonstrated that the increased expressions of glycolysis markers, including LDHA, were certified in both CaP- CaP-RR cell lines and xenograft tumors. Knock-down or inhibition of LDHA combined with RT increase the radiosensitivity in PC3-RR cells, induce more apoptosis and autophagy compared to each single treatment alone. Conclusions: We have successfully identified protein signatures using a CaP-RR xenograft animal model. The glycolysis pathway protein LDHA is a promising candidate to develop combination therapy to overcome CaP radioresistance. Citation Format: Jingli Hao, Peter H. Graham, Lei Chang, Jie Ni, Valerie Wasinger, Julia Beretov, Joseph Bucci, Paul Cozzi, Yong Li. Identification of lactate dehydrogenase A (LDHA) as a potential therapeutic target for prostate cancer radiotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2001. doi:10.1158/1538-7445.AM2015-2001

Sirtuin-3 (SIRT3) is an important mitochondria NAD+-dependent deacetylase that is known to target mitochondrial proteins for deacetylation and regulates a variety of cellular functions. We have previously shown that SIRT3 was overexpressed in human melanoma cells and tissues and its genetic knockdown resulted in a significant antiproliferative response in human melanoma cells (In: AACR Annual Meeting: Proceedings; 2014 April 5-April 9, San Diego; CA, Abstract # 3516). Our data suggested that small molecule inhibitors of SIRT3 may be developed for the management of melanoma. In this study, we determined the anti-proliferative efficacy of a newly identified small molecule SIRT3 inhibitor, 4′-bromo-resveratrol, in human melanoma cell lines (G361, SK-MEL-28 and SK-MEL-2). Treatment of melanoma cells with 4-bromoresveratrol (0, 12.5, 25, 50, 100, and 200 × 10-3 mM; for 24, 48 and 72 hrs) resulted in a significant decrease in cell proliferation in a dose- and time- dependent manner. In addition, 4′-bromo-resveratrol treatment also resulted in a marked decrease in the growth, viability, clonogenic survival and migration of melanoma cells. Further, the anti-proliferative effects of 4′-bromo-resveratrol were accompanied by a marked downregulation of proliferating cell nuclear antigen (PCNA) protein as well as SIRT3 mRNA and protein. Furthermore, 4′-bromo-resveratrol treatment to melanoma cells resulted in 1) significant induction of apoptosis in melanoma cells, as evident from increase in PARP cleavage, decrease in pro-caspase-3, pro-caspase-8; and 2) G0/G1 phase cell cycle arrest. We also observed a marked increase in WAF-1/p21 protein levels and decreases in cyclin D1 and cdk2 protein levels as a result of 4′-bromo-resveratrol treatment in melanoma cells. Finally, we also found that 4′-bromo-resveratrol caused a metabolic reprograming in human melanoma cells as shown by decreases in the levels of lactate production, glucose uptake, NAD+/NADH ratio, which were accompanied by downregulation in protein levels of lactate dehydrogenase A (LDHA) and glucose transporter 1 (Glut1) in melanoma cells. Collectively, our data suggest that the small molecule SIRT3 inhibitors, including 4′-bromo-resveratrol, may be developed for melanoma management. However, detailed mechanistic studies as well as in vivo validation studies in appropriate animal model(s) are needed to ascertain the clinical potential of 4′-bromo-resveratrol and/or other SIRT3 inhibitors. Citation Format: Jasmine George, Minakshi Nihal, Chandra K. Singh, Nihal Ahmad. Small molecule SIRT3 inhibitor 4′-bromo-resveratrol inhibits proliferation, promotes apoptosis and causes metabolic reprograming of human melanoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1127. doi:10.1158/1538-7445.AM2015-1127

Background: Head and Neck (H&N) cancer is one of the most prevalent neoplasms worldwide. It is well accepted that H&N cancer development is a multistep process resulting from internal and external cellular/molecular and physiological challenges leading to a number of genetic, epigenetic and metabolic abnormalities. In comparison with mitochondrial energy production in normal cells, previous studies have shown that cancer cells preferentially utilize the by-products of glycolysis, regardless of oxygen status, as an energy source. In view of this physiological variance, many investigations demonstrated that manipulations of glycolytic enzymes could potentially modulate cell malignancy of various cancers. Lactate dehydrogenase A (LDHA), one of the glycolytic enzymes essential for conversion of pyruvate into lactate, has been previously emphasized for its potential cancer-promoting effect; nevertheless, LDHA-mediated regulation for cellular malignancy and its underlying molecular mechanisms during H&N carcinogenesis is still poorly understood. Methods: Current experimental scheme was two-folds. It was initiated by histological analysis for LDHA expression in normal and tumorous tongue epithelium from animals treated with oral carcinogen 4-Nitroquinoline 1-oxide (4-NQO) to define potential role of LDHA between normal and cancerous oral tissues. Further in vitro establishment of genetic (shRNA) and chemical (oxamate) induced LDHA deficient H&N cancer cells were carried out and multifaceted cancer phenotypes including cell growth/survival, cell motility, cell differentiation and sensitivity to chemotherapeutic drug cisplatin were examined in LDHA deficient cells. Results: 4-NQO treatment stimulated tongue epithelial hyperplasia as histological analysis showed progressive dysplasia/carcinoma correlated with longer (12-week) 4-NQO treatment. LDHA expression was detected in basal layer of normal mouse tongue epithelium and enriched in suprabasal layer in 4-NQO induced hyperplastic/dysplastic tongue precancerous epithelium as well as aggressive tongue carcinoma suggesting that LDHA plays an important role during tongue carcinogenesis. In agreement with in vivo data, LDHA is highly expressed in H&N cancer cells from different origins (tongue, oropharyngeal or oral squamous cells). LDHA deficient head and neck H&N cancer cells were successfully established and showed (1) decreased cell growth; (2) downregulated cell migration, invasion and anchorage-independent growth; (3) greater expression of differentiation marker involucrin and (4) increased cisplatin sensitivity in comparison with control cells revealing LDHA-mediated tumorigenic regulations. Conclusions: Present study confirmed that suppression of glycolytic enzymes LDHA could downregulate cell malignancy in H&N cancer cells. The outcomes are of great potential to develop the metabolism mediated anti-cancer treatment for H&N cancer patients. Citation Format: Tsai-Ying Chen, Wan-Jung Chang, Chang-Yi Chen, Wan-Chun Li. Regulatory role of lactate dehydrogenase A during head and neck carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1186. doi:10.1158/1538-7445.AM2015-1186

