Breast Cancer Cell Metabolism Research Articles

Dichloroacetate enhances apoptotic cell death via oxidative damage and attenuates lactate production in metformin-treated breast cancer cells.

The unique metabolism of breast cancer cells provides interest in exploiting this phenomenon therapeutically. Metformin, a promising breast cancer therapeutic, targets complex I of the electron transport chain leading to an accumulation of reactive oxygen species (ROS) that eventually lead to cell death. Inhibition of complex I leads to lactate production, a metabolic byproduct already highly produced by reprogrammed cancer cells and associated with a poor prognosis. While metformin remains a promising cancer therapeutic, we sought a complementary agent to increase apoptotic promoting effects of metformin while attenuating lactate production possibly leading to greatly improved efficacy. Dichloroacetate (DCA) is a well-established drug used in the treatment of lactic acidosis which functions through inhibition of pyruvate dehydrogenase kinase (PDK) promoting mitochondrial metabolism. Our purpose was to examine the synergy and mechanisms by which these two drugs kill breast cancer cells. Cell lines were subjected to the indicated treatments and analyzed for cell death and various aspects of metabolism. Cell death and ROS production were analyzed using flow cytometry, Western blot analysis, and cell counting methods. Images of cells were taken with phase contrast microscopy or confocal microscopy. Metabolism of cells was analyzed using the Seahorse XF24 analyzer, lactate assays, and pH analysis. We show that when DCA and metformin are used in combination, synergistic induction of apoptosis of breast cancer cells occurs. Metformin-induced oxidative damage is enhanced by DCA through PDK1 inhibition which also diminishes metformin promoted lactate production. We demonstrate that DCA and metformin combine to synergistically induce caspase-dependent apoptosis involving oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in expanding breast cancer therapies.

Dinuclear berenil-platinum (II) complexes as modulators of apoptosis in human MCF-7 and MDA-MB231 breast cancer cells.

The metabolism of alkylating agents is accompanied by the generation of reactive oxygen species. The aim of this study was to treat estrogen receptor-positive and estrogen receptor-negative human breast cancer cells, MCF-7 and MDA-MB-231, respectively, with cisplatin and five different berenil-platinum (II) complexes, and then to investigate the oxidative modifications of DNA, lipid and protein, and to compare them with the profile of various pro- and antiapoptotic proteins. Changes in the levels of 8-hydroxy-2'-deoxyguanosine, 4-hydroxynonenal, carbonyl groups, dityrosine, active caspases 3, 8 and 9, as well as the expression of Bcl-2, Bax, cytochrome c, and p53 were subsequently examined. Activities of superoxide dismutase, catalase and glutathione, vitamin C levels were also investigated. Cellular reactions to cisplatin and the berenil-platinum (II) derivatives were more pronounced in MCF-7 cells as compared with the MDA-MB231 cells. Furthermore, the berenil-platinum (II) derivatives were found to be more effective than cisplatin. All of the complexes reduced the activity of superoxide dismutase and catalase, and also lowered the levels of non-enzymatic antioxidants. Increased level of lipid, protein as well as DNA damage markers was also observed after berenil-platinum (II) derivatives treatment. Similarly, the increase in the levels of the proapoptotic factors, were detected in MCF-7 and MDA-MB231 cells. Incubation of examined cells with the berenil-platinum (II) complexes also led to the increase in the levels of active caspases 3, 8 and 9. In conclusion, the results of the present study demonstrated that berenil-platinum (II) complexes more efficiently mediate cellular oxidative modifications and proapoptotic metabolism, particularly in MCF-7 cells, compared to cisplatin. Pt2(isopropylamine)4berenil2 and Pt2(piperidine)4(berenil)2 notably affected the cellular metabolism of estrogen-positive breast cancer cells. Thus, these complexes may be valuable for the design of additional anticancer drugs.

Novel drugs that target the estrogen-related receptor alpha: their therapeutic potential in breast cancer.

