Tumormetabolizmus

Napjaink egyik fontos kérdése, hogy a daganatsejtekben eddig megfi gyelt és mára egyre jobban jellemzett onkogén hatások (genetikai és epigenetikai) eredményeként bekövetkező anyagcsere-változások és a primeren jelentkező és ezen keresztül létrejövő metabolikus át- vagy újraprogramozás (metabolic reprogramming) hogyan befolyásolja a tumorok kialakulását, progresszióját és terápiás válaszkészségét. A különböző tumorsejtek metabolikus változásai egyedi variációkat jelentenek, és ezek alapján adott daganatok (akár szövettani típustól független) eltérő metabolikus profi llal rendelkezhetnek, típusonként eltérő tápanyagigénnyel és tápanyag-hasznosítási útvonallal (glikolízis, glutaminolízis, zsírsav-oxidáció, autofágia stb.) jellemezhetőek. Előbbi változások biológiai következményei számos ponton befolyásolják a daganatsejtek túlélési, proliferációs, metasztatikus viselkedését és mikrokörnyezeti változásait. Abban az esetben, ha adott metabolikus fenotípusok megjelennek, vagy irreverzibilis változásokat eredményeznek az adott tumorsejtekben, akkor olyan kezelések, amelyek ezeket a folyamatokat érintik, várható terápiás sikereket eredményezhetnek

A bioenergetikai profil vizsgálata 14C-glükóz és 14C-acetát oxidációjának összehasonlításával tumorsejtekben és tumoros szervezetben

A tumorsejtek anyagcseréjét a változó mikrokörnyezeti tényezõk mellett a tumornövekedést támogató genetikai mechanizmusok is befolyásolják. Napjainkban egyre nagyobb az igény a humán tumorok terápiás válaszreakcióinak vagy metabolikus profiljukra épülõ szubtípusainak vizsgálatára. A tumorsejtek és a tumort hordozó gazdaszervezet metabolikus/bioenergetikai jellegének tájékozódó igényû tanulmányozására alkalmas módszernek tartható a 14C-glükóz és 14C-acetát oxidációjának vizsgálata. Munkánkban radioaktívan jelölt bioenergetikai szubsztrátokból felszabaduló CO2 meghatározásával a tumorsejtek (in vitro sejtvonalak, illetve primer humán lymphocyták és leukaemiasejtek) és a tumoros szervezet (SCID, C57Bl/6) metabolikus aktivitását vizsgáltuk in vitro és in vivo. Megállapítottuk, hogy a szolid tumorból származó tumorsejtek többsége fokozottabban oxidálja a glükózt, mint az acetátot, míg a vérbõl izolált AML-, CML- és CLL-sejtek az acetátot oxidálták nagyobb mértékben, mint a glükózt in vitro. In vivo vizsgálatainkban azt tapasztaltuk, hogy a bioenergetikai szubsztrátok intravénás vagy per os adagolásakor kimutatható a tumorok hatása a gazdaszervezet glükóz-, illetve acetátoxidációjára. Elsõ adatot szolgáltattunk az emberi tumort hordozó gazdaszervezetek metabolikus profiljának változásáról xenograft modellen. Összefoglalva, eredményeink szerint több bioenergetikai szubsztrát oxidációjának összehasonlítása informatív módszer lehet a tumorsejtek in vitro, illetve a tumorok és a gazdaszervezet in vivo metabolikus profiljának vizsgálatában

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 β-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

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

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

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.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.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).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

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

Abstract 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

Additional file 2: Figure S2. of Rapamycin (mTORC1 inhibitor) reduces the production of lactate and 2-hydroxyglutarate oncometabolites in IDH1 mutant fibrosarcoma cells

The representative photos of p-mTOR, p-S6, LDH-A and Gls immunostainings in HT-1080 xenograft tumours. The expressions of p-mTOR, p-S6, LDH-A and Gls were studied in control and Rapamune treated xenograft tumours (DAB-brown staining; magnification 400X) (PDF 209 kb)