Hexokinase II Research Articles

Abstract 1038: Exploiting ROS and metabolic differences to selectively killing cisplatin resistant lung cancer

Abstract Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the KRAS mutation status, share one common parameter which is increased reactive oxygen species (ROS) when compared to their parental cells counterpart. Importantly, we found that CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism (OXMET) for energy and biosynthesis. Thus, alteration in mitochondria which resulted in metabolic changes and higher basal levels of ROS may be one of the major factors contributing to cisplatin resistance. We have investigated mitochondrial morphology in parental vs. CR cells using transmission electron microscopy (TEM). Our data clearly showed that CR cells possessed significantly higher number of mitochondria per total cell area (n = 4; p = 0.0006). CR cells also consumed 2-4 fold more oxygen when compared to their parental cells. To further confirm that oxidative phosphorylation was utilized more in CR cells, we assayed mitochondrial membrane potential (MMP) using TMRE staining. MMP was significantly higher in CR cells suggesting that CR cells possess highly active mitochondria. Key glycolytic enzymes hexokinase II (HKII) and lactate dehydrogenase A (LDHA), as well as lactate production were decreased in all CR cell lines which corresponded to the resistance to glycolytic inhibitor, 2DG (n = 8; p<0.03). Moreover, CR cells were highly sensitive to glutamine deprivation. Glutamine withdrawal for 72h resulted in 75-80% cell death in CR cells while only 10-15% cell death occurred in parental cells. CR also take up twice as much L-[G-3H] glutamine when compared to its parental counterpart (n = 4, p<0.05). Here, using Riluzole (the FDA approved drug that interferes with cystine/glutamate pump and results in reduced intracellular glutathione (GSH) levels), can selectively kill CR cells. ROS levels were increased (2-3X) more in resistant cells after Riluzole treatment while no significant change occurred in parental cells. The ID50 dosage of parental cells were 3-4 fold more than theirs CR cell counterparts and no cytotoxicity were found in normal lung fibroblast (n = 8; p<0.05). However, antioxidant, N-acetylcysteine (NAC, 0.1mM), cannot completely abort cell death by Riluzole suggesting other mechanisms are involved. Importantly, Riluzole is also effective in 2 mouse xenograft models (H460CR (KRAS mutant) and SC (wt)). Tumors completely disappeared in wild type mice and were significant reduced in KRAS mutant mice (n = 5 per treatment group; p<0.002). We conclude that addiction to glutamine instead of glucose and higher basal ROS levels seen in CR cells can make them hypersensitive to Riluzole. Repurpose this drug should be considered for future treatment of CR lung cancer patients. Supported by Department of Veterans Affairs, CDA2 award (1K2BX001289) and Woman Cancer Association Fund. Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Sumedh Shah, Shu-Mei Chen, Vy Dinh, Macus T. Kuo, Lynn G. Feun, Niramol Savaraj. Exploiting ROS and metabolic differences to selectively killing cisplatin resistant lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1038.

Alterations in glucose metabolism proteins responsible for the Warburg effect in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is the most frequent esophageal tumor in the world. ESCC presents late diagnosis, highly aggressive behavior and poor survival. Changes in tumor cell energy metabolism appear to have a prominent role in malignant transformation. Tumor cells consume glucose avidly and produce lactic acid, even under normoxia. Among the factors that may contribute to the stimulation of glycolysis in tumor cells, there are changes in the glycolytic pathway enzymes such as: pyruvate kinase M1 and M2 (PKM2 and PKM1), hexokinase II (HKII), glucose transporter isoform 1 (GLUT-1), and transcription factor induced by hypoxia (HIF1α), responsible for the transcription of proteins cited. The objective of this study is to evaluate the alterations of these proteins and their association with clinicopathological data in ESCC. We performed immunohistochemistry to determine HIF-1α, GLUT-1, PKM1, PKM2, HK2 and Ki67-expression in ESCC patients and controls. Also, we used RT-qPCR to evaluated mRNA expression of GLUT-1 in esophageal mucosa of individuals without cancer, but are alcohol drinkers and tobacco smokers. Our results showed the exclusively expression of GLUT-1 in tumors cells and dysplastic samples. We also observed a compartmentalization of the expression of PKM1 and PKM2 in relation to tumor cells and stroma associated to tumor areas. All of the proteins evaluated, excepted GLUT-1, were frequently detected in normal mucosa. No correlations between clinicopathological features and protein expressions were observed. GLUT-1 expression appears in initial tumor lesions and is maintained through ESCC evolution. We reported for the first time PKM1 staining in normal esophagus and ESCC, being mostly present in more differentiated cells.

