Corinna Seliger

Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

Background An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein. Methods Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays. Results Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells. Conclusion We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.

Diabetes, use of antidiabetic drugs, and the risk of glioma

BACKGROUND Prior epidemiologic studies suggest inverse relations between diabetes and glioma risk, but the underlying mechanisms, including use of antidiabetic drugs, are unknown. METHODS We therefore performed a matched case-control analysis using the Clinical Practice Research Datalink (CPRD). We identified incident glioma cases diagnosed between 1995 and 2012 and matched each case with 10 controls on age, gender, calendar time, general practice, and years of active history in the CPRD. We performed conditional logistic regression to estimate odds ratios (ORs) with 95% CIs, adjusted for body mass index and smoking. RESULTS We identified 2005 cases and 20 050 controls. Diabetes was associated with decreased risk of glioma (OR = 0.74; 95% CI = 0.60-0.93), particularly glioblastoma (OR = 0.69; 95% CI = 0.51-0.94). Glioblastoma risk reduction was markedly pronounced among diabetic men (OR = 0.60; 95% CI = 0.40-0.90), most apparently for those with diabetes of long-term duration (OR for >5 vs 0 y = 0.46; 95% CI = 0.26-0.82) or poor glycemic control (OR for HbA1c ≥ 8 vs <6.5% = 0.20; 95% CI = 0.06-0.70). In contrast, the effect of diabetes on glioblastoma risk was absent among women (OR = 0.85; 95% CI = 0.53-1.36). No significant associations with glioma were found for use of metformin (OR for ≥ 30 vs 0 prescriptions = 0.72; 95% CI = 0.38-1.39), sulfonylureas (OR = 0.71; 95% CI = 0.39-1.30), or insulin (OR = 0.79; 95% CI = 0.37-1.69). CONCLUSIONS Antidiabetic treatment appears to be unrelated to glioma, but long-term diabetes duration and increased HbA1c both show decreased glioma risk. Stronger findings in men than women suggest low androgen levels concurrent with diabetes as a biologic mechanism.

Ibuprofen and Diclofenac Restrict Migration and Proliferation of Human Glioma Cells by Distinct Molecular Mechanisms

Background Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with anti-tumorigenic effects in different tumor entities. For glioma, research has generally focused on diclofenac; however data on other NSAIDs, such as ibuprofen, is limited. Therefore, we performed a comprehensive investigation of the cellular, molecular, and metabolic effects of ibuprofen and diclofenac on human glioblastoma cells. Methods Glioma cell lines were treated with ibuprofen or diclofenac to investigate functional effects on proliferation and cell motility. Cell cycle, extracellular lactate levels, lactate dehydrogenase-A (LDH-A) expression and activity, as well as inhibition of the Signal Transducer and Activator of Transcription 3 (STAT-3) signaling pathway, were determined. Specific effects of diclofenac and ibuprofen on STAT-3 were investigated by comparing their effects with those of the specific STAT-3 inhibitor STATTIC. Results Ibuprofen treatment led to a stronger inhibition of cell growth and migration than treatment with diclofenac. Proliferation was affected by cell cycle arrest at different checkpoints by both agents. In addition, diclofenac, but not ibuprofen, decreased lactate levels in all concentrations used. Both decreased STAT-3 phosphorylation; however, diclofenac led to decreased c-myc expression and subsequent reduction in LDH-A activity, whereas treatment with ibuprofen in higher doses induced c-myc expression and less LDH-A alteration. Conclusions This study indicates that both ibuprofen and diclofenac strongly inhibit glioma cells, but the subsequent metabolic responses of both agents are distinct. We postulate that ibuprofen may inhibit tumor cells also by COX- and lactate-independent mechanisms after long-term treatment in physiological dosages, whereas diclofenac mainly acts by inhibition of STAT-3 signaling and downstream modulation of glycolysis.

