Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Fusicoccin A,a Phytotoxic Carbotricyclic Diterpene Glucoside of Fungal Origin,Reduces Proliferation and Invasion of Glioblastoma Cells by Targeting Multiple Tyrosine Kinases

Glioblastoma multiforme (GBM) is a deadly cancer that possesses an intrinsic resistance to pro-apoptotic insults, such as conventional chemotherapy and radiotherapy, and diffusely invades the brain parenchyma, which renders it elusive to total surgical resection. We found that fusicoccin A, a fungal metabolite from Fusicoccum amygdali, decreased the proliferation and migration of human GBM cell lines in vitro, including several cell lines that exhibit varying degrees of resistance to pro-apoptotic stimuli. The data demonstrate that fusicoccin A inhibits GBM cell proliferation by decreasing growth rates and increasing the duration of cell division and also decreases two-dimensional (measured by quantitative video microscopy) and three-dimensional (measured by Boyden chamber assays) migration. These effects of fusicoccin A treatment translated into structural changes in actin cytoskeletal organization and a loss of GBM cell adhesion. Therefore, fusicoccin A exerts cytostatic effects but low cytotoxic effects (as demonstrated by flow cytometry). These cytostatic effects can partly be explained by the fact that fusicoccin inhibits the activities of a dozen kinases, including focal adhesion kinase (FAK), that have been implicated in cell proliferation and migration. Overexpression of FAK, a nonreceptor protein tyrosine kinase, directly correlates with the invasive phenotype of aggressive human gliomas because FAK promotes cell proliferation and migration. Fusicoccin A led to the down-regulation of FAK tyrosine phosphorylation, which occurred in both normoxic and hypoxic GBM cell culture conditions. In conclusion, the current study identifies a novel compound that could be used as a chemical template for generating cytostatic compounds designed to combat GBM.     

AICAR inhibits cancer cell growth and triggers cell-type distinct effects on OXPHOS biogenesis, oxidative stress and Akt activation

The AMP-activated protein kinase agonist AICAR mimics a low intracellular energy state and inhibits the proliferation of cancer cells by different mechanisms, which may depend on the bioenergetic signature of these cells. AICAR can also stimulate mitochondrial biogenesis in myoblasts, neurons and HeLa cells. Yet, whether the reactivation of oxidative phosphorylation biogenesis by AICAR contributes to the growth arrest of cancer cells remains undetermined. To investigate this possibility, we looked at the impact of 24- and 48-hour treatments with 750 μM AICAR on human cancer cell lines (HeLa, DU145, and HEPG2), non-cancer cells (EM64, FM14, and HLF), embryonic cells (MRC5) and Rho(0) cells. We determined the bioenergetic profile of these cells and assessed the effect of AICAR on oxidative phosphorylation biogeneis, cell viability and cell proliferation, ROS generation, mitochondrial membrane potential and apoptosis induction. We also followed possible changes in metabolic regulators such as Akt and Hif1-α stabilization which might participate to the anti-proliferative effect of AICAR. Our results demonstrated a strong and cancer-specific anti-growth effect of AICAR that may be explained by three different modes according to cell type: the first mode included stimulation of the mitochondrial apoptotic pathway however with compensatory activation of Akt and upregulation of oxidative phosphorylation. In the second mode of action of AICAR Akt phosphorylation was reduced. In the third mode of action, apoptosis was activated by different pathways. The sensitivity to AICAR was higher in cells with a low steady-state ATP content and a high proliferation rate.     

Metabolic preconditioning of cells with AICAR-riboside: Improved cryopreservation and cell-type specific impacts on energetics and proliferation