Re-programming tumour cell metabolism to treat cancer: no lone target for lonidamine.

Tumour cell metabolism is very different from normal cell metabolism; cancer cells re-programme the metabolic pathways that occur in normal cells in such a manner that it optimizes their proliferation, growth and survival. Although this metabolic re-programming obviously operates to the advantage of the tumour, it also offers unique opportunities for effective cancer therapy. Molecules that target the tumour cell-specific metabolic pathways have potential as novel anti-cancer drugs. Lonidamine belongs to this group of molecules and is already in use in some countries for cancer treatment. It has been known for a long time that lonidamine interferes with energy production in tumour cells by inhibiting hexokinase II (HKII), a glycolytic enzyme. However, subsequent studies have uncovered additional pharmacological targets for the drug, which include the electron transport chain and the mitochondrial permeability transition pore, thus expanding the pharmacological effects of the drug on tumour cell metabolism. A study by Nancolas et al. in a recent issue of the Biochemical Journal identifies two additional new targets for lonidamine: the pyruvate transporter in the mitochondria and the H+-coupled monocarboxylate transporters in the plasma membrane (PM). It is thus becoming increasingly apparent that the anti-cancer effects of lonidamine do not occur through a single target; the drug works at multiple sites. Irrespective of the molecular targets, what lonidamine does in the end is to undo what the tumour cells have done in terms of re-programming cellular metabolism and mitochondrial function.

Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma.

We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XFe-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0.007); and drug resistance to doxorubicin (p = 0.008) of the TFAM-KD clone were significantly higher than those of the NT clone. Bioenergetically, the TFAM-KD clone expressed lower mOCRB (p = 0.009) but higher ECARB (p = 0.037) than did the NT clone. We conclude that a reduction of mtDNA copy number and decrease of respiratory function of mitochondria in RCC might be compensated for by an increase of enzymes and factors that are involved in the upregulation of glycolysis to confer RCC more invasive and a drug-resistant phenotype in vitro.

Dual time-point FDG PET/CT and FDG uptake and related enzymes in lymphadenopathies: preliminary results.

The purpose of this study was to determine the ability of dual time-point (DTP) PET/CT with (18)F-FDG to discriminate between malignant and benign lymphadenopathies. The relationship between DTP FDG uptake and glucose metabolism/hypoxia markers in lymphadenopathies was also assessed. Patients with suspected lymphoma or recently diagnosed treatment-naive lymphoma were prospectively enrolled for DTP FDG PET/CT (scans 60min and 180min after FDG administration). FDG-avid nodal lesions were segmented to yield volume and standardized uptake values (SUV), including SUVmax, SUVmean, cSUVmean (with partial volume correction), total lesion glycolysis (TLG) and cTLG (with partial volume correction). Expression of glucose transporter-1 (GLUT-1), hexokinase-II (HK-II), glucose-6-phosphatase (G6Pase) and hypoxia-inducible factor-1alpha (HIF-1alpha) were assessed with immunohistochemistry and enzyme activity was determined for HK and G6Pase. FDG uptake was assessed in 203 lesions (146 malignant and 57 benign). Besides volume, there were significant increases over time for all parameters, with generally higher levels in the malignant lesions. The retention index (RI) was not able to discriminate between malignant and benign lesions. Volume, SUVmax, TLG and cTLG for both scans were able to discriminate between the two groups statistically, but without complete separation. Glucose metabolism/hypoxia markers were assessed in 15 lesions. TLG and cTLG were correlated with GLUT-1 expression on the 60-min scan. RI-max and RI-mean and SUVmax, SUVmean and cSUVmean on the 60-min scan were significantly correlated with HK-II expression. RI was not able to discriminate between malignant and benign lesions, but some of the SUVs were able to discriminate on the 60-min and 180-min scans. Furthermore, FDG uptake was correlated with GLUT-1 and HK-II expression.

