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Metabolic characteristics of kidney cancers have mainly been obtained from the most frequent clear cell renal cell carcinoma (CCRCC) studies. Moreover, the bioenergetic perturbances that affect metabolic adaptation possibilities of papillary renal cell carcinoma (PRCC) have not yet been detailed. Therefore, our study aimed to analyze the in situ metabolic features of PRCC vs. CCRCC tissues and compared the metabolic characteristics of PRCC, CCRCC, and normal tubular epithelial cell lines. The protein and mRNA expressions of the molecular elements in mammalian target of rapamycin (mTOR) and additional metabolic pathways were analyzed in human PRCC cases compared to CCRCC. The metabolic protein expression pattern, metabolite content, mTOR, and metabolic inhibitor sensitivity of renal carcinoma cell lines were also studied and compared with tubular epithelial cells, as “normal” control. We observed higher protein expressions of the “alternative bioenergetic pathway” elements, in correlation with the possible higher glutamine and acetate consumption in PRCC cells instead of higher glycolytic and mTOR activity in CCRCCs. Increased expression of certain metabolic pathway markers correlates with the detected differences in metabolite ratios, as well. The lower lactate/pyruvate, lactate/malate, and higher pyruvate/citrate intracellular metabolite ratios in PRCC compared to CCRCC cell lines suggest that ACHN (PRCC) have lower Warburg glycolytic capacity, less pronounced pyruvate to lactate producing activity and shifted OXPHOS phenotype. However, both studied renal carcinoma cell lines showed higher mTOR activity than tubular epithelial cells cultured in vitro, the metabolite ratio, the enzyme expression profiles, and the higher mitochondrial content also suggest increased importance of mitochondrial functions, including mitochondrial OXPHOS in PRCCs. Additionally, PRCC cells showed significant mTOR inhibitor sensitivity and the used metabolic inhibitors increased the effect of rapamycin in combined treatments. Our study revealed in situ metabolic differences in mTOR and metabolic protein expression patterns of human PRCC and CCRCC tissues as well as in cell lines. These underline the importance in the development of specific new treatment strategies, new mTOR inhibitors, and other anti-metabolic drug combinations in PRCC therapy.

Nowadays, extracellular vesicles (EVs) raise a great interest as they are implicated in intercellular communication between cancer and stromal cells. Our aim was to understand how vesicular NME1 and NME2 released by breast cancer cells influence the tumour microenvironment. As a model, we used human invasive breast carcinoma cells overexpressing NME1 or NME2, and first analysed in detail the presence of both isoforms in EV subtypes by capillary Western immunoassay (WES) and immunoelectron microscopy. Data obtained by both methods showed that NME1 was present in medium-sized EVs or microvesicles, whereas NME2 was abundant in both microvesicles and small-sized EVs or exosomes. Next, human skin-derived fibroblasts were treated with NME1 or NME2 containing EVs, and subsequently mRNA expression changes in fibroblasts were examined. RNAseq results showed that the expression of fatty acid and cholesterol metabolism-related genes was decreased significantly in response to NME1 or NME2 containing EV treatment. We found that FASN (fatty acid synthase) and ACSS2 (acyl-coenzyme A synthetase short-chain family member 2), related to fatty acid synthesis and oxidation, were underexpressed in NME1/2-EV-treated fibroblasts. Our data show an emerging link between NME-containing EVs and regulation of tumour metabolism.

Up-regulation of the long non-coding RNA LINC00152 can contribute to cancer development, proliferation and invasion, including colorectal cancer, however, its mechanism of action in colorectal carcinogenesis and progression is only insufficiently understood. In this work we correlated LINC00152 expression with promoter DNA methylation changes in colorectal tissues along the normal-adenoma-carcinoma sequence and studied the effects of LINC00152 silencing on the cell cycle regulation and on the whole transcriptome in colon carcinoma cells using cell and molecular biology techniques. LINC00152 was significantly up-regulated in adenoma and colorectal cancer (p

Folic acid (FA) is a synthetic form of vitamin B9, generally used as a nutritional supplement and an adjunctive medication in cancer therapy. FA is involved in genetic and epigenetic regulation; therefore, it has a dual modulatory role in established neoplasms. We aimed to investigate the effect of short-term (72 h) FA supplementation on colorectal cancer; hence, HT-29 and SW480 cells were exposed to different FA concentrations (0, 100, 10,000 ng/mL). HT-29 cell proliferation and viability levels elevated after 100 ng/mL but decreased for 10,000 ng/mL FA. Additionally, a significant (p ≤ 0.05) improvement of genomic stability was detected in HT-29 cells with micronucleus scoring and comet assay. Conversely, the FA treatment did not alter these parameters in SW480 samples. RRBS results highlighted that DNA methylation changes were bidirectional in both cells, mainly affecting carcinogenesis-related pathways. Based on the microarray analysis, promoter methylation status was in accordance with FA-induced expression alterations of 27 genes. Our study demonstrates that the FA effect was highly dependent on the cell type, which can be attributed to the distinct molecular background and the different expression of proliferation- and DNA-repair-associated genes (YWHAZ, HES1, STAT3, CCL2). Moreover, new aspects of FA-regulated DNA methylation and consecutive gene expression were revealed.

Despite advancements in cancer management, tumor relapse and metastasis are associated with poor outcomes in many cancers. Over the past decade, oncogene-driven carcinogenesis, dysregulated cellular signaling networks, dynamic changes in the tissue microenvironment, epithelial-mesenchymal transitions, protein expression within regulatory pathways, and their part in tumor progression are described in several studies. However, the complexity of metabolic enzyme expression is considerably under evaluated. Alterations in cellular metabolism determine the individual phenotype and behavior of cells, which is a well-recognized hallmark of cancer progression, especially in the adaptation mechanisms underlying therapy resistance. In metabolic symbiosis, cells compete, communicate, and even feed each other, supervised by tumor cells. Metabolic reprogramming forms a unique fingerprint for each tumor tissue, depending on the cellular content and genetic, epigenetic, and microenvironmental alterations of the developing cancer. Based on its sensing and effector functions, the mechanistic target of rapamycin (mTOR) kinase is considered the master regulator of metabolic adaptation. Moreover, mTOR kinase hyperactivity is associated with poor prognosis in various tumor types. In situ metabolic phenotyping in recent studies highlights the importance of metabolic plasticity, mTOR hyperactivity, and their role in tumor progression. In this review, we update recent developments in metabolic phenotyping of the cancer ecosystem, metabolic symbiosis, and plasticity which could provide new research directions in tumor biology. In addition, we suggest pathomorphological and analytical studies relating to metabolic alterations, mTOR activity, and their associations which are necessary to improve understanding of tumor heterogeneity and expand the therapeutic management of cancer.