The incidence of breast cancer continues to rise: 1.7 million women were diagnosed with and 521,000 women died from breast cancer in 2012. This review considers first current treatment options: surgery; radiotherapy; and systemic endocrine, anti-biological, and cytotoxic therapies. Clinical management includes prevention, early detection by screening, treatment with curative intent, management of chronic disease, and palliative control of advanced breast cancer. Next, the potential of novel drugs that target DNA repair, growth factor dependence, intracellular and intercellular signal transduction, and cell cycle are considered. Estrogen-related receptor alpha has attracted attention as a therapeutic target in triple-negative breast cancers with de novo resistance to, and in breast cancers with acquired resistance to, endocrine therapies such as antiestrogens and aromatase inhibitors. Estrogen-related receptor alpha is an orphan receptor and transcription factor. Its activity is regulated by coregulator proteins and posttranslational modification. It is an energy sensor that controls adaptation to energy demand and may facilitate glycolytic metabolism and mitochondrial oxidative respiration in breast cancer cells. Estrogen-related receptor alpha increases breast cancer cell migration, proliferation, and tumor development. It is expressed at high levels in estrogen receptor-negative tumors, and is proposed to activate estrogen-responsive genes in endocrine-resistant tumors. The structures and functions of the ligand-binding domains of estrogen receptor alpha and estrogen-related receptor alpha, their ability to bind estrogens, phytoestrogens, and synthetic ligands, and the effects of ligand agonists, antagonists, and inverse agonists on biological activity, are evaluated. Synthetic ligands of estrogen-related receptor alpha have activity in preclinical models of metabolic disorders, diabetes, osteoporosis, and oncology. The clinical settings in which these novel drugs might have utility in the management of advanced breast cancer, and biomarkers for stratification of patients likely to benefit, are discussed. Finally, the potential side effects of the novel drugs on metabolism, osteoporosis, osteo-metastasis, and cachexia are considered.

Docosahexaenoic acid attenuates breast cancer cell metabolism and the Warburg phenotype by targeting bioenergetic function.

Docosahexaenoic acid (DHA; C22:6n-3) depresses mammary carcinoma proliferation and growth in cell culture and in animal models. The current study explored the role of interrupting bioenergetic pathways in BT-474 and MDA-MB-231 breast cancer cell lines representing respiratory and glycolytic phenotypes, respectively and comparing the impacts of DHA with a non-transformed cell line, MCF-10A. Metabolic investigation revealed that DHA supplementation significantly diminished the bioenergetic profile of the malignant cell lines in a dose-dependent manner. DHA enrichment also resulted in decreases in hypoxia-inducible factor (HIF-1α) total protein level and transcriptional activity in the malignant cell lines but not in the non-transformed cell line. Downstream targets of HIF-1α, including glucose transporter 1 (GLUT 1) and lactate dehydrogenase (LDH), were decreased by DHA treatment in the BT-474 cell line, as well as decreases in LDH protein level in the MDA-MB-231 cell line. Glucose uptake, total glucose oxidation, glycolytic metabolism, and lactate production were significantly decreased in response to DHA supplementation; thereby enhancing metabolic injury and decreasing oxidative metabolism. The DHA-induced metabolic changes led to a marked decrease of intracellular ATP levels by 50% in both cancer cell lines, which mediated phosphorylation of metabolic stress marker, AMPK, at Thr172. These findings show that DHA contributes to impaired cancer cell growth and survival by altering cancer cell metabolism, increasing metabolic stress and altering HIF-1α-associated metabolism, while not affecting non-transformed MCF-10A cells. This study provides rationale for enhancement of current cancer prevention models and current therapies by combining them with dietary sources, like DHA.

Estradiol Stimulates Glucose Metabolism via 6-Phosphofructo-2-kinase (PFKFB3)

Estradiol (E2) administered to estrogen receptor-positive (ER(+)) breast cancer patients stimulates glucose uptake by tumors. Importantly, this E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and, as a result, downstream metabolic regulators of E2 are expected to have utility as targets for the development of anti-breast cancer agents. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) control glycolytic flux via their product, fructose-2,6-bisphosphate (F26BP), which activates 6-phosphofructo-1-kinase (PFK-1). We postulated that E2 might promote PFKFB3 expression, resulting in increased F26BP and glucose uptake. We demonstrate that PFKFB3 expression is highest in stage III lymph node metastases relative to normal breast tissues and that exposure of human MCF-7 breast cancer cells to E2 causes a rapid increase in [(14)C]glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, F26BP. Importantly, selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor markedly reduces the E2-mediated increase in F26BP, [(14)C]glucose uptake, and glycolysis. Furthermore, co-treatment of MCF-7 cells with the PFKFB3 inhibitor and the anti-estrogen ICI 182,780 synergistically induces apoptotic cell death. These findings demonstrate for the first time that the estrogen receptor directly promotes PFKFB3 mRNA transcription which, in turn, is required for the glucose metabolism and survival of breast cancer cells. Importantly, these results provide essential preclinical information that may allow for the ultimate design of combinatorial trials of PFKFB3 antagonists with anti-estrogen therapies in ER(+) stage IV breast cancer patients.

Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress

BackgroundA variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis.ResultsAcidosis increased both glutaminolysis and fatty acid β-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes.ConclusionsAcidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific.

Effects of dinuclear berenil-platinum(II) complexes on fibroblasts redox status

Platinum(II) complex anticarcinogenic mechanisms are associated with changes in the cellular redox status of cancer as well as healthy cells. Therefore, the goal of the present study was to investigate oxidative modifications in cellular components following fibroblast exposure to novel dinuclear berenil-platinum(II) complexes. ROS levels, antioxidant parameters level/activity, and damage to DNA, lipids, and proteins, including pro-apoptotic and anti-apoptotic factors in human skin fibroblasts following berenil-platinum(II) complex treatments i.e. Pt2(isopropylamine)4(berenil)2, Pt2(piperazine)4(berenil)4, Pt2(2-picoline)4(berenil)2, Pt2(3-picoline)4(berenil)2, and Pt2(4- picoline)4(berenil)2 were examined. Treatment of fibroblasts with platinum(II) complexes has shown that all compounds enhance total ROS and superoxide anion generation as well as change the activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase and decrease in the level of non-enzymatic antioxidants (GSH, vitamin C, E and A). Such a situation is conducive to oxidative stress formation and oxidative modifications of cellular macromolecules and to increase in the expression of proapoptotic proteins. Pt2(isopropylamine)4(berenil)2 elicited the most damage, which resulted in oxidative modification of cellular components. The therapeutic use of this complex would cause considerable side effects in patients, therefore the agent lacks drug potential; however Pt2(piperazine)4(berenil)2 and Pt2(2-picoline)4(berenil)2 exhibited reduced redox and increased apoptotic profiles compared to cisplatin. Results of this paper and preliminary data show that Pt2(2-picoline)4(berenil)2 is less dangers than cisplatin to fibroblasts and more disruptive than cisplatin to breast cancer cell metabolism, and therefore it is a promising candidate for use in future anticancer drug strategies.

WNT5A-mediated -catenin-independent signalling is a novel regulator of cancer cell metabolism

WNT5A has been identified as an important ligand in the malignant progression of a number of tumours. Although WNT5A signalling is often altered in cancer, the ligand’s role as either a tumour suppressor or oncogene varies between tumour types and is a contemporary issue for investigators of β-catenin-independent WNT signalling in oncology. Here, we report that one of the initial effects of active WNT5A signalling in malignant melanoma cells is an alteration in cellular energy metabolism and specifically an increase in aerobic glycolysis. This was found to be at least in part due to an increase in active Akt signalling and lactate dehydrogenase (LDH) activity. The clinical relevance of these findings was strengthened by a strong correlation (P < 0.001) between the expression of WNT5A and LDH isoform V in a cohort of melanocytic neoplasms. We also found effects of WNT5A on energy metabolism in breast cancer cells, but rather than promoting aerobic glycolysis as it does in melanoma, WNT5A signalling increased oxidative phosphorylation rates in breast cancer cells. These findings support a new role for WNT5A in the metabolic reprogramming of cancer cells that is a context- dependent event.