MYC, which is overexpressed in a variety of human cancers, drives aerobic glycolysis, also called the Warburg effect. The Warburg effect results in regeneration of NAD+ from increased conversion of pyruvate to lactate, such that decreased lactate dehydrogenase A (LDHA) activity results in NAD+ depletion and can result in cell death. Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme that is involved in the NAD+ salvage pathway, was reported to be directly upregulated by Myc. Increased NAD+ synthesis thus helps to replenish NAD+ along with LDHA. In this regard, we have previously reported the synergy of inhibiting LDHA and NAMPT in causing lymphoma xenograft regression. Targeting NAMPT by its inhibitor FK866 has been tested in clinical trials. However, the toxicity of FK866 in normal cells is not negligible, with thrombocytopenia being reported. Toxicity, however, could be diminished if therapy is administered at specific times of the day (termed chronotherapy) when normal cells are least dependent on NAMPT, whose levels oscillate every 24 hours. We have previously found that Myc upregulates Rev-erbα, which inhibits and disrupts oscillation of the circadian rhythm gene ARNTL (protein name Bmal1) in the osteosarcoma cell line U2OS. However, although NAMPT was shown oscillating in normal mouse liver and other tissues, how the oscillation of NAMPT is affected in cancer especially under Myc overexpression is still poorly understood. Here we show Myc induces Nampt mRNA and protein in mouse hepatocellular carcinoma (mHCC) cell line that has been engineered tetracycline inducible Myc transgene expression vector. Inhibition of NAMPT by FK866 induces perturbation of circadian gene expression in mHCC cells, including Rev-erbα, Per1 and Cry1. The alteration of circadian gene expression can be rescued by nicotinamide, suggesting that the circadian clock is affected by NAD+/NADH ratio. Interestingly, Nampt mRNA oscillates in mHCC cell lines in the absence of Myc and the oscillation was lost when Myc is induced, suggesting a therapeutic opportunity could exist in the window between circadian regulation of the ebb-and-flow of normal cell NAMPT level and the sustained, non-circadian cancer cell NAMPT level. Our observation that Myc disrupts the oscillation its target gene NAMPT provides a conceptual framework for metabolic chronotherapy that could potentially lead to better cancer treatment strategies that reduce side effects. We thank the following founding sources: NIH R01CA57341, LLS 6106-14. Citation Format: Annie L. Hsieh, Brian J. Altman, Anand Venkataraman, David I. Bellovin, Dean W. Felsher, John B. Hogenesch, Chi V. Dang. Oncogenic Myc disrupts NAMPT circadian oscillation in mouse hepatocellular carcinoma cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1419. doi:10.1158/1538-7445.AM2014-1419

Lactate Dehydrogenase A (LDHA) is an attractive candidate for targeting glycolysis-addicted tumors. However, due to the inherent plasticity of metabolic networks in cells, there is concern that the benefit of targeting LDHA may be transient and that resistance will quickly emerge. To identify predictive features of LHDA inhibitor sensitivity and to understand how cells adapt to long-term LHDA inhibition, we screened a large panel (∼500) of tumor cell lines with GNE-140, a newly developed LHDA inhibitor. We found that approximately 15% of lines were inherently sensitive to the LDHA inhibitor, with sensitivity correlating with increased expression of glycolysis genes and inversely correlating with expression of oxidative phosphorylation genes. Despite the metabolic plasticity of cells, the timing of acquired resistance to LDHA inhibitors was comparable with other targeted agents. Under long-term LDHAi treatment, glycolytic cells acquired resistance by increased oxidative phosporylation (OX-PHOS) in a mechanism dependent on the AMPK stress response pathway; targeting either AMPK, downstream kinases, or OX-PHOS using tool compounds synergized with and prevented acquired resistance to GNE-140. Taken together, our data suggests that targeting anaerobic glycolysis may benefit a subset of patients across indications and that combinations with agents that block AMPK signaling or the mitochondria will be effective at delaying tumor relapse. Citation Format: Aaron Boudreau, David Peterson, John Moffat, Bonnie Liu, Mandy Kwong, Min Gao, Hans Purkey, Thomas O'Brien, Georgia Hatzivassiliou, Anneleen Daemen, Marie Evangelista. Resistance to LDHA inhibitors requires signaling through the AMPK/mTOR/S6K pathway leading to increased oxidative phosphorylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1423. doi:10.1158/1538-7445.AM2014-1423

