Glycolytic Phenotype Of Cancer Cells Research Articles

Correlation of PKM2 and CD44 Protein Expression with Poor Prognosis in Platinum-Treated Epithelial Ovarian Cancer: A Retrospective Study.

CD44, a surface marker for cancer stem cells, interacts with PKM2, a key regulator of aerobic glycolysis, and enhances the glycolytic phenotype of cancer cells leading to antioxidant protection and macromolecules’ synthesis. To clarify the clinical importance of this “cross-talk” as a mechanism of drug resistance, we assessed the expression both of PKM2 and of CD44 in cancer cells of patients with epithelial ovarian cancer (EOC) treated with platinum-based treatment. One hundred and seventy-one patients with EOC were assessed for PKM2mRNA expression and PKM2 and CD44 proteins detection. Associations with progression-free survival (PFS) and overall survival (OS) were assessed with Kaplan–Meier and adjusted Cox regression models. PKM2mRNA and protein as well as CD44 protein were detectable in the majority of patients. Positive correlation between PKM2 and CD44 protein expression was observed (Spearman rho = 0.2, p = 0.015). When we used the median to group patients into high versus low expression, high PKM2mRNA and protein levels were significantly associated with lower progression-free survival (PFS; p = 0.003 and p = 0.002, respectively) and shorter overall survival (OS; p ≤ 0.001 and p = 0.001, respectively). However, high CD44 protein expression was significantly correlated only with shorter OS (p = 0.004). Moreover, patients with both high PKM2 and CD44 protein levels experienced shorter PFS and OS (p = 0.007 and p = 0.003, respectively) compared to patients with low expression of both proteins. Finally, higher PKM2mRNA and protein expression as well as CD44 protein expression (HR: 2.16; HR: 1.82; HR: 1.01, respectively) were independent prognostic factors for decreased median OS (mOS), whereas only PKM2 protein expression (HR: 1.95) was an independent prognostic factor for decreased median PFS (mPFS). In conclusion, PKM2 expression is a negative prognostic factor in EOC patients, but the interaction between CD44 and PKM2 that may be implicated in EOC platinum-resistance needs further investigation.

Mitoketoscins: Novel mitochondrial inhibitors for targeting ketone metabolism in cancer stem cells (CSCs)

Previous studies have now well-established that epithelial cancer cells can utilize ketone bodies (3-hydroxybutyrate and aceto-acetate) as mitochondrial fuels, to actively promote tumor growth and metastatic dissemination. The two critical metabolic enzymes implicated in this process are OXCT1 and ACAT1, which are both mitochondrial proteins. Importantly, over-expression of OXCT1 or ACAT1 in human breast cancer cells is sufficient to genetically drive tumorigenesis and/or lung metastasis, validating that they indeed behave as metabolic “tumor promoters”. Here, we decided to target these two enzymes, which give cancer cells the ability to recycle ketone bodies into Acetyl-CoA and, therefore, to produce increased ATP. Briefly, we used computational chemistry (in silico drug design) to select a sub-set of potentially promising compounds that spatially fit within the active site of these enzymes, based on their known 3D crystal structures. These libraries of compounds were then phenotypically screened for their effects on total cellular ATP levels. Positive hits were further validated by metabolic flux analysis. Our results indicated that four of these compounds effectively inhibited mitochondrial oxygen consumption. Two of these compounds also induced a reactive glycolytic phenotype in cancer cells. Most importantly, using the mammosphere assay, we showed that these compounds can be used to functionally inhibit cancer stem cell (CSC) activity and propagation. Finally, our molecular modeling studies directly show how these novel compounds are predicted to bind to the active catalytic sites of OXCT1 and ACAT1, within their Coenzyme A binding site. As such, we speculate that these mitochondrial inhibitors are partially mimicking the structure of Coenzyme A. Thus, we conclude that OXCT1 and ACAT1 are important new therapeutic targets for further drug development and optimization. We propose that this new class of drugs should be termed “mitoketoscins”, to reflect that they were designed to target ketone re-utilization and mitochondrial function.