89 : A role for CCL5 in breast cancer cell metabolism

The inflammatory chemokine, CCL5, activates its cognate receptor, CCR5, to initiate a number of responses including chemotaxis, cell growth and apoptosis. We have shown that CCL5-mediated mTOR activation influences T cell chemotaxis by initiating the translation of proteins implicated in cell migration, including MMP-9 and cyclin D1. Rapid production of these proteins may prime T cells for efficient migration. Recently, we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis, enhanced ATP production and activation of the nutrient sensing AMPK pathway, all associated with enabling CCL5-mediated chemotaxis. Viewed altogether, our data suggest that in addition to influencing cell growth/apoptosis and providing migrational cues for T cells, CCL5-CCR5 interactions increase energy supplies to match the energy demands of chemotaxis. Several studies have demonstrated a pivotal role for CCL5–CCR5 interactions in breast cancer progression: CCL5 is highly expressed in high grade tumors and is a predictor of rapid disease progression in stage II and III breast cancer patients. Conversely, patients with benign breast disorders express low levels of CCL5. Breast cancer cell lines respond to and migrate towards CCL5, and secrete physiological levels of CCL5 in culture. We have shown that at physiological levels, CCL5 enhances proliferation of the breast cancer cell line, MCF-7.CCR5. CCL5 induces the formation of the eIF4F translation initiation complex through an mTOR-dependent process and initiates mRNA translation for proteins associated with cell proliferation, thereby providing CCR5-positive breast cancer cells with a proliferative advantage. Cancer cells are more dependent on glycolysis than non-dividing cells for ATP generation and metabolite flux. Glycolysis is associated with nucleotide synthesis and lactate acidification of the microenvironment, promoting angiogenesis and tumor invasion of the basement membrane. Accordingly, we conducted studies to investigate whether CCL5 activation of CCR5 will influence metabolic events that would enhance tumor growth, tissue invasion and metastases. We provide evidence that in both primary mouse breast cancer tumors and in a panel of breast cancer cell lines, CCL5–CCR5 interactions lead to increased proliferation associated with CCL5–CCR5 mediated increased glucose uptake, increased ATP production, and enhanced glycolysis, associated with extracellular acidification. These data suggest that targeting CCL5–CCR5 interactions that invoke tumor metabolism may be an effective therapeutic strategy to limit tumor growth.

Effects of sorafenib on energy metabolism in breast cancer cells: role of AMPK–mTORC1 signaling

In this study, we investigated the effects and the underlying molecular mechanisms of the multi-kinase inhibitor sorafenib in a panel of breast cancer cell lines. Sorafenib inhibited cell proliferation and induced apoptosis through the mitochondrial pathway. These effects were neither correlated with modulation of MAPK and AKT pathways nor dependent on the ERα status. Sorafenib promoted an early perturbation of mitochondrial function, inducing a deep depolarization of mitochondrial membrane, associated with drop of intracellular ATP levels and increase of ROS generation. As a response to this stress condition, the energy sensor AMPK was rapidly activated in all the cell lines analyzed. In MCF-7 and SKBR3 cells, AMPK enhanced glucose uptake by up-regulating the expression of GLUT-1 glucose transporter, as also demonstrated by AMPKα1 RNA interference, and stimulated aerobic glycolysis thus increasing lactate production. Moreover, the GLUT-1 inhibitor fasentin blocked sorafenib-induced glucose uptake and potentiated its cytotoxic activity in SKBR3 cells. Persistent activation of AMPK by sorafenib finally led to the impairment of glucose metabolism both in MCF-7 and SKBR3 cells as well as in the highly glycolytic MDA-MB-231 cells, resulting in cell death. This previously unrecognized long-term effect of sorafenib was mediated by AMPK-dependent inhibition of the mTORC1 pathway. Suppression of mTORC1 activity was sufficient for sorafenib to hinder glucose utilization in breast cancer cells, as demonstrated by the observation that the mTORC1 inhibitor rapamycin induced a comparable down-regulation of GLUT-1 expression and glucose uptake. The key role of AMPK-dependent inhibition of mTORC1 in sorafenib mechanisms of action was confirmed by AMPKα1 silencing, which restored mTORC1 activity conferring a significant protection from cell death. This study provides insights into the molecular mechanisms driving sorafenib anti-tumoral activity in breast cancer, and supports the need for going on with clinical trials aimed at proving the efficacy of sorafenib for breast cancer treatment.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

BackgroundRecent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells.MethodsIn this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed.ResultsBoth Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer.ConclusionsWe conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect.