Triple-negative breast cancer (TNBC) represents 20-25% of sporadic breast cancers, lacks ER, PR, and overexpressed Her2 – and thus has no targeted treatment options. TNBC is the most clinically challenging subtype with exceptionally poor prognosis, high recurrence and metastases and currently represents an unmet medical need. While classified pathologically by negative criteria, TNBC is a heterogeneous group of breast cancers in need of improved molecular classification. Numerous studies have linked EGFR signaling to TNBC, but paradoxically, TNBC is refractory to the well-used EGFR inhibitors that have been efficacious in other cancers. In addition, Wnt signaling has been linked to TNBC, but its relationship to EGFR signaling and inhibitor resistance has not been explored. In the current work, the HBP1 transcriptional repressor may be a new and unappreciated bridge to both TNBC pathways. Previously, we have shown that decreases in HBP1: 1) triggered an increase in both Wnt and EGFR signaling to regulate proliferation and senescence; 2) were coincident with exceptional increased tumor growth and invasiveness in preclinical models of breast cancer; and 3) were associated with a poor breast cancer prognosis. While altered metabolism has been investigated in many cancers, the mechanisms by which EGFR and Wnt signaling might trigger a metabolic reprogramming have not been investigated in TNBC. Our data suggests that HBP1 may be a new integrating factor. Our new data show that HBP1 levels are significantly reduced in TNBC relative to other breast cancer subtypes in clinical specimens. We derived a new combined EGFR and HBP1 gene signature that predicted 90+% of TNBC patients in 4 large patient databases and additionally predicted a poor patient prognosis in non-TNBC patients. To understand how HBP1 decreases trigger a poor prognosis, a whole genome analysis revealed a surprising change in 300+ genes to enact a Warburg-like metabolic reprogramming. The Warburg effect is characterized by increased glycolytic flux with increased biosynthesis of amino acids, lipids, and nucleic acids'all to provide for the increased growth and proliferation demands of a tumor cell. The specific pattern was an increase in certain isoforms involved in glycolysis and TCA cycle and a decrease in the expression of genes involved in oxidative phosphorylation. We used gene-, biochemistry- and NMR-based approaches to investigate the metabolic alterations upon changes in HBP1 expression in TNBC cells and tumors. Our results show that hexokinase 2 (HK2), Pyruvate Kinase-M2 (PKM2), Lactate Dehydrogenase A (LDHA), malate dehydrogenase 2 (MDH2) are amongst the metabolic enzymes that are altered with HBP1 expression and with increased Wnt signaling. HK2, PKM2, LDHA and MDH2 have all been previously associated with the Warburg effect. The NMR and biochemical analysis showed that lactate and fumarate are amongst the metabolites that are altered. The results here provide important metabolomics information to impact MRI/MRS studies. While breast MRIs are used for assessing tumor morphology, MRS (magnetic resonance spectroscopy) methods can non-invasively visualize metabolic spectra in tumors or metastases. The metabolic changes from our studies potentially focus MRS/MRI applications for the future discovery of non-invasive MRS biomarkers to improve TNBC classification and treatment efficacy. (Supported by a grant to ASY and JB from the Dept. of Defense). Citation Format: Amy S. Yee, Kurtz Eric Paulson, Kai Wang, Wesley Field, Brian Pedro, Davis Vigneault, James B. Baleja. The HBP1 transcriptional repressor: An unexpected window on breast cancer metabolism in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B030.

Rationale: Lactate dehydrogenase A (LDHA), which catalyzes the conversion of pyruvate to lactate, serves as a poor prognostic serum marker for many different types of human cancers; however, it is not known whether this is simply the result of LDHA being released from dying cancer cells in patients with high tumor burden or whether LDHA could also be a signaling molecule that portends poor clinical outcome. Based on the observation that extracellular LDHA was identified as the key survival factor for cardiomyocytes exposed to oxidative stress, we sought to determine whether extracellular LDHA can act as a cancer cell survival factor and hence contributes to poor clinical outcome. We reasoned that LDHA released from the damaged cells could activate intracellular signaling pathways in surrounding tumor cells by means of membrane receptors, ultimately protecting those surrounding tumor cells from oxidative stress-induced cell death. Methods: To investigate our hypothesis, recombinant LDHA and its isoform LDHB was produced by transforming E. coli strain BL21(DE3) with pET-SUMO expression vector (Clontech) containing full-length human LDHA or LDHB cDNA. The resulting recombinant proteins were purified using TALON Metal Affinity Resin and used for 16h pre-treatment of Ramos human Burkitt's lymphoma and A6L human pancreatic cancer cells cultured in full serum containing medium. The amount of recombinant proteins relative to serum albumin is estimated to be less 1% and hence any effects of LDHA could not be simply due to non-specific protein concentration effect. Following the pre-treatment procedure, these cancer cells were subjected to oxidative stress induced by hydrogen peroxide (H2O2) at 100 μM for 24h. Cells were then counted in a hemacytometer using trypan blue dye to exclude dead cells. Results: We observed that while LDHB had no effect on cell death induced by H2O2 as compared to the non-pretreatment group, LDHA can act as a protective factor against oxidative stress induced by H2O2, and ultimately cell death. We also investigated the mechanism involved in the protective role of LDHA by using recombinant LDHA as a molecular probe against membrane proteins that were extracted from cancer cells. Conclusion: Our study defines the novel role and mechanism of LDHA protein as a survival factor in cancer cells, in addition to its enzymatic activity, which may explain the prognostic significance of extracellular LDHA. Furthermore, this study can lead to the identification of new membrane receptor(s) for LDHA which may provide new targets for cancer therapy. Citation Format: Brad Poore, Joshua Kee Park, Iman Afif, Laura Shingleton, Chi Van Dang, Anne Le. Novel role and mechanism of the LDHA protein as a survival factor for cancer cells. [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 852. doi:10.1158/1538-7445.AM2013-852