Statin use and risk of glioma: population-based case–control analysis

Statins have been reported to decrease the incidence of cancer, but the risk of glioma among statin users has been investigated in only two prior observational studies, both of them suggesting a modest protective effect of statins. We conducted a matched case–control study using data from the UK-based Clinical Practice Research Datalink to analyse use of statins among 2469 cases with glioma and 24,690 controls. We performed conditional logistic regression analysis to calculate relative risks, estimated as odds ratios (ORs) with 95 % confidence intervals (CIs) adjusting for multiple confounding factors. As compared with non-use of statins, use of statins was not associated with risk of glioma (OR for ≥90 prescriptions=0.75; 95 % CI 0.48–1.17). Our findings do not support previous sparse evidence of a possible inverse association between statin use and glioma risk.

Stattic and metformin inhibit brain tumor initiating cells by reducing STAT3-phosphorylation.

Glioblastoma (GBM) is the most common and malignant type of primary brain tumor and associated with a devastating prognosis. Signal transducer and activator of transcription number 3 (STAT3) is an important pathogenic factor in GBM and can be specifically inhibited with Stattic. Metformin inhibits GBM cell proliferation and migration. Evidence from other tumor models suggests that metformin inhibits STAT3, but there is no specific data on brain tumor initiating cells (BTICs). We explored proliferation and migration of 7 BTICs and their differentiated counterparts (TCs) after treatment with Stattic, metformin or the combination thereof. Invasion was measured in situ on organotypic brain slice cultures. Protein expression of phosphorylated and total STAT3, as well as AMPK and mTOR signaling were explored using Western blot. To determine functional relevance of STAT3 inhibition by Stattic and metformin, we performed a stable knock-in of STAT3 in selected BTICs. Inhibition of STAT3 with Stattic reduced proliferation in all BTICs, but only in 4 out of 7 TCs. Migration and invasion were equally inhibited in BTICs and TCs. Treatment with metformin reduced STAT3-phosphorylation in all investigated BTICs and TCs. Combined treatment with Stattic and metformin led to significant additive effects on BTIC proliferation, but not migration or invasion. No additive effects on TCs could be detected. Stable STAT3 knock-in partly attenuated the effects of Stattic and metformin on BTICs. In conclusion, metformin was found to inhibit STAT3-phosphorylation in BTICs and TCs. Combined specific and unspecific inhibition of STAT3 might represent a promising new strategy in the treatment of glioblastoma.

Response-predictive gene expression profiling of glioma progenitor cells in vitro.

Background High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist. Methods In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a) to enrich specimens for brain tumor initiating cells and (b) to confront cells with a therapeutic agent before expression profiling. Results As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro. Conclusion For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.

Metabolic syndrome in relation to risk of meningioma

Background Meningioma is a frequent primary intracranial tumor, the etiology of which is potentially related to adiposity. Metabolic syndrome (MetS) is an increasingly common disease characterized by having at least three of the following conditions: central adiposity, arterial hypertension, dyslipidemia, and insulin resistance. Only one prior study investigated MetS in relation to meningioma risk and found a positive association between the two. Results Among 2,027 cases and 20,269 controls, body mass index was positively associated with meningioma (p-value for trend < 0.0001). Arterial hypertension was also associated with an increased risk of meningioma (OR = 1.34; 95% CI = 1.20– 1.49). By comparison, high-density lipoprotein, triglycerides, fasting serum glucose, and use of ACE-inhibitors, AT-II inhibitors, beta-blockers, diuretics, calcium antagonists, nitrates, or statins were not associated with risk of meningioma. Materials and Methods We conducted a matched case-control analysis using data from the U.K.-based Clinical Practice Research Datalink (CPRD) to analyse medical conditions and treatments related to MetS in cases with meningioma and meningioma-free controls. We identified all cases with an incident diagnosis of meningioma between 1995 and 2015 and matched each to ten controls on age, sex, calendar time, general practice, and number of years of active history in the CPRD prior to the index date. Exposures were assessed using computerised records. We conducted conditional logistic regression analysis to determine relative risks, estimated as odds ratios (ORs) with 95% confidence intervals (CIs), adjusted for confounding factors. Conclusions Obesity and arterial hypertension are positively associated with risk of meningioma. Further studies are needed to better understand potential underlying biologic mechanisms.