Ablation of CD44 induces glycolysis-to-oxidative phosphorylation transition via modulation of the c-Src-Akt-LKB1-AMPKα pathway.

Cluster of differentiation 44 (CD44) is a transmembrane glycoprotein that has been identified as a cancer stem cell marker in various cancer cells. Although many studies have focused on CD44 as a cancer stem cell marker, its effect on cancer cell metabolism remains unclear. To investigate the role of CD44 on cancer cell metabolism, we established CD44 knock-down cells via retroviral delivery of shRNA against CD44 in human breast cancer cells. Silencing of CD44 decreased the glycolytic phenotype of cancer cells, affecting glucose uptake, ATP production, and lactate production. We also found that ablation of the CD44-induced lactate dehydrogenase (LDH) isoenzyme results in a shift to LDH1 due to LDHA down-regulation and LDHB up-regulation, implying the importance of LDH isoenzyme modulation on cancer metabolism. The expression of glycolysis-related proteins including hypoxia inducible factor-1α (HIF-1α) and LDHA was decreased by CD44 silencing. These effects were due to the up-regulation of liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK)α activity by reduction in c-Src and Akt activity in CD44 knock-down cells. Finally, induction of LKB1/AMPKα activity blocked the expression of HIF-1α and its target gene, LDHA. Inversely, LDHB expression was repressed by HIF-1α. Collectively, these results indicate that the CD44 silencing-induced metabolic shift is mediated by the regulation of c-Src/Akt/LKB1/AMPKα/HIF-1α signaling in human breast cancer cells.

PDK2-mediated alternative splicing switches Bnip3 from cell death to cell survival.

Herein we describe a novel survival pathway that operationally links alternative pre-mRNA splicing of the hypoxia-inducible death protein Bcl-2 19-kD interacting protein 3 (Bnip3) to the unique glycolytic phenotype in cancer cells. While a full-length Bnip3 protein (Bnip3FL) encoded by exons 1-6 was expressed as an isoform in normal cells and promoted cell death, a truncated spliced variant of Bnip3 mRNA deleted for exon 3 (Bnip3Δex3) was preferentially expressed in several human adenocarcinomas and promoted survival. Reciprocal inhibition of the Bnip3Δex3/Bnip3FL isoform ratio by inhibiting pyruvate dehydrogenase kinase isoform 2 (PDK2) in Panc-1 cells rapidly induced mitochondrial perturbations and cell death. The findings of the present study reveal a novel survival pathway that functionally couples the unique glycolytic phenotype in cancer cells to hypoxia resistance via a PDK2-dependent mechanism that switches Bnip3 from cell death to survival. Discovery of the survival Bnip3Δex3 isoform may fundamentally explain how certain cells resist Bnip3 and avert death during hypoxia.

PKM2 and CD44 tumoral expression to predict the effectiveness of front-line paclitaxel and carboplatin chemotherapy in epithelial ovarian cancer.

e16564 Background: CD44, a surface marker for cancer stem cells interacts with PKM2, a key regulator of aerobic glycolysis and enhances the glycolytic phenotype of cancer cells that, subsequently, ...