Introduction: Amplification and overexpression of c-Myc is very common in human cancers, including ovarian cancer. Heightened aerobic glycolysis (the Warburg effect) is a key metabolic hallmark of cancer and is thought to be under the control of c-Myc. JQ-1 is a selective small-molecure BET bromodomain inhibitor, and BET inhibition by JQ-1 leads to the downregulation of MYC transcription. Thus, JQ-1 functions as a novel c-Myc inhibitor and has been found to potently suppress tumor growth in hematologic malignancies. Thus, we aim to assess the impact of JQ-1 on the proliferation of human ovarian cancer cell lines and further explore its effects on cellular metabolism, especially the aerobic glycolysis pathway and its key enzyme—lactate dehydrogenase A (LDHA). Methods: Three human ovarian cancer cell lines (HEY and SKOV3) were used in this study. Cell proliferation was assessed by MTT assay after exposure to JQ-1 (generously provided by the laboratory of Dr. James E. Bradner). Cell cycle progression was evaluated by Cellometer. Apoptosis was assessed by Annexin V-FITC assay. Expression of c-Myc, p21, phosphorylated-LDHA and pan-LDHA were detected by Western blot analysis. Cellular glucose uptake, ATP production, reactive oxygen species (ROS), LDHA activity and lactate production were determined by ELISA assay using the corresponding commercial kits. Mitochondrial membrane potential changes were measured by JC-1 staining via Cellometer. Results: JQ-1 suppressed cellular proliferation (IC50 = 360 nM for HEY, p = 0.00001; IC50 = 3500 nM for SKOV3, p = 0.0007) via G1 cell cycle arrest in the ovarian cancer cell lines, but only caused moderate apoptosis. Western blot analysis demonstrated that JQ-1 increased p21 expression and decreased expression of c-Myc and phosphorylation of LDHA. In parallel, treatment with JQ-1 decreased LDHA activity, glucose uptake, mitochondrial membrane potential, lactate and ATP production and increased ROS levels in the ovarian cancer cell lines. Conclusions: We find that targeting c-Myc by JQ-1 significantly suppresses proliferation through G1 arrest in ovarian cancer cell lines. In addition, JQ-1 had multiple influences on cancer metabolism, particularly in the aerobic glycolysis pathway. JQ-1 reduced both the expression and activity of LDHA, inhibited lactate production and decreased the energy supply of the ovarian cancer cells. This evidence suggests that JQ-1 may be a promising targeted therapy for ovarian cancer through its effects on both cell proliferation and metabolism. Citation Format: Haifeng Qiu, Chunxiao Zhou, Amanda L. Jackson, Joshua E. Kilgore, Victoria L. Bae-Jump. JQ-1, a novel c-Myc inhibitor, suppressed cell proliferation and metabolism through the downregulation of lactate dehydrogenase A in ovarian cancer cells. [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 2418. doi:10.1158/1538-7445.AM2013-2418

Cetuximab is an anti-human epidermal growth factor receptor (EGFR) monoclonal antibody currently approved for treatment of patients with head and neck squamous cell carcinoma (HNSCC) in combination with conventional radiotherapy and chemotherapy. Previous studies showed that cetuximab downregulates the alpha subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1α) through inhibition of EGFR-mediated downstream cell signaling and that downregulation of HIF-1α is required for cetuximab-induced antiproliferative effects; however, the underlying mechanism has not been established. In this study, we explored the mechanism from the perspective of cancer metabolism. HIF-1 plays a critical role in reprogramming cancer metabolism towards aerobic glycolysis (i.e., the Warburg effect), which is critically important for supplying cancer cells with the biomass needed for cell proliferation. We found that cetuximab downregulated lactate dehydrogenase A (LDH-A) and inhibited glycolysis in cetuximab-sensitive cells in a HIF-1α downregulation-dependent manner. HNSCC cells with acquired resistance to cetuximab expressed a high level of HIF-1α and were highly glycolytic. Overexpression of a HIF-1α mutant conferred resistance to cetuximab-induced G1-phase cell cycle arrest, which could be overcome by knockdown of LDH-A expression. Addition of a small molecule LDH-A inhibitor markedly enhanced the response of cetuximab-resistant cells to cetuximab. Cetuximab had no noticeable inhibitory effect on glycolysis in nontransformed cells, in which glycolysis is followed by oxidative phosphorylation in the mitochondria. These findings provide novel mechanistic insights into cetuximab-induced antitumor effects from the perspective of cancer metabolism and suggest novel strategies for enhancing the response of cancer patients to cetuximab therapy. Citation Format: Haiquan Lu, Xinqun Li, Zhen Fan. Cetuximab downregulates lactate dehydrogenase-A, leading to inhibition of glycolysis and reversal of the Warburg effect in HNSCC cells. [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 5409. doi:10.1158/1538-7445.AM2013-5409

The Warburg effect is a distinct cancer hallmark that shifts the metabolic pathway in cancer cells through elevated aerobic glycolysis for energy supply and biomass. However, the mechanism of early stage metabolic dysregulation still remains unclear. 14-3-3ζ was found to be over-expressed in the early stages of breast disease and cancer (Atypical ductal hyperplasia and Ductal carcinoma in situ) and in >40% of breast cancer patients, correlating with poor patient prognosis. We found that 14-3-3ζ over-expression in non-transformed human mammary epithelial cells (HMECs), MCF10A and MCF12A, positively correlated with changes in the cellular glycolytic activity (increased glucose uptake, increased lactate production and reduced oxygen consumption). Additionally, 14-3-3ζ over-expression in HMECs induced soft agar colony formation and disorganized acini in three dimensional (3D) culture. Interestingly, we identified that lactate dehydrogenase A (LDHA) was up-regulated in these 14-3-3ζ overexpressing HMECs. LDHA knockdown in 14-3-3ζ overexpressing cells reduced aerobic glycolysis, decreased soft agar colonies and partially rescued the abnormal acinar structures, indicating LDHA upregulation contributed to 14-3-3ζ-mediated aerobic glycolysis and early transformation. Taken together, our data provide new insight into metabolic deregulation during the early stages of breast cancer transformation. Citation Format: Chia-Chi Chang, Jia Xu, Hai Wang, Chenyu Zhang, Jian Zhang, Sumaiyah K. Rehman, Ping Li, Dihua Yu. Increased glycolysis by 14-3-3ζ-mediated LDHA up-regulation contributes to transformation of early mammary epithelial cells. [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 5427. doi:10.1158/1538-7445.AM2013-5427

