Glycolysis In Breast Cancer Cells Research Articles

The miR-186-3p/EREG axis orchestrates tamoxifen resistance and aerobic glycolysis in breast cancer cells.

Tamoxifen resistance is one of the major challenges for its medical uses in estrogen receptor (ER)-positive breast cancer. Aerobic glycolysis, an anomalous characteristic of glucose metabolism in cancer cells, has been shown to associate with the resistance to chemotherapeutic agents. It remains, however, largely unclear whether and how tamoxifen resistance contributes to aerobic glycolysis in breast cancer. Here, we report that tamoxifen resistance is associated with enhanced glycolysis in ER-positive breast cancer cells. We demonstrate that EREG, an agonist of EGFR, has an important role in enhancing glycolysis via activating EGFR signaling and its downstream glycolytic genes in tamoxifen-resistant breast cancer cells. We further show that EREG is a direct target of miR-186-3p and that downregulation of miR-186-3p by tamoxifen results in EREG upregulation in tamoxifen-resistant breast cancer cells. Importantly, systemic delivery of cholesterol-modified agomiR-186-3p to mice bearing tamoxifen-resistant breast tumors effectively attenuates both tumor growth and [18F]-fluoro-deoxyglucose ([18F]-FDG) uptake. Together, our results reveal a novel molecular mechanism of resistance to hormone therapies in which the miR-186-3p/EREG axis orchestrates tamoxifen resistance and aerobic glycolysis in ER-positive breast cancer, suggesting targeting miR-186-3p as a promising strategy for therapeutic intervention in endocrine-resistant breast tumors.

Extracellular vesicle-packaged HIF-1α-stabilizing lncRNA from tumour-associated macrophages regulates aerobic glycolysis of breast cancer cells.

Metabolic reprogramming is a hallmark of cancer. Here, we demonstrate that tumour-associated macrophages (TAMs) enhance the aerobic glycolysis and apoptotic resistance of breast cancer cells via the extracellular vesicle (EV) transmission of a myeloid-specific lncRNA, HIF-1α-stabilizing long noncoding RNA (HISLA). Mechanistically, HISLA blocks the interaction of PHD2 and HIF-1α to inhibit the hydroxylation and degradation of HIF-1α. Reciprocally, lactate released from glycolytic tumour cells upregulates HISLA in macrophages, constituting a feed-forward loop between TAMs and tumour cells. Blocking EV-transmitted HISLA inhibits the glycolysis and chemoresistance of breast cancer in vivo. Clinically, HISLA expression in TAMs is associated with glycolysis, poor chemotherapeutic response and shorter survival of patients with breast cancer. Our study highlights the potential of lncRNAs as signal transducers that are transmitted between immune and tumour cells via EVs to promote cancer aerobic glycolysis.

Betulinic acid suppresses breast cancer aerobic glycolysis via caveolin-1/NF-κB/c-Myc pathway

Emerging evidence has suggested that targeting glycolysis may be a promising strategy for cancer treatment. Betulinic acid (BA) is a natural pentacyclic terpene that has been reported to be active in inhibiting various malignancies. Here, we showed that BA could inhibit aerobic glycolysis activity in breast cancer cell lines MCF-7 and MDA-MB-231 by hampering lactate production, glucose uptake and extracellular acidification rate (ECAR), as well as suppressing aerobic glycolysis-related proteins including c-Myc, lactate dehydrogenase A (LDH-A) and p-PDK1/PDK1 (pyruvate dehydrogenase kinase 1). Mechanistic studies validated Caveolin-1 (Cav-1) as one of key targets of BA in suppressing aerobic glycolysis, as BA administration resulted in Cav-1 upregulation, whereas silencing Cav-1 abrogated the inhibitory effect of BA on aerobic glycolysis. Further investigations demonstrated that BA suppressed aerobic glycolysis in breast cancer cells by regulating the Cav-1/NF-κB/c-Myc pathway. More meaningfully, BA significantly inhibited breast cancer growth and glycolytic activity in both the transgenic MMTV-PyVT+/− breast cancer spontaneous model and the zebrafish breast cancer xenotransplantation model without any detectable side effects in vivo. Taken together, our study sheds novel insights into BA as a promising candidate drug for suppressing aerobic glycolysis, highlighting Cav-1 as a potential molecular target of BA and aerobic glycolysis regulation.