Differential association of STAT3 and HK-II expression in hepatitis B virus- and hepatitis C virus-related hepatocellular carcinoma.

STAT3 and hexokinase II (HK-II) are involved in viral infection and carcinogenesis of various cancers including hepatocellular carcinoma (HCC). The roles of STAT3 and HK-II in hepatitis B virus (HBV)- and hepatitis C virus (HCV)-related HCC remain largely unclear. This study examined STAT3 and HK-II expression in HBV- and HCV-related HCC, HBV-related liver fibrosis, and normal control liver by using tissue microarray and immunohistochemical method. Results showed that STAT3 expression in HBV-related HCC, HCV-related HCC, and HBV-related liver fibrosis was significantly higher than in control liver (P < 0.001, P = 0.016, and P = 0.005, respectively) and had no significant differences between these three diseased liver tissues. The HK-II expression in HBV-related HCC was significantly higher than that in HCV-related HCC, HBV-related liver fibrosis, and control liver (P = 0.007, P = 0.029, and P = 0.008, respectively) but had no significant elevation in and no significant differences between HCV-related HCC, HBV-related liver fibrosis, and control liver. The HK-II expression was significantly correlated to STAT3 expression in HBV-related HCC (P = 0.022), but no correlation was observed in HCV-related HCC, HBV-related liver fibrosis, and control liver. In conclusion, STAT3 expression is upregulated in both HBV- and HCV-related HCC, while HK-II is predominantly upregulated and correlated to STAT3 in HBV-related HCC. These differential expression and association may suggest the distinct roles of STAT3 and HK-II in hepatocarcinogenesis of HBV and HCV infection. Studies are needed to confirm the relationship of STAT3 and HK-II and to examine the underlying mechanisms. J. Med. Virol. 88:1552-1559, 2016. © 2016 Wiley Periodicals, Inc.

Inhibition of Sirolimus on the growth of pancreatic carcinoma and its effect on the expression of glucose transporter and hexokinase Ⅱ

To investigate whether Sirolimus could affect the glycolytic catabolism pathways of pancreatic carcinoma through the control of hypoxia induced factor (HIF-1α) to inhibit the growth of tumor, and explore the potential mechanism of targeting the signaling pathways of mTOR for the treatment of pancreatic carcinoma. Sirolimus was applied to treat the pancreatic carcinoma in nude mice orthotopic transplantation model, its difference with the control group was compared; RT-PCR and Western blot were used to measure the mRNA and protein expression of mTOR, HIF-1α, Glucose carrier protein 1 (GLUT-1) and Hexokinase Ⅱ (HK-Ⅱ), respectively; the changes of activity of HK-Ⅱ in the tumor was determined. The tumor mass of the control group (1.97±0.21)g was significantly larger than that of the Sirolimus group (0.38±0.10)g (P<0.01), and the volume of the control group (1.40±0.15) mm(3) was significantly larger than that of the Sirolimus group (0.27±0.07) mm(3) (P<0.01). The expressions of mTOR, GLUT-1 and HK-Ⅱ mRNA in the control group were higher than those of the Sirolimus group (P<0.05), while no significant change was observed in the expression of HIF-1α (P>0.05); the expressions of p-mTOR, HIF-1α, GLUT-1 and HK-Ⅱ proteins in the control group were higher than those of the Sirolimus group (P<0.05). The activity of HK-Ⅱ in the control group was higher than that of the Sirolimus group (P<0.05). Sirolimus could affect the expression of GLUT-1 and HK-Ⅱ in pancreatic carcinoma through the effects of HIF-1α to inhibit tumor growth, indicating that blocking the mTOR pathway could control the glycolytic metabolism pathways of pancreatic carcinoma, which may become the new strategy for the treatment of pancreatic carcinoma.