In species whose evolutionary history has provided natural tolerance to dehydration and freezing, metabolic depression is often a pre-requisite for survival. We tested the hypothesis that preconditioning of mammalian cells with 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside (AICAR) to achieve metabolic depression will promote greater survivorship during cryopreservation. AICAR is used extensively to stimulate AMP-activated protein kinase (AMPK), which can result in downregulation of biosynthetic processes. We showed that the metabolic interconversion of AICAR was cell-type dependent. Accumulation of 5-aminoimidazole-4-carboxamide-1b-d-ribofuranosyl-5′-monophosphate (ZMP), as well as other metabolites that possess multiple phosphates (i.e., ZDP, ZTP), varied approximately 3.5-fold across the cell lines tested. AICAR treatment also significantly influenced the concentrations of cellular adenylates (ATP, ADP, and AMP). Depression of cell metabolism and proliferation with AICAR treatment differed among cell lines. Proliferation for a given cell line was negatively correlated with the fold-increase achieved in the ‘effective adenylate ratio’ ([AMP] + [ZMP])/[ATP]) after AICAR treatment. Metabolic preconditioning with AICAR promoted a significant increase in viability post-freezing in J774.A1 macrophages, HepG2/C3A cells and primary hepatocytes but not in NIH/3T3 fibroblasts or OMK cells. The effect of AICAR on viability after freezing was positively correlated (r² = 0.94) with the fold-increase in the ‘effective adenylate ratio’. Thus for each cell line, the greater the depression of metabolism and proliferation due to preconditioning with AICAR, the greater was the survivorship post-freezing.     

PDK4 Protein Promotes Tumorigenesis through Activation of cAMP-response Element-binding Protein (CREB)-Ras Homolog Enriched in Brain (RHEB)-mTORC1 Signaling Cascade

Mechanistic target of rapamycin (mTOR) integrates multiple extracellular and intracellular signals to regulate cell growth and survival. Hyperactivation of mTOR has been observed in various cancers. Regulation of mTOR activity is thus of importance in physiological processes and tumor development. Here, we present pyruvate dehydrogenase kinase 4 (PDK4) as a novel regulator of mTORC1 signaling. mTORC1 activity was augmented with PDK4 overexpression and reduced by PDK4 suppression in various cell lines. Furthermore, PDK4 bound to cAMP-response element-binding protein (CREB) and prevented its degradation. The enhanced CREB consequently transactivated the expression of Ras homolog enriched in brain (RHEB), a direct key activator of mTORC1, independent of AMP-activated protein kinase or tuberous sclerosis complex protein 2. PDK4 potentiated the mTORC1 effectors hypoxia-inducible factor 1α and pyruvate kinase isozymes M2 and promoted aerobic glycolysis (Warburg effect). Knockdown of PDK4 suppressed the tumor development of cancer cells with activated mTORC1. The abundance of PDK4 dictated the responsiveness of cells to the mTOR inhibitor, rapamycin. Combinatory suppression of mTOR and PDK4 exerted synergistic inhibition on cancer cell proliferation. Therefore, PDK4 promotes tumorigenesis through activation of the CREB-RHEB-mTORC1 signaling cascade.     

Lyn Facilitates Glioblastoma Cell Survival under Conditions of Nutrient Deprivation by Promoting Autophagy

Members of the Src family kinases (SFK) can modulate diverse cellular processes, including division, death and survival, but their role in autophagy has been minimally explored. Here, we investigated the roles of Lyn, a SFK, in promoting the survival of human glioblastoma tumor (GBM) cells in vitro and in vivo using lentiviral vector-mediated expression of constitutively-active Lyn (CA-Lyn) or dominant-negative Lyn (DN-Lyn). Expression of either CA-Lyn or DN-Lyn had no effect on the survival of U87 GBM cells grown under nutrient-rich conditions. In contrast, under nutrient-deprived conditions (absence of supplementation with L-glutamine, which is essential for growth of GBM cells, and FBS) CA-Lyn expression enhanced survival and promoted autophagy as well as inhibiting cell death and promoting proliferation. Expression of DN-Lyn promoted cell death. In the nutrient-deprived GBM cells, CA-Lyn expression enhanced AMPK activity and reduced the levels of pS6 kinase whereas DN-Lyn enhanced the levels of pS6 kinase. Similar results were obtained in vitro using another cultured GBM cell line and primary glioma stem cells. On propagation of the transduced GBM cells in the brains of nude mice, the CA-Lyn xenografts formed larger tumors than control cells and autophagosomes were detectable in the tumor cells. The DN-Lyn xenografts formed smaller tumors and contained more apoptotic cells. Our findings suggest that on nutrient deprivation in vitro Lyn acts to enhance the survival of GBM cells by promoting autophagy and proliferation as well as inhibiting cell death, and Lyn promotes the same effects in vivo in xenograft tumors. As the levels of Lyn protein or its activity are elevated in several cancers these findings may be of broad relevance to cancer biology.     