C-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer.

PurposeRecent studies have shown that long non-coding RNA (lncRNA) play an important role in cancer metabolism and development. The lncRNA small nucleolar RNA host gene 7 (SNHG7) was reported to be upregulated in colorectal cancer and contribute to its progression. In the current study, we investigated the role of lncRNA-SNHG7 in breast cancer and explored the underlying mechanism.MethodsWe monitored the expression of lncRNA-SNHG7 in breast cancer tissues and breast cancer cell lines. We evaluated the effects of lncRNA-SNHG7 on cell proliferation and glycolysis in breast cancer cells by knocking down or overexpressing lncRNA-SNHG7. We searched for the potential microRNA (miRNA) target of lncRNA-SNHG7 and evaluated the effects of the target miRNA on glycolysis. We evaluated the potential regulation of lncRNA-SNHG7 by c-Myc.ResultsLncRNA-SNHG7 was up-regulated in both breast cancer tissues and breast cancer cell lines. Knocking down lncRNA-SNHG7 inhibited breast cancer cell proliferation while overexpressing lncRNA-SNHG7 enhanced cell proliferation. Knocking down lncRNA-SNHG7 resulted in decreased expression of lactate dehydrogenase A (LDHA) and decreased glycolysis. LncRNA-SNHG7 targeted miR-34a-5p to regulate LDHA expression and glycolysis. c-Myc bound to promoter of lncRNA-SNHG7 and positively regulated lncRNA-SNHG7 expression.ConclusionWe demonstrated that c-Myc regulated glycolysis through the lncRNA-SNHG7/miR-34a-5p/LDHA axis in breast cancer cells.

GPR119 agonist enhances gefitinib responsiveness through lactate-mediated inhibition of autophagy

BackgroundLigand-dependent activation of the G-protein coupled receptor 119 (GPR119) lowers blood glucose via glucose-dependent insulin secretion and intestinal glucagon-like peptide-1 production. However, the function of GPR119 in cancer cells has not been studied.MethodsGPR119 expression was assessed by real-time qPCR and immunohistochemistry in human breast cancer cell lines and breast cancer tissues. Cell proliferation and cell cycle analyses were performed by Incucyte® live cell analysis system and flow cutometry, respectively. Autophagy activity was estimeated by western blottings and LC3-GFP transfection.ResultsmRNA or protein expression of GPR119 was detected in 9 cancer cell lines and 19 tissue samples. Cotreatment with GPR119 agonist (MBX-2982 or GSK1292263) significantly potentiated gefitinib-induced cell growth inhibition in gefitinib-insensitive MCF-7 and MDA-MB-231 breast cancer cells. We observed that caspase-3/7 activity was enhanced with the downregulation of Bcl-2 in MCF-7 cells exposed to MBX-2982. Gefitinib-induced autophagy is related with cancer cell survival and chemoresistance. GPR119 agonists inhibit gefitinib-induced autophagosome formation in MCF-7 and MDA-MB-231 cells. MBX-2982 also caused a metabolic shift to enhanced glycolysis accompanied by reduced mitochondrial oxidative phosphorylation. MBX-2982 increased intracellular (~ 2.5 mM) and extracellular lactate (~ 20 mM) content. Gefitinib-mediated autophagy was suppressed by 20 mM lactate in MCF-7 cells.ConclusionsGPR119 agonists reduced mitochondrial OXPHOS and stimulated glycolysis in breast cancer cells, with consequent overproduction of lactate that inhibited autophagosome formation. Because autophagy is crucial for the survival of cancer cells exposed to TKIs, GPR119 agonists potentiated the anticancer effects of TKIs.

PKM2 promotes glucose metabolism through a let-7a-5p/Stat3/hnRNP-A1 regulatory feedback loop in breast cancer cells.