Reversal of the Warburg effect in breast cancer cells by a novel agent.

e13566 Background: It is well known that most cancer cells display a Warburg effect, a state of active glycolysis with lactate production under aerobic conditions. NF-κB has been shown to play a role in modulating cancer metabolism and control the balance between glycolysis and mitochondrial respiration. We have previously shown that panepoxydone (PP), a compound isolated from an edible mushroom, interferes with NF-κB mediated signal transduction. Additionally, we have shown that PP decreases cell viability and proliferation in breast cancer cells. To further characterize the antitumor effect of PP, here we have evaluated it ability to modulate cancer metabolism. Methods: The metabolic action of PP was evaluated in estrogen receptor positive (MCF-7) and triple negative (MDAMB-231) breast cancer cell lines. Cytotoxicity was determined by the cell titer glow assay and expressed as IC50. Metabolic activity, expressed as basal oxygen consumption rate (OCR, an indicator of oxidative phosphorylation) and extracellular acidification rate (ECAR, an indicator of lactate acid production), was measured in real-time using the XF24 Extracellular Flux Analyzer. Mitochondrial membrane potential (MMP) alteration was determined through flow cytometry and LDH-A expression was analyzed via western blot. Results: Significant antitumor activity with PP was seen in both MCF-7 (IC50 5 µM) and MDAMB-231 cells (IC50 15 µM). This correlated to a significant dose-dependent reduction in OCAR in MCF-7 (2-fold, p&lt;0.05) and MDAMB-231 cells (3.5-fold, p&lt;0.01). However, while a decrease in ECAR (1.5-fold, p&lt;0.05), along with decreased LDHA expression was noted in the 231 cells (3.5-fold, p&lt;0.01), this was not seen with the MCF-7 cells. A similar trend was noted with increased damaged mitochondria noted in 231-cells, but again no significant difference in MCF-7 cells. Conclusions: Taken together, these results indicate that PP has the potential to modulate metabolism in breast cancer cells, especially triple negative cells, as noted by its ability to inhibit glycolysis. The inhibition of glycolysis has become an important strategy in treating cancer and development of novel glycolytic inhibitors, such as PP, warrants its further investigation in breast cancer treatment.

Abstract 3562: Genome-wide analysis of the vitamin D receptor (VDR) binding sites reveals vitamin D role modulating autophagy and metabolism in luminal-like breast cancer cells.

Abstract The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), regulates gene expression through the vitamin D receptor (VDR). Even though 1,25D3 is still best known as a regulator of bodily calcium homeostasis, 1,25D3 has a plethora of physiological actions which include immune and nervous system modulation, cellular proliferation and differentiation. Epidemiological and model studies have revealed the cancer-protective properties of 1,25D3 in colon, prostate and notably in breast. We are actively investigating the anti-proliferative properties of 1,25D3 in a panel of human breast cancer cell lines. We found that 1,25D3 induces autophagy in several luminal-like breast cancer cell models, but not in basal-like or mesenchymal-like models. We performed VDR and signaling partner RXR whole-genome ChIP-seq analysis to uncover all of the direct 1,25D3 target genes involved in regulation of metabolism and autophagy. Our studies suggest that energy regulation as an important aspect of 1,25D3 anti-proliferative properties in luminal-like breast cancer cells. Citation Format: Luz E. Tavera, Thomas Westerling, Myles Brown. Genome-wide analysis of the vitamin D receptor (VDR) binding sites reveals vitamin D role modulating autophagy and metabolism in luminal-like breast 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 3562. doi:10.1158/1538-7445.AM2013-3562

Abstract 551: The requirement of Runx2-dependent Akt phosphorylation in breast cancer cell survival and metastasis.