Ovarian cancer is characterized by genomic and microenvironmental heterogeneity, both of which contribute to tumor progression, metastasis, and drug resistance. Hypoxia, or low oxygen tension, is one microenvironmental factor that influences genomic instability and promotes angiogenesis and metastasis. We investigated the gene expression levels of hypoxia-regulated genes in matched ovarian serious carcinoma primary and peritoneal metastasis from five patients. The hypoxia gene set used was based on a published metagene hypoxia signature established using breast and head and neck cancer samples by Buffa et al, 2010. To ensure that the genes selected were also hypoxia-regulated in ovarian tumor cells, we grew the Hey and Ovcar-3 ovarian cancer cell lines under normoxia, 1.0% and 0.2% oxygen for 4, 16, and 48 hours. RNA was extracted and cDNA prepared for quantitative real-time polymerase chain reaction (qRT-PCR). Each sample was analyzed in triplicate and normalized for loading using ribosomal protein, large, PO (RPLP0). Relative gene expression was calculated by comparing hypoxia treated samples to the normoxic control grown for the same length of time. The Hey cells were also grown in an intraperitoneal xenograft model and tumor tissue harvested following 22 days of growth. Primary tumor gene expression was normalized to the Hey cell line grown in normoxia. The metastatic tissue was also compared relative to each matched primary sample. From an initial list of 16 genes, we identified 9 genes that demonstrated robust increases in gene expression across treatment conditions in vitro in both OVCAR 3 and Hey cells: vascular endothelial growth factor (VEGF-A), solute carrier family 2 (SLC2A1; Glut-1), phosphoglycerate mutase 1 (PGAM1),lactate dehydrogenase A (LDHA), prolyl 4-hydroxylase, alpha polypeptide (P4HA1), adrenomedullin (ADM), v-yc myelocytomatosis viral related oncogene ( NDRG1; N-myc), aldolase A (ALDOA), carbonic anhydrase 9 (CA9). Xenograft tissue showed high overexpression of all 9 of these hypoxia regulated genes when normalized to the Hey normoxia cell line control grown in vitro, with CA9 levels being maximally elevated. On average, primary tumors showed highest expression levels of VEGF and CA9 when normalized to the in vitro Hey cell normoxia control. When the primary tumor samples were compared to matched metastatic tissue there was a strong correlation between expression patterns of VEGF-A and SLC2A1. Two of five matched samples showed over 3 fold elevation in VEGF and SL2A1 in the metastatic tissue relative to primary tumor. Our data suggests that there may be significant heterogeneity between the primary and metastatic tumors which could lead to sampling bias when investigating hypoxia regulated genes or gene products such as VEGF-A and SL2A1 as prognostic or therapeutic biomarkers. Citation Format: Amanda F. Baker, Scott Malm, Cindy Laughren, Setsuko K. Comparison of a hypoxia gene signature in matched ovarian primary tumors and peritoneal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A6.

Background and Aim: Tumor cells depend on metabolic alterations for their continued growth and survival, and such changes make cancer cells peculiarly addicted to the rapacious uptake of glucose. The Warburg effect is such a metabolic feature of cancers that helps to preferentially metabolize pyruvate via glycolytic pathway to lactate by lactate dehydrogenase A (LDHA). Our recent findings indicated that LDHA is required not only for tumor initiation but for tumor maintenance and progression (Le et al., PNAS, 2010). Here, we investigated the therapeutic potential of LDHA inhibition in pancreatic cancer, and attempt to delineate the factors responsible for tumor response. Methods: We evaluated the in vivo efficacy of FX11, a small molecule inhibitor of LDHA, in a panel of pancreatic cancer xenografts with annotated mutational status. Non-invasive quantitative assessment of lactate production was measured by real-time hyperpolarization experiments with 1-13C-labeled pyruvate using a DNP polarizer (HyperSense). [18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET) combined with computed tomography (CT) imaging was conducted to evaluate the effect of FX11 treatment on glucose metabolism. Liquid chromatography - mass spectrometry (LC-MS) was used to quantify the tumor metabolites. Ki-67 and TUNEL staining were performed to determine the effect of FX11 treatment on apoptosis and tumor cell proliferation. Results: p53-deficient pancreatic cancer xenografts showed higher baseline metabolic activity and FX11 treatment down-regulated the tumor metabolic activity. Real-time imaging of pyruvate to lactate conversion using nuclear magnetic resonance (NMR) spectroscopy revealed that FX11 treatment inhibits pyruvate to lactate conversion in p53-deficient pancreatic cancer xenografts. Importantly, p53 promotes the expression of TP53-induced glycolysis regulator (TIGAR) and loss of p53 in tumors results in reduced TIGAR levels. The metabolic profiles of p53-deficient versus p53-proficient pancreatic cancer xenografts were remarkably different. FX11 treatment attenuates tumor progression, induces apoptosis and reduces tumor cell proliferation, preferentially in p53-deficient pancreatic cancer xenografts. Conclusions: Because the Warburg effect is characteristic of virtually all cancers and p53 is frequently mutated in vast majority of human cancers, our finding that p53, a key regulator of glycolysis and a major factor deciding the therapeutic outcome of targeting the Warburg effect may have broad clinical implications. Our findings may help to identify patient subsets that may be particularly responsive to LDHA targeted agents in clinical trials. Acknowledgement: Stand Up To Cancer-AACR Dream Team Translational Cancer Research Grant No. SU2C-AACR DT0509. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-61. doi:1538-7445.AM2012-LB-61