The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression.

Cancer cells heavily rely on the glycolytic pathway regardless of oxygen tension. Hexokinase II (HKII) catalyses the first irreversible step of glycolysis and is often overexpressed in cancer cells. 3-Bromopyruvate (3BP) has been shown to primarily target HKII, and is a promising anti-cancer compound capable of altering critical metabolic pathways in cancer cells. Abnormal vasculature within tumours leads to heterogeneous microenvironments, including glucose availability, which may affect drug sensitivity. The aim of the present study was to elucidate the mechanisms by which 3BP acts on colorectal cancer (CRC) cells with focus on the HKII/Akt signalling axis. High HKII-expressing cell lines were more sensitive to 3BP than low HKII-expressing cells. 3BP-induced rapid Akt phosphorylation at site Thr-308 and cell death via both apoptotic and necrotic mechanisms. Cells grown under lower glucose concentrations showed greater resistance towards 3BP. Cells with HKII knockdown showed no changes in 3BP sensitivity, suggesting the effects of 3BP are independent of HKII expression. These results emphasize the importance of the tumour microenvironment and glucose availability when considering therapeutic approaches involving metabolic modulation.

Long-term prognostic implications and therapeutic target role of hexokinase II in patients with nasopharyngeal carcinoma.

Tumor cells preferentially use anaerobic glycolysis rather than oxidative phosphorylation to generate energy. Hexokinase II (HK-II) is necessary for anaerobic glycolysis and displays aberrant expression in malignant cells. The current study aimed to evaluate the role of HK-II in the survival and biological function of nasopharyngeal carcinoma (NPC). Our study demonstrated that high expression of HK-II was associated with poor survival outcomes in NPC patients. When using 3-BrOP (an HK-II inhibitor) to repress glycolysis, cell proliferation and invasion were attenuated, accompanied by the induction of apoptosis and cell cycle arrest at the G1 stage. Furthermore, 3-BrOP synergized with cisplatin (DDP) to induce NPC cell death. Collectively, we provided that the aberrant expression of HK-II was associated with the malignant phenotype of NPC. A combined treatment modality that targets glycolysis with DDP holds promise for the treatment of NPC patients.

Hexokinase II expression is correlated with colorectal cancer prognosis

Abstract Background Enhanced glycolysis is observed during tumorigenesis, with an upregulation in key glycolytic enzymes. Hexokinase II (HKII) catalyzes the first irreversible step of glycolysis and is often overexpressed in tumors. Abnormal vasculature within tumors leads to regional ischemic conditions that promote tumor progression. The aims of this study were to assess the expression of HKII in colorectal cancer tissues, and to correlate HKII expression with clinical parameters and patient outcome. Materials and method Sections from 60 FFPE primary colorectal cancer tumor samples were dual fluorescence immunostained for HKII and carbonic anhydrase IX (CAIX; serving as an ischemic marker) and assessed using semi-quantitative immunofluorescence. Associations of HKII and CAIX levels with patient characteristics, tumor pathology and clinical outcome were studied using univariate analysis. Results HKII expression was found in neoplastic cells of non-ischemic regions of tumors and within the tumor stroma. CAIX expression was found primarily in neoplastic cells, and was associated with patient age (OR=4.04, ≥70 vs. Conclusion We identified low overall HKII expression to be associated with the outcome of colon cancer. However, the correlation between stromal HKII expression and worse survival in colorectal cancer patients warrant further investigation. MicroAbstract This study assessed the prognostic value of hexokinase II expression in colorectal cancer. In total, 60 FFPE primary colorectal cancer tumor samples were immunostained and correlated with patient characteristics, tumor pathology and clinical outcome. Interestingly, low overall HKII expression was correlated with worse patient outcome. Separately, stromal HKII staining was correlated with worse patient outcome.

