Marie-Julie Nokin

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. DOI: http://dx.doi.org/10.7554/eLife.19375.001

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.

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-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.

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.

PO-242 Myoferlin controls mitochondrial structure and metabolism in pancreatic ductal adenocarcinoma, and affects tumor aggressiveness

Introduction Pancreatic ductal adenocarcinoma (pdac) is the most common type of pancreatic cancer, and the third leading cause of cancer related death. therapeutic options remain very limited and are still based on classical chemotherapies. cell fraction can survive to the chemotherapy and is responsible for tumor relapse. it appears that these cells rely on oxydative phosphorylation (oxphos) for survival. Myoferlin, a membrane protein involved in cell fusion was recently shown by our laboratory to be overexpressed in pancreatic cancer. Material and methods We used pancreatic cancer cell lines depleted in myoferlin to assess mitochondrial function with an extracellular flux analyser. pancreas cancer samples from the institutional biobank with matched pet scan data were used to correlate myoferlin abundance and glycolysis.results and discussionsin the present study, we discovered that myoferlin was more expressed in cell lines undergoing (oxphos) than in glycolytic cell lines. in the former cell lines, we showed that myoferlin silencing reduced oxphos activity and forced cells to switch to glycolysis. the decrease in oxphos activity is associated with mitochondrial condensation and network disorganization. an increase of dynamin-related protein (drp)-1 phosphorylation in myoferlin-depleted cells led us to suggest mitochondrial fission, reducing cell proliferation, atp production and inducing autophagy and ros accumulation. electron microscopy observation revealed mitophagy, suggesting mitochondrial alterations. To confirm the clinical importance of myoferlin in pdac, we showed that low myoferlin expression was significantly correlated to high overall survival. myoferlin staining of pdac sections was negatively correlated with several 18fdg pet indices indicating that glycolytic lesions had less myoferlin. these observations are fully in accordance with our in vitro data.conclusionas the mitochondrial function was associated with cell chemoresistance, the metabolic switch induced by myoferlin silencing could open up a new perspective in the development of therapeutic strategies. among them, targeting functional domains (c2, dysf, …) of myoferlin should be a priority.

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.

PO-225 Dual role of methylglyoxal, a glycolysis side-product, in cancer

Introduction Methylglyoxal (MG) is spontaneously produced during glycolysis. MG reacts with proteins, lipids and DNA, thus inducing a dicarbonyl stress. Glyoxalase 1 (GLO1) detoxifies MG into D-Lactate. High MG is notably associated with diabetes and cancer as the latter presents a deregulated energy metabolism where tumour cells use glycolysis rather than mitochondrial respiration. The impact of MG on cancer is ambiguous as some studies reported an anti-tumour and others a pro-tumour effect upon GLO1 silencing. Material and methods In this study, we aimed to evaluate the effect of MG stress on a panel of cancer cell lines: MDA-MB-231 known to be glycolytic and MCF7 cells known to be more oxidative breast cancer cells and U87-MG and U251 glioblastoma cell lines. We characterised glycolysis, MG production and detoxification under different conditions in these cell lines. U87-MG tumour growth upon MG treatment was evaluated using the chick chorioallantoic membrane tumour model (CAM). Results and discussions We observed that only MDA-MB-231 and U87-MG cell lines are able to increase their glycolysis flux upon glucose stimulation. In these conditions, both cell lines increased their MG production and detoxification via the glyoxalase system. Under exogenous MG treatment, both glycolytic cell lines showed increased expression of both NRF2 and its target gene GLO1, whereas no ROS accumulation was detected. Upon GLO1 activity inhibition, an alternative MG detoxification system, the aldo-keto reductase (AKR) enzymes, is significantly increased at least in U87-MG cells. Finally, U87-MG cells xenografted on CAM and treated with MG showed an increased growth at low MG doses while high doses reduced tumour growth. Both effects could be reversed by co-treatment with a potent MG-scavenger, L-Carnosine. Conclusion Our data demonstrate for the first time that cancer cells are not equal when facing MG stress. Their response to MG is mainly dependent upon energy metabolism and detoxification capacity (including GLO1 and AKRs). Reduced glutathione level is also an important feature to consider. Ongoing studies will help identify the molecular mecanisms underlying MG dual effect in cancer.

PO-219 Methylglyoxal-induced dicarbonyl stress: role in melanoma progression and response to therapy

Introduction Methylglyoxal (MG) is an endogenous dicarbonyl spontaneously produced during glycolysis able to react with proteins, lipids and DNA, inducing a carbonyl stress. Glyoxalase 1 (GLO1) detoxifies MG into D-Lactate. High MG is notably associated with diabetes and cancer. Melanoma is the most deadly form of skin cancer. Therapy is notably based on the inhibition of the MAPK pathway, often over activated. Unfortunately, BRAF and MEK inhibitors are briefly efficient as tumours rapidly develop resistance mechanisms. Melanoma tumours are generally highly glycolytic and therefore inevitably produce high amounts of MG, accumulating Advanced Glycation End products (AGEs). Phenformin, a metformin analogue with MG-scavenging properties, improves the therapeutic effect of BRAF inhibition. This observation is in good accordance with our hypothesis that MG could be involved in progression and resistance of melanoma. Material and methods This study aims to assess the metabolic profile of various human melanoma cell lines comprising BRAF and/or MEK inhibitors sensitive and resistant cells. First, we plan to explore thoroughly dicarbonyl stress status in these cell lines and in patient tissues. Next, we will subject melanoma cells to anti-carbonyl stress agents such as l-carnosine and aminoguanidine alone or in combination with MAPK pathway inhibitors and assess their effect on tumour cell survival. Results and discussions IHC staining of argpyrimidines, MG-derived AGEs, in a collection of melanoma samples showed that MG carbonyl stress is a constant feature in melanoma. MG AGEs were detectable in both BRAF inhibitors sensitive and resistant cell lines: A375 and A2058, respectively. Interestingly, the treatment of these cells with exogenous MG induced different responses: A375 cells increased their MG detoxification system and their metabolic activity as assessed by their increased GLUT1 and GLUT3 glucose transporters and PGC1alpha mitochondrial regulator. Whereas, A2058 resistant melanoma cells showed a decrease of both GLO1 activity and the metabolic markers investigated. Conclusion Ongoing experiments will help to understand these phenotypes and to further characterise a serie of WT and BRAF mutated melanoma cells. We plan to assess carbonyl stress in a larger human melanoma collection to investigate the correlation with tumour stage, metastasis, overall survival and resistance to therapy. Finally, we will test the efficacy of a combined therapy using BRAF and/or MEK inhibitors and MG scavenger molecules such as carnosine.