CDKN2A/B deletions are strongly associated with meningioma progression: a meta-analysis of individual patient data.

Homozygous CDKN2A/B deletion has been associated with an increased risk of recurrence in meningiomas. However, the evidence is confined to a limited number of studies, and the importance of heterozygous CDKN2A/B deletions remains insufficiently investigated. Hence, the present meta-analysis reconstructs individual patient data (IPD) and reconstructs the probabilities of progression-free survival (PFS) stratified by CDKN2A/B status. IPD of PFS rates were extracted from published Kaplan-Meier plots using the R package IPDfromKM in R studio (RStudio, Boston, MA, USA). Reconstructed Kaplan-Meier Plots of the pooled IPD data were created. One-stage and two-stage meta-analyses were performed. Hazard ratios (HR) were used as effective measures. Of 181 records screened, four articles with 2521 participants were included. The prevalence of homozygous CDKN2A/B deletions in the included studies was 0.049 (95% CI 0.040-0.057), with higher tumor grades associated with a significantly greater proportion of CDKN2A/B deletions. The reconstructed PFS curves for the pooled cohort showed that the median PFS time of patients with a CDKN2A/B wild-type status, heterozygous or homozygous CDKN2A/B deletion was 180.0 (95% CI 145.7-214.3), 26.1 (95% CI 23.3-29.0), and 11.00 (95% CI 8.6-13.3) months, respectively (p < 0.0001). Both hetero- or homozygous CDKN2A/B deletions were significantly associated with shortened time to meningioma progression. One-stage meta-analysis showed that hetero- (HR: 5.5, 95% CI 4.0-7.6, p < 0.00001) and homozygous CDKN2A/B deletions (HR: 8.4, 95% CI 6.4-11.0, p < 0.00001) are significantly associated with shortened time to meningioma progression. Multivariable Cox regression analysis of progression in a subgroup with available covariates (age, sex, WHO grade, and TERT status) and also two-stage meta-analysis confirmed and validated the results of the one-stage analysis that both heterozygous and homozygous CDKN2A/B deletions are of prognostic importance. Further large-scale studies of WHO grade 2 and 3 meningiomas are needed to validate the importance of heterozygous CDKN2A/B deletions with consideration of established factors.

Viability of Glioblastoma Cells and Fibroblasts in the Presence of Imidazole-Containing Compounds.

The naturally occurring dipeptide carnosine (ß-alanyl-L-histidine) specifically attenuates tumor growth. Here, we ask whether other small imidazole-containing compounds also affect the viability of tumor cells without affecting non-malignant cells and whether the formation of histamine is involved. Patient-derived fibroblasts and glioblastoma cells were treated with carnosine, L-alanyl-L-histidine (LA-LH), ß-alanyl-L-alanine, L-histidine, histamine, imidazole, ß-alanine, and L-alanine. Cell viability was assessed by cell-based assays and microscopy. The intracellular release of L-histidine and formation of histamine was investigated by high-performance liquid chromatography coupled to mass spectrometry. Carnosine and LA-LH inhibited tumor cell growth with minor effects on fibroblasts, and L-histidine, histamine, and imidazole affected viability in both cell types. Compounds without the imidazole moiety did not diminish viability. In the presence of LA-LH but not in the presence of carnosine, a significant rise in intracellular amounts of histidine was detected in all cells. The formation of histamine was not detectable in the presence of carnosine, LA-LH, or histidine. In conclusion, the imidazole moiety of carnosine contributes to its anti-neoplastic effect, which is also seen in the presence of histidine and LA-LH. Despite the fact that histamine has a strong effect on cell viability, the formation of histamine is not responsible for the effects on the cell viability of carnosine, LA-LH, and histidine.

De novo variants in ATP2B1 lead to neurodevelopmental delay.

Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.

RNA sequencing of glioblastoma tissue slice cultures reveals the effects of treatment at the transcriptional level.