Inhibition of alanine aminotransferase in silico and in vivo promotes mitochondrial metabolism to impair malignant growth

Cancer cells commonly exhibit increased nonoxidative D-glucose metabolism whereas induction of mitochondrial metabolism may impair malignant growth. We have first used an in silico method called elementary mode analysis to identify inhibition of ALAT (L-alanine aminotransferase) as a putative target to promote mitochondrial metabolism. We then experimentally show that two competitive inhibitors of ALAT, L-cycloserine and β-chloro-L-alanine, inhibit L-alanine production and impair D-glucose uptake of LLC1 Lewis lung carcinoma cells. The latter inhibition is linked to an initial energy deficit, as quantified by decreased ATP content, which is then followed by an activation of AMP-activated protein kinase and subsequently increased respiration rates and mitochondrial production of reactive oxygen species, culminating in ATP replenishment in ALAT-inhibited LLC1 cells. Moreover, we observe altered phosphorylation of p38 MAPK (mitogen-activated protein kinase 14), ERK (extracellular signal-regulated kinase 1/2), and Rb1 (retinoblastoma 1) proteins, as well as decreased expression of Cdc25a (cell decision cycle 25 homolog A) and Cdk4 (cyclin-dependent kinase 4). Importantly, these sequelae of ALAT inhibition culminate in similarly reduced anchorage-dependent and anchorage-independent growth rates of LLC1 cells, together suggesting that inhibition of ALAT efficiently impairs cancer growth by counteracting the Warburg effect due to compensatory activation of mitochondrial metabolism.     

Docosahexaenoic acid (DHA) sensitizes brain tumor cells to etoposide-induced apoptosis

In this study, we investigated whether DHA, a nutritionally important n-3 unsaturated fatty acid, modulated the sensitivity of brain tumor cells to the anticancer drug, etoposide (VP16). Medulloblastoma (MB) cell lines, Daoy and D283, and glioblastoma (GBM) cell lines, U138 and U87, were exposed to DHA or VP16 alone or in combination. The effects on cell proliferation and the induction of apoptosis were determined by using MTS and Hoechest 33342/PI double staining. U87 and U138 cells were found to be insensitive to the addition of DHA and VP16, whereas the two MB cell lines showed high sensitivity. DHA or VP16 alone showed little effect on cell proliferation or death in either the MB or GBM cell lines, but pretreatment with DHA enhanced the responsiveness to VP16 in the MB cell lines. To understand the mechanisms of combined DHA and VP16 on MB cells, pathway specific oligo array analyses were performed to dissect possible signaling pathways involved. The addition of DHA and VP16, in comparison to VP16 added alone, resulted in marked suppression in the expression of several genes involved in DNA damage repair, cell proliferation, survival, invasion, and angiogenesis, including PRKDC, Survivin, PIK3R1, MAPK14, NFκB1, NFκBIA, BCL2, CD44, and MAT1. These results suggest (1) that the effects of DHA and VP16 in brain tumor cells are mediated in part by the down regulation of events involved in DNA repair and the PI3K/MAPK signaling pathways and (2) that brain tumors genotypically mimicked by MB cells may benefit from therapies combining DHA with VP16.     

CDC7-dependent transcriptional regulation of RAD54L is essential for tumorigenicity and radio-resistance of glioblastoma

Accumulating evidence indicates that cell division cycle 7-related protein kinase(CDC7) plays an essential role in tumor cells and it could induces cell proliferation and could be related to prognosis in multiple types of cancer. However, the biological role and molecular mechanism of CDC7 in GBM still remains unclear. In this study, we identified that CDC7 expression was enriched in glioblastoma (GBM) tumors and was functionally required for tumor proliferation and its expression was associated to poor prognosis in GBM patients. Mechanically, CDC7 induced radio resistance in GBM cells and CDC7 knock down increased cell apoptosis when combined with radiotherapy. Moreover, CDC7 regulated The DNA repair/recombination protein 54L (RAD54L) expression via regulation of RAD54L promoter activity. Therapeutically, we found that CDC7 inhibitor attenuated tumor growth both in vitro and in vivo. Collectively, CDC7 promotes proliferation, induces radio resistance in GBM, and could become a potential therapeutic target for GBM.     