Tumor cells metabolize more glucose to lactate in aerobic or hypoxic conditions than normal cells. Pyruvate kinase isoenzyme type M2 (PKM2) is crucial for tumor cell aerobic glycolysis. We established a role for let-7a-5p/Stat3/hnRNP-A1/PKM2 signaling in breast cancer cell glucose metabolism. PKM2 depletion via small interfering RNA (siRNA) inhibits cell proliferation and aerobic glycolysis in breast cancer cells. Signal transducer and activator of transcription 3 (Stat3) promotes upregulation of heterogeneous nuclear ribonucleoprotein (hnRNP)-A1 expression, hnRNP-A1 binding to pyruvate kinase isoenzyme (PKM) pre messenger RNA, and the subsequent formation of PKM2. This pathway is downregulated by the microRNA let-7a-5p, which functionally targets Stat3, whereas hnRNP-A1 blocks the biogenesis of let-7a-5p to counteract its ability to downregulate the Stat3/hnRNP-A1/PKM2 signaling pathway. The downregulation of Stat3/hnRNP-A1/PKM2 by let-7a-5p is verified using a breast cancer. These results suggest that let-7a-5p, Stat3, and hnRNP-A1 form a feedback loop, thereby regulating PKM2 expression to modulate glucose metabolism of breast cancer cells. These findings elucidate a new pathway mediating aerobic glycolysis in breast cancers and provide an attractive potential target for breast cancer therapeutic intervention.

Abstract 2418: PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis

Abstract Metabolic reprogramming is a hallmark of cancer. Herein we discover that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator-associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondrial calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol-1,4,5-trisphosphate receptors (InsP3Rs), methylated PKM2 inhibits the influx of calcium from the endoplasmic reticulum to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticles perturbs the metabolic energy balance in cancer cells, leading to a decrease in cell proliferation, migration and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to InsP3R-dependent mitochondrial functions Citation Format: Fabao Liu, Fengfei Ma, Yuyuan Wang, Ling Hao, Hao Zeng, Chenxi Jia, Yidan Wang, Peng Liu, Irene M. Ong, Baobin Li, Guojun Chen, Jiaoyang Jiang, Shaoqin Gong, Lingjun Li, Wei Xu. PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis [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 2418.

HMGB2 is associated with malignancy and regulates Warburg effect by targeting LDHB and FBP1 in breast cancer

BackgroundHigh-mobility group box 2 (HMGB2) is implicated in tumorigenesis in various cancers. However, the clinical significance of HMGB2 signaling in human breast cancer progression remains unknown.MethodsWe investigated HMGB2 expression in 185 cases of primary breast cancer and matched normal breast tissue specimens, and explored the underlying mechanisms of altered HMGB2 expression as well as the impact of this altered expression on breast cancer growth and on aerobic glycolysis using in vitro and animal models of breast cancer.ResultsHMGB2 was more highly expressed in tumor-cell nuclei of breast cancer cells than in the adjacent normal breast tissues (P < 0.05). Higher HMGB2 expression correlated with larger tumor size (P = 0.003) and advanced tumor stage (P = 0.033). A Cox proportional hazards model revealed that HMGB2 expression was an independent prognostic factor for breast cancer after radical resection (P < 0.05). Experimentally, knockdown of HMGB2 expression by stable transfected shRNA significantly decreased the growth and glycolysis of breast cancer cells both in vitro and in mouse models. Mechanically, promotion of breast cancer progression by HMGB2 directly and significantly correlated with activation of LDHB expression and inactivation of FBP1 expression.ConclusionsThese results disclose a novel role for HMGB2 in reprogramming the metabolic process in breast cancer cells by targeting LDHB and FBP1 and provide potential prognostic predictors for breast cancer patients.

Abstract B021: Inhibition of β-catenin impairs triple-negative breast cancer (TNBC) cell migration and invasion by modulating aerobic glycolysis components

