Methylglyoxal: a novel upstream regulator of DNA methylation.

BACKGROUND: Aerobic glycolysis, also known as the Warburg effect, is predominantly upregulated in a variety of solid tumors, including breast cancer. We have previously reported that methylglyoxal (MG), a very reactive by-product of glycolysis, unexpectedly enhanced the metastatic potential in triple negative breast cancer (TNBC) cells. MG and MG-derived glycation products have been associated with various diseases, such as diabetes, neurodegenerative disorders, and cancer. Glyoxalase 1 (GLO1) exerts an anti-glycation defense by detoxifying MG to D-lactate. METHODS: Here, we used our validated model consisting of stable GLO1 depletion to induce MG stress in TNBC cells. Using genome-scale DNA methylation analysis, we report that this condition resulted in DNA hypermethylation in TNBC cells and xenografts. RESULTS: GLO1-depleted breast cancer cells showed elevated expression of DNMT3B methyltransferase and significant loss of metastasis-related tumor suppressor genes, as assessed using integrated analysis of methylome and transcriptome data. Interestingly, MG scavengers revealed to be as potent as typical DNA demethylating agents at triggering the re-expression of representative silenced genes. Importantly, we delineated an epigenomic MG signature that effectively stratified TNBC patients based on survival. CONCLUSION: This study emphasizes the importance of MG oncometabolite, occurring downstream of the Warburg effect, as a novel epigenetic regulator and proposes MG scavengers to reverse altered patterns of gene expression in TNBC.

Comparative Analysis and Isoform-Specific Therapeutic Vulnerabilities of KRAS Mutations in Non-Small Cell Lung Cancer.

PURPOSE: Activating missense mutations of KRAS are the most frequent oncogenic driver events in lung adenocarcinoma (LUAD). However, KRAS isoforms are highly heterogeneous, and data on the potential isoform-dependent therapeutic vulnerabilities are still lacking. EXPERIMENTAL DESIGN: We developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability of KRAS-mutant isoforms. In parallel, we analyzed clinicopathologic and genomic data from 3,560 patients with non-small cell lung cancer (NSCLC) to survey allele-specific features associated with oncogenic KRAS mutations. RESULTS: In isogenic cell lines expressing different mutant KRAS isoforms, we identified isoform-specific biochemical, biological, and oncogenic properties both in vitro and in vivo. These exclusive features correlated with different therapeutic responses to MEK inhibitors, with KRAS G12C and Q61H mutants being more sensitive compared with other isoforms. In vivo, combined KRAS G12C and MEK inhibition was more effective than either drug alone. Among patients with NSCLCs that underwent comprehensive tumor genomic profiling, STK11 and ATM mutations were significantly enriched among tumors harboring KRAS G12C, G12A, and G12V mutations. KEAP1 mutation was significantly enriched among KRAS G12C and KRAS G13X LUADs. KRAS G13X-mutated tumors had the highest frequency of concurrent STK11 and KEAP1 mutations. Transcriptomic profiling revealed unique patterns of gene expression in each KRAS isoform, compared with KRAS wild-type tumors. CONCLUSIONS: This study demonstrates that KRAS isoforms are highly heterogeneous in terms of concurrent genomic alterations and gene-expression profiles, and that stratification based on KRAS alleles should be considered in the design of future clinical trials.

mTOR Inhibitors Prevent CMV Infection through the Restoration of Functional αß and γδ T cells in Kidney Transplantation.