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.

Monolayer cultures, the less standard three-dimensional (3D) culturing systems, and xenografts are the main tools used in current basic and drug development studies of cancer research. The aim of biofabrication is to design and construct a more representative in vivo 3D environment, replacing two-dimensional (2D) cell cultures. Here, we aim to provide a complex comparative analysis of 2D and 3D spheroid culturing, and 3D bioprinted and xenografted breast cancer models. We established a protocol to produce alginate-based hydrogel bioink for 3D bioprinting and the long-term culturing of tumour cells in vitro. Cell proliferation and tumourigenicity were assessed with various tests. Additionally, the results of rapamycin, doxycycline and doxorubicin monotreatments and combinations were also compared. The sensitivity and protein expression profile of 3D bioprinted tissue-mimetic scaffolds showed the highest similarity to the less drug-sensitive xenograft models. Several metabolic protein expressions were examined, and the in situ tissue heterogeneity representing the characteristics of human breast cancers was also verified in 3D bioprinted and cultured tissue-mimetic structures. Our results provide additional steps in the direction of representing in vivo 3D situations in in vitro studies. Future use of these models could help to reduce the number of animal experiments and increase the success rate of clinical phase trials.

Cancer hypoxia, recognized as one of the most important hallmarks of cancer, affects gene expression, metabolism and ultimately tumor biology-related processes. Major causes of cancer hypoxia are deficient or inappropriate vascularization and systemic hypoxia of the patient (frequently induced by anemia), leading to a unique form of genetic reprogramming by hypoxia induced transcription factors (HIF). However, constitutive activation of oncogene-driven signaling pathways may also activate hypoxia signaling independently of oxygen supply. The consequences of HIF activation in tumors are the angiogenic phenotype, a novel metabolic profile and the immunosuppressive microenvironment. Cancer hypoxia and the induced adaptation mechanisms are two of the major causes of therapy resistance. Accordingly, it seems inevitable to combine various therapeutic modalities of cancer patients by existing anti-hypoxic agents such as anti-angiogenics, anti-anemia therapies or specific signaling pathway inhibitors. It is evident that there is an unmet need in cancer patients to develop targeted therapies of hypoxia to improve efficacies of various anti-cancer therapeutic modalities. The case has been opened recently due to the approval of the first-in-class HIF2α inhibitor.

Synthetic vitamin B9, also known as folic acid (FA), is an extensively used nutritional supplement as well as an adjunctive medication in cancer therapy. However, there is increasing evidence that FA can promote the progression of established colorectal cancers (CRC); therefore, great care is required in the case of its application. In order to gain knowledge about the underlying mechanisms, we analyzed the genomic and the epigenomic effect of different FA supplies on two CRC cell lines with distinct molecular backgrounds. HT-29 and SW480 cells were kept in FA-free media (0 ng/mL) or treated with 100 ng/mL and 10000 ng/mL FA for 72 hours. Firstly, cell proliferation and cell viability alterations were determined with Supforhodamine B and AlamarBlue assays; then, cell cycle analysis was performed using fluorescence-activated cell sorting (FACS). Global DNA methylation level was investigated with the pyrosequencing of long interspersed nuclear element 1 (LINE-1) retrotransposons. Micronucleus scoring performed on DAPI- and anti-γ-H2AX-stained slides, as well as Comet assay, were used to detect the impact of FA on genome integrity. Finally, we analyzed gene expression alterations with Human Transcriptome Array (HTA) 2.0. Our data revealed that 100 ng/mL FA induced significant (p≤0.05) elevation (HT-29100: 128.4±24.9%) of HT-29 cell proliferation compared to the other two circumstances (HT-290: 101.3±13.5%; HT-2910000: 86.1±20.8%). The tendency of cell viability was analogous to the results detected during cell proliferation analyses (HT-290: 91.6±13.3%; HT-29100: 115.8±30.9%; HT-2910000: 64.1±20.2%). The genomic stability of FA-supplemented HT-29 samples was improved in a significant manner (p≤0.05), based on the results of micronucleus scoring (HT-290: 0.56±0.05%; HT-29100: 0.17±0.05%; HT-2910000: 0.25±0.09%) and Comet assay (HT-290: 37.4±3.5%; HT-29100: 31.2±3.4%; HT-2910000: 20.1±3.6%). However, in SW480 cells, remarkable alterations were not detected concerning these parameters. Fundamental differences were observed between the two cell lines in the case of cell cycle (HT-29: G0/1 phase dominance; SW480: S phase dominance) and global DNA methylation analysis (HT-29: 59.1±1.0; SW480: 49.0±0.2), but exposing them to different FA doses did not lead to significant changes. Gene expression alterations were more diverse, as genes involved in carcinogenesis were either up- (HES1, SLC7A11) or downregulated (CCL2) for FA supplementation. We concluded that the effect of FA was considerably influenced by the cell type and the applied FA concentration. Thereby, translating our in vitro results to patient care, we would emphasize the importance of genetic and epigenetic investigations coupled with the choice of proper FA dose upon CRC diagnosis to achieve the best disease outcome. Citation Format: Sára Zsigrai, Alexandra Kalmár, Barbara Kinga Barták, Zsófia Brigitta Nagy, Krisztina Andrea Szigeti, Gábor Valcz, Titanilla Dankó, Anna Sebestyén, Gábor Barna, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Folic acid has a cell type- and dose-dependent effect on the genome and the epigenome of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3730.