12. Prevention of cisplatin-induced nephrotoxicity by dichloroacetate

Background There is a continuing need to improve the efficacy of anticancer agents and to reduce the toxicity associated with them. Recently, reversal of the glycolytic phenotype of cancer cells with novel agents such as DCA (dichloroacetate) has been recognised as an important new target for cancer therapy. DCA is currently being assessed as a novel chemotherapeutic in clinical trials. It is anticipated that DCA will be used in combination with other well-established chemotherapeutic drugs. It is therefore critically important to determine the effects of DCA on therapeutic efficacy, and off-target effects of existing frontline anti-cancer drugs. We have recently discovered that DCA induces glutamate cysteine ligase, the rate-limiting step in glutathione (GSH) synthesis, a major cellular antioxidant. We hypothesised that the ability of DCA to stimulate GSH-synthesising capacity could reduce cisplatin-induced nephrotoxicity, which is thought to be mediated in part by oxidative stress. Aims To determine whether DCA can attenuate cisplatin-induced nephrotoxicity, and to determine if DCA influences the anti-cancer properties of cisplatin. Methods 120 Balb/c mice were injected subcutaneously with the syngeneic 4T1 breast cancer cell line and then co-treated with DCA and cisplatin weekly for 1 month. Controls included treatment with cisplatin alone, DCA alone, or normal saline injections. Serum blood urea nitrogen (BUN) and creatinine were measured and kidney damage was assessed histologically. Tumour size was monitored throughout. Results and conclusions DCA prevented increases in serum creatinine, BUN, and renal proximal tubule apoptosis evident in cisplatin-only treated mice. The tumour size in mice in the co-treated group decreased at the same rate as the cisplatin-only treated mice. We have thus concluded that DCA largely prevents the development of cisplatin-induced nephrotoxicity, and does not attenuate its anti-cancer properties.

Abstract B155: VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and demonstrates context-dependent cytotoxicity to cancer cells.

Abstract Pyruvate dehydrogenase kinase (PDK) regulates the activity of the pyruvate dehydrogenase complex (PDC) through phosphorylation of three serine residues on the E1α subunit, resulting in decreased activity. The expression of all four mammalian isoforms of PDK have been shown to be up-regulated either under tumour relevant conditions (PDK-1 & PDK-3 by hypoxia) or by the loss of function of common tumour suppressor genes (PDK-2 by p53; PDK-4 by pRB). Furthermore, PDK-1 expression has been clinically correlated with poor prognosis in Gastric, HNSCC and Colon cancer. Previous studies employing RNAi have provided evidence for a survival role for PDK-1 as well as its importance in maintaining the glycolytic phenotype of cancer cells. DCA, a weak inhibitor of PDK, has also been used to study the role of PDK in cancer; however, interpretation of these studies has been complicated by conflicting data and the lack of specificity of this agent for PDK. Here we report the discovery of a novel, potent and selective pan-isoform inhibitor of PDK, VER-246608. Consistent with a PDK mediated MOA, treatment of PC-3 cells with VER-246608 resulted in increased PDC activity and oxygen consumption as well as reduced lactate production and glucose consumption. Interestingly, modulation of glycolytic activity required compound concentrations which achieved > 90 % reduction in E1α phosphorylation and this was only observed under glucose depleted conditions. Under normal culture conditions, VER-246608 showed little cytotoxicity to cancer cells. We hypothesised that the supraphysiological glucose concentrations present in cell culture media may limit the effect of PDK inhibition due to elevated intracellular pyruvate levels. We therefore performed cytotoxicity studies under conditions of limited nutrient availability. We found that combined depletion of glucose and glutamine or serum alone resulted in enhanced cytotoxicity vs normal media; however, hypoxic conditions had no effect. Furthermore, this differential cytotoxicity correlated with reduced pyruvate levels in the compound treated cells cultured in the above ‘austere’ conditions. VER-246608 also inhibited the growth of tumour spheroids with a potency that was comparable to cells grown in 2D culture. In addition, combination treatment studies revealed that VER-246608 potentiated anti-cancer agents which are known to influence mitochondrial function. In contrast, the lipoamide binding site inhibitor, Nov3r, showed no evidence of cytotoxicity under any of the above conditions and was ineffective in altering glycolytic activity. These studies suggest that PDK inhibition may be effective under the nutrient depleted conditions found in the tumour microenvironment and that combination treatments should be explored to reveal the full potential of this therapeutic strategy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B155. Citation Format: Jonathan Moore, Anna Staniszewska, Terence Shaw, Jalanie D'Alessandro, Ben Davis, Alan Surgenor, Lisa Baker, Natalia Massanova, James Murray, Alba Macias, Paul Brough, Mike Wood, Patrick C. Mahon. VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and demonstrates context-dependent cytotoxicity to cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B155.