One of the major challenges in cancer research is finding models that closely resemble tumors within patients. Human tissue slice cultures are a promising approach to provide a model of the patient's tumor biology ex vivo. Recently, it was shown that these slices can be successfully analyzed by whole transcriptome sequencing as well as automated histochemistry, increasing their usability as preclinical model. Glioblastoma multiforme (GBM) is a highly malignant brain tumor with poor prognosis and little is known about its genetic background and heterogeneity regarding therapy success. In this study, tissue from the tumors of 25 patients with primary GBM was processed into slice cultures and treated with standard therapy (irradiation and temozolomide). Total RNA sequencing and automated histochemistry were performed to enable analysis of treatment effects at a transcriptional and histological level. Slice cultures from long-term survivors (overall survival [OS] > 24 months) exhibited more apoptosis than cultures from patients with shorter OS. Proliferation within these slices was slightly increased in contrast to other groups, but not significantly. Among all samples, 58 protein-coding genes were upregulated and 32 downregulated in treated vs. untreated slice cultures. In general, an upregulation of DNA damage-related and cell cycle checkpoint genes as well as enrichment of genotoxicity pathways and p53-dependent signaling was found after treatment. Overall, the current study reproduces knowledge from former studies regarding the feasibility of transcriptomic analyses and automated histology in tissue slice cultures. We further demonstrate that the experimental data merge with the clinical follow-up of the patients, which improves the applicability of our model system.

Erythrocytes Prevent Degradation of Carnosine by Human Serum Carnosinase.

The naturally occurring dipeptide carnosine (ß-alanyl-l-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography-mass spectrometry. In addition, we studied carnosine's effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase.

Surfactant protein C is associated with perineuronal nets and shows age-dependent changes of brain content and hippocampal deposits in wildtype and 3xTg mice.

Surfactant protein C (SP-C) modulates cerebrospinal fluid (CSF) rheology. During ageing, its declining levels are accompanied by an increased burden of white matter lesions. Pulmonary SP-C intermediates harbouring the BRICHOS-domain prevent protein misfolding in the lungs. Thus, cerebral SP-C intermediates may counteract cerebral ß-amyloidosis, a hallmark of Alzheimer's disease (AD). However, data on the molecular neuroanatomy of SP-C and its alterations in wildtype and triple transgenic (3xTg) mice, featuring essential elements of AD-neuropathology, are lacking. Therefore, this study investigated SP-C-containing structures in murine forebrains and their spatial relationships with vascular, glial and neuronal components of the neurovascular unit. Fluorescence labelling demonstrated neuronal SP-C in the medial habenula, the indusium griseum and the hippocampus. Glial counterstaining elucidated astrocytes in the corpus callosum co-expressing SP-C and S100ß. Notably, perineuronal nets were associated with SP-C in the nucleus reticularis thalami, the lateral hypothalamus and the retrosplenial cortex. In the hippocampus of aged 3xTg mice, an increased number of dot-like depositions containing SP-C and Reelin, but devoid of BRICHOS-immunoreactivity were observed apart from AD-like lesions. Wildtype and 3xTg mice revealed an age-dependent increase of such deposits markedly pronounced in about 24-month-old 3xTg mice. SP-C levels of the intracellular and extracellular compartments in each group revealed an inverse correlation of SP-C and Reelin, with reduced SP-C and increased Reelin in an age-dependent fashion especially in 3xTg mice. Taken together, extracellular SP-C, as modulator of glymphatic clearance and potential ligand of PNs, declines in 3xTg mice, which show an accumulation of extracellular Reelin depositions during ageing.

Antifibrotic Effects of Amyloid-Beta and Its Loss in Cirrhotic Liver.

The function and regulation of amyloid-beta (Aß) in healthy and diseased liver remains unexplored. Because Aß reduces the integrity of the blood-brain barrier we have examined its potential role in regulating the sinusoidal permeability of normal and cirrhotic liver. Aß and key proteins that generate (beta-secretase 1 and presenilin-1) and degrade it (neprilysin and myelin basic protein) were decreased in human cirrhotic liver. In culture, activated hepatic stellate cells (HSC) internalized Aß more efficiently than astrocytes and HSC degraded Aß leading to suppressed expression of α-smooth muscle actin (α-SMA), collagen 1 and transforming growth factor ß (TGFß). Aß also upregulated sinusoidal permeability marker endothelial NO synthase (eNOS) and decreased TGFß in cultured human liver sinusoidal endothelial cells (hLSEC). Liver Aß levels also correlate with the expression of eNOS in transgenic Alzheimer's disease mice and in human and rodent cirrhosis/fibrosis. These findings suggest a previously unexplored role of Aß in the maintenance of liver sinusoidal permeability and in protection against cirrhosis/fibrosis via attenuation of HSC activation.

Non-enzymatic reaction of carnosine and glyceraldehyde-3-phosphate accompanies metabolic changes of the pentose phosphate pathway.

OBJECTIVES: Carnosine (ß-alanyl-l-histidine) is a naturally occurring dipeptide that selectively inhibits cancer cell growth, possibly by influencing glucose metabolism. As its precise mode of action and its primary targets are unknown, we analysed carnosine's effect on metabolites and pathways in glioblastoma cells. MATERIALS AND METHODS: Glioblastoma cells, U87, T98G and LN229, were treated with carnosine, and metabolites were analysed by gas chromatography coupled with mass spectrometry. Furthermore, mitochondrial ATP production was determined by extracellular flux analysis and reaction products of carnosine were investigated using mass spectrometry. RESULTS: Carnosine decreased the intracellular abundance of several metabolites indicating a reduced activity of the pentose phosphate pathway, the malate-aspartate shuttle and the glycerol phosphate shuttle. Mitochondrial respiration was reduced in U87 and T98G but not in LN229 cells, independent of whether glucose or pyruvate was used as substrate. Finally, we demonstrate non-enzymatic reaction of carnosine with dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. However, glycolytic flux from glucose to l-lactate appeared not to be affected by the reaction of carnosine with the metabolites. CONCLUSIONS: Carnosine reacts non-enzymatically with glycolytic intermediates reducing the activity of the pentose phosphate pathway which is required for cell proliferation. Although the activity of the malate-aspartate and the glycerol phosphate shuttle appear to be affected, reduced mitochondrial ATP production under the influence of the dipeptide is cell-specific and appears to be independent of the effect on the shuttles.

Deep sequencing and automated histochemistry of human tissue slice cultures improve their usability as preclinical model for cancer research.

Cancer research requires models closely resembling the tumor in the patient. Human tissue cultures can overcome interspecies limitations of animal models or the loss of tissue architecture in in vitro models. However, analysis of tissue slices is often limited to histology. Here, we demonstrate that slices are also suitable for whole transcriptome sequencing and present a method for automated histochemistry of whole slices. Tumor and peritumoral tissue from a patient with glioblastoma was processed to slice cultures, which were treated with standard therapy including temozolomide and X-irradiation. Then, RNA sequencing and automated histochemistry were performed. RNA sequencing was successfully accomplished with a sequencing depth of 243 to 368 x 106 reads per sample. Comparing tumor and peritumoral tissue, we identified 1888 genes significantly downregulated and 2382 genes upregulated in tumor. Treatment significantly downregulated 2017 genes, whereas 1399 genes were upregulated. Pathway analysis revealed changes in the expression profile of treated glioblastoma tissue pointing towards downregulated proliferation. This was confirmed by automated analysis of whole tissue slices stained for Ki67. In conclusion, we demonstrate that RNA sequencing of tissue slices is possible and that histochemical analysis of whole tissue slices can be automated which increases the usability of this preclinical model.

Carnosine increases efficiency of temozolomide and irradiation treatment of isocitrate dehydrogenase-wildtype glioblastoma cells in culture.

Aim: The naturally occurring dipeptide carnosine (CAR) has been considered for glioblastoma therapy. As CAR also protects against ionizing irradiation (IR), we investigated whether it may counteract standard therapy consisting of postsurgery IR and treatment with temozolomide (TMZ). Materials & methods: Four isocitrate dehydrogenase-wildtype primary cell cultures were exposed to different doses of IR and different concentrations of TMZ and CAR. After exposure, viability under the different conditions and combinations of them was determined. Results: All cultures responded to treatment with TMZ and IR with reduced viability. CAR further decreased viability when TMZ and IR were combined. Conclusion: Treatment with CAR does not counteract glioblastoma standard therapy. As the dipeptide also protects nontumor cells from IR, it may reduce deleterious side effects of treatment.

The Chk1 inhibitor SAR-020106 sensitizes human glioblastoma cells to irradiation, to temozolomide, and to decitabine treatment.

BACKGROUND: Glioblastoma is the most common and aggressive brain tumour in adults with a median overall survival of only 14 months after standard therapy with radiation therapy (IR) and temozolomide (TMZ). In a novel multimodal treatment approach we combined the checkpoint kinase 1 (Chk1) inhibitor SAR-020106 (SAR), disrupting homologue recombination, with standard DNA damage inducers (IR, TMZ) and the epigenetic/cytotoxic drug decitabine (5-aza-2'-deoxycitidine, 5-aza-dC). Different in vitro glioblastoma models are monitored to evaluate if the impaired DNA damage repair may chemo/radiosensitize the tumour cells. METHODS: Human p53-mutated (p53-mut) and -wildtype (p53-wt) glioblastoma cell lines (p53-mut: LN405, T98G; p53-wt: A172, DBTRG) and primary glioblastoma cells (p53-mut: P0297; p53-wt: P0306) were treated with SAR combined with TMZ, 5-aza-dC, and/or IR and analysed for induction of apoptosis (AnnexinV and sub-G1 assay), cell cycle distribution (nuclear PI staining), DNA damage (alkaline comet or gH2A.X assay), proliferation inhibition (BrdU assay), reproductive survival (clonogenic assay), and potential tumour stem cells (nestinpos/GFAPneg fluorescence staining). Potential treatment-induced neurotoxicity was evaluated on nestin-positive neural progenitor cells in a murine entorhinal-hippocampal slice culture model. RESULTS: SAR showed radiosensitizing effects on the induction of apoptosis and on the reduction of long-term survival in p53-mut and p53-wt glioblastoma cell lines and primary cells. In p53-mut cells, this effect was accompanied by an abrogation of the IR-induced G2/M arrest and an enhancement of IR-induced DNA damage by SAR treatment. Also TMZ and 5-aza-dC acted radioadditively albeit to a lesser extent. The multimodal treatment achieved the most effective reduction of clonogenicity in all tested cell lines and did not affect the ratio of nestinpos/GFAPneg cells. No neurotoxic effects were detected when the number of nestin-positive neural progenitor cells remained unchanged after multimodal treatment. CONCLUSION: The Chk1 inhibitor SAR-020106 is a potent sensitizer for DNA damage-induced cell death in glioblastoma therapy strongly reducing clonogenicity of tumour cells. Selectively enhanced p53-mut cell death may provide stronger responses in tumours defective of non-homologous end joining (NHEJ). Our results suggest that a multimodal therapy involving DNA damage inducers and DNA repair inhibitors might be an effective anti-tumour strategy with a low risk of neurotoxicity.

Carnosine inhibits glioblastoma growth independent from PI3K/Akt/mTOR signaling.

Glioblastoma is a high-grade glioma with poor prognosis even after surgery and standard therapy. Here, we asked whether carnosine (ß-alanyl-L-histidine), a naturally occurring dipeptide, exert its anti-neoplastic effect on glioblastoma cells via PI3K/Akt/mTOR signaling. Therefore, glioblastoma cells from the lines U87 and T98G were exposed to carnosine, to the mTOR inhibitor rapamycin and to the PI3K inhibitor Ly-294,002. Pyruvate dehydrogenase kinase (PDK4) expression, known to be a target of PI3K/Akt/mTOR, and which is also affected by carnosine, was analyzed by RT-qPCR, and reporter gene assays with the human PDK4 promoter were performed. Cell viability was assessed by cell-based assays and mTOR and Akt phosphorylation by Western blotting. Rapamycin and Ly-294,002 increased PDK4 mRNA expression in both cell lines but significance was only reached in U87. Carnosine significantly increased expression in both lines. A significant combinatorial effect of carnosine was only detected in U87 when the dipeptide was combined with Ly-294,002. Reporter gene assays revealed no specific effect of carnosine on the human PDK4 promoter, whereas both inhibitors increased reporter gene expression. Rapamycin reduced phosphorylation of mTOR, and Ly-294,002 that of Akt. A significant reduction of Akt phosphorylation was observed in the presence of carnosine in U87 but not in T98G, and carnosine had no effect on mTOR phosphorylation. Cell viability as determined by ATP in cell lysates was reduced only in the presence of carnosine. We conclude that carnosine's anti-neoplastic effect is independent from PI3K/Akt/mTOR signaling. As the dipeptide reduced viability in tumor cells that do not respond to PI3K or mTOR inhibitors, it appears to be worth to further investigate the mechanisms by which carnosine exerts its anti-tumor effect and to consider it for therapy, especially as it is a naturally occurring compound that has already been used for the treatment of other diseases without indication of side-effects.

The proton-coupled oligopeptide transporters PEPT2, PHT1 and PHT2 mediate the uptake of carnosine in glioblastoma cells.

The previous studies demonstrated that carnosine (ß-alanyl-L-histidine) inhibits the growth of tumor cells in vitro and in vivo. Considering carnosine for the treatment of glioblastoma, we investigated which proton-coupled oligopeptide transporters (POTs) are present in glioblastoma cells and how they contribute to the uptake of carnosine. Therefore, mRNA expression of the four known POTs (PEPT1, PEPT2, PHT1, and PHT2) was examined in three glioblastoma cell lines, ten primary tumor cell cultures, in freshly isolated tumor tissue and in healthy brain. Using high-performance liquid chromatography coupled to mass spectrometry, the uptake of carnosine was investigated in the presence of competitive inhibitors and after siRNA-mediated knockdown of POTs. Whereas PEPT1 mRNA was not detected in any sample, expression of the three other transporters was significantly increased in tumor tissue compared to healthy brain. In cell culture, PHT1 expression was comparable to expression in tumor tissue, PHT2 exhibited a slightly reduced expression, and PEPT2 expression was reduced to normal brain tissue levels. In the cell line LN405, the competitive inhibitors ß-alanyl-L-alanine (inhibits all transporters) and L-histidine (inhibitor of PHT1/2) both inhibited the uptake of carnosine. SiRNA-mediated knockdown of PHT1 and PHT2 revealed a significantly reduced uptake of carnosine. Interestingly, despite its low expression at the level of mRNA, knockdown of PEPT2 also resulted in decreased uptake. In conclusion, our results demonstrate that the transporters PEPT2, PHT1, and PHT2 are responsible for the uptake of carnosine into glioblastoma cells and full function of all three transporters is required for maximum uptake.

Assessment of ApoC1, LuzP6, C12orf75 and OCC-1 in cystic glioblastoma using MALDI-TOF mass spectrometry, immunohistochemistry and qRT-PCR.

Mass spectrometric analysis of glioblastoma cyst fluids has disclosed a protein peak with m/z 6424-6433. Among the proteins, potentially generating this peak are ApoC1 and LuzP6. To further elucidate protein expression of glioblastoma cells, we analyzed MALDI-TOF results of cyst fluid, performed immunohistochemistry and mRNA analysis. MALDI-TOF protein extraction from 24 glioblastoma cyst fluids was performed with a weak cation exchange. 50 glioblastoma samples were stained with two custom-made antibodies against LuzP6 and commercial antibodies against ApoC1, C12orf75 and OCC-1 and analyzed. For mRNA detection, 16 tissue samples were stored in RNAlater, extracted using the miRNeasy kit and reversely transcribed. For 12 patients, synopsis of results from all three examinations was possible. MALDI-TOF confirmed the peak at 6433 Da in 75% of samples. Immunohistochemically, LuzP6 was detected in 92% (LuzP61-29) and 96% (LuzP630-58) of samples and ApoC1 in 66%. Mean mRNA levels were highest for ApoC1, followed by LuzP6. No correlation between mRNA expression, immunohistochemical staining and intensity of the MALDI-TOF peaks was found. An unequivocal identification of one protein as the source for the 6433 peak is not possible, but our results point to ApoC1 and LuzP6 as the underlying proteins.

Carnosine's inhibitory effect on glioblastoma cell growth is independent of its cleavage.

The naturally occurring dipeptide carnosine (ß-alanyl-L-histidine) inhibits the growth of tumor cells. As its component L-histidine mimics the effect, we investigated whether cleavage of carnosine is required for its antineoplastic effect. Using ten glioblastoma cell lines and cell cultures derived from 21 patients suffering from this malignant brain tumor, we determined cell viability under the influence of carnosine and L-histidine. Moreover, we determined expression of carnosinases, the intracellular release of L-histidine from carnosine, and whether inhibition of carnosine cleavage attenuates carnosine's antineoplastic effect. We observed a significantly higher response of the cells to L-histidine than to carnosine with regard to cell viability in all cultures. In addition, we detected protein and mRNA expression of carnosinases and a low but significant release of L-histidine in cells incubated in the presence of 50 mM carnosine (p < 0.05), which did not correlate with carnosine's effect on viability. Furthermore, the carnosinase 2 inhibitor bestatin did not attenuate carnosine's effect on viability. Interestingly, we measured a ~ 40-fold higher intracellular abundance of L-histidine in the presence of 25 mM extracellular L-histidine compared to the amount of L-histidine in the presence of 50 mM carnosine, both resulting in a comparable decrease in viability. In addition, we also examined the expression of pyruvate dehydrogenase kinase 4 mRNA, which was comparably influenced by L-histidine and carnosine, but did not correlate with effects on viability. In conclusion, we demonstrate that the antineoplastic effect of carnosine is independent of its cleavage.

D,L-Methadone does not improve radio- and chemotherapy in glioblastoma in vitro.

PURPOSE: Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. Median survival of glioblastoma patients under standard therapy including radiotherapy and chemotherapy using temozolomide (TMZ) is 14.6 months. As cell culture experiments combining D,L-methadone with doxorubicin demonstrated an increased reduction of cell viability of glioblastoma cells, the opioid has been discussed as a drug for the treatment of GBM. Despite lack of clinical and experimental evidence that D,L-methadone in combination with standard therapy will be beneficial, an increasing number of tumor patients medicating themselves with D,L-methadone present to the hospitals in Germany. METHODS: As a first step towards understanding whether D,L-methadone may increase the efficacy of standard therapy, we used a cell culture model of primary GBM and fibroblast cell cultures derived from GBM patients. The cultures were treated with different concentrations of D,L-methadone in combination with X-irradiation, TMZ or both. Cell viability was determined by measuring ATP in cell lysates and dehydrogenase activity in living cells. RESULTS: When only treated with D,L-methadone, 1 µM of the opioid was sufficient to reduce viability of fibroblasts, whereas 10 µM was needed to significantly reduce glioblastoma cell viability. In addition, D,L-methadone did not improve the anti-neoplastic effects of X-irradiation, temozolomide or both. CONCLUSIONS: As D,L-methadone reduces glioblastoma cell viability only when concentrations are used that had been reported to be toxic to patients and as there were no interactions observable combining it with standard therapy, a recommendation for the use of D,L-methadone in glioblastoma therapy cannot be given.

ß-Catenin and Yes-Associated Protein 1 Cooperate in Hepatoblastoma Pathogenesis.

Hepatoblastoma (HB), the most common pediatric primary liver neoplasm, shows nuclear localization of ß-catenin and yes-associated protein 1 (YAP1) in almost 80% of the cases. Co-expression of constitutively active S127A-YAP1 and ΔN90 deletion-mutant ß-catenin (YAP1-ΔN90-ß-catenin) causes HB in mice. Because heterogeneity in downstream signaling is being identified owing to mutational differences even in the ß-catenin gene alone, we investigated if co-expression of point mutants of ß-catenin (S33Y or S45Y) with S127A-YAP1 led to similar tumors as YAP1-ΔN90-ß-catenin. Co-expression of S33Y/S45Y-ß-catenin and S127A-YAP1 led to activation of Yap and Wnt signaling and development of HB, with 100% mortality by 13 to 14 weeks. Co-expression with YAP1-S45Y/S33Y-ß-catenin of the dominant-negative T-cell factor 4 or dominant-negative transcriptional enhanced associate domain 2, the respective surrogate transcription factors, prevented HB development. Although histologically similar, HB in YAP1-S45Y/S33Y-ß-catenin, unlike YAP1-ΔN90-ß-catenin HB, was glutamine synthetase (GS) positive. However, both ΔN90-ß-catenin and point-mutant ß-catenin comparably induced GS-luciferase reporter in vitro. Finally, using a previously reported 16-gene signature, it was shown that YAP1-ΔN90-ß-catenin HB tumors exhibited genetic similarities with more proliferative, less differentiated, GS-negative HB patient tumors, whereas YAP1-S33Y/S45Y-ß-catenin HB exhibited heterogeneity and clustered with both well-differentiated GS-positive and proliferative GS-negative patient tumors. Thus, we demonstrate that ß-catenin point mutants can also collaborate with YAP1 in HB development, albeit with a distinct molecular profile from the deletion mutant, which may have implications in both biology and therapy.

Carnosine influences transcription via epigenetic regulation as demonstrated by enhanced histone acetylation of the pyruvate dehydrogenase kinase 4 promoter in glioblastoma cells.

Carnosine (ß-alanyl-L-histidine) affects a plethora of signaling pathways and genes in different biological systems. Although known as a radical scavenger, not all of these effects can simply be ascribed to its chemical nature. As previous experiments pointed towards the possibility that carnosine affects epigenetic regulation via histone acetylation, we investigated this hypothesis using the glioblastoma cell lines U87 and T98G in which carnosine's anti-neoplastic effect is accompanied by increased expression of pyruvate dehydrogenase kinase 4. Viability and expression of PDK4 was analyzed after incubation in carnosine and different histone deacetylase inhibitors (HDACi) using cell-based assays and qRT-PCR. In addition, chromatin immunoprecipitation (ChIP) experiments were performed and the global influence of carnosine on histone H3 acetylation was analyzed by Western blot. Carnosine as well as the HDACi used increased expression of PDK4. In addition, all compounds reduced cell viability, although differences were observed with regard to magnitude and required concentrations. ChIP analysis revealed increased acetylation of histone H3 in the PDK4 promoter of U87 and T98G cells (~ 1.3- and ~ 1.7-fold, respectively) 6 h after the addition of carnosine (50 mM) followed by increased expression of PDK4 mRNA. Western blots did not detect a general increase of H3 acetylation at a genome-wide scale under the influence of carnosine. Our experiments for the first time demonstrate that carnosine influences epigenetic regulation via increased histone acetylation.

Carnosine selectively inhibits migration of IDH-wildtype glioblastoma cells in a co-culture model with fibroblasts.

BACKGROUND: Glioblastoma (GBM) is a tumor of the central nervous system. After surgical removal and standard therapy, recurrence of tumors is observed within 6-9 months because of the high migratory behavior and the infiltrative growth of cells. Here, we investigated whether carnosine (ß-alanine-l-histidine), which has an inhibitory effect on glioblastoma proliferation, may on the opposite promote invasion as proposed by the so-called "go-or-grow concept". METHODS: Cell viability of nine patient derived primary (isocitrate dehydrogenase wildtype; IDH1R132H non mutant) glioblastoma cell cultures and of eleven patient derived fibroblast cultures was determined by measuring ATP in cell lysates and dehydrogenase activity after incubation with 0, 50 or 75 mM carnosine for 48 h. Using the glioblastoma cell line T98G, patient derived glioblastoma cells and fibroblasts, a co-culture model was developed using 12 well plates and cloning rings, placing glioblastoma cells inside and fibroblasts outside the ring. After cultivation in the presence of carnosine, the number of colonies and the size of the tumor cell occupied area were determined. RESULTS: In 48 h single cultures of fibroblasts and tumor cells, 50 and 75 mM carnosine reduced ATP in cell lysates and dehydrogenase activity when compared to the corresponding untreated control cells. Co-culture experiments revealed that after 4 week exposure to carnosine the number of T98G tumor cell colonies within the fibroblast layer and the area occupied by tumor cells was reduced with increasing concentrations of carnosine. Although primary cultured tumor cells did not form colonies in the absence of carnosine, they were eliminated from the co-culture by cell death and did not build colonies under the influence of carnosine, whereas fibroblasts survived and were healthy. CONCLUSIONS: Our results demonstrate that the anti-proliferative effect of carnosine is not accompanied by an induction of cell migration. Instead, the dipeptide is able to prevent colony formation and selectively eliminates tumor cells in a co-culture with fibroblasts.

The Wnt/ß-catenin pathway determines the predisposition and efficiency of liver-to-pancreas reprogramming.

Transdifferentiation (TD) is the direct reprogramming of adult cells into cells of alternate fate and function. We have previously shown that liver cells can be transdifferentiated into beta-like, insulin-producing cells through ectopic expression of pancreatic transcription factors (pTFs). However, the efficiency of the process was consistently limited to <15% of the human liver cells treated in culture. The data in the current study suggest that liver-to-pancreas TD is restricted to a specific population of liver cells that is predisposed to undergo reprogramming. We isolated TD-predisposed subpopulation of liver cells from >15 human donors using a lineage tracing system based on the Wnt response element, part of the pericentral-specific promoter of glutamine synthetase. The cells, that were propagated separately, consistently exhibited efficient fate switch and insulin production and secretion in >60% of the cells upon pTF expression. The rest of the cells, which originated from 85% of the culture, resisted TD. Both populations expressed the ectopic pTFs with similar efficiencies, followed by similar repression of hepatic genes. Our data suggest that the TD-predisposed cells originate from a distinct population of liver cells that are enriched for Wnt signaling, which is obligatory for efficient TD. In TD-resistant populations, Wnt induction is insufficient to induce TD. An additional step of chromatin opening enables TD of these cells. CONCLUSION: Liver-to-pancreas TD occurs in defined predisposed cells. These cells' predisposition is maintained by Wnt signaling that endows the cells with the plasticity needed to alter their transcriptional program and developmental fate when triggered by ectopic pTFs. These results may have clinical implications by drastically increasing the efficacy of TD in future clinical uses. (Hepatology 2018).