In spite of tremendous developments in breast cancer treatment, the relatively high incidence of relapsing cases indicates a great need to find new therapeutic strategies in recurrent, metastatic and advanced cases. The bioenergetic needs of growing tumours at the primary site or in metastases&mdash, accumulating genomic alterations and further heterogeneity&mdash, are supported by metabolic rewiring, an important hallmark of cancer. Adaptation mechanisms as well as altered anabolic and catabolic processes balance according to available nutrients, energy, oxygen demand and overgrowth or therapeutic resistance. Mammalian target of rapamycin (mTOR) hyperactivity may contribute to this metabolic plasticity and progression in breast carcinomas. We set out to assess the metabolic complexity in breast cancer cell lines and primary breast cancer cases. Cellular metabolism and mTOR-related protein expression were characterised in ten cell lines, along with their sensitivity to specific mTOR and other metabolic inhibitors. Selected immunohistochemical reactions were performed on ~100 surgically removed breast cancer specimens. The obtained protein expression scores were correlated with survival and other clinicopathological data. Metabolic and mTOR inhibitor mono-treatments had moderate antiproliferative effects in the studied cell lines in a subtype-independent manner, revealing their high adaptive capacity and survival/growth potential. Immunohistochemical analysis of p-S6, Rictor, lactate dehydrogenase A, glutaminase, fatty acid synthase and carnitine palmitoyltransferase 1A in human samples identified high mTOR activity and potential metabolic plasticity as negative prognostic factors for breast cancer patients, even in subtypes generally considered as low-risk. According to our results, breast cancer is characterised by considerable metabolic diversity, which can be targeted by combining antimetabolic treatments and recent therapies. Alterations in these pathways may provide novel targets for future drug development in breast cancer. We also propose a set of immunostainings for scoring metabolic heterogeneity in individual cases in order to select patients who may benefit from more accurate follow-up and specific therapies.

Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylome analyses, DNA stability assessments and exome sequencing. SAM reduced cell number and increased senescence by causing S phase arrest, besides, multiple EMT-related genes (e.g., TGFB1) were downregulated in both cell lines. Alteration in the global DNA methylation level was not observed, but certain methylation changes in gene promoters were detected. SAM-induced &gamma, H2AX elevation could be associated with activated DNA repair pathway showing upregulated gene expression (e.g., HUS1). Remarkable genomic stability elevation, namely, decreased micronucleus number and comet tail length was observed only in SW480 after treatment. SAM has the potential to induce senescence, DNA repair, genome stability and to reduce CRC progression. However, the different therapeutic responses of HT-29 and SW480 to SAM emphasize the importance of the molecular characterization of CRC cases prior to methyl donor supplementation.

mTOR activation has been observed in rhabdomyosarcoma (RMS), however, mTOR complex (mTORC) 1 inhibition has had limited success thus far. mTOR activation alters the metabolic pathways, which is linked to survival and metastasis. These pathways have not been thoroughly analyzed in RMSs. We performed immunohistochemistry on 65 samples to analyze the expression of mTOR complexes (pmTOR, pS6, Rictor), and several metabolic enzymes (phosphofructokinase, lactate dehydrogenase-A, &beta, F1-ATPase, glucose-6-phosphate dehydrogenase, glutaminase). RICTOR amplification, as a potential mechanism of Rictor overexpression, was analyzed by FISH and digital droplet PCR. In total, 64% of the studied primary samples showed mTOR activity with an mTORC2 dominance (82%). Chemotherapy did not cause any relevant change in mTOR activity. Elevated mTOR activity was associated with a worse prognosis in relapsed cases. RICTOR amplification was not confirmed in any of the cases. Our findings suggest the importance of the Warburg effect and the pentose-phosphate pathway beside a glutamine demand in RMS cells. The expression pattern of the studied mTOR markers can explain the inefficacy of mTORC1 inhibitor therapy. Therefore, we suggest performing a detailed investigation of the mTOR profile before administering mTORC1 inhibitor therapy. Furthermore, our findings highlight that targeting the metabolic plasticity could be an alternative therapeutic approach.

In geosciences, high precision isotope ratio determination provides essential information about processes in geological systems. Novel ambitions evolve closer to biological applications. Copper is an essential metal for human body taking part of several cellular processes (e.g. respiratory chain, enzyme function, iron metabolism, elimination of reactive oxygen species, cell signalling pathways etc). However, the disorder of copper homeostasis causes serious diseases like Wilson disease (Cu accumulation in liver caused by genetical disorder) and it could also promote tumour growth by supporting angiogenesis and metastasis formation [Denoyer et al., 2015]. Despite numerous experiments, focusing on copper concentration determination in different tumour tissues (e.g. breast, lung cancer, etc.) hoping to assist in tumour diagnosis, the results are not convincing enough. However, previous studies on hepatocellular cancer and oral squamous cell carcinoma showed that tumour tissue appears to be relatively enriched in 65Cu compared to normal tissue whereas the δ65Cu in blood of tumorous patient decreased according to data obtained from control population [Balter et al., 2015, Lobo et al., 2017]. Our main aim is to elaborate a method to understand better the change in 63Cu/65Cu stable isotope ratio during tumour growth. In this approach, we present our first results on copper isotope ratio determination in a xenograft mouse model. Our model was established in SCID (severe combined immunodeficiency disease) mice by injecting human cancer cells (1x107 cells) subcutaneously. After the tumour reached approximately 2-3 cm diameter, the tumour mass was cut it in small, equal pieces and transplanted further into 10 mice increasing the experimental set-up homogeneity. All the animals were sacrificed by cardiac puncture under deep terminal anaesthesia within four weeks. Tumour and organs were removed by ceramic knife then were frozen with liquid nitrogen and stored at -80°C. We measured the copper concentration and δ65Cu in the tumour tissue, blood, liver, kidney and brain. A clean laboratory ambience was chosen to perform the sample preparation processes decreasing the environmental contamination. Separation of copper from other biologically essential element (Na, Mg, Fe, Zn) interfering the copper isotope measurement is a serious condition of the preparation [Lauwens et al., 2017]. Effects of sodium (23Na40Ar+) and magnesium (25Mg40Ar+) on copper isotope ratio were solved by choosing not the peak center but the interference-free plateau. Our measurements have been carried out on a Thermo Neptune PLUS multicollector mass spectrometer equipped with 9 moveable Faraday detectors, 3 amplifiers with a resistance of 1013 Ohm, and 6 amplifiers with a resistance of 1011 Ohm, in wet plasma conditions. The mass spectrometric measurement of the copper isotope ratio is doped either with Ni or Ga reference material which have a well-known isotope ratios. References:Balter V. et al. PNAS 2015; 112: 982−985.Denoyer D. et al. Metallomics 2015; 7: 1459−76.Lauwens S. et al. J. Anal. At. Spectrom. 2017; 32: 597−608.Lobo L. et al. Talanta 2017; 165: 92−97.