BACKGROUND: The reported association of mTOR-inhibitor (mTORi) treatment with a lower incidence of cytomegalovirus (CMV) infection in kidney transplant recipients (KTR) who are CMV seropositive (R+) remains unexplained. METHODS: The incidence of CMV infection and T-cell profile was compared between KTRs treated with mTORis and mycophenolic acid (MPA), and in vitro mTORi effects on T-cell phenotype and functions were analyzed. RESULTS: In KTRs who were R+ and treated with MPA, both αß and γδ T cells displayed a more dysfunctional phenotype (PD-1+, CD85j+) at day 0 of transplantation in the 16 KTRs with severe CMV infection, as compared with the 17 KTRs without or with spontaneously resolving CMV infection. In patients treated with mTORis (n=27), the proportion of PD-1+ and CD85j+ αß and γδ T cells decreased, when compared with patients treated with MPA (n=44), as did the frequency and severity of CMV infections. mTORi treatment also led to higher proportions of late-differentiated and cytotoxic γδ T cells and IFNγ-producing and cytotoxic αß T cells. In vitro, mTORis increased proliferation, viability, and CMV-induced IFNγ production of T cells and decreased PD-1 and CD85j expression in T cells, which shifted the T cells to a more efficient EOMESlow Hobithigh profile. In γδ T cells, the mTORi effect was related to increased TCR signaling. CONCLUSION: Severe CMV replication is associated with a dysfunctional T-cell profile and mTORis improve T-cell fitness along with better control of CMV. A dysfunctional T-cell phenotype could serve as a new biomarker to predict post-transplantation infection and to stratify patients who should benefit from mTORi treatment. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Proportion of CMV Seropositive Kidney Transplant Recipients Who Will Develop a CMV Infection When Treated With an Immunosuppressive Regimen Including Everolimus and Reduced Dose of Cyclosporine Versus an Immunosuppressive Regimen With Mycophenolic Acid and Standard Dose of Cyclosporine A (EVERCMV), NCT02328963.

Role for the metalloproteinase ADAM28 in the control of airway inflammation, remodelling and responsiveness in asthma.

Background: Asthma is characterized by morphological modifications of the airways (inflammation and remodelling) and bronchial hyperresponsiveness. Mechanisms linking these two key features of asthma are still poorly understood. ADAM28 (a disintegrin and metalloproteinase 28) might play a role in asthma pathophysiology. ADAM28 exists as membrane-bound and soluble forms and is mainly expressed by lymphocytes and epithelial cells. Methods: ADAM28-/- mice and ADAM28+/+ counterparts were sensitized and exposed to ovalbumin (OVA). Airway responsiveness was measured using the flexiVent® system. After sacrifice, bronchoalveolar lavage (BAL) was performed and lungs were collected for analysis of airway inflammation and remodelling. Results: The expression of the soluble form of ADAM28 was lower in the lungs of OVA-exposed mice (as compared to PBS-exposed mice) and progressively increased in correlation with the duration of allergen exposure. In lungs of ADAM28-/- mice exposed to allergens, the proportion of Th2 cells among CD 4 + cells and the number of B cells were decreased. Bronchial responsiveness was lower in ADAM28-/- mice exposed to allergens and similar to the responsiveness of sham-challenged mice. Similarly, features of airway remodelling (collagen deposition, smooth muscle hyperplasia, mucous hyperplasia) were significantly less developed in OVA-exposed ADAM28-/- animals in sharp contrasts to ADAM28+/+. In addition, we report the first evidence of ADAM28 RNA expression by lung fibroblasts and we unveil a decreased capacity of lung fibroblasts extracted from OVA-exposed ADAM28-/- mice to proliferate as compared to those extracted from OVA-exposed ADAM28+/+ suggesting a direct contribution of this enzyme to the modulation of airway remodelling. Conclusion: These results suggest that ADAM28 might be a key contributor to the pathophysiology of asthma.

Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy.