Metabolic changes in primary tumors have a significant impact on tumor progression and on the development of the metastatic phenotype. In cancer patients, serum total lactate dehydrogenase (LDH) levels are often increased, and the gene for LDH-A protein, is frequently upregulated in tumors (Koukourakis, Giatromanolaki et al. 2003). These features have been linked to poor prognosis, and a greater metastatic potential has been reported in patients with high LDH serum levels. Since LDH-A protein is required for the maintenance and progression of many tumors (Fantin, St-Pierre et al. 2006), it is also becoming a potential target for cancer therapy (Le, Cooper et al. 2010; Seth, Grant et al. 2011). We investigated the relationship between LDH-A expression and lactate production in two isogenic breast cancer lines (67NR and 4T1) (Serganova, Rizwan et al. 2011). These two cell lines were derived from a single mammary tumor that developed spontaneously in a BALB/c mouse. Each subclone was shown to have different phenotypic properties (Aslakson and Miller 1992). LDH-A expression, lactate concentration, glucose utilization and oxygen consumption were measured in cells, and the potential relationship between tumor lactate levels (measured by magnetic resonance spectroscopic imaging (MRSI)) and tumor glucose utilization (measured by [18F] 2-deoxy-2-fluoro-D-glucose positron emission tomography ([18F]FDG-PET)) was assessed in orthotopic breast tumors derived from these cell lines. We show a substantial difference in LDH-A expression between 67NR and 4T1 cells under normoxia and hypoxia. We also show that small orthotopic 4T1 tumors generate ten-fold more lactate than corresponding 67NR tumors. The high lactate levels in small primary 4T1 tumors are associated with intense pimonidazole staining (a hypoxia indicator). Less intense hypoxia staining was observed in the larger 67NR tumors, and is consistent with the gradual increase and plateau of lactate concentration in enlarging 67NR tumors. We show that lactate-MRSI has a greater dynamic range than [18F]FDG-PET and may be a more sensitive measure to evaluate the aggressive and metastatic potential of primary breast tumors. We hypothesized that tumor lactate levels monitored by MRSI will reflect LDH-A enzymatic activity and tumor phenotype. We also suggest that lactate-MRSI is a more sensitive measure than [18F]FDG-PET, and could be used in the clinic to evaluate the aggressive potential of primary breast tumors. Aslakson, C. J. and F. R. Miller (1992). Cancer Res 52(6): 1399-1405. Fantin, V. R., J. St-Pierre, et al. (2006). Cancer Cell 9(6): 425-434. Koukourakis, M. I., A. Giatromanolaki, et al. (2003). Br J Cancer 89(5): 877-885.Le, A., C. R. Cooper, et al. (2010). Proc Natl Acad Sci U S A 107(5): 2037-2042. Serganova, I., A. Rizwan, et al. (2011). Clin Cancer Res 17(19): 6250-6261. Seth, P., A. Grant, et al. (2011). Neoplasia 13(1): 60-71. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1003. doi:1538-7445.AM2012-1003

Heat shock transcription factor 1 (HSF1) is the master regulator of the heat shock responses in eukaryotes. Upon exposure to a variety of stresses, HSF1 activates its target genes, subsequently activating the heat shock responses. Although not well understood, the functions of HSF1 are far beyond the classical induction of heat shock responses. Recent reports have shown that HSF1 plays an important role in cancer development. We investigated the role and mechanism of HSF1 in regulating oncogene-mediated dysregulated cellular metabolism and cell growth in cancer cells. We found that oncogene ErbB2-overexpressing cancer cells possessed significantly higher level of glycolysis when compared to ErbB2-low expressing cells, and the downregulation of ErbB2 markedly decreased glycolysis. Overexpression of ErbB2 increased the expression of HSF1 and two critical glycolysis-regulating molecules, lactate dehydrogenase A (LDH-A) and Hexokinase (HK). ErbB2 activated HSF1, indicated by the increased HSF1 trimer formation and DNA binding activity, and promoted the rate of HSF1 protein translation. HSF1 bound to the promoter of LDH-A and transcriptionally activated LDH-A. Meanwhile, the downregulation of HSF1 reduced the expression of LDH-A and subsequently decreased cancer cell glycolysis and growth. These results demonstrate that in cancer cells, ErbB2 promotes glycolysis and cell growth through HSF1-mediated upregulation of LDH-A. Trastuzumab (Herceptin) and Paclitaxel (Taxol) are important drugs in treatment of breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. We investigated the antitumor effects of trastuzumab and Taxol in combination with glycolysis inhibitors in breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of HSF1 and LDH-A in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab and Taxol resistance. Strikingly, we found that combining trastuzumab or Taxol with glycolysis inhibition synergistically inhibited both drug-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. These results show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance. This work was supported by NIH Grant RO1CA149646, The Vincent F. Kilborn, Jr. Cancer Research Foundation, and Radiumhospitalets Legater Project 334003. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3208. doi:1538-7445.AM2012-3208

Pancreatic cancers have one of the highest fatality rates among all cancers and their response to therapies is highly variable. We have recently shown that inhibition of lactate dehydrogenase A (LDHA) and glutaminase (GLS) through small drug-like molecules causes pancreatic tumor regression. These studies indicate that targeting the metabolic reprogramming of cancer cells is a promising new approach to pancreatic cancer therapy. In order to translate these metabolic inhibitors to the clinic, our goal is to characterize and enable targeted selection of patients based on predicted metabolic response. Although genetic mutations may ultimately be the prime causes of early stage cancer, they can also lead to changes in cancer metabolism which enable cancer cells to proliferate and survive even under the hypoxic and nutrient-deprived conditions often encountered in the tumor microenvironment. The biochemical changes in cancer cells, which determine their behavior in terms of growth, survival and mobility, represent a functional readout of responses to external agents. We hypothesize that the metabolites of tumors that are sensitive to metabolic inhibitors are measurably different from those of resistant ones. We can therefore propose these metabolites as potential functional predictors of response. The proposed research will also study the metabolic reprogramming of cancer cells in the tumor microenvironment that can affect pancreatic cancer sensitivity to LDHA and GLS inhibition. The proposed experiments will use Metabolomics technology to trace the metabolic prototypes of pancreatic orthotopic xenografts to identify the metabolic signature of FX11 or BPTES-sensitive tumors as compared to resistant ones. For an analysis of the effects due to the tumor hypoxic microenvironment, we will use our innovative tool, the HypoxCR reporter system, which can simultaneously detect hypoxic cells and proliferating cells to define the effects of the pancreatic cancer tumor microenvironment on their sensitivity to metabolic inhibitors. This will be the first evaluation of the hypoxic tumor microenvironment in regards to how it affects the response of subfractions of pancreatic orthotopic xenograft tumor cells to metabolic inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 784A. doi:1538-7445.AM2012-784A