Warburg effect increases steady-state ROS condition in cancer cells through decreasing their antioxidant capacities (Anticancer effects of 3-bromopyruvate through antagonizing Warburg effect)

Cancer cells undergo an increased steady-state ROS condition compared to normal cells. Among the major metabolic differences between cancer cells and normal cells is the dependence of cancer cells on glycolysis as a major source of energy even in the presence of oxygen (Warburg effect). In Warburg effect, glucose is catabolized to lactate that is extruded through monocarboxylate transporters to the microenvironment of cancer cells, while in normal cells, glucose is metabolized into pyruvate that is not extruded. Pyruvate is a potent antioxidant, while lactate has no antioxidant effect. Pyruvate in normal cells may be further metabolized to acetyl CoA and then through Krebs cycle with production of antioxidant intermediates e.g. citrate, malate and oxaloacetate together with the reducing equivalents (NADH.H+). Through activity of mitochondrial transhydrogenase, NADH.H+ replenishes NADPH.H+, coenzyme of glutathione reductase which replenishes reduced form of glutathione (potent antioxidant). This enhances antioxidant capacities of normal cells, while cancer cells exhibiting Warburg effect may be deprived of all that antioxidant capabilities due to loss of extruded lactate (substrate for Krebs cycle). Although intrinsic oxidative stress in cancer cells is high, it may be prevented from reaching progressively increasing levels that are cytotoxic to cancer cells. This may be due to some antioxidant effects exerted by hexokinase II (HK II) and NADPH.H+ produced through HMP shunt. Glycolytic phenotype in cancer cells maintains a high non-toxic oxidative stress in cancer cells and may be responsible for their malignant behavior. Through HK II, glycolysis fuels the energetic arm of malignancy, the mitotic arm of malignancy (DNA synthesis through HMP shunt pathway) and the metastatic arm of malignancy (hyaluronan synthesis through uronic acid pathway) in addition to the role of phosphohexose isomerase (autocrine motility factor). All those critical three arms start with the substrate G6P that is a direct product of HK II. 3-bromopyruvate (3BP, inhibitor of HK II) may prove as a promising anticancer and antimetastatic agent based on antagonizing the Warburg effect and disturbing the malignant behavior in cancer cells.

Phase I study of BPM 31510 in advanced solid tumors: Updated analysis of a novel treatment with promising activity.

3015 Background: BPM 31510 is a novel small molecule that targets the metabolic machinery of the cancer microenvironment to reverse the aerobic glycolytic phenotype of cancer cells. Effector downstream signaling results in re-capitulation of BCL-2 mediated apoptosis and disruption in tumor vasculature by modulation of VEGF. (NR Narain et al., Proceedings of AACR Meeting Abstracts 2011). Methods: A standard 3+3 phase I, dose-escalation study design was used in patients with advanced solid tumors refractory to standard treatment. Primary objectives were establishment of the maximum tolerated dose (MTD) and safety/pharmacokinetic (PK) correlates. Secondary objectives included exploratory pharmacodynamics (PD) and preliminary efficacy (RECIST-1.1) of BPM 31510 in sequential cohorts of 3 to 6 pts. Results: At time of submission, 34 patients with advanced cancer who had failed multiple chemotherapeutic regimens had been enrolled in 7 dose cohorts (ranging from 5.6 mg/kg to 78.2 mg/kg). Patients received a median of 2 cycles (1-7). 2 patients have had grade 3 elevation in PT/INR, otherwise there have been no grade 3/4 treatment related toxicities to date. The pharmacokinetics of BPM 31510 are linear and there were no sex differences in the parameters normalized by dose and body surface area. Tmax and Cmax are associated with the end of the infusion. The values for t1/2 ranged from 2.18 to 13.3 hr, with little or no dependence of t1/2 on dose. Objective tumor responses have been noted at the dose of 58.6mg/kg with 1 partial response (myxoid liposarcoma) and 1 minor response (pleomorphic sarcoma). Six patients (19%) have had disease stabilization (> 4 months). Conclusions: Interim data from this phase I study indicate that BPM 31510 is well tolerated with no dose limiting toxicities to date. A partial response and minor response were observed and correlates with dose escalation. Taken together, there is strong rationale for further clinical development of this compound as an anti-cancer agent.