Abstract In the present study, we investigated the role of β-catenin on aerobic glycolysis in breast cancer cells and further evaluated its effects on triple-negative breast cancer (TNBC) cell migration and invasion. We used two metastatic TNBC cell lines, i.e., MDA-MB-468 and MDA-MB-231, and treated them with β-catenin antagonists (WNT5A and XAV939). Evaluation of glycolytic markers in WNT5A-expressing cell lines showed selective downregulation in the expression of β-catenin, platelet type-phosphofructokinase (PFKP), and lactate secretion, without affecting cell proliferation. Of the three phosphofructokinase isoform, we found that only PFKP expression was associated with decreased survival in breast cancer patients. We also found that the cause of PKFP and lactate downregulation was mediated by inhibition of β-catenin signaling, which is in accordance with the finding that β-catenin signaling positively regulates lactate production. Functionally, we showed that specific downregulation of β-catenin resulted in reduced lactate production accompanied by impaired TNBC cells migration and invasion. Finally, we demonstrated that apart from its inhibitory effect on lactate production, inhibition of β-catenin can also impair TNBC cell migration induced by exogenous lactate via downregulating monocarboxylate transporter 1 (MCT1). Overall, we demonstrated that inhibition of β-catenin-PFKP signaling axis impaired lactate production as well as its uptake from tumor microenvironment (via MCT1), thereby impairing TNBC cells' migration. Citation Format: Chandra P. Prasad, Tommy Andersson. Inhibition of β-catenin impairs triple-negative breast cancer (TNBC) cell migration and invasion by modulating aerobic glycolysis components [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B021.

WITHDRAWN: Lack of miR-216b-5p, miR-33a-5p or miR-122-5p in MSCs exosomes promotes breast cancer glycolysis by targeting HK2, LDHA and PKM2

// Jiyi Xia 1, * , Xiaolan Yu 2, * , Xiguang Mao 3 , Hongyuan An 4 , Xiaoping Tang 5 , Xiaoyan Wang 5 , Tao He 1 and Yong Liu 6 1 Cancer Research Institute of SouthWest Medical University, Luzhou, Sichuan, China 2 Department of Obstetrics and Gynecology, Affiliated TCM Hospital of SouthWest Medical University, Luzhou, Sichuan, China 3 Department of Obstetrics and Gynecology, Affiliated Hospital of SouthWest Medical University, Luzhou, Sichuan, China 4 The Second Ward of Surgery (Breast Surgery), Luzhou People’s Hospital, Luzhou, Sichuan, China 5 Experimental Medicine Center, The Affiliated Hospital of SouthWest Medical University, Luzhou, Sichuan, China 6 Department of Pathology, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, China * These authors have contributed equally to this work Correspondence to: Tao He, email: hetaipeace@163.com Yong Liu, email: liuyong_lz@126.com Keywords: MSC; exosomes; breast cancer; miRNA Received: April 27, 2017 Accepted: October 28, 2017 Published: January 02, 2018 ABSTRACT Exosomes contain many important components including miRNAs for cancer-stromal communication. Mesenchymal stromal cells support tumor development, however, the low expression of miRNAs in exosomes and their regulation roles from mesenchymal stromal cells are still unclear. In this study, we hypothesized that mesenchymal stromal cells-derived exosomes promote breast cancer cell glycolysis. Our results showed that mesenchymal stromal cells-derived exosomes promoted cell proliferation and glycolysis of breast cancer cells. Furthermore, sequencing of exosomal RNAs revealed miR-216b-5p, miR-33a-5p and miR-122-5p were lack of expression in exosomes from mesenchymal stromal cells. MiR-216b-5p, miR-33a-5p and miR-122-5p overexpression in breast cancer cells suppressed glycolysis by targeting HK2, LDHA and PKM2 respectively. These results provided valuable insights and mechanism of mesenchymal stromal cells-secreted exosomes on the glycolysis of breast cancer.

Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop.

Aberrant energy metabolism is critical for cancer progression. Tumor-associated macrophages (TAMs) can stimulate tumor angiogenesis and enhance cancer metastasis; however, the metabolic interaction between cancer cells and macrophages characterized by lactate shuttles remains unclear. Here, we showed that lactate activated human macrophages to a TAM-like phenotype and stimulated the secretion of CCL5 by activation of Notch signaling in macrophages. Reciprocally, CCL5 increased cell migration, induced cancer cell EMT, and promoted aerobic glycolysis in breast cancer cells, suggesting a positive metabolic feedback loop in the co-culture system. Inhibition of CCR5, the cognate receptor of CCL5, or neutralization of CCL5, broke the metabolic loop and decreased cancer cell migration and EMT. Inhibition of aerobic glycolysis significantly reduced breast cancer cell EMT, indicated that aerobic glycolysis was necessary for the invasive phenotype of cancer cells. We further showed that TGF-β signaling regulated the expression of CCR5 in the co-culture system, and CCL5 induced glycolysis by mediation of AMPK signaling. The expression of CCL5-CCR5 axis was highly associated with macrophage infiltration, TGF-β and p-AMPK in clinical samples. CCL5-CCR5 axis promoted breast cancer metastasis in vivo. Our findings suggested a pivotal role of CCL5-CCR5 axis in the metabolic communication between cancer cells and macrophages.