A Potential Role for the Inhibition of PI3K Signaling in Glioblastoma Therapy

Glioblastoma multiforme (GBM) is the most common primary brain tumor and among the most difficult to treat malignancies per se. In almost 90% of all GBM alterations in the PI3K/Akt/mTOR have been found, making this survival cascade a promising therapeutic target, particular for combination therapy that combines an apoptosis sensitizer, such as a pharmacological inhibitor of PI3K, with an apoptosis inducer, such as radio- or chemotherapy. However, while in vitro data focusing mainly on established cell lines has appeared rather promising, this has not translated well to a clinical setting. In this study, we analyze the effects of the dual kinase inhibitor PI-103, which blocks PI3K and mTOR activity, on three matched pairs of GBM stem cells/differentiated cells. While blocking PI3K-mediated signaling has a profound effect on cellular proliferation, in contrast to data presented on two GBM cell lines (A172 and U87) PI-103 actually counteracts the effect of chemotherapy. While we found no indications for a potential role of the PI3K signaling cascade in differentiation, we saw a clear and strong contribution to cellular motility and, by extension, invasion. While blocking PI3K-mediated signaling concurrently with application of chemotherapy does not appear to be a valid treatment option, pharmacological inhibitors, such as PI-103, nevertheless have an important place in future therapeutic approaches.     

BET bromodomain proteins are required for glioblastoma cell proliferation

Epigenetic proteins have recently emerged as novel anticancer targets. Among these, bromodomain and extra terminal domain (BET) proteins recognize lysine-acetylated histones, thereby regulating gene expression. Newly described small molecules that inhibit BET proteins BRD2, BRD3, and BRD4 reduce proliferation of NUT (nuclear protein in testis)-midline carcinoma, multiple myeloma, and leukemia cells in vitro and in vivo. These findings prompted us to determine whether BET proteins may be therapeutic targets in the most common primary adult brain tumor, glioblastoma (GBM). We performed NanoString analysis of GBM tumor samples and controls to identify novel therapeutic targets. Several cell proliferation assays of GBM cell lines and stem cells were used to analyze the efficacy of the drug I-BET151 relative to temozolomide (TMZ) or cell cycle inhibitors. Lastly, we performed xenograft experiments to determine the efficacy of I-BET151 in vivo. We demonstrate that BRD2 and BRD4 RNA are significantly overexpressed in GBM, suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. I-BET151 treatment enriched cells at the G1/S cell cycle transition. Importantly, I-BET151 is as potent at inhibiting GBM cell proliferation as TMZ, the current chemotherapy treatment administered to GBM patients. Since I-BET151 inhibits GBM cell proliferation by arresting cell cycle progression, we propose that BET protein inhibition may be a viable therapeutic option for GBM patients suffering from TMZ resistant tumors.     

Nimbolide inhibits IGF‐I‐mediated PI3K/Akt and MAPK signalling in human breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

The insulin‐like growth factor I (IGF‐I) signalling pathway contributes a major role on various cancer cell proliferation, survival and cell cycle. The present study was aimed to investigate the effect of nimbolide on IGF signalling and cell cycle arrest in MCF‐7 and MDA‐MB‐231 breast cancer cell lines. The protein expression of IGF signalling molecules and cell cycle protein levels was assessed by western blot analysis. In order to study the interaction of nimbolide on IGF‐1 signalling pathway, IGF‐I and phosphoinositide 3‐kinase (PI3K) inhibitor (LY294002) were used to treat MCF‐7 and MDA‐MB‐231 cells. Further, the cell cycle arrest was analysed by flow cytometry. The protein expression of IGF signalling molecules was significantly decreased in nimbolide‐treated breast cancer cells. PI3K inhibitor and IGF‐I with nimbolide treatment notably inhibited phosphorylated Akt. The cell cycle arrest was observed at the G0/G1 phase, and accumulation of apoptotic cells was observed in nimbolide‐treated breast cancer cell lines. Nimbolide also increased the protein expression of p21 and decreased the cyclins in both the cell lines. Nimbolide decreases the proliferation of breast cancer cells by modulating the IGF signalling molecules, which could be very useful for the breast cancer treatment. Copyright © 2014 John Wiley & Sons, Ltd.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

DW-MRI as a Predictive Biomarker of Radiosensitization of GBM through Targeted Inhibition of Checkpoint Kinases

PURPOSE: The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2)-mediated increased DNA repair capability, which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate diffusion-weighted magnetic resonance imaging (DW-MRI) as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. EXPERIMENTAL DESIGN: We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. DW-MRI and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. RESULTS: AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53 wild-type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. CONCLUSION: DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA damage-induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM.     