Abstract LB-163: Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells

Abstract An increased glycolytic flux accompanied by activation of the pentose phosphate pathway (PPP) is implicated in chemoresistance of cancer cells. In this study, we found that CD44, a cell surface marker for cancer stem cells, interacts with pyruvate kinase M2 (PKM2) and thereby enhances the glycolytic phenotype of cancer cells that are either deficient in p53 or exposed to hypoxia. CD44 ablation by RNA interference increased metabolic flux to mitochondrial respiration and concomitantly inhibited entry into glycolysis and the PPP. Such metabolic changes induced by CD44 ablation resulted in marked depletion of cellular reduced glutathione (GSH) and increased the intracellular level of reactive oxygen species (ROS) in glycolytic cancer cells. Furthermore, CD44 ablation enhanced the effect of chemotherapeutic drugs in p53-deficient or hypoxic cancer cells. Taken together, our findings suggest that metabolic modulation by CD44 is a potential therapeutic target for glycolytic cancer cells that manifest drug resistance. 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-163. doi:1538-7445.AM2012-LB-163

Modulation of Glucose Metabolism by CD44 Contributes to Antioxidant Status and Drug Resistance in Cancer Cells

An increased glycolytic flux accompanied by activation of the pentose phosphate pathway (PPP) is implicated in chemoresistance of cancer cells. In this study, we found that CD44, a cell surface marker for cancer stem cells, interacts with pyruvate kinase M2 (PKM2) and thereby enhances the glycolytic phenotype of cancer cells that are either deficient in p53 or exposed to hypoxia. CD44 ablation by RNA interference increased metabolic flux to mitochondrial respiration and concomitantly inhibited entry into glycolysis and the PPP. Such metabolic changes induced by CD44 ablation resulted in marked depletion of cellular reduced glutathione (GSH) and increased the intracellular level of reactive oxygen species in glycolytic cancer cells. Furthermore, CD44 ablation enhanced the effect of chemotherapeutic drugs in p53-deficient or hypoxic cancer cells. Taken together, our findings suggest that metabolic modulation by CD44 is a potential therapeutic target for glycolytic cancer cells that manifest drug resistance.

Glucose metabolism in cancer cells

Cancer cells alter their metabolism in order to support their rapid proliferation and expansion across the body. In particular, tumor cells, rather than fueling glucose in the oxidative phosphorylation pathway, generally use glucose for aerobic glycolysis. In this review, we discuss some of the mechanisms thought to be responsible for the acquisition of a glycolytic phenotype in cancer cells and how the switch towards glycolysis represents a selective growth advantage. Glucose deprivation can activate oncogenes and these can upregulate proteins involved in aerobic glycolysis. In turn, proteins implicated in increased glycolysis can render tumor cells more resistant to apoptosis. Aerobic glycolysis induces acidification of the tumor environment, favoring the development of a more aggressive and invasive phenotype. Altering the pH around tumors might represent a way to hamper tumor development as suggested by a recent work demonstrating that bicarbonate, which increases the pH of tumors, prevented spontaneous metastatization. The acquisition of a glycolytic phenotype by transformed cells confers a selective growth advantage to these cells. Interfering with aerobic glycolysis, therefore, represents a potentially effective strategy to selectively target cancer cells.