Most proliferating and tumor cells generate the energy required to support rapid cell division by aerobic glycolysis instead of oxidative phosphorylation (Warburg effect). By extension, in order to meet the increased requirements of proliferation, they often display essential changes in energy metabolism and nutrient uptake pathways. Several oncogenes, including Myc HIF-1α, and AKT, drive this switch directly, by targeting Hexokinase II, Lactate Dehydrogenase A, Pyruvate Kinase M2, and Pyruvate Dehydrogenase Kinase 1. The AMP-activated protein kinase (AMPK) is a highly conserved protein kinase complex that plays a central role in the regulation of cellular energy homeostasis. The fact that AMPK is an evolutionarily conserved energy sensor and plays a role both downstream and upstream of defined tumor suppressors, suggests that it may function as a tumor suppressor; however, the exact contribution of AMPK to tumor progression is unclear. We have generated a Pten−/- mouse model in which the PI3K pathway is selectively activated in the thyroid epithelial cells, leading to hyperplasia which progresses to follicular carcinoma. The early hyperplastic lesions exhibit a dramatic down-regulation of the expression levels of many tricarboxylic acid cycle proteins due to reduced mRNA expression, which results in enhanced compensatory glycolysis (over 3-fold increase in lactate production in mutants vs. wt). In addition, the expression of most mitochondrially-encoded genes is heavily down-regulated, and none of the genes and proteins classically associated with the Warburg effect is found deregulated in the mutant thyroid cells. In this context, while AMPK is found to be activated in wt thyroids through phosphorylation on Thr172, AMPK is inactivated in mutant thyroids through PI3K-dependent phosphorylation on Ser485. Pten−/− mice treated with 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, IP 500 mg/kg/day for 4 weeks) display a restoration of the metabolic and mitochondrially-encoded gene expression, as well as a significant decrease in the lactate production. Pharmacological PI3K inhibition in human tumor-derived thyroid cancer cell lines increases the expression levels of both TCA cycle and OXPHOS genes. Moreover, it also causes AMPK reactivation through Thr172 phosphorylation with a concomitant reduction in pAMPK Ser485. Based on these compelling data, we propose a novel mechanism leading to a Warburg-like effect in preneoplastic lesions: PI3K activation initiates through AMPK inactivation a coordinated rearrangement of the expression of metabolic genes, which favors aerobic glycolysis at the expense of TCA/OXPHOS. Further elucidation of this process will lead to innovative targeted approaches to selectively disrupt tumor progression, while preserving normal metabolism in non mutated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5141. doi:1538-7445.AM2012-5141

Aspirin, a non-steroidal anti-inflammatory drug has been consistently associated with a reduced risk of colon cancer; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate, and post-transnationally modify cellular proteins in HCT-116 human cancer cells to understand the potential mechanisms by which it may exert anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immune-affinity purification, mass spectrometry and immunoblotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeletal proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde 3 phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiments showed that aspirin also acetylated glucose 6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. In vitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6-phosphate dehydrogenase activity. Selective inhibition of glucose 6-phosphate dehydrogenase and other key proteins through acetylation may represent important mechanisms by which aspirin may exert its anti-cancer effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1638. doi:1538-7445.AM2012-1638

Most cancer cells exhibit a peculiar metabolic behavior, producing energy by aerobic glycolysis instead of oxidative phosphorylation (Warburg effect). Several oncogenes directly drive this switch modulating the levels and activity of, among others, Hexokinase II, Lactate Dehydrogenase A, Pyruvate Kinase M2, and Pyruvate Dehydrogenase Kinase 1. Anaplastic thyroid carcinoma (ATC) is the most aggressive form of thyroid cancer, and often derives from pre-existing well-differentiated tumors. Despite a relatively low prevalence, it accounts for a disproportionate number of thyroid cancer-related deaths, due to its resistance to any therapeutic approach. We have engineered the first mouse model of ATC by combining in the mouse thyroid follicular cells two molecular hallmarks of human ATC: activation of PI3K (via Pten deletion) and inactivation of p53. By 9 months of age, over 75% of the compound mutant mice develop aggressive, undifferentiated thyroid tumors that evolve from pre-existing follicular hyperplasia and carcinoma. Expression of the Foxe1, Pax8, and Nkx2–1 transcription factors, as well as of the thyroid-specific genes Duox2, Tpo, Tg, Tshr, and Slc5a5 (Nis) is reduced to almost undetectable levels in the anaplastic tumors, resembling the significant deregulation found in human ATC. Strikingly, the undifferentiated areas of all the tumors analyzed lose the expression of the epithelial marker E-cadherin, acquire the expression of the mesenchymal marker Vimentin, and display high levels of phosphorylated Smad2, strongly suggesting that elevated Tgf-β signaling contributes to the establishment and maintenance of EMT (epithelial-mesenchymal transition). Furthermore, similar to the human tumors, [Pten, p53]thyr−/− cells and tumors undergo the glycolytic switch known as Warburg effect. They display an up-regulation in the expression of Hexokinase II, Pyruvate Kinase M2, and Hypoxia-inducible factor 1α, and an increase in the amount of lactate levels. ATCs show an increase in glucose uptake by positron emission tomography (PET) and established cell lines derived from these tumors exhibit a high sensitivity to the therapeutic use of glycolytic inhibitors such as 3-bromopyruvate and 2-deoxyglucose, which synergize with standard chemotherapy. Taken together, this model represents a novel, powerful tool to understand the biology of human thyroid anaplastic carcinomas, and provide a compelling rationale for targeting glycolysis to increase chemotherapy response in ATC patients. The elucidation of this process will lead to innovative targeted approaches to selectively disrupt tumor progression, while preserving normal metabolism in non mutated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B138.