Abstract Breast cancer is not only most commonly diagnosed in women worldwide but its metastasis shows serious life threatening consequences. The cell survival and mitogenesis via phosphatidyl inositol 3’ kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling pathways are critical for breast cancer cell survival, proliferation, metabolism and metastasis. A de-regulation of osteogenic Runt-related transcription factor-2 (Runx2) is recently implicated in mammary carcinogenesis. We found that Runx2 differentially regulates the serine/threonine kinase Akt phosphorylation (pAkt), a downstream effecter of PI3K signaling, in non-tumorigenic and invasive mammary epithelial cell lines. The decline in pAkt upon Runx2 suppression in invasive MDA-MB-231 cells was completely abrogated by PI3K inhibitor LY294002. In contrast, the MAPK inhibitor, U0126, showed partial restoration of pAkt upon Runx2 depletion suggesting that Runx2-mediated crosstalk stimulates feedback mechanism within the PI3K and MAPK signaling pathways. The expression analysis of kinases and phosphatases functional in pAkt regulation revealed that the decline in pAkt upon Runx2 knockdown in MDA-MB-231 cells is due to the reduction in PI3K p85 subunit and mammalian target of rapamycin complex-2 (mTORC2). Additionally, we find altered expression of FOXO1, a downstream molecule of PI3K/Akt signaling, due to Runx2-mediated regulation of pAkt levels in MCF-10A and MDA-MB-231 cell lines. To examine the phenotypic effects of Runx2 and PI3K/Akt pathway crosstalk, we evaluated the Runx2-depleted MDA-MB-231 cells for survival under stress conditions. These studies revealed an altered morphology coupled with increased rounding and sub-G1 population in response to glucose- and serum-deprivation upon Runx2 knockdown. The increase in sub-G1 population was restored by glucose and serum re-constitution in the culture media. An increase in Annexin V staining, decline in pAkt levels and cleavage of caspase-3 confirmed the induction of apoptotic cell death. To understand the Runx2-mediated regulation of PI3K/Akt signaling pathway in breast cancer metastasis, the control or Runx2-depleted MDA-MB-231 cells were inoculated intra-cardiacally into immunodeficient mice. The tumors at lung and bone metastatic sites were harvested, FACS-sorted and examined for pAkt levels. Interestingly, to this end, the in-vivo metastatic tumor-derived Runx2 knockdown MDA-MB-231 cells showed an increase in pAkt levels in contrast to in-vitro cultured Runx2 knockdown MDA-MB-231 cells. The data suggests that the mechanism of pAkt activation might involve a dynamic requirement of transcriptional control and feedback regulation within PI3K/Akt and MAPK signaling pathways during different phases of tumor growth in primary and metastatic microenvironment. Citation Format: Manish Tandon, Zujian Chen, Jitesh Pratap. The requirement of Runx2-dependent Akt phosphorylation in breast cancer cell survival and metastasis. [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 551. doi:10.1158/1538-7445.AM2013-551

Metabolic control analysis of respiration in human cancer tissue

Bioenergetic profiling of cancer cells is of great potential because it can bring forward new and effective therapeutic strategies along with early diagnosis. Metabolic Control Analysis (MCA) is a methodology that enables quantification of the flux control exerted by different enzymatic steps in a metabolic network thus assessing their contribution to the system‘s function. Our main goal is to demonstrate the applicability of MCA for in situ studies of energy metabolism in human breast and colorectal cancer cells as well as in normal tissues. We seek to determine the metabolic conditions leading to energy flux redirection in cancer cells. A main result obtained is that the adenine nucleotide translocator exhibits the highest control of respiration in human breast cancer thus becoming a prospective therapeutic target. Additionally, we present evidence suggesting the existence of mitochondrial respiratory supercomplexes that may represent a way by which cancer cells avoid apoptosis. The data obtained show that MCA applied in situ can be insightful in cancer cell energetic research.

Abstract P6-01-05: Enhancement of18F-FDG uptake and glycolysis by epidermal growth factor via PI3K activation in T47D breast cancer cells

Abstract It is well known that the binding of epidermal growth factor (EGF) to EGF receptors (EGFR) stimulates proliferation of breast cancer cells via activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathways. There was a report that glucose consumption and lactate production were increased in MDA-MB-468 breast cancer cells following EGF exposure. However, the effect of EGF in enhancing glucose uptake and signaling pathways that are involved have not been clearly revealed. We thus investigated the role of EGF in stimulation of18F-FDG uptake in T47D breast cancer cells, and further elucidated the molecular mechanisms that are involved. Exposure of T47D cells to 100 ng/mL EGF for 24 h caused a substantial augmentation of18F-FDG uptake to even greater extents to 310.8% ± 30.1% of control cells (P &amp;lt; 0.001), and this effect of EGF showed dose- and time-dependency. Increased glucose uptake by EGF occurred through enhanced membrane GLUT-1 expression as well as hexokinase activity. Inhibition experiments with cycloheximide showed that the metabolic effect by EGF required new protein biosynthesis. Stimulation of glucose uptake by EGF was dependent on sufficient EGFR expression because18F-FDG uptake in MCF-7 cells weakly expressing EGFR was not affected by EGF treatment. Although EGF was also a stimulator of T47D cell proliferation (137.3 ± 7.3% of basal levels at 24 h, P &amp;lt; 0.001), the metabolic effect occurred in a manner that clearly preceded and surpassed the proliferative effect. EGF-stimulated proliferation was significantly inhibited by the EGFR inhibitor BIBX1382, the PI3K inhibitor LY294002 and the specific MAPK inhibitor PD98059. Unlike proliferation, the ability of EGF to augment18F-FDG uptake was found to be dependent on PI3K but not on MAPK activity. These findings yield the insight into our understanding of the role of EGF and the molecular basis that are involved in glucose metabolism of breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-01-05.