Reversal of the glycolytic phenotype with dichloroacetate in a mouse mammary adenocarcinoma model

Dichloroacetate (DCA) has recently been proposed as a novel non-toxic anticancer agent that can reverse the glycolytic phenotype in cancer cells through the inhibition of pyruvate dehydrogenase kinase. Aims and Methods Three mouse cancer cell lines (V14, 4T1 and B16) were used to examine the histological changes caused by DCA in vivo. V14 or 4T1 mammary adenocarcinoma cells were injected subcutaneously and B16 melanoma cells were injected intravenously. Mice were treated with 0.5–2.0 g/L DCA in the drinking water. Results Growth of established V14 tumours was halted by DCA treatment (28% reduction in size, 1.5 g/L for 9 days, n = 8 per group, p = 0.08). Treatment from the day of cell injection resulted in fewer tumours being established (5/8 sites in treated mice vs 16/16 untreated, p = 0.03) and complete regression of one tumour. Histological assessment found DCA treatment resulted in increased tumour-infiltrating lymphocytes (TILs) but no change in mitotic rate, fibrosis or necrosis. Tumours from the 4T1 or B16 cell lines showed no growth inhibition and no evidence of TILs, correlating with a lack of sensitivity to growth inhibition in vitro. Conclusions In V14 tumours DCA appears to prevent or delay breast cancer formation either through growth inhibition or enhanced immune function.

18. Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivo

Dichloroacetate (DCA) has recently been proposed as a novel and relatively non-toxic anticancer agent that can reverse the glycolytic phenotype in cancer cells through the inhibition of pyruvate dehydrogenase kinase. Over 90% of all tumours show an increased glycolysis and this response could contribute to tumour progression, metastasis and resistance to various chemotherapy agents. Reversing the glycolytic phenotype could potentially slow down tumour growth, induce apoptosis and delay metastasis and is therefore a potential anti-cancer strategy. Aims and Methods Rat breast adenocarcinoma cell lines were used to examine the effect of DCA both in vitroand in vivo. Results The growth of several breast cancer cell lines (MCF-7, T47D, 13762 MAT and V14 cells) was found to be inhibited by DCA in vitro.Further examination of 13762 MAT breast adenocarcinoma cells found that reversal of the glycolytic phenotype by DCA correlated with the inhibition of proliferation without any increase in cell death. This was despite a small but significant increase in caspase 3/7 activity, which may sensitise cancer cells to other apoptotic triggers. In vivo,DCA caused a 58% reduction in the number of lung metastases observed macroscopically after injection of 13762 MAT cells into the tail vein of rats (p = 0.0001, n>9 per group). The lesions in the DCA treated rats developed less tumoral necrosis, had a higher mitotic count and a greater lymphocytic infiltration than the control group. Conclusion These results demonstrate the anti-cancer potential of DCA and the reversing the glycolytic phenotype.

Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivo

The glycolytic phenotype is a widespread phenomenon in solid cancer forms, including breast cancer. Dichloroacetate (DCA) has recently been proposed as a novel and relatively non-toxic anti-cancer agent that can reverse the glycolytic phenotype in cancer cells through the inhibition of pyruvate dehydrogenase kinase. We have examined the effect of DCA against breast cancer cells, including in a highly metastatic in vivo model. The growth of several breast cancer cell lines was found to be inhibited by DCA in vitro. Further examination of 13762 MAT rat mammary adenocarcinoma cells found that reversal of the glycolytic phenotype by DCA correlated with the inhibition of proliferation without any increase in cell death. This was despite a small but significant increase in caspase 3/7 activity, which may sensitize cancer cells to other apoptotic triggers. In vivo, DCA caused a 58% reduction in the number of lung metastases observed macroscopically after injection of 13762 MAT cells into the tail vein of rats (P = 0.0001, n > or = 9 per group). These results demonstrate that DCA has anti-proliferative properties in addition to pro-apoptotic properties, and can be effective against highly metastatic disease in vivo, highlighting its potential for clinical use.