Cancer cells take up more glucose than normal tissue and favor aerobic glycolysis, generating lactate through a NADH-dependent enzyme, lactate dehydrogenase A (LDH-A). This is the last step of glycolysis that permits the regeneration of NAD+, which is needed as an electron acceptor to maintain cytosolic glucose catabolism. Therefore, most tumor cells are reliant on lactate production for their survival. LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production, and expression of LDH-A was previously implicated to be involved in tumour initiation and growth. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. Our phospho-proteomics studies revealed that oncogenic fibroblast growth factor (FGF) receptor type 1 (FGFR1) tyrosine kinase directly phosphorylates LDH-A. Structural and biochemical studies revealed that phosphorylation at Y10 and Y83 activates LDH-A by promoting the formation of active, tetrameric LDH-A and binding of LDH-A substrate NADH, respectively. Moreover, we found that LDH-A is commonly phosphorylated at Y10 in diverse human cancer cells by multiple oncogenic tyrosine kinases including BCR-ABL, FLT3 and JAK2, which represents an acute molecular mechanism underlying increased lactate production in cancer cells. Furthermore, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, show decreased cell proliferation and ATP levels under hypoxia, increased mitochondrial respiration to sustain glycolysis by providing NAD+, and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation activates LDH-A to promote the Warburg effect and tumor growth by regulating NADH/NAD+ redox homeostasis in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2798. doi:10.1158/1538-7445.AM2011-2798

Most cancer cells exhibit a peculiar metabolic behavior, producing energy by aerobic glycolysis instead of oxidative phosphorylation (Warburg effect). Several oncogenes directly drive this switch modulating the levels and activity of, among others, Hexokinase II, Lactate Dehydrogenase A, Pyruvate Kinase M2, and Pyruvate Dehydrogenase Kinase 1. We have generated a Pten-/- mouse model in which the PI3K pathway is selectively activated in the thyroid epithelial cells, leading to hyperplasia which later progresses to follicular carcinoma. Using proteomics to identify proteins differentially expressed in early hyperplastic lesions, we have found that the levels of many tricarboxylic acid cycle proteins are down-regulated in mutant thyroids. Down-regulation of these proteins is due to reduced mRNA expression, and results in reduced oxidative phosphorylation and enhanced glycolysis (over 3-fold increase in lactate production in mutants vs. wt). Furthermore, although mutant thyroids cells have a normal number of mitochondria, the expression of most mitochondrially-encoded genes is heavily down-regulated. Strikingly, none of the genes and proteins classically associated with the Warburg effect is found deregulated in the mutant thyroid cells. Analysis of the expression of transcription factors and co-activators involved in cellular metabolism revealed that the decrease in TCA cycle and OXPHOS genes is due to a reduction in ERRα, ERRγ, PPARα and PPARγ levels. Conversely, none of the co-activators analyzed (PRC1, PGC-1α, and PGC-1β) exhibited changes in mRNA expression. Pharmacological and genetic PI3K inhibition in mouse tumor-derived thyroid cancer cell lines increased the expression levels of both TCA cycle OXPHOS genes. Moreover, it also increased mtDNA amount, suggesting that the transition from hyperplasia to cancer involves additional alterations of the metabolic machinery. Likewise, PI3K inhibition induced a marked decrease in lactate secretion. Finally, these thyroid cancer cell lines exhibited high sensitivity to glycolysis inhibitors such as 3-bromopyruvate. Based on these compelling data, we propose a novel mechanism leading to the Warburg effect in preneoplastic lesions: PI3K activation initiates a coordinated rearrangement of the expression of metabolic genes, which favors aerobic glycolysis at the expense of TCA/OXPHOS. The elucidation of this process will lead to innovative targeted approaches to selectively disrupt tumor progression, while preserving normal metabolism in non mutated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2011-1261

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 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) 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 therapeutic 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 therapeutic targeting of cancer energy metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5453.

The expression and activity of lactate dehydrogenase A (LDH-A) in malignant tissue is considered a prognostic marker of poor outcome in many tumors. A limitation of tissue LDH-A measurements is the requirement of tissue for assay. The development of new non-invasive methods is highly demanded for clinical evaluation. One mechanistic link between tumor cell metabolism and the activity of lactate dehydrogenase A (LDH-A) is based on the HIF-1 transcription factor and its upregulation in tumors. HIF-1 binds to the LDH-A promoter and increases LDH-A expression; this in turn results in increased lactic acid production. The objective of this study was to detect the metabolic changes during tumor growth using a multi-modality imaging approach. To monitor HIF-1 pathway activity by bioluminescence imaging (BLI), we cloned an external Gaussia luciferase (exGLuc) reporter gene into an -IRES-GFP cassette, and placed the cassette in a self-inactivated retroviral vector under the control of an enhancer, containing multiple copies of the binding sites for the HIF-1 transcription factor. 18F-fluorodeoxyglucose (FDG)/microPET imaging was used for the detection of glucose metabolism in tumors, while lactate production was measured by proton magnetic resonance spectroscopic imaging (MRI/S). To validate this multi-modality imaging approach, human glioblastoma U87MG cells were transduced with the HIF-1 reporter vector and the HIF-1 reporter response was validated in vitro by exposure cells to hypoxia (1% O2) and to CoCl2 or DFO (deferoxamine mesylate). Despite a high basal level of HIF-1 transcriptional activity in the U87MG reporter cells, a strong correlation between oxygen deprivation and the level of exGLuc/GFP expression (indicating HIF-1 transcriptional activation) was detected, and this increase corresponded with a significant increase in LDH-A expression, assessed by immunoblotting. In vivo experiments using U87 reporter-xenografts also demonstrated a correspondence between the levels of HIF-1 expression and lactate production assessed by MRI/S. The measurement of lactate production using MRI/S with consecutive BLI imaging of HIF-1 activity and 18F fluorodeoxyglucose (FDG)/microPET confirmed the feasibility of a multi-modality imaging approach to study the link between the HIF-1 activity and a lactate production, as well as the level of glucose (FDG) uptake in growing xenografts. This multi-modal imaging paradigm can provide new and relevant information, and the opportunity to initiate and monitor more effective treatment strategies to arrest tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5234.