Oestrogen-related receptors in breast cancer: control of cellular metabolism and beyond

Oestrogen-related receptors (ERRs) are orphan nuclear receptors that were initially investigated in breast cancer because of their structural relationship to oestrogen receptors. Recent data have shown that the ERRs control vast gene networks that are involved in glycolysis, glutaminolysis, oxidative phosphorylation, nutrient sensing and biosynthesis pathways. In the context of breast cancer, the ERRs affect cellular metabolism in a manner that promotes a Warburg-like phenotype. The ERRs also modulate breast cancer cell metabolism, growth and proliferation through the regulation of key oncoproteins. We discuss the value but also the implications of the complexity of targeting the ERRs for the development of cancer therapeutics.

The Effect of Nitric Oxide on Mitochondrial Metabolism and Proliferation in Breast Cancer Cells

The Effect of Nitric Oxide on Mitochondrial Metabolism and Proliferation in Breast Cancer Cells

The lipid phenotype of breast cancer cells characterized by Raman microspectroscopy: towards a stratification of malignancy.

Although molecular classification brings interesting insights into breast cancer taxonomy, its implementation in daily clinical care is questionable because of its expense and the information supplied in a single sample allocation is not sufficiently reliable. New approaches, based on a panel of small molecules derived from the global or targeted analysis of metabolic profiles of cells, have found a correlation between activation of de novo lipogenesis and poorer prognosis and shorter disease-free survival for many tumors. We hypothesized that the lipid content of breast cancer cells might be a useful indirect measure of a variety of functions coupled to breast cancer progression. Raman microspectroscopy was used to characterize metabolism of breast cancer cells with different degrees of malignancy. Raman spectra from MDA-MB-435, MDA-MB-468, MDA-MB-231, SKBR3, MCF7 and MCF10A cells were acquired with an InVia Raman microscope (Renishaw) with a backscattered configuration. We used Principal Component Analysis and Partial Least Squares Discriminant Analyses to assess the different profiling of the lipid composition of breast cancer cells. Characteristic bands related to lipid content were found at 3014, 2935, 2890 and 2845 cm−1, and related to lipid and protein content at 2940 cm−1. A classificatory model was generated which segregated metastatic cells and non-metastatic cells without basal-like phenotype with a sensitivity of 90% and a specificity of 82.1%. Moreover, expression of SREBP-1c and ABCA1 genes validated the assignation of the lipid phenotype of breast cancer cells. Indeed, changes in fatty acid unsaturation were related with the epithelial-to-mesenchymal transition phenotype. Raman microspectroscopy is a promising technique for characterizing and classifying the malignant phenotype of breast cancer cells on the basis of their lipid profiling. The algorithm for the discrimination of metastatic ability is a first step towards stratifying breast cancer cells using this rapid and reagent-free tool.

P-Hydroxybenzoate esters metabolism in MCF7 breast cancer cells

Parabens are among the most frequently used preservatives to inhibit microbial growth and extend the shelf life of a range of consumer products. The objective of the present study was to gain insight into the metabolism of parabens in breast cancer cells (MCF7) since they have demonstrated estrogenic activity towards these cells and have been detected in breast cancer tissues. The toxicity of parabens to MCF7 cells was determined using MTT assays. Hydrolysis of methyl-, butyl and benzyl-paraben to p-hydroxybenzoic acid was analyzed in cultured MCF7 cells and in cellular homogenates. Glucuronidation and sulfoconjugation were studied in MCF7 homogenates, and parabens were analyzed by HPLC. Methyl-paraben was shown to be far less toxic than butyl and benzyl-paraben. Parabens were completely stable in MCF7 homogenates whereas p-nitrophenyl acetate, a substrate type, underwent hydrolysis. MCF7 cell homogenates did not express glucuronidation and sulfoconjugation activities toward parabens. The higher stability of parabens may explain their accumulation in breast cancer tissue as previously reported in the literature.