Serine/Threonine Kinase CHEK1-Dependent Transcriptional Regulation of RAD54L Promotes Proliferation and Radio Resistance in Glioblastoma

Accumulating evidence indicates that Checkpoint kinase 1 (CHEK1) plays an essential role in tumor cells and that it could induce cell proliferation and could be related to prognosis in multiple types of cancer. However, the biological role and molecular mechanism of CHEK1 in GBM still remain unclear. In this study, we identified that CHEK1 expression was enriched in glioblastoma (GBM) tumors and was functionally required for tumor proliferation and that its expression was associated to poor prognosis in GBM patients. Mechanically, CHEK1 induced radio resistance in GBM cells, and CHEK1 knockdown increased cell apoptosis when combined with radiotherapy via regulation of the DNA repair/recombination protein 54L (RAD54L) expression. Therapeutically, we found that CHEK1 inhibitor attenuated tumor growth both in vitro and in vivo. Collectively, CHEK1 promotes proliferation, induces radio resistance in GBM, and could become a potential therapeutic target for GBM.     

Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo

Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.     

Downregulation of the Werner syndrome protein induces a metabolic shift that compromises redox homeostasis and limits proliferation of cancer cells

The Werner syndrome protein (WRN) is a nuclear protein required for cell growth and proliferation. Loss‐of‐function mutations in the Werner syndrome gene are associated with the premature onset of age‐related diseases. How loss of WRN limits cell proliferation and induces replicative senescence is poorly understood. Here, we show that WRN depletion leads to a striking metabolic shift that coordinately weakens the pathways that generate reducing equivalents for detoxification of reactive oxygen species and increases mitochondrial respiration. In cancer cells, this metabolic shift counteracts the Warburg effect, a defining characteristic of many malignant cells, resulting in altered redox balance and accumulation of oxidative DNA damage that inhibits cell proliferation and induces a senescence‐like phenotype. Consistent with these findings, supplementation with antioxidant rescues at least in part cell proliferation and decreases senescence in WRN‐knockdown cancer cells. These results demonstrate that WRN plays a critical role in cancer cell proliferation by contributing to the Warburg effect and preventing metabolic stress.     

Serine/threonine protein phosphatase 6 modulates the radiation sensitivity of glioblastoma

Increasing the sensitivity of glioblastoma cells to radiation is a promising approach to improve survival in patients with glioblastoma multiforme (GBM). This study aims to determine if serine/threonine phosphatase (protein phosphatase 6 (PP6)) is a molecular target for GBM radiosensitization treatment. The GBM orthotopic xenograft mice model was used in this study. Our data demonstrated that the protein level of PP6 catalytic subunit (PP6c) was upregulated in the GBM tissue from about 50% patients compared with the surrounding tissue or control tissue. Both the in vitro survival fraction of GBM cells and the patient survival time were highly correlated or inversely correlated with PP6c expression (R²=0.755 and −0.707, respectively). We also found that siRNA knockdown of PP6c reduced DNA-dependent protein kinase (DNA-PK) activity in three different GBM cell lines, increasing their sensitivity to radiation. In the orthotopic mice model, the overexpression of PP6c in GBM U87 cells attenuated the effect of radiation treatment, and reduced the survival time of mice compared with the control mice, while the PP6c knocking-down improved the effect of radiation treatment, and increased the survival time of mice. These findings demonstrate that PP6 regulates the sensitivity of GBM cells to radiation, and suggest small molecules disrupting or inhibiting PP6 association with DNA-PK is a potential radiosensitizer for GBM.     

Proviral Insertion in Murine Lymphomas 2 (PIM2) Oncogene Phosphorylates Pyruvate Kinase M2 (PKM2) and Promotes Glycolysis in Cancer Cells

Pyruvate kinase M2 (PKM2) is a key player in the Warburg effect of cancer cells. However, the mechanisms of regulating PKM2 are not fully elucidated. Here, we identified the protein-serine/threonine kinase PIM2, a known oncogene, as a novel binding partner of PKM2. The interaction between PIM2 and PKM2 was confirmed by multiple biochemical approaches in vitro and in cultured cells. Importantly, we found that PIM2 could directly phosphorylate PKM2 on the Thr-454 residue, resulting in an increase of PKM2 protein levels. Compared with wild type, PKM2 with the phosphorylation-defective mutation displayed a reduced effect on glycolysis, co-activating HIF-1α and β-catenin, and cell proliferation, while enhancing mitochondrial respiration of cancer cells. These findings demonstrate that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target.