BACKGROUND: High-mobility group box 1 (HMGB1) is a multifunctional redox-sensitive protein involved in various intracellular (eg, chromatin remodeling, transcription, autophagy) and extracellular (inflammation, autoimmunity) processes. Regarding its role in cancer development/progression, paradoxical results exist in the literature and it is still unclear whether HMGB1 mainly acts as an oncogene or a tumor suppressor. METHODS: HMGB1 expression was first assessed in tissue specimens (n=359) of invasive breast, lung and cervical cancer and the two distinct staining patterns detected (nuclear vs cytoplasmic) were correlated to the secretion profile of malignant cells, patient outcomes and the presence of infiltrating immune cells within tumor microenvironment. Using several orthotopic, syngeneic mouse models of basal-like breast (4T1, 67NR and EpRas) or non-small cell lung (TC-1) cancer, the efficacy of several HMGB1 inhibitors alone and in combination with immune checkpoint blockade antibodies (anti-PD-1/PD-L1) was then investigated. Isolated from retrieved tumors, 14 immune cell (sub)populations as well as the activation status of antigen-presenting cells were extensively analyzed in each condition. Finally, the redox state of HMGB1 in tumor-extruded fluids and the influence of different forms (oxidized, reduced or disulfide) on both dendritic cell (DC) and plasmacytoid DC (pDC) activation were determined. RESULTS: Associated with an unfavorable prognosis in human patients, we clearly demonstrated that targeting extracellular HMGB1 elicits a profound remodeling of tumor immune microenvironment for efficient cancer therapy. Indeed, without affecting the global number of (CD45+) immune cells, drastic reductions of monocytic/granulocytic myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes, a higher M1/M2 ratio of macrophages as well as an increased activation of both DC and pDC were continually observed following HMGB1 inhibition. Moreover, blocking HMGB1 improved the efficacy of anti-PD-1 cancer monoimmunotherapy. We also reported that a significant fraction of HMGB1 encountered within cancer microenvironment (interstitial fluids) is oxidized and, in opposite to its reduced isoform, oxidized HMGB1 acts as a tolerogenic signal in a receptor for advanced glycation endproducts-dependent manner. CONCLUSION: Collectively, we present evidence that extracellular HMGB1 blockade may complement first-generation cancer immunotherapies by remobilizing antitumor immune response.

Downregulation of Glutamine Synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1-driven acute lymphoblastic leukemia.

The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.

Targeting Infrequent Driver Alterations in Non-Small Cell Lung Cancer.

The discovery of oncogenic driver mutations led to the development of targeted therapies with non-small cell lung cancer (NSCLC) being a paradigm for precision medicine in this setting. Nowadays, the number of clinical trials focusing on targeted therapies for uncommon drivers is growing exponentially, emphasizing the medical need for these patients. Unfortunately, similar to what is observed with most targeted therapies directed against a driver oncogene, the clinical response is almost always temporary and acquired resistance to these drugs invariably emerges. Here, we review the biology of infrequent genomic actionable alterations in NSCLC as well as the current and emerging therapeutic options for these patients. Mechanisms leading to acquired drug resistance and future challenges in the field are also discussed.

Inhibition of DDR1 enhances in vivo chemosensitivity in KRAS-mutant lung adenocarcinoma.

Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive biomarkers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DDR1 is upregulated during chemotherapy both in vitro and in vivo. Moreover, analysis of a cohort of patients with LUAD suggested that high DDR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DDR1 inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD.

Methylglyoxal Scavengers Resensitize KRAS-Mutated Colorectal Tumors to Cetuximab.

The use of cetuximab anti-epidermal growth factor receptor (anti-EGFR) antibodies has opened the era of targeted and personalized therapy in colorectal cancer (CRC). Poor response rates have been unequivocally shown in mutant KRAS and are even observed in a majority of wild-type KRAS tumors. Therefore, patient selection based on mutational profiling remains problematic. We previously identified methylglyoxal (MGO), a by-product of glycolysis, as a metabolite promoting tumor growth and metastasis. Mutant KRAS cells under MGO stress show AKT-dependent survival when compared with wild-type KRAS isogenic CRC cells. MGO induces AKT activation through phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin 2 (mTORC2) and Hsp27 regulation. Importantly, the sole induction of MGO stress in sensitive wild-type KRAS cells renders them resistant to cetuximab. MGO scavengers inhibit AKT and resensitize KRAS-mutated CRC cells to cetuximab in vivo. This study establishes a link between MGO and AKT activation and pinpoints this oncometabolite as a potential target to tackle EGFR-targeted therapy resistance in CRC.

Methylglyoxal, a glycolysis metabolite, triggers metastasis through MEK/ERK/SMAD1 pathway activation in breast cancer.