TNFα driven HIF-1α-hexokinase II axis regulates MHC-I cluster stability through actin cytoskeleton

Hypoxia-inducible Factor-1α (HIF-1α)-regulated expression of Hexokinase-II (HKII) remains a cornerstone in the maintenance of high metabolic demands subserving various pro-tumor functions including immune evasion in gliomas. Since inflammation-induced HIF-1α regulates Major Histocompatibility Complex class I (MHC-I) gene expression, and as cytoskeletal dynamics affect MHC-I membrane clusters, we investigated the involvement of HIF-1α-HKII axis in Tumor Necrosis Factor-α (TNFα)-mediated MHC-I membrane cluster stability in glioma cells and the involvement of actin cytoskeleton in the process. TNFα increased the clustering and colocalization of MHC-I with cortical actin in a HIF-1α dependent manner. siRNA mediated knockdown of HIF-1α as well as enzymatic inhibition of HK II by Lonidamine, delocalized mitochondrially bound HKII. This altered subcellular HKII localization affected TNFα-induced cofilin activation and actin turnover, as pharmacological inhibition of HKII by Lonidamine decreased Actin-related protein 2 (ARP2)/cofilin interaction. Photobleaching studies revealed destabilization of TNFα- induced stable MHC-I membrane clusters in the presence of Lonidamine and ARP2 inhibitor CK666. This work highlights how TNFα triggers a previously unknown function of metabolic protein HKII to influence an immune related outcome. Our study establishes the importance of inflammation induced HIF-1α in integrating two crucial components- the metabolic and immune, through reorganization of cytoskeleton.

Reversal of Warburg Effect and Reactivation of Oxidative Phosphorylation by Differential Inhibition of EGFR Signaling Pathways in Non-Small Cell Lung Cancer.

One of the hallmarks of cancer cells is the excessive conversion of glucose to lactate under normoxic conditions, also known as the Warburg effect. Here, we tested whether the targeted inhibition of EGFR may revert this effect and reactivate mitochondrial oxidative phosphorylation in non-small cell lung cancer (NSCLC). Sensitive (HCC827) and resistant (H1975 and H1993) NSCLC cells were treated with a panel of EGFR or MET inhibitors, and then tested for changes of EGFR signaling, glycolytic cascade, and mitochondrial function. Silencing of key glycolytic enzymes was then performed with targeted siRNAs. Furthermore, tumor-bearing nude mice treated with EGFR inhibitors were evaluated with (18)F-FDG PET/CT and tumors were analyzed for glycolytic and mitochondrial proteins. Effective inhibition of EGFR signaling in NSCLC cells induced a dramatic reduction of hexokinase II (HKII) and phospho-pyruvate kinase M2 (p-PKM2, Tyr105) levels as well as an upregulation of mitochondrial complexes subunits (OXPHOS). Accordingly, a decreased lactate secretion and increased intracellular ATP levels were also observed in response to EGFR inhibitors. Downregulation of HKII and PKM2 by targeted siRNA transfection did not cause upregulation of OXPHOS but enhanced the effects of EGFR TKIs. Conversely, selective inhibition of AKT and ERK1/2 caused OXPHOS upregulation and glycolysis inhibition, respectively. Similar findings were obtained in tumors from animals treated with appropriate EGFR inhibitors. Our findings indicate that EGFR inhibitors may reactivate oxidative phosphorylation of cancer cells and provide a mechanistic clue for the rational combination of agents targeting EGFR-dependent proliferation and glucose metabolism in cancer therapy.