Clinical Reasoning: Pneumocephalus and pneumorrhachis in a 67-year-old man

A 67-year-old man was admitted to the medical intensive care unit at a hospital in eastern Bavaria. The patient presented with back pain, fever (38.7°C, 101.7°F), disorientation regarding time, place, and his own identity, and drowsiness, all of which had developed in recent weeks. Apart from these findings, the neurologic examination was unremarkable. One day before admission, the patient had been placed on an IV broad-spectrum antibiotic regimen of ceftriaxone and combined piperacillin and tazobactam in response to increased infection parameters. In addition to the mans febrile temperature, his leukocyte count was elevated to 12,500/μL; however, examinations of urea, electrolytes, blood sugar, and liver and thyroid functions yielded unremarkable findings. In the absence of a specific focus from the internal medicine perspective, the possibility of meningoencephalitis was considered. A cranial CT study detected diffuse subarachnoid pneumocephalus with generalized accumulation of gas in the cranial cavities (figure 1). There was no recognizable point of entry for the gas.

Use of metformin and survival of patients with high-grade glioma: Metformin and glioma

High‐grade glioma (HGG) is associated with poor prognosis. Drug repurposing evolves as new modality to improve standard therapy. The antidiabetic drug metformin has been found to inhibit glioma cell growth in vitro and in vivo. The aim of the present retrospective cohort study was to evaluate the survival of patients with HGG with or without treatment with metformin, based on a large cohort of a cancer registry. The analysis included 1,093 patients with HGG diagnosed between 1998 and 2013 from the population‐based clinical cancer registry Regensburg (Germany), which covers 2.1 Mio inhabitants and 98% of all cancer diagnoses. We performed multivariable adjusted Cox‐regression analyses. Hazard Ratios (HRs) with 95% Confidence Intervals (CIs) for overall survival (OS) and progression‐free survival (PFS) of patients with HGG with or without treatment with metformin were obtained. Use of metformin was associated with a significantly better overall and progression‐free survival of patients with WHO grade III glioma (HR for OS = 0.30; 95% CI = 0.11–0.81, HR for PFS = 0.29; 95% CI = 0.11–0.78), while there were no significant relations with OS (HR = 0.83; 95% CI = 0.57–1.20) or PFS (HR = 0.85; 95% CI = 0.59–1.22) in patients with WHO grade IV glioma. In conclusion, use of metformin is associated with better overall and progression‐free survival of patients with WHO grade III. Possible underlying mechanisms include the higher prevalence of IDH mutations in WHO grade III glioma, which might sensitize to the metabolic drug metformin.

Diabetes, use of metformin, and the risk of meningioma

Background Metformin is a commonly used oral antidiabetic agent that has been associated with decreased cancer risk. However, data regarding the association between metformin use and the risk of meningioma are unavailable. Methods We conducted a matched case-control analysis using data from the U.K.-based Clinical Practice Research Datalink (CPRD) to analyse diabetes status, duration of diabetes, glycemic control, and use of metformin, sulfonylureas, and insulin in relation to the risk of meningioma. We conducted conditional logistic regression analyses to determine relative risks, estimated as odds ratios (ORs) with 95% confidence intervals (CIs) and adjusted for body mass index, smoking, history of arterial hypertension, myocardial infarction, and use of estrogens (among women). Results We identified 2,027 meningioma cases and 20,269 controls. For diabetes there was the suggestion of an inverse association with meningioma (OR = 0.89; 95%CI = 0.74–1.07), which was driven by an inverse relation among women (OR = 0.78; 95%CI = 0.62–0.98), in whom we also noted a suggestive inverse association with duration of diabetes (p-value for trend = 0.071). For metformin there was a suggestive positive relation, particularly after matching on duration of diabetes and HbA1c level (OR = 1.64; 95%CI = 0.89–3.04). Sulfonylureas showed no clear association (OR = 0.91; 95%CI = 0.46–1.80). For insulin there was the suggestion of an inverse relation, in particular when comparing a high vs. low number of prescriptions (OR = 0.58; 95%CI = 0.18–1.83). Conclusion Further studies are needed to solidify a possible inverse association between diabetes and meningioma risk and to clarify the role of antidiabetics in this context.