BACKGROUND: Elevated aerobic glycolysis rate is a biochemical alteration associated with malignant transformation and cancer progression. This metabolic shift unavoidably generates methylglyoxal (MG), a potent inducer of dicarbonyl stress through the formation of advanced glycation end products (AGEs). We have previously shown that the silencing of glyoxalase 1 (GLO1), the main MG detoxifying enzyme, generates endogenous dicarbonyl stress resulting in enhanced growth and metastasis in vivo. However, the molecular mechanisms through which MG stress promotes metastasis development remain to be unveiled. METHODS: In this study, we used RNA sequencing analysis to investigate gene-expression profiling of GLO1-depleted breast cancer cells and we validated the regulated expression of selected genes of interest by RT-qPCR. Using in vitro and in vivo assays, we demonstrated the acquisition of a pro-metastatic phenotype related to dicarbonyl stress in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cellular models. Hyperactivation of MEK/ERK/SMAD1 pathway was evidenced using western blotting upon endogenous MG stress and exogenous MG treatment conditions. MEK and SMAD1 regulation of MG pro-metastatic signature genes in breast cancer cells was demonstrated by RT-qPCR. RESULTS: High-throughput transcriptome profiling of GLO1-depleted breast cancer cells highlighted a pro-metastatic signature that establishes novel connections between MG dicarbonyl stress, extracellular matrix (ECM) remodeling by neoplastic cells and enhanced cell migration. Mechanistically, we showed that these metastasis-related processes are functionally linked to MEK/ERK/SMAD1 cascade activation in breast cancer cells. We showed that sustained MEK/ERK activation in GLO1-depleted cells notably occurred through the down-regulation of the expression of dual specificity phosphatases in MG-stressed breast cancer cells. The use of carnosine and aminoguanidine, two potent MG scavengers, reversed MG stress effects in in vitro and in vivo experimental settings. CONCLUSIONS: These results uncover for the first time the key role of MG dicarbonyl stress in the induction of ECM remodeling and the activation of migratory signaling pathways, both in favor of enhanced metastatic dissemination of breast cancer cells. Importantly, the efficient inhibition of mitogen-activated protein kinase (MAPK) signaling using MG scavengers further emphasizes the need to investigate their therapeutic potential across different malignancies.

Methylglyoxal, a potent inducer of AGEs, connects between diabetes and cancer.

Diabetes is one of the most frequent diseases throughout the world and its incidence is predicted to exponentially progress in the future. This metabolic disorder is associated with major complications such as neuropathy, retinopathy, atherosclerosis, and diabetic nephropathy, the severity of which correlates with hyperglycemia, suggesting that they are triggered by high glucose condition. Reducing sugars and reactive carbonyl species such as methylglyoxal (MGO) lead to glycation of proteins, lipids and DNA and the gradual accumulation of advanced glycation end products (AGEs) in cells and tissues. While AGEs are clearly implicated in the pathogenesis of diabetes complications, their potential involvement during malignant tumor development, progression and resistance to therapy is an emerging concept. Meta-analysis studies established that patients with diabetes are at higher risk of developing cancer and show a higher mortality rate than cancer patients free of diabetes. In this review, we highlight the potential connection between hyperglycemia-associated AGEs formation on the one hand and the recent evidence of pro-tumoral effects of MGO stress on the other hand. We also discuss the marked interest in anti-glycation compounds in view of their strategic use to treat diabetic complications but also to protect against augmented cancer risk in patients with diabetes.

mTOR Inhibition via Displacement of Phosphatidic Acid Induces Enhanced Cytotoxicity Specifically in Cancer Cells.