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis.

Metabolic reprogramming is a hallmark of cancer. Herein we discovered that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondria calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol 1, 4, 5-trisphosphate receptors (IP3Rs), methylated PKM2 inhibits the influx of calcium from endoplasmic reticulum (ER) to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticle perturbs metabolic energy balance in cancer cells, leading to decrease of cell proliferation, migration, and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to IP3R-dependent mitochondrial functions.

Abstract 1485: Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination

Abstract Cancer deaths, including breast cancer, are caused by metastasis of the malignant tumors to distant locations. However, current methods of detection cannot distinguish pre-invasive breast cancer from noninvasive breast tumor or benign breast disease. Population-wide mammographic screenings have led to increased detection of ductal carcinoma in situ or DCIS, noninvasive, proliferative cells contained by the basement membrane of the terminal ductal lobular unit. DCIS is usually not associated with metastasis and/or cancer death. Each year, in the US alone, about 1.5 million of women who have been diagnosed with DCIS or a suspicious lump/lesion by mammography will require resection or breast biopsy after diagnosis for further pathologic analysis. However, ~80-85% of biopsies result in noninvasive breast disease or benign findings. As a result, a considerable number of patients suffer from side effects caused by breast biopsy and/or overtreatment. Therefore, there is an urgent need to find a biomarker for pre-invasive breast cancer. The ability of cancer cells to produce lactate through aerobic glycolysis (the Warburg effect) is a consistent hallmark of cancer, including breast cancer. Recent advancements in liquid chromatography-mass spectrometry (LC-MS) technology have significantly improved the sensitivity of this method compared to traditional NMR or GC-MS-based technologies, which make it feasible to detect very low concentrations of small molecules or metabolites. We have recently established a positional isotopic labeling and LC-MS-based targeted metabolomics method that can directly measure the conversion from [1-13C]glucose to [3-13C]lactate through glycolysis. Our results show that metastatic breast cancer cells exhibit a dramatically increased production of [3-13C]lactate from [1-13C]glucose even under aerobic conditions as compared to low- or noninvasive breast cancer cell lines. We found that the rate of aerobic glycolysis is closely correlated with glucose uptake and lactate production in breast cancer cells. We have also observed significantly elevated production of [3-13C]lactate in serum samples of early stage metastatic mammary tumors developed in mice. Since elevated levels of lactate are closely correlated to increased tumor aggressiveness, these results suggest that monitoring of lactate production from glycolysis by targeted metabolomics may provide a biomarker for pre-invasive breast cancer. These results will pave the way for further exploration of the elevated production of lactate as a promising biomarker for pre-invasive breast cancer and for assessment of therapeutic response in clinical trials. Citation Format: Da-Qing Yang, Margot Cleary. Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination [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 1485. doi:10.1158/1538-7445.AM2017-1485

Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

Alterations of cellular metabolism play a central role in the development and progression of cancer. Oroxylin A, an active flavonoid of a Chinese traditional medicinal plant, was previously shown to modulate glycolysis in cancer cells. However, the mechanism by which oroxylin A regulates glycolysis is still not well defined. Here, we show that oroxylin A inhibits glycolysis in breast cancer cells via the Sirtuin 3 (SIRT3)-mediated destabilization of hypoxia-inducible factor 1α (HIF1α), which controls glycolytic gene expression. Oroxylin A promotes superoxide dismutase (SOD2) gene expression through SIRT3-regulated DNA-binding activity of FOXO3a and increases the activity of SOD2 by promoting SIRT3-mediated deacetylation. In vivo, oroxylin A inhibits the growth of transplanted human breast tumors associated with glycolytic suppression. These data indicate that oroxylin A inhibits glycolysis-dependent proliferation of breast cancer cells, through the suppression of HIF1α stabilization via SIRT3 activation, providing preclinical information for the cancer therapies of SIRT3 stimulation.

Abstract A06: Obesity induces a metabolic switch toward aerobic glycolysis in breast cancer cells in vitro and in vivo

Abstract Background: The prevalence of obesity is steadily increasing worldwide, impacting the incidence of a variety of diseases, including breast cancer (BCa). Although epidemiological studies show a strong association between elevated body mass index (BMI) and a heightened risk of postmenopausal BCa, the underlying etiology remains relatively unknown. One potential mechanism by which obesity contributes to BCa progression and recurrence is through induction of obesity-induced stress (OBIS). Reactive oxygen species (ROS) are byproducts of metabolism, and excessive production or accumulation of ROS is known to contribute to early tumor initiating events through redox modulation of p53 transcriptional activity. Objectives: Recent studies indicate that p53 is a key regulator of glycolysis. As the obese state is known to promote aerobic glycolysis, the preferred mode of energy production by most transformed cells, and because approximately 40% of human breast cancers harbor p53 mutations, the aim of this study was to determine the impact of obesity on rates of glucose consumption and genotoxic stress in the context of p53 status. Results: Using the MMTV-Wnt1 transgenic mouse model of spontaneous breast cancer harboring a heterozygous deletion mutation for p53, we determined the impact of diet-induced obesity (DIO) on markers of aerobic glycolysis and oxidative stress. Interestingly, thioredoxin interacting protein (TXNIP), a regulator of glucose homeostasis, was found to be induced in the normal mammary fat pad (MFP) of obese p53-heterozygous (p53-het) mice versus p53-wild type (p53-wt) MFPs. Conversely, MCF-7 breast cancer cells grown in vitro in serum from these obese mice showed a 50% decrease in TXNIP expression in comparison to cells cultured in serum from the non-obese mice. These findings are consistent with TXNIP's putative role as a tumor suppressor gene. Further, glucose transporter 1 (GLUT-1), a basal glucose transporter required to sustain cellular respiration, was upregulated 5-fold in the MFP of obese p53-het mice compared to MFP taken from non-obese p53-het mice, confirming that obesity regulates glucose uptake in the MFP independently of p53 status. Additionally, the obese p53-het mice displayed elevated serum levels of 8-isoprostane, a biomarker of oxidative stress. Additional in vitro studies demonstrate that MCF-7 cells cultured in serum derived from obese versus non-obese C57BL/6 mice (Ob-serum and non-Ob serum) displayed heightened levels of ROS and significant modulation of HIF-1α, a ROS-inducible transcription factor known to regulate glycolysis. Ob-serum also enhanced lactate dehydrogenase activity and inhibited expression of pyruvate dehydrogenase (PDH), concurrently promoting glycolysis and bypassing oxidative phosphorylation (OXPHOS). This metabolic shift was associated with the accumulation of γ-H2AX foci, a known indicator of DNA double-strand breaks (DSBs). Finally, we observed that a combined shRNA-mediated knockdown of TXNIP and p53 in MCF-7 cells resulted in a dramatic induction of DSBs, and this was exacerbated by exposure to Ob-serum. Potential Impact: Based on these findings, we conclude that obesity may enhance the rate of metabolic reprogramming in breast cancer cells, resulting in OBIS and accumulation of ROS; thereby promoting disease progression by enhancing genotoxic stress in cells harboring a p53 mutation. Citation Format: David Antonio Cavazos, Karrie Wheatly, Stephen Hursting. Obesity induces a metabolic switch toward aerobic glycolysis in breast cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A06.

Hypoxia-induced IL-32β increases glycolysis in breast cancer cells

IL-32β is highly expressed and increases the migration and invasion of gastric, lung, and breast cancer cells. Since IL-32 enhances VEGF production under hypoxic conditions, whether IL-32β is regulated by hypoxia was examined. Hypoxic conditions and a mimetic chemical CoCl2 enhanced IL-32β production. When cells were treated with various inhibitors of ROS generation to prevent hypoxia-induced ROS function, IL-32β production was suppressed by both NADPH oxidase and mitochondrial ROS inhibitors. IL-32β translocated to the mitochondria under hypoxic conditions, where it was associated with mitochondrial biogenesis. Thus, whether hypoxia-induced IL-32β is associated with oxidative phosphorylation (OXPHOS) or glycolysis was examined. Glycolysis under aerobic and anaerobic conditions is impaired in IL-32β-depleted cells, and the hypoxia-induced IL-32β increased glycolysis through activation of lactate dehydrogenase. Src is also known to increase lactate dehydrogenase activity, and the hypoxia-induced IL-32β was found to stimulate Src activation by inhibiting the dephosphorylation of Src. These findings revealed that a hypoxia-ROS-IL-32β-Src-glycolysis pathway is associated with the regulation of cancer cell metabolism.