RRAD inhibits aerobic glycolysis, invasion, and migration and is associated with poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer worldwide. However, the mechanism underlying the HCC development remains unclear. Ras-related associated with diabetes (RRAD) is a small Ras-related GTPase which has been implicated in metabolic disease and several types of cancer, yet its functions in HCC remain unknown. A tissue microarray constructed by 90 paired HCC tissues and adjacent non-cancerous liver tissues was used to examine the protein levels of RRAD, and the messenger RNA (mRNA) expression of RRAD was also detected in a subset of this cohort. The prognostic significance of RRAD was estimated by the Kaplan-Meier analysis and Cox regression. The glucose utilization assay and lactate production assay were performed to measure the role of RRAD in HCC glycolysis. The effect of RRAD in HCC invasion and metastasis was analyzed by transwell assays. Our results suggested that the expression of RRAD was downregulated in HCC tissues compared to the adjacent non-tumorous liver tissues both in mRNA and protein levels and lower RRAD expression served as an independent prognostic indicator for the survival of HCC patients. Moreover, RRAD inhibited hepatoma cell aerobic glycolysis by negatively regulating the expression of glucose transporter 1 (GLUT1) and hexokinase II (HK-II). In addition, RRAD inhibition dramatically increased hepatoma cell invasion and metastasis. In conclusion, our study revealed that RRAD expression was decreased in HCC tumor tissues and predicted poor clinical outcome for HCC patients and played an important role in regulating aerobic glycolysis and cell invasion and metastasis and may represent potential targets for improving the treatment of HCC.

Suppression of miR-181a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression.

BackgroundLow survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated.ResultsH2O2 (600 μM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2.ConclusionsThese findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

Abstract 1180: Hexokinase II plays a pivotal role in colorectal cancer cell proliferation and survival

Abstract The Warburg Effect describes the widely observed metabolic phenotype of cancer cells and their heavy reliance on the glycolytic pathway for energy production regardless of oxygen tension. This is a result of alterations to metabolic signaling pathways leading to an upregulation in key glycolytic enzymes. Hexokinase II (HKII) catalyzes the first irreversible step of glycolysis and is often overexpressed in tumors. 3-bromopyruvate (3BP) has been shown to primarily target HKII, and is a promising anti-cancer compound capable of targeting critical metabolic pathways in cancer cells. Here we examine the importance of HKII to cell proliferation and survival and also whether its inhibition could provide a therapeutic strategy in human colorectal cancer (CRC). Initially, established human CRC cell lines (HCT116, Caco2, SW480, DLD-1) were screened for HKII mRNA and protein expression. Cell proliferation and survival following HKII inhibition via both siRNA and 3BP treatment was determined using crystal violet staining, flow cytometry and western blotting. Sensitivity to 3BP was correlated with HKII expression amongst the cell lines screened. We next examined the pro-survival AKT signaling pathway via western blotting and found that AKT phosphorylation was dependent on 3BP dose and was residue-specific. Immunofluorescence was then used to examine subcellular HKII localization. Following 3BP treatment, mitochondrial localization of HKII was lost. As a result of tumor vascular abnormalities, regions within a tumor are exposed to transient and/or chronic ischemia, resulting in altered oxygen and nutrient availablity. To examine the effects of glucose availability on 3BP sensitivity, media glucose was gradually reduced in a step-wise manner and HKII expression, cell proliferation and cell survival were evaluated in CRC cell lines. Cells grown under reduced or normal glucose conditions were less sensitive to 3BP exposure than cells grown under high glucose conditions, and responses correlated with changes in HKII expression. Using immunostaining, we examined the expression of HKII in both normoxic and ischemic regions in FFPE samples of human CRC. These results were correlated to tumor stage and grade, along with patient outcome. Results from this study further define the role of HKII in colorectal cancer progression and show that 3-bromopyruvate could be useful as an anti-cancer therapy against CRC tumors with high expression of HKII. Citation Format: Nelson Ho, Brenda L. Coomber. Hexokinase II plays a pivotal role in colorectal cancer cell proliferation and survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1180. doi:10.1158/1538-7445.AM2015-1180

Insulin and hypoxia-inducible factor-1 cooperate in pancreatic cancer cells to increase cell viability.