The mTOR is a central regulator of cell growth and is highly activated in cancer cells to allow rapid tumor growth. The use of mTOR inhibitors as anticancer therapy has been approved for some types of tumors, albeit with modest results. We recently reported the synthesis of ICSN3250, a halitulin analogue with enhanced cytotoxicity. We report here that ICSN3250 is a specific mTOR inhibitor that operates through a mechanism distinct from those described for previous mTOR inhibitors. ICSN3250 competed with and displaced phosphatidic acid from the FRB domain in mTOR, thus preventing mTOR activation and leading to cytotoxicity. Docking and molecular dynamics simulations evidenced not only the high conformational plasticity of the FRB domain, but also the specific interactions of both ICSN3250 and phosphatidic acid with the FRB domain in mTOR. Furthermore, ICSN3250 toxicity was shown to act specifically in cancer cells, as noncancer cells showed up to 100-fold less sensitivity to ICSN3250, in contrast to other mTOR inhibitors that did not show selectivity. Thus, our results define ICSN3250 as a new class of mTOR inhibitors that specifically targets cancer cells.Significance: ICSN3250 defines a new class of mTORC1 inhibitors that displaces phosphatidic acid at the FRB domain of mTOR, inducing cell death specifically in cancer cells but not in noncancer cells. Cancer Res; 78(18); 5384-97. ©2018 AACR.

Myoferlin controls mitochondrial structure and activity in pancreatic ductal adenocarcinoma, and affects tumor aggressiveness.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death. Therapeutic options remain very limited and are based on classical chemotherapies. Energy metabolism reprogramming appears as an emerging hallmark of cancer and is considered a therapeutic target with considerable potential. Myoferlin, a ferlin family member protein overexpressed in PDAC, is involved in plasma membrane biology and has a tumor-promoting function. In the continuity of our previous studies, we investigated the role of myoferlin in the context of energy metabolism in PDAC. We used selected PDAC tumor samples and PDAC cell lines together with small interfering RNA technology to study the role of myoferlin in energetic metabolism. In PDAC patients, we showed that myoferlin expression is negatively correlated with overall survival and with glycolytic activity evaluated by 18F-deoxyglucose positron emission tomography. We found out that myoferlin is more abundant in lipogenic pancreatic cancer cell lines and is required to maintain a branched mitochondrial structure and a high oxidative phosphorylation activity. The observed mitochondrial fission induced by myoferlin depletion led to a decrease of cell proliferation, ATP production, and autophagy induction, thus indicating an essential role of myoferlin for PDAC cell fitness. The metabolic phenotype switch generated by myoferlin silencing could open up a new perspective in the development of therapeutic strategies, especially in the context of energy metabolism.

Methylglyoxal-derived stress: An emerging biological factor involved in the onset and progression of cancer.

Cancer is a disease characterised by uncontrolled growth and proliferation of cells. Tumours primarily show a higher rate of glucose uptake for lactate production even in the presence of functional mitochondria. An important metabolic consequence is intracellular formation of glucose-derived carbonyl reactive species such as methylglyoxal (MG). It has become clear that MG is the most potent glycation agent in our body, leading to alterations of proteins and DNA, and cellular dysfunction. In recent years, emerging evidence indicates that MG plays a role in the development of cancer. This review will examine studies regarding the effects of MG on cancer onset and progression and discuss their controversies. Finally, the utilisation of inhibitors and MG scavengers will be addressed in the context of MG-mediated stress blockade for cancer therapy.

Hormetic potential of methylglyoxal, a side-product of glycolysis, in switching tumours from growth to death.

Metabolic reprogramming toward aerobic glycolysis unavoidably favours methylglyoxal (MG) and advanced glycation end products (AGEs) formation in cancer cells. MG was initially considered a highly cytotoxic molecule with potential anti-cancer value. However, we have recently demonstrated that MG enhanced tumour growth and metastasis. In an attempt to understand this dual role, we explored MG-mediated dicarbonyl stress status in four breast and glioblastoma cancer cell lines in relation with their glycolytic phenotype and MG detoxifying capacity. In glycolytic cancer cells cultured in high glucose, we observed a significant increase of the conversion of MG to D-lactate through the glyoxalase system. Moreover, upon exogenous MG challenge, glycolytic cells showed elevated amounts of intracellular MG and induced de novo GLO1 detoxifying enzyme and Nrf2 expression. Thus, supporting the adaptive nature of glycolytic cancer cells to MG dicarbonyl stress when compared to non-glycolytic ones. Finally and consistent with the pro-tumoural role of MG, we showed that low doses of MG induced AGEs formation and tumour growth in vivo, both of which can be reversed using a MG scavenger. Our study represents the first demonstration of a hormetic effect of MG defined by a low-dose stimulation and a high-dose inhibition of tumour growth.