A novel miR-155/miR-143 cascade controls glycolysis by regulatinghexokinase 2in breast cancer cells

Cancer cells preferentially metabolize glucose through aerobic glycolysis. This phenomenon, known as the Warburg effect, is an anomalous characteristic of glucose metabolism in cancer cells. Chronic inflammation is a key promoting factor of tumourigenesis. It remains, however, largely unexplored whether and how pro-tumourigenic inflammation regulates glucose metabolism in cancer cells. Here, we show that pro-inflammatory cytokines promote glycolysis in breast cancer cells, and that the inflammation-induced miR-155 functions as an important mediator in this process. We further show that miR-155 acts to upregulate hexokinase 2 (hk2), through two distinct mechanisms. First, miR-155 promotes hk2 transcription by activation of signal transducer and activator of transcription 3 (STAT3), a transcriptional activator for hk2. Second, via targeting C/EBPβ (a transcriptional activator for mir-143), miR-155 represses mir-143, a negative regulator of hk2, thus resulting in upregulation of hk2 expression at the post-transcriptional level. The miR-155-mediated hk2 upregulation also appears to operate in other types of cancer cells examined. We suggest that the miR-155/miR-143/HK2 axis may represent a common mechanism linking inflammation to the altered metabolism in cancer cells.

Progestins activate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in breast cancer cells

PFKFB (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose-2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is extensively involved in cell proliferation owing to its key role in carbohydrate metabolism. In the present study we analyse its mechanism of regulation by progestins in breast cancer cells. We report that exposure of T47D cells to synthetic progestins (ORG2058 or norgestrel) leads to a rapid increase in Fru-2,6-P2 concentration. Our Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a PRE (progesterone-response element) to which PR (progesterone receptor) binds and thus transactivates PFKFB3 gene transcription. PR expression in the PR-negative cell line MDA-MB-231 induces endogenous PFKFB3 expression in response to norgestrel. Direct binding of PR to the PRE box (-3490nt) was confirmed by ChIP (chromatin immunoprecipiation) experiments. A dual mechanism affecting PFKFB3 protein and gene regulation operates in order to assure glycolysis in breast cancer cells. An immediate early response through the ERK (extracellular-signal-regulated kinase)/RSK (ribosomal S6 kinase) pathway leading to phosphorylation of PFKFB3 on Ser461 is followed by activation of mRNA transcription via cis-acting sequences on the PFKFB3 promoter.

Estrogen and retinoic acid antagonistically regulate several microRNA genes to control aerobic glycolysis in breast cancer cells

In addition to estrogen receptor modulators, retinoic acid and other retinoids are promising agents to prevent breast cancer. Retinoic acid and estrogen exert antagonistic regulations on the transcription of coding genes and we evaluated here whether these two compounds have similar effects on microRNAs. Using an integrative approach based on several bioinformatics resources together with experimental validations, we indeed found that retinoic acid positively regulates miR-210 and miR-23a/24-2 expressions and is counteracted by estrogen. Conversely, estrogen increased miR-17/92 and miR-424/450b expressions and is inhibited by retinoic acid. In silico functional enrichment further revealed that this combination of transcriptional/post-transcriptional regulations fully impacts on the molecular effects of estrogen and retinoic acid. Besides, we unveiled a novel effect of retinoic acid on aerobic glycolysis. We specifically showed that it increases extracellular lactate production, an effect counteracted by the miR-210 and the miR-23a/24-2, which simultaneously target lactate dehydrogenase A and B mRNAs. Together our results provide a new framework to better understand the estrogen/retinoic acid antagonism in breast cancer cells.