The aim of the present study was to investigate whether interstitial insulin and cancer-induced hypoxia-inducible factor-1 (HIF-1) cooperate in pancreatic cancer cells. A population of 45 nude mice were divided into one intact control group and six pancreatic tumor-carrier groups. Pancreatic tumors were generated using HIF-1-positive wild-type MiaPaCa2 (wt-MiaPaCa2) pancreatic cancer cells in three groups of carriers and MiaPaCa2 cells transfected with small interfering RNA against HIF-1α (si-MiaPaCa2 cells) in the other three carrier groups. To vary the intrapancreatic insulin levels, tumor-carrying mice were subjected to one of the following conditions: i) Untreated, ii) single injection of the β-cell toxin streptozotosin prior to cancer cell transplantation and iii) daily injection of insulin following cancer cell transplantation. After 12 weeks, tumor viability was assessed by histological analysis. Western blotting of the tumor grafts was performed to determine the protein expression levels of insulin receptor (IR) and two downstream proteins, hexokinase-II (HK-II) and vascular endothelial growth factor (VEGF). Histologically, the greatest viability was observed in wt-MiaPaCa2 tumors with carriers that remained untreated. These tumors also exhibited greater IR expression than their si-MiaPaCa2 counterparts, indicating that HIF-1 is necessary for basal expression of IR. However, IR expression was increased in wt-MiaPaCa2 and si-MiaPaCa2 tumors when the carriers were treated with exogenous insulin. This indicates that the insulin-induced IR expression was independent of HIF-1. Notably, the insulin-induced IR expression was associated with increased HK-II and VEGF expression in wt-MiaPaCa2 tumors but not si-MiaPaC2 tumors. Therefore, the present study proposes that insulin and HIF-1 may cooperate to increase pancreatic cancer cell viability. Furthermore, the HIF-1 signaling pathway is required for insulin-induced HK-II and VEGF expression, as well as basal IR expression levels in pancreatic cancer cells.

Identification of a mitochondrial-binding site on the N-terminal end of hexokinase II.

Hexokinase II (HKII) is responsible for the first step in the glycolysis pathway by adding a phosphate on to the glucose molecule so it can proceed down the pathway to produce the energy for continuous cancer cell growth. Tumour cells overexpress the HKII enzyme. In fact, it is the overexpression of the HKII enzyme that makes the diagnosis of cancer possible when imaged by positron emission tomography (PET). HKII binds to the voltage-dependent anion channel (VDAC) located on the mitochondrial outer membrane (MOM). When bound to the MOM, HKII is blocking a major cell death pathway. Thus, HKII is responsible for two characteristics of cancer cells, rapid tumour growth and inability of cancer cells to undergo apoptosis. One method to identify novel compounds that may interfere with the HKII-VDAC-binding site is to create a molecular model using the crystal structure of HKII. However, the amino acid(s) responsible for HKII binding to VDAC are not known. Therefore, a series of truncations and point mutations were made to the N-terminal end of HKII to identify the binding site to VDAC. Deletions of the first 10 and 20 amino acids indicated that important amino acid(s) for binding were located within the first 10 amino acids. Next, a series of point mutations were made within the first 10 amino acids. It is clear from the immunofluorescence images and immunoblot results that mutating the fifth amino acid from histidine to proline completely abolished binding to the MOM.

SIRT3 Is Crucial for Maintaining Skeletal Muscle Insulin Action and Protects Against Severe Insulin Resistance in High-Fat-Fed Mice.

Protein hyperacetylation is associated with glucose intolerance and insulin resistance, suggesting that the enzymes regulating the acetylome play a role in this pathological process. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has been linked to energy homeostasis. Thus, it is hypothesized that the dysregulation of the mitochondrial acetylation state, via genetic deletion of SIRT3, will amplify the deleterious effects of a high-fat diet (HFD). Hyperinsulinemic-euglycemic clamp experiments show, for the first time, that mice lacking SIRT3 exhibit increased insulin resistance due to defects in skeletal muscle glucose uptake. Permeabilized muscle fibers from HFD-fed SIRT3 knockout (KO) mice showed that tricarboxylic acid cycle substrate–based respiration is decreased while fatty acid–based respiration is increased, reflecting a fuel switch from glucose to fatty acids. Consistent with reduced muscle glucose uptake, hexokinase II (HKII) binding to the mitochondria is decreased in muscle from HFD-fed SIRT3 KO mice, suggesting decreased HKII activity. These results show that the absence of SIRT3 in HFD-fed mice causes profound impairments in insulin-stimulated muscle glucose uptake, creating an increased reliance on fatty acids. Insulin action was not impaired in the lean SIRT3 KO mice. This suggests that SIRT3 protects against dietary insulin resistance by facilitating glucose disposal and mitochondrial function.

Tripeptidyl peptidase II links intracellular amino acids homeostasis with immune response through glycolytic pathway (IRM9P.463)

Abstract Proteasomes and lysosomes constitute the major cellular proteolytic systems that not only remove unfolded, damaged, or unnecessary proteins, but also provide an essential supply of free amino acids for energy and new protein synthesis. Tripeptidyl peptidase II (TPP2) is a required component of the proteasome-ubiquitin protein degradation pathway. The patients lack of TPP2 manifest a combined immunodeficiency with recurrent infections, severe autoimmunity, and neurodevelopmental delay. We observed a marked increase in lysosome number and lysosomal proteolytic activity in patient cells lacking TPP2 activity. Upregulated lysosomes compensated for deficient amino acid homeostasis when TPP2 was absent. We confirmed these results in Tpp2 knockout mice and in normal human cells treated with butabindide, a selective chemical inhibitor of TPP2. However, we also found that the overabundant lysosomes exerted a pathogenic effect in which key glycolytic enzymes including hexokinase II (HKII), were consumed. The resulting decreased glycolytic potential in TPP2 defective cells selectively attenuated the production of several immunomodulatory cytokines such as IFN-γ and IL-1β, whose production require aerobic glycolysis. Taken together, our data reveal the vital role of TPP2 in amino acid supply and unveil the intimate homeostatic balance between lysosome number, amino acid level, and glycolysis in mammalian cells.

TGFβ Triggers miR-143/145 Transfer From Smooth Muscle Cells to Endothelial Cells, Thereby Modulating Vessel Stabilization.

The miR-143/145 cluster is highly expressed in smooth muscle cells (SMCs), where it regulates phenotypic switch and vascular homeostasis. Whether it plays a role in neighboring endothelial cells (ECs) is still unknown. To determine whether SMCs control EC functions through passage of miR-143 and miR-145. We used cocultures of SMCs and ECs under different conditions, as well as intact vessels to assess the transfer of miR-143 and miR-145 from one cell type to another. Imaging of cocultured cells transduced with fluorescent miRNAs suggested that miRNA transfer involves membrane protrusions known as tunneling nanotubes. Furthermore, we show that miRNA passage is modulated by the transforming growth factor (TGF) β pathway because both a specific transforming growth factor-β (TGFβ) inhibitor (SB431542) and an shRNA against TGFβRII suppressed the passage of miR-143/145 from SMCs to ECs. Moreover, miR-143 and miR-145 modulated angiogenesis by reducing the proliferation index of ECs and their capacity to form vessel-like structures when cultured on matrigel. We also identified hexokinase II (HKII) and integrin β 8 (ITGβ8)-2 genes essential for the angiogenic potential of ECs-as targets of miR-143 and miR-145, respectively. The inhibition of these genes modulated EC phenotype, similarly to miR-143 and miR-145 overexpression in ECs. These findings were confirmed by ex vivo and in vivo approaches, in which it was shown that TGFβ and vessel stress, respectively, triggered miR-143/145 transfer from SMCs to ECs. Our results demonstrate that miR-143 and miR-145 act as communication molecules between SMCs and ECs to modulate the angiogenic and vessel stabilization properties of ECs.