Methylglyoxal-Mediated Stress Correlates with High Metabolic Activity and Promotes Tumor Growth in Colorectal Cancer.

Cancer cells generally rely on aerobic glycolysis as a major source of energy. Methylglyoxal (MG), a dicarbonyl compound that is produced as a side product during glycolysis, is highly reactive and induces the formation of advanced glycation end-products that are implicated in several pathologies including cancer. All mammalian cells have an enzymatic defense against MG composed by glyoxalases GLO1 and GLO2 that converts MG to d-lactate. Colorectal cancer (CRC) is one of the most frequently occurring cancers with high morbidity and mortality. In this study, we used immunohistochemistry to examine the level of MG protein adducts, in a series of 102 CRC human tumors divided into four clinical stages. We consistently detected a high level of MG adducts and low GLO1 activity in high stage tumors compared to low stage ones suggesting a pro-tumor role for dicarbonyl stress. Accordingly, GLO1 depletion in CRC cells promoted tumor growth in vivo that was efficiently reversed using carnosine, a potent MG scavenger. Our study represents the first demonstration that MG adducts accumulation is a consistent feature of high stage CRC tumors. Our data point to MG production and detoxification levels as an important molecular link between exacerbated glycolytic activity and CRC progression.

Methylglyoxal, a glycolysis side-product, induces Hsp90 glycation and YAP-mediated tumor growth and metastasis.

Metabolic reprogramming toward aerobic glycolysis unavoidably induces methylglyoxal (MG) formation in cancer cells. MG mediates the glycation of proteins to form advanced glycation end products (AGEs). We have recently demonstrated that MG-induced AGEs are a common feature of breast cancer. Little is known regarding the impact of MG-mediated carbonyl stress on tumor progression. Breast tumors with MG stress presented with high nuclear YAP, a key transcriptional co-activator regulating tumor growth and invasion. Elevated MG levels resulted in sustained YAP nuclear localization/activity that could be reverted using Carnosine, a scavenger for MG. MG treatment affected Hsp90 chaperone activity and decreased its binding to LATS1, a key kinase of the Hippo pathway. Cancer cells with high MG stress showed enhanced growth and metastatic potential in vivo. These findings reinforce the cumulative evidence pointing to hyperglycemia as a risk factor for cancer incidence and bring renewed interest in MG scavengers for cancer treatment.

New role of osteopontin in DNA repair and impact on human glioblastoma radiosensitivity.

Glioblastoma (GBM) represents the most aggressive and common solid human brain tumor. We have recently demonstrated the importance of osteopontin (OPN) in the acquisition/maintenance of stemness characters and tumorigenicity of glioma initiating cells. Consultation of publicly available TCGA database indicated that high OPN expression correlated with poor survival in GBM patients. In this study, we explored the role of OPN in GBM radioresistance using an OPN-depletion strategy in U87-MG, U87-MG vIII and U251-MG human GBM cell lines. Clonogenic experiments showed that OPN-depleted GBM cells were sensitized to irradiation. In comet assays, these cells displayed higher amounts of unrepaired DNA fragments post-irradiation when compared to control. We next evaluated the phosphorylation of key markers of DNA double-strand break repair pathway. Activating phosphorylation of H2AX, ATM and 53BP1 was significantly decreased in OPN-deficient cells. The addition of recombinant OPN prior to irradiation rescued phospho-H2AX foci formation thus establishing a new link between DNA repair and OPN expression in GBM cells. Finally, OPN knockdown improved mice survival and induced a significant reduction of heterotopic human GBM xenograft when combined with radiotherapy. This study reveals a new function of OPN in DNA damage repair process post-irradiation thus further confirming its major role in GBM aggressive disease.

Targeting osteopontin suppresses glioblastoma stem-like cell character and tumorigenicity in vivo.

Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence.