Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1.

Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.

The SDHB Arg230His mutation causing familial paraganglioma alters glycolysis in a new Caenorhabditis elegans model.

The conserved B-subunit of succinate dehydrogenase (SDH) participates in the tricarboxylic acid cycle (TCA) cycle and mitochondrial electron transport. The Arg230His mutation in SDHB causes heritable pheochromocytoma/paraganglioma (PPGL). In Caenorhabditiselegans, we generated an in vivo PPGL model (SDHB-1 Arg244His; equivalent to human Arg230His), which manifests delayed development, shortened lifespan, attenuated ATP production and reduced mitochondrial number. Although succinate is elevated in both missense and null sdhb-1(gk165) mutants, transcriptomic comparison suggests very different causal mechanisms that are supported by metabolic analysis, whereby only Arg244His (not null) worms demonstrate elevated lactate/pyruvate levels, pointing to a missense-induced, Warburg-like aberrant glycolysis. In silico predictions of the SDHA-B dimer structure demonstrate that Arg230His modifies the catalytic cleft despite the latter's remoteness from the mutation site. We hypothesize that the Arg230His SDHB mutation rewires metabolism, reminiscent of metabolic reprogramming in cancer. Our tractable model provides a novel tool to investigate the metastatic propensity of this familial cancer and our approach could illuminate wider SDH pathology.This article has an associated First Person interview with the first author of the paper.

Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

Pheochromocytoma/paragangliomas (Pheo/PGL) are rare endocrine cancers with strong genetic background. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) predispose patients to malignant disease with limited therapeutic options and poor prognosis. Using a host of cellular and molecular biology techniques in 2D and 3D cell culture formats we show that SDH inhibition had cell line specific biological and biochemical consequences. Based on our studies performed on PC12 (rat chromaffin cell line), Hela (human cervix epithelial cell line), and H295R (human adrenocortical cell line) cells, we demonstrated that chromaffin cells were not affected negatively by the inhibition of SDH either by siRNA directed against SDHB or treatment with SDH inhibitors (itaconate and atpenin A5). Cell viability and intracellular metabolite measurements pointed to the cell line specific consequences of SDH impairment and to the importance of glutamate metabolism in chromaffin cells. A significant increase in glutaminase-1 (GLS-1) expression after SDH impairment was observed in PC12 cells. GLS-1 inhibitor BPTES was capable of significantly decreasing proliferation of SDH impaired PC12 cells. Glutaminase-1 and SDHB expressions were tested in 35 Pheo/PGL tumor tissues. Expression of GLS1 was higher in the SDHB low expressed group compared to SDHB high expressed tumors. Our data suggest that the SDH-associated malignant potential of Pheo/PGL is strongly dependent on GLS-1 expression and glutaminases may be novel targets for therapy.

Inhibition of Metabolic Shift can Decrease Therapy Resistance in Human High-Grade Glioma Cells.

The high-grade brain malignancy, glioblastoma multiforme (GBM), is one of the most aggressive tumours in central nervous system. The developing resistance against recent therapies and the recurrence rate of GBMs are extremely high. In spite several new ongoing trials, GBM therapies could not significantly increase the survival rate of the patients as significantly. The presence of inter- and intra-tumoral heterogeneity of GBMs arise the problem to find both the pre-existing potential resistant clones and the cellular processes which promote the adaptation mechanisms such as multidrug resistance, stem cell-ness or metabolic alterations, etc. In our work, the in situ metabolic heterogeneity of high-grade human glioblastoma cases were analysed by immunohistochemistry using tissue-microarray. The potential importance of the detected metabolic heterogeneity was tested in three glioma cell lines (grade III-IV) using protein expression analyses (Western blot and WES Simple) and therapeutic drug (temozolomide), metabolic inhibitor treatments (including glutaminase inhibitor) to compare the effects of rapamycin (RAPA) and glutaminase inhibitor combinations in vitro (Alamar Blue and SRB tests). The importance of individual differences and metabolic alterations were observed in mono-therapeutic failures, especially the enhanced Rictor expressions after different mono-treatments in correlation to lower sensitivity (temozolomide, doxycycline, etomoxir, BPTES). RAPA combinations with other metabolic inhibitors were the best strategies except for RAPA+glutaminase inhibitor. These observations underline the importance of multi-targeting metabolic pathways. Finally, our data suggest that the detected metabolic heterogeneity (the high mTORC2 complex activity, enhanced expression of Rictor, p-Akt, p-S6, CPT1A, and LDHA enzymes in glioma cases) and the microenvironmental or treatment induced metabolic shift can be potential targets in combination therapy. Therefore, it should be considered to map tissue heterogeneity and alterations with several cellular metabolism markers in biopsy materials after applying recently available or new treatments.

The Effects of Different mTOR Inhibitors in EGFR Inhibitor Resistant Colon Carcinoma Cells.

Several monoclonal antibodies and inhibitors targeting signalling pathways are being used in personalised medicine. Anti-EGFR antibodies seem to be effective, however, therapy resistance often occurs in colon carcinoma cases. mTOR inhibitors (mTORIs) could have a potential role in the breakthrough of therapy resistance. The mTOR activity related protein expression patterns and the in vitro effects of EGFR inhibitors (EGFRIs), mTORIs and their combinations were studied in different colon carcinoma cell lines (with different genetic backgrounds). Alamar Blue test and flow cytometry were used to analyse the in vitro proliferation and apoptotic effects of cetuximab, gefitinib, cisplatin, rapamycin, PP242 and NVP-BEZ235. The expressions of mTOR activity related proteins (p-70S6K, p-S6, Rictor, p-mTOR, Raptor) were studied by Western blot, immunocytochemistry and Duolink staining. The EGFRI resistance of the studied colon carcinoma cell lines related to their known mutations were confirmed, neither gefitinib nor cetuximab inhibited the proliferation or induced apoptosis in vitro. Individual differences in Rictor and Raptor expressions were detected by Western blot and immunocytochemistry beside elevated mTOR activity of these different colon carcinoma cell lines. These expression patterns correlated to the mTORIs sensitivity differences, moreover, mTORIs could enhance the effects of EGFRIs and other in vitro treatments. Our results suggest that mTORI combinations could be helpful in both EGFRI and platinum-based therapy of colon carcinomas. Moreover, we suggest determining both mTOR complex activity and mutations in Akt/mTOR signalling pathways for selecting the appropriate mTORIs and patients in potential future combination treatments.

Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells.

BACKGROUND: Glioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, thus gliomas are mainly incurable diseases. Tumour cells can adapt rapidly to alterations during therapeutic treatments related to their metabolic rewiring and profound heterogeneity in tissue environment. Renewed interests aim to develop effective treatments targeting angiogenesis, kinase activity and/or cellular metabolism. mTOR (mammalian target of rapamycin), whose hyper-activation is characteristic for many tumours, promotes metabolic alterations, macromolecule biosynthesis, cellular growth and survival. Unfortunately, mTOR inhibitors with their lower toxicity have not resulted in appreciable survival benefit. Analysing mTOR inhibitor sensitivity, other metabolism targeting treatments and their combinations could help to find potential agents and biomarkers for therapeutic development in glioma patients. METHODS: In vitro proliferation assays, protein expression and metabolite concentration analyses were used to study the effects of mTOR inhibitors, other metabolic treatments and their combinations in glioma cell lines. Furthermore, mTOR activity and cellular metabolism related protein expression patterns were also investigated by immunohistochemistry in human biopsies. Temozolomide and/or rapamycin treatments altered the expressions of enzymes related to lipid synthesis, glycolysis and mitochondrial functions as consequences of metabolic adaptation; therefore, other anti-metabolic drugs (chloroquine, etomoxir, doxycycline) were combined in vitro. RESULTS: Our results suggest that co-targeting metabolic pathways had tumour cell dependent additive/synergistic effects related to mTOR and metabolic protein expression patterns cell line dependently. Drug combinations, especially rapamycin + doxycycline may have promising anti-tumour effect in gliomas. Additionally, our immunohistochemistry results suggest that metabolic and mTOR activity alterations are not related to the recent glioma classification, and these protein expression profiles show individual differences in patients' materials. CONCLUSIONS: Based on these, combinations of different new/old drugs targeting cellular metabolism could be promising to inhibit high adaptation capacity of tumour cells depending on their metabolic shifts. Relating to this, such a development of current therapy needs to find special biomarkers to characterise metabolic heterogeneity of gliomas.

GABA, glutamine, glutamate oxidation and succinic semialdehyde dehydrogenase expression in human gliomas.

BACKGROUND: Bioenergetic characterisation of malignant tissues revealed that different tumour cells can catabolise multiple substrates as salvage pathways, in response to metabolic stress. Altered metabolism in gliomas has received a lot of attention, especially in relation to IDH mutations, and the associated oncometabolite D-2-hydroxyglutarate (2-HG) that impact on metabolism, epigenetics and redox status. Astrocytomas and oligodendrogliomas, collectively called diffuse gliomas, are derived from astrocytes and oligodendrocytes that are in metabolic symbiosis with neurons; astrocytes can catabolise neuron-derived glutamate and gamma-aminobutyric acid (GABA) for supporting and regulating neuronal functions. METHODS: Metabolic characteristics of human glioma cell models - including mitochondrial function, glycolytic pathway and energy substrate oxidation - in relation to IDH mutation status and after 2-HG incubation were studied to understand the Janus-faced role of IDH1 mutations in the progression of gliomas/astrocytomas. The metabolic and bioenergetic features were identified in glioma cells using wild-type and genetically engineered IDH1-mutant glioblastoma cell lines by metabolic analyses with Seahorse, protein expression studies and liquid chromatography-mass spectrometry. RESULTS: U251 glioma cells were characterised by high levels of glutamine, glutamate and GABA oxidation. Succinic semialdehyde dehydrogenase (SSADH) expression was correlated to GABA oxidation. GABA addition to glioma cells increased proliferation rates. Expression of mutated IDH1 and treatment with 2-HG reduced glutamine and GABA oxidation, diminished the pro-proliferative effect of GABA in SSADH expressing cells. SSADH protein overexpression was found in almost all studied human cases with no significant association between SSADH expression and clinicopathological parameters (e.g. IDH mutation). CONCLUSIONS: Our findings demonstrate that SSADH expression may participate in the oxidation and/or consumption of GABA in gliomas, furthermore, GABA oxidation capacity may contribute to proliferation and worse prognosis of gliomas. Moreover, IDH mutation and 2-HG production inhibit GABA oxidation in glioma cells. Based on these data, GABA oxidation and SSADH activity could be additional therapeutic targets in gliomas/glioblastomas.

In situ analysis of mTORC1/2 and cellular metabolism-related proteins in human Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare progressive cystic lung disease with features of a low-grade neoplasm. It is primarily caused by mutations in TSC1 or TSC2 genes. Sirolimus, an inhibitor of mTOR complex 1 (mTORC1), slows down disease progression in some, but not all patients. Hitherto, other potential therapeutic targets such as mTOR complex 2 (mTORC2) and various metabolic pathways have not been investigated in human LAM tissues. The aim of this study was to assess activities of mTORC1, mTORC2 and various metabolic pathways in human LAM tissues through analysis of protein expression. Immunohistochemical analysis of p-S6 (mTORC1 downstream protein), Rictor (mTORC2 scaffold protein) as well as GLUT1, GAPDH, ATPB, GLS, MCT1, ACSS2 and CPT1A (metabolic pathway markers) were performed on lung tissue from 11 patients with sporadic LAM. Immunoreactivity was assessed in LAM cells with bronchial smooth muscle cells as controls. Expression of p-S6, Rictor, GAPDH, GLS, MCT1, ACSS2 and CPT1A was significantly higher in LAM cells than in bronchial smooth muscle cells (P<.01). No significant differences were found between LAM cells and normal bronchial smooth muscle cells in GLUT1 and ATPB expression. The results are uniquely derived from human tissue and indicate that, in addition to mTORC1, mTORC2 may also play an important role in the pathobiology of LAM. Furthermore, glutaminolysis, acetate utilization and fatty acid ß-oxidation appear to be the preferred bioenergetic pathways in LAM cells. mTORC2 and these preferred bioenergetic pathways appear worthy of further study as they may represent possible therapeutic targets in the treatment of LAM.

Lithocholic acid, a bacterial metabolite reduces breast cancer cell proliferation and aggressiveness.

Our study aimed at finding a mechanistic relationship between the gut microbiome and breast cancer. Breast cancer cells are not in direct contact with these microbes, but disease could be influenced by bacterial metabolites including secondary bile acids that are exclusively synthesized by the microbiome and known to enter the human circulation. In murine and bench experiments, a secondary bile acid, lithocholic acid (LCA) in concentrations corresponding to its tissue reference concentrations (< 1 µM), reduced cancer cell proliferation (by 10-20%) and VEGF production (by 37%), aggressiveness and metastatic potential of primary tumors through inducing mesenchymal-to-epithelial transition, increased antitumor immune response, OXPHOS and the TCA cycle. Part of these effects was due to activation of TGR5 by LCA. Early stage breast cancer patients, versus control women, had reduced serum LCA levels, reduced chenodeoxycholic acid to LCA ratio, and reduced abundance of the baiH (7α/ß-hydroxysteroid dehydroxylase, the key enzyme in LCA generation) gene in fecal DNA, all suggesting reduced microbial generation of LCA in early breast cancer.

Rapamycin (mTORC1 inhibitor) reduces the production of lactate and 2-hydroxyglutarate oncometabolites in IDH1 mutant fibrosarcoma cells.

BACKGROUND: Multiple studies concluded that oncometabolites (e.g. D-2-hydroxyglutarate (2-HG) related to mutant isocitrate dehydrogenase 1/2 (IDH1/2) and lactate) have tumour promoting potential. Regulatory mechanisms implicated in the maintenance of oncometabolite production have great interest. mTOR (mammalian target of rapamycin) orchestrates different pathways, influences cellular growth and metabolism. Considering hyperactivation of mTOR in several malignancies, the question has been addressed whether mTOR operates through controlling of oncometabolite accumulation in metabolic reprogramming. METHODS: HT-1080 cells - carrying originally endogenous IDH1 mutation - were used in vitro and in vivo. Anti-tumour effects of rapamycin were studied using different assays. The main sources and productions of the oncometabolites (2-HG and lactate) were analysed by 13C-labeled substrates. Alterations at protein and metabolite levels were followed by Western blot, flow cytometry, immunohistochemistry and liquid chromatography mass spectrometry using rapamycin, PP242 and different glutaminase inhibitors, as well. RESULTS: Rapamycin (mTORC1 inhibitor) inhibited proliferation, migration and altered the metabolic activity of IDH1 mutant HT-1080 cells. Rapamycin reduced the level of 2-HG sourced mainly from glutamine and glucose derived lactate which correlated to the decreased incorporation of 13C atoms from 13C-substrates. Additionally, decreased expressions of lactate dehydrogenase A and glutaminase were also observed both in vitro and in vivo. CONCLUSIONS: Considering the role of lactate and 2-HG in regulatory network and in metabolic symbiosis it could be assumed that mTOR inhibitors have additional effects besides their anti-proliferative effects in tumours with glycolytic phenotype, especially in case of IDH1 mutation (e.g. acute myeloid leukemias, gliomas, chondrosarcomas). Based on our new results, we suggest targeting mTOR activity depending on the metabolic and besides molecular genetic phenotype of tumours to increase the success of therapies.

[Measuring 14C-glucose and 14C-acetate oxidation in tumour cells and tumorous host organism]. / A bioenergetikai profil vizsgálata 14C-glükóz és 14C-acetát oxidációjának összehasonlításával tumorsejtekben és tumoros szervezetben.

Tumour cell metabolism can be influenced by alterations of the extracellular microenvironment and the tumour-promoting genetically changed mechanisms. There is increasing interest to introduce appropriate bioenergetic assays to describe the therapeutic effect and metabolic subtypes of tumours in clinical oncology. The analysis of 14C-glucose and 14C-acetate oxidation could be a suitable method to examine the metabolic/bioenergetic profiles of tumour cells and tumorous host organisms. The metabolic activity of tumour cells (in vitro cell lines, primary human lymphocytes and leukaemia cells) and the tumourous host organism were examined in vitro and in vivo by detecting the released CO2 levels derived from the radioactive carbon atom labelled energy substrates. We have found that the most cancer cells of solid tumours oxidised glucose more intensively than acetate. It was interesting that AML, CML and CLL cells isolated from blood preferred acetate as an energy substrate in vitro. Furthermore, based on our observations, tumours affected the glucose or acetate oxidation of the organism when applying bioenergetic substrates per os or iv. We provided the first data about the alterations in metabolic profiles of the tumour bearing organism in xenograft models. In summary, according to our results, comparison of the energy substrate oxidation can be an indicative method related to the metabolic profile analysis of tumour cells in vitro and tumorous host organism in vivo.