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.

Metabolic alteration is characteristic during tumour growth and therapy, however, targeting metabolic rewiring could overcome therapy resistance. mTOR hyperactivity, autophagy and other metabolic processes, including mitochondrial functions, could be targeted in breast cancer progression. We investigated the growth inhibitory mechanism of rapamycin + doxycycline treatment in human breast cancer model systems. Cell cycle and cell viability, including apoptotic and necrotic cell death, were analysed using flow cytometry, caspase activity measurements and caspase-3 immunostainings. mTOR-, autophagy-, necroptosis-related proteins and treatment-induced morphological alterations were analysed by WesTM, Western blot, immunostainings and transmission electron microscopy. The rapamycin + doxycycline combination decreased tumour proliferation in about 2/3rd of the investigated cell lines. The continuous treatment reduced tumour growth significantly both in vivo and in vitro. The effect after short-term treatment was reversible, however, autophagic vacuoles and degrading mitochondria were detected simultaneously, and the presence of mitophagy was also observed after the long-term rapamycin + doxycycline combination treatment. The rapamycin + doxycycline combination did not cause apoptosis or necrosis/necroptosis, but the alterations in autophagy- and mitochondria-related protein levels (LC3-B-II/I, p62, MitoTracker, TOM20 and certain co-stainings) were correlated to autophagy induction and mitophagy, without mitochondria repopulation. Based on these results, we suggest considering inducing metabolic stress and targeting mTOR hyperactivity and mitochondrial functions in combined anti-cancer treatments.

BACKGROUND: In spite of therapeutic approaches, liver cancer is still one of the deadliest type of tumor in which tumor microenvironment may play an active role in the outcome of the disease. Decorin, a small leucine-rich proteoglycan is not only responsible for assembly and maintenance of the integrity of the extracellular matrix, but a natural inhibitor of cell surface receptors, thus it exerts antitumorigenic effects. Here we addressed the question whether this effect of decorin is independent of the tumor phenotypes including differentiation, proliferation and invasion. METHOD: Four hepatoma cell lines HepG2, Hep3B, HuH7 and HLE, possessing different molecular backgrounds, were selected to investigate. After proliferation tests, pRTK arrays, WB analyses, and immunofluorescent examinations were performed on decorin treated and control cells for comparison. RESULTS: Significant growth inhibitory potential of decorin on three out of four hepatoma cell lines was proven, however the mode of its action was different. Induction of p21WAF1/CIP1, increased inactivation of c-myc and β-catenin, and decrease of EGFR, GSK3β and ERK1/2 phosphorylation levels were observed in HepG2 cells, pathways already well-described in literature. However, in the p53 deficient Hep3B and HuH7, InsR and IGF-1R were the main receptors transmitting signals. In harmony with its receptor status, Hep3B cells displayed high level of activated AKT. As the cell line is retinoblastoma mutant, ATR/Chk1/Wee1 system might hinder the cell cycle in G2/M phase via phosphorylation of CDK1. In Huh7 cells, all RTKs were inhibited by decorin followed by downregulation of AKT. Furthermore, HuH7 cell line responded with concentration-dependent ERK activation and increased phospho-c-myc level. Decorin had only a non-significant effect on the proliferation rate of HLE cell line. However, it responded with a significant decrease of pAKT, c-myc and β-catenin activity. In this special cell line, the inhibition of TGFβ may be the first step of the protective effect of decorin. CONCLUSIONS: Based on our results decorin may be a candidate therapeutic agent in the battle against liver cancer, but several questions need to be answered. It is certain that decorin is capable to exert its suppressor effect in hepatoma cells without respect to their phenotype and molecular background.

Loss of liver kinase B (LKB1, encoded by STK11) has been observed in about one-third of lung adenocarcinomas (ADCs) and can cause the inactivation of its major target AMP-activated protein kinase (AMPK) and subsequently, activation of the mammalian target of rapamycin (mTOR) pathway. In addition to its inhibitory effects on tumor growth and proliferation, AMPK may affect acetyl-CoA homeostasis and induces histone acetylation through regulating the activity and nuclear translocation of acyl-CoA synthetase short-chain family member 2 (ACSS2) and ATP citrate lyase (ACLY). Nuclear and/or cytoplasmic expression of LKB1/AMPK/mTOR pathway markers (LKB1, p-AMPKα, p-S6), enzymes involved in acetate and lipid metabolism (p-ACC, ACSS2, ACLY), acetyl-histone H3 (ac-H3), and immune regulatory proteins (PD-L1, COX-2) was assessed in lung ADCs (N=100) by immunohistochemistry. The expression of mRNAs coding for proteins involved in antitumor immunity was also analyzed in STK11-mutant and wild-type cases with using TCGA data. Associations with the clinicopathological parameters as well as the antiproliferative effects of mTOR and metabolic inhibitors (rapamycin, metformin, phenformin, and ACSS2 inhibitor) and a histone deacetylase (HDAC) inhibitor (valproic acid) were also studied in ADC cell lines harboring KRAS and/or STK11 mutations. In cases with low or no LKB1 expression, we observed significantly lower expression of p-AMPKα, p-ACC, ac-H3, ACSS2, and ACLY (P < .01). PD-L1 expression was also slightly decreased in these samples. The expression of most of the studied mRNAs, particularly those involved in innate immunity, was also lower in STK11 mutant cases. In our in vitro studies, rapamycin had a significant antiproliferative effect on KRAS- and/or STK11-mutant cell lines with high mTOR activity. The ACSS2 inhibitor and valproic acid were not effective as monotherapies, however, valproic acid significantly increased the antitumoral effects of rapamycin in STK11-mutant cells similarly to phenformin. Our results suggest that histone acetylation levels are lower in ADCs with LKB1 loss, perhaps by lower expression and decreased nuclear translocation of ACSS2 and ACLY. HDAC inhibitors may have the benefit to maintain histone acetylation in STK11-mutant ADCs, therefore, restoring the expression of tumor suppressors or proteins involved in antitumor immunity, which are silenced in cancer cells. Simultaneous inhibition of mTOR and HDACs may exhibit significant antitumor properties, especially in cancer cells with STK11 mutation and high mTOR activity. Supported by the Hungarian Respiratory Foundation, National Bionics Program of National Research, Development and Innovation Fund of Hungary (ED_17-1-2017-0009), and grants of the Hungarian National Research, Development and Innovation Office (NKFI-FK-128404). Citation Format: Ildiko Krencz, Daniel Sztankovics, Titanilla Danko, Gabor Petovari, Eniko Vetlenyi, Regina Raffay, Judit Moldvay, Judit Papay, Anna Sebestyen. LKB1 loss-associated changes in histone acetylation are potential therapeutic targets in lung adenocarcinoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-046.

Global DNA hypomethylation can be observed along with the aging of normal cells, and it is also related to tumor initiation and progression. S-adenosylmethionine (SAM) is a universal methyl donor molecule, used as a dietary supplement. SAM is involved in DNA methylation processes, thereby it may have a favorable effect on gene expression of cancer-associated genes through epigenetic modifications, but may also influence DNA folding during repair processes. Our aim was to analyze the effect of SAM treatment on global and promoter-specific DNA methylation level, gene expression, DNA integrity, cell cycle and the proliferation of two different colorectal cancer cell lines (HT-29, SW480). HT-29 and SW480 cells were treated with SAM in different concentrations (0, 0.5, 1 mmol/l) for 48 hours. Global DNA methylation status was analyzed by bisulfite pyrosequencing of long interspersed nuclear element-1 (LINE-1) retrotransposons. Promoter-specific DNA methylation alterations were determined by Reduced Representation Bisulfite Sequencing (RRBS) method. Gene expression changes were detected using Human Transcriptome Array 2.0 (HTA 2.0). DNA integrity analysis was performed with γH2AX ELISA, immunostaining and Comet Assay. Flow cytometry measurement and Sulforhodamine B (SRB) assay were assessed for cell cycle and proliferation determination. Global and promoter-specific DNA methylation alterations, as well as decreased expression (p< 0.05) of genes, that are involved in epithelial-mesenchymal transition were observed after SAM treatment. Increased phosphorylation of H2AX (74.9, 166.5, 200.6 pM) and decreased micronucleus number (1.47, 0.76, 0.45% of cells) were referred to the activation of reparative processes, that was supported by the changes of comet tail lengths. Proportion of cells was decreased in the G0/G1 (48.4, 28.5, 20.4%) phase; however, it was increased in both S (45.7, 61.7, 67.0%) and G2/M (6.0, 10.7, 12.5%) phases. Significant (p< 0.05) decrease of cell proliferation (99.5, 78.0, 70.6%) was also detected with SRB assay. SAM is able to alter the DNA methylation pattern of tumor cells and can induce DNA repair. Activation of these processes can lead to cell cycle arrest, decreased proliferation, and inhibition of epithelial-mesenchymal transition. Tumor cells could be targeted by SAM through different pathways; therefore, it may enhance the effect of chemotherapeutic agents. Citation Format: Sára Zsigrai, Alexandra Kalmár, Krisztina Andrea Szigeti, Zsófia Brigitta Nagy, Barbara K. Barták, Gábor Valcz, Orsolya Galamb, Titanilla Dankó, Zsolt Tulassay, Péter Igaz, Béla Molnár. Alteration of DNA methylation, DNA repair and epithelial-mesenchymal transition in colorectal cancer cell lines by S-adenosylmethionine treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 164.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13046-018-0946-5) contains supplementary material, which is available to authorized users.

Figure S3. Vigabatrin abolished the pro-proliferative effect of GABA a., The effect of GABA (5 mM), vigabatrin (0.6 mM) and GABA+vigabatrin on the proliferation of U251 wt glioma cells. SRB and Alamar Blue (AB) proliferation assays were used in 24-h treated cell cultures; b., Alterations in cell numbers (U251 wt cells) followed in every 4-day passage using 3-week continuous treatment, the average cell numbers were calculated from triplicates. (PDF 198 kb)

Figure S2. Extracellular 2-HG levels after 13C-substrate labellings detected by LC-MS in U251 IDH1m cells. a., 2-HG pool after 24 h following 13C-substrates incubation: 4 mM U-13C-glutamine labelled intra- and extracellular 2-HG. b., 10 mM U-13C-glucose labelled extracellular 2-HG in D5030. c., 10 mM 2-13C-acetate labelled 2-HG in D5030. Unlabelled 2-HG did not contain incorporated 13C atoms, M + 1/2/3/4/5 = mass number increased with 1/2/3/4 or 5 13C atoms in 2-HG from different labellings (the low rate of M + 4 is not visible in the figure). The labelling conditions were given in the legends of Fig 3. (PDF 197 kb)

Figure S1. Simplified scheme of the examined bioenergetic pathways in the studied IDH1 wild-type and mutant glioma cells. In our study, different energy substrate oxidation and metabolic enzymes relating to the following bioenergetic pathways were analysed regarding the role of IDH1 mutation. Energy substrate oxidation: glucose, glutamine, citrate, GABA, acetate, malate, lactate and glutamine were measured by Seahorse technique. Energy metabolites - succinate, fumarate, malate, citrate, α-ketoglutarate, glutamate and 2-hydroxyglutarate were determined by liquid chromatography-mass spectrometry. Several protein expressions were measured by Western blot analysis or immunohistochemistry - Glycolysis: hexokinase 2 (HK2), phosphofructokinase P (PFKP), Glutaminolysis: alanine, serine, cysteine-preferring transporter 2 (ASCT2), glutaminase (Gls), GABA shunt: GABA transporter (GAT1), succinic semialdehyde dehydrogenase (SSADH), acetate consumption: acetyl-CoA synthetase 2 (ACSS2). Other abbreviations can be found in the figure: GLUT1: glucose transporter 1, IDH: isocitrate dehydrogenase, LDH: lactate dehydrogenase, MCT1: monocarboxylate transporter 1, OAC: oxaloacetate, SSA: succinic semialdehyde. (PDF 348 kb)

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. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0544-y) contains supplementary material, which is available to authorized users.

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).

Adding 2-HG, lactate did not influence the anti-proliferative effect of rapamycin treatment in HT-1080 cells. The in vitro anti-proliferative effect of rapamycin (50 ng/ml), Na-L-lactate (5 mM) and D-2-HG (0.5 mM) combination treatments were studied by Alamar Blue test after 48–72 h (the results of a representative experiment n = 6, *:p

Introduction The mTOR (mammalian target of rapamycin) is a receptor tyrosine kinase which plays an important role in cell growth regulation, proliferation and tumour cell survival in cancer cells. Differences of mTOR activity were described in tumours. mTOR dependent and independent metabolic activity of tumour cells could be essential in survival and therapy resistance of breast cancer. Changes of mTOR and metabolic activity of breast cancer could correlate to the patients’ treatment outcome. Material and methods In our work, the correlations between the metabolic phenotype and mTOR activity of breast cancer cell lines were studied. Furthermore, the expressions of mTOR activity related proteins and several metabolic transporter, enzymes were studied by Western blot, ICC and IHC using breast cancer cell lines and human tissue samples. The metabolic phenotypes were also analysed using LC-MS. Moreover, the effect of mTOR, metabolic inhibitors and chemotherapeutics were also studied in vitro. Results and discussions Subtypes independent differences in mTORC1/C2 complex and metabolic activities were detected in breast cancer cell lines. Certain cell lines were characterised with high glucose uptake, high mTORC1 complex activity and high lactate production, while other cell lines showed balanced mTORC1 and mTORC2 activity, well-functioning oxidative phosphorylation capacity and higher levels of TCA metabolites which were related to intact mitochondrial functions. The metabolic phenotype and mTORC1/C2 activity correlated to the detected mTOR inhibitors, metabolic inhibitors and chemotherapeutic sensitivity of breast cancer cell lines in vitro. The metabolic and mTOR activity differences can be observed in human tissue samples independently of breast carcinoma cell subtypes, as well. Conclusion Differences of metabolic and mTOR related protein levels may explain the different therapeutic sensitivity. We suggest evaluating the metabolic phenotype and mTOR activity in breast cancer subtypes, which may help to introduce therapeutic drug combinations and predict therapy resistance. Supported by STIA-KF-17(SA), Bolyai-590/2015(SA), UNKP-17-3(HZ)−17-3(KI)−17-2(DT) and EFOP-3.6.3-VEKOP-16-2017-00009.

Absztrakt A hasüregi sebészeti beavatkozásokat követő adhaesio kialakulását számos műtéti körülmény segíti elő – pl. vérzés, idegen anyag bejutása, szövetsérülés, -roncsolódás, ischaemia, hypoxia. Képződése műtéti típustól függően akár 95%-os is lehet. Az adhaesio sok esetben elősegíti további posztoperatív szövődmények kialakulását. Ez lehet krónikus hasi fájdalom, a szervek megfelelő motilitásának akadályozása, de nőknél akár infertilitást is okozhat. Éppen ezért fontos a képződés mechanizmusának pontos ismerete, illetve a megfelelő állatmodell kidolgozása, amely lehetővé teszi a különböző prevenciós módszerek vizsgálatait. Az adhaesioképződésben kulcsszerepet játszó molekulákat, működésük és egymásra hatásuk mechanizmusát szakirodalmi adatok összefoglalásával állítottuk össze. Az adhaesiós állatmodell kidolgozásához hím Wistar-patkányokat használtunk. Az adhaesio kialakításához a következő módszereket alkalmaztuk: pontszerű vérzés előidézése, különböző idegen anyagok beültetése, ischaemizált terület létrehozása, kisebb és jelentősebb szöveti roncsolás. Modelljeinkben a pontszerű vérzések és ischaemizált hasfali területek esetén nem látható adhaesio. Idegen anyagok használatánál a behelyezett anyag felületétől függő méretű adhaesio képződött. Kisebb sérülés esetén szintén jól működött az adhaesio képződését gátló mechanizmus. Az adhaesiós kísérleteink közül a durva szövetsérülést okozó típus a legmegbízhatóbb, az adhaesioképződés minden állatban megfigyelhető, ezért további vizsgálatainkhoz jól használható modell. A bonyolult mechanizmusban a kulcsszereplők megismerése és a megfelelő modell kialakítása elengedhetetlen feltétele a megfelelő prevenciós módszerek kidolgozásának.

Growing evidence suggests that deregulation of signalling elements of Notch and mammalian target of rapamycin (mTOR) pathways contribute to tumorigenesis. These signals play important roles in cellular functions and malignancies. Their tumorigenic role in T-cell acute lymphoblastic leukaemia (T-ALL) is well known; however, their potential interactions and functions are poorly characterized in Hodgkin lymphoma (HL). The aim of our study was to characterize mTOR and Notch signalling elements in HL cell lines (DEV, L1236, KMH2) and human biopsies and to investigate their cross-talk in the tumorous process. High mTOR activity and constitutive NOTCH1 activation was confirmed in HL cell lines, without any known oncogenic mutations in key elements, including those common to both pathways. The anti-tumour effect of Notch inhibitors are well known from several preclinical models but resistance and side effects occur in many cases. Here, we tested mTOR and Notch inhibitors and their combinations in gamma-secretase inhibitor (GSI) resistant HL cells in vitro and in vivo. mTOR inhibitor alone or in combination was able to reduce tumour growth; furthermore, it was more effective in xenograft models in vivo. Based on these results, we suggest that constitutively activated NOTCH1 may be a potential target in HL therapy; furthermore, mTOR inhibitors may be effective for decreasing tumour growth if resistance to Notch inhibitors develop.

Introduction Long non-coding RNAs (lncRNAs) contribute to different cancers including colorectal cancer (CRC). Previous studies have shown altered LINC00152 expression in CRC, but the detailed mechanism of its effects during CRC development and progression is not well studied. We aimed to study the effects of LINC00152 silencing on the cell cycle regulation and whole transcriptome in colon carcinoma cells. We also analysed the DNA methylation alterations caused by LINC00152 knockdown. Material and methods LINC00152 were silenced in SW480 colon carcinoma cells using Stealth siRNAs. Flow cytometric cell cycle analysis was performed using propidium-iodide DNA staining. Cyclin D1 protein expression was detected using flow cytometry after labelling with anti-cyclin D1 antibody. The effect of LINC00152 silencing to genome-wide gene expression was studied on Human Transcriptome Array 2.0 microarrays. DNA methylation alterations after LINC00152 knockdown were evaluated using Reduced Representation Bisulfite Sequencing (RRBS) method using NextSeq500 device. Results and discussions Silencing of LINC00152 significantly suppressed cell growth compared to negative control cells (p 1). Using RRBS, DNA methylation alterations after LINC00152 silencing could be detected genome-widely including hypomethylation in SFRP2 and ALDH1A3 gene promoters. Conclusion Our results suggest that LINC00152 lncRNA can contribute to CRC pathogenesis by facilitating cell proliferation through up-regulation of several oncogenes/metastatic genes in WNT, Notch and TP53 pathways and of cyclin D1 cell cycle progression gene, furthermore, by affecting the promoter methylation status of certain CRC associated genes.

Introduction Kidney transplantation improves survival and life quality for patients with end-stage renal disease (ESRD), however, immunosuppression leads to increased risk of cancer, including renal cell carcinoma (RCC). The PI3K/Akt/mTOR pathway has a fundamental role in renal physiology and its inhibitors are used as immunosuppressive as well as antitumour agents. Recent studies suggest that immunosuppressive regimen, especially calcineurin inhibitors, may affect mTOR pathway activation. The aim of our study was to analyse the expression of mTORC1 and mTORC2-related proteins in RCCs developed in kidneys of transplant recipients and healthy individuals as controls. Material and methods Expression of p-mTOR (catalytic subunit of mTORC1 and mTORC2), p-S6 (mTORC1-related protein) and Rictor (mTORC2-related protein) was assessed in RCC tissues obtained from 46 kidney transplant recipients (15 clear cell RCCs, 31 papillary RCCs) and 46 healthy individuals (28 clear cell RCCs, 18 papillary RCCs) using IHC on tissue microarrays. Tissues from patients with ESRD (n=10) and normal kidney (n=3) were also studied. Results and discussions In normal kidney, expression of p-S6 and p-mTOR was weak in tubular epithelial cells. In contrast, expression of Rictor was high in the epithelium of both proximal and distal tubules. In ESRD, expression of p-mTOR, p-S6, and Rictor appeared to be higher in transplant recipients (n=6) compared to those who have not received immunosuppression (n=4). Expression of p-mTOR and Rictor was significantly higher in RCCs of diseased native kidneys of renal transplant recipients compared to RCCs developed in healthy individuals (p 0.01 and p 0.001, respectively) providing information about mTORC2 activation in post-transplant RCCs. According to previous observations, expression of p-S6 was significantly higher in clear cell RCCs as compared to papillary RCCs in both post-transplant and non-transplant patients (p 0.01 and p 0.05, respectively). Conclusion Our results highlighted the role of mTORC2 in the pathobiology of post-transplant RCCs. There are insufficient evidences for the protective role of mTORC1 inhibitors as immunosuppressive agents against post-transplant malignancies. Use of dual mTORC1/C2 inhibitors in the antitumour and immunosuppressive therapy needs further investigation and may represent a promising opportunity in the treatment or prevention of post-transplant RCCs. Supported by the UNKP-17–3, Bolyai Found of Hungarian Academy Sciences, and Semmelweis University Innovation Found STIA-KF-17.

Introduction IDH mutations are expressed in 80% of low grade gliomas and secondary glioblastomas with a favourable prognosis comparing to non IDH mutant gliomas [According to WHO (2016)] classification, IDH1 mutation is an important marker to classify gliomas. Our main interest is to study the role of certain metabolic alterations in glioma models, especially mitochondrial functions, glycolytic activity, IDH1 mutations and its regulation. Material and methods U87 MG, U373 MG and U251 MG and U251 MG IDH1 R132H (kind gift of Dr. W. Leenders) overexpressing cells were used in vitro . Cellular oxygen consumption, glycolytic activity, energy substrates oxidation were measured by Seahorse technique. Furthermore, the amount and the activity of several proteins related mTOR (mammalian target of rapamycin) and other metabolic pathways were studied. The proliferation and apoptosis rates of in vitro cell cultures were also studied. We analysed extra- and intracellular metabolite levels (citrate, succinate, fumarate, alfa-ketoglutarate, malate, 2-hydroxyglutarate – D-2-HG, glutamate) using LC-MS measurements. Addressing the question what are the sources of D-2-HG in IDH1 mutant U251 MG cells, we fed the cells with 13 C-labelled energy substrates. Results and discussions The glycolytic activity and oxygen consumption rates of the four cell lines show differences. The wild type U251 MG cell line has higher glycolytic activity and lower basal respiration than its IDH1 mutant variant glioma cell line pair. Addition of D-2-HG (72 hour) to wild type U251 MG cells increased the basal respiration and decreased the glycolytic activity of U251 MG cells. Using various bioenergetic substrates, it has been shown that U251 MG cells can oxidise glutamine, glutamate and malate at significantly higher level than IDH1 mutant U251 MG cells. This may be due to the alternative use of glutamine, we found that glutamine is the main source of D-2-HG production in these IDH1 mutant glioma cells. Furthermore, differences were found in the expression pattern of proteins which regulate energy metabolism in the studied glioma cells. Conclusion Based on the results of the examined glioma cells, IDH1 mutant cells showed a lower glycolytic capacity, a higher respiration and altered glutamine metabolism, which could be therapeutic targets in the future. Supported by UNKP-17–2, UNKP-17–3, Richter Centenarium Foundation, Bolyai-590/2015 and STIA-KF-17.

In spite of the improved efficacy of therapy, it still fails in 15-20 % of childhood acute lymphoblastic leukemia (ALL) patients. Recently, altered expression of certain miRNAs (miRs) have been described in ALL with potential effect on prognosis. Presence of certain miRs (miRNA-16, -21, -24, -29b, -128b, -142-3p, -155, -223) was characterized in human lymphoma and leukemia cells by real-time PCR. Expression of miRs in pediatric ALL patients (n = 24) was measured before chemotherapy, at conventional response checkpoints and at relapse. Correlation between altered miR expression and response to prednisolone at day 8 of therapy and long term prognosis was statistically analysed. Overexpression of "oncomiR/inflammamiR"-21 - which is characteristic in different tumors-was missing in human ALL cells. However, higher expression of miR-128b and lower expression of miR-223 is generally characteristic for human ALL cell lines and ALL cells isolated from pediatric patients. Correlation was shown between miR-128b expression and prognosis, prednisolone response and survival data in childhood ALL. Expression of miR-128b and miR-223-both are leukemia specific-changed in parallel with percentage of bone marrow blasts in remission and during relapse. Therefore, we suggest that overexpression of miR-128b and downregulation of miR-223 shows a significant correlation with treatment response and prognosis in childhood ALL.

A huge number of factors play a significant role in the process of adhesion formation, like bleeding, the presence of foreign bodies, tissue injury, tissue destruction, ischemia and hypoxia. Adhesions are present in 95% of the cases following abdominal surgery. As a result of adhesions a large number of postoperative complications can occur, such as abdominal pain, bowel motility disturbances and infertility. Hence, it is important to know the precise mechanism of adhesion formation process and establish a suitable animal model to investigate the underlying mechanisms. Molecules which play a part in the process of adhesion formation were collected from the international literature. Male Wistar rats were used to create the adhesion model. Bleeding, implantation of foreign bodies, creation of ischemic areas and tissue destructions were carried out. Within this experiment the tiny bleeding and ischemic areas did not result in adhesion formation. The adhesion formation due to foreign body implantation depends on the type of the materials. Due to the inhibitory mechanism of adhesion formation there was no adhesion detectable due to tiny peritoneal destruction. The most reliable model was the one when gross tissue destruction of the abdominal wall was applied and the resulting bleeding initiated the adhesion formation process. It is also extremely important to know the key participants in the complex process of adhesion formation. This reliable model can help to work out the proper method of prevention.A hasüregi sebészeti beavatkozásokat követő adhaesio kialakulását számos műtéti körülmény segíti elő – pl. vérzés, idegen anyag bejutása, szövetsérülés, -roncsolódás, ischaemia, hypoxia. Képződése műtéti típustól függően akár 95%-os is lehet. Az adhaesio sok esetben elősegíti további posztoperatív szövődmények kialakulását. Ez lehet krónikus hasi fájdalom, a szervek megfelelő motilitásának akadályozása, de nőknél akár infertilitást is okozhat. Éppen ezért fontos a képződés mechanizmusának pontos ismerete, illetve a megfelelő állatmodell kidolgozása, amely lehetővé teszi a különböző prevenciós módszerek vizsgálatait. Az adhaesioképződésben kulcsszerepet játszó molekulákat, működésük és egymásra hatásuk mechanizmusát szakirodalmi adatok összefoglalásával állítottuk össze. Az adhaesiós állatmodell kidolgozásához hím Wistar-patkányokat használtunk. Az adhaesio kialakításához a következő módszereket alkalmaztuk: pontszerű vérzés előidézése, különböző idegen anyagok beültetése, ischaemizált terület létrehozása, kisebb és jelentősebb szöveti roncsolás. Modelljeinkben a pontszerű vérzések és ischaemizált hasfali területek esetén nem látható adhaesio. Idegen anyagok használatánál a behelyezett anyag felületétől függő méretű adhaesio képződött. Kisebb sérülés esetén szintén jól működött az adhaesio képződését gátló mechanizmus. Az adhaesiós kísérleteink közül a durva szövetsérülést okozó típus a legmegbízhatóbb, az adhaesioképződés minden állatban megfigyelhető, ezért további vizsgálatainkhoz jól használható modell. A bonyolult mechanizmusban a kulcsszereplők megismerése és a megfelelő modell kialakítása elengedhetetlen feltétele a megfelelő prevenciós módszerek kidolgozásának.

There is increasing attention for PI3K/Akt/mTOR pathway as a potential target in tumor therapy. The high activity of mTOR is a characteristic feature of many tumors including hematological malignancies. However, the pathological characterisation of mTOR activity of lymphoid malignancies - especially the activity of different complexes – is still incomplete. We studied the mTOR activity related phospho-proteins and the elements of mTOR complexes by using different techniques (ELISA, Western blot, different immunohistochemical stainings, flow cytometry) depending on the tumor sample type (fresh/fixed human tissues, isolated human cells from blood or bone marrow, cell lines). The expression and activity results were compared in order to find correlation with clinical data or to mTOR inhibitor sensitivity in vitro. The proliferation and apoptotic effects after in vitro treatments were detected by Alamar Blue assay and flow cytometry. We established the in situ measurement for the amount of mTORC1, C2 complexes by Duolink stainings to compare the effects of different mTOR inhibitors to the mTORC1/C2 complex availability in lymphoma cell cultures. Our studies described high mTOR activity in all acute lymphoid leukemia (ALL) cases and in the majority of mantle cell lymphomas, anaplastic large cell lymphomas, Burkitt lymphomas, diffuse large B cell lymphomas (DLBCL), Hodgkin lymphomas (HL) and in certain cutan T lymphomas. Further characterisation of HLs, DLBCLs and ALL cases showed that high mTOR activity especially with the presence of mTORC2 is a sign of unfavorable prognosis. mTOR activity related to p-S6 and p-4EBP1 expression (ELISA and flow cytometry) at diagnosis was significantly higher in patients with poor prognosis in ALL cases. In DLBCL patient's the results showed that high mTOR activity with Rictor overexpression significantly correlated to the worse prognosis and shorter survival. We also detected the sign of high mTOR activity in more than 90% of HLs (HLs usually with favorable prognosis), however, in these cases Rictor was not found (the mTOR activity related to mTORC1). In the different lymphoma cell lines increased mTOR activity was detected and we could describe different Rictor, Raptor and mTOR-Rictor complex expression. Moreover, the inhibitor sensitivity of the cell lines and the different expression of the elements related to mTORC1 and C2 correlated. Our results confirmed the increased mTOR activity related to mTORC1 and/or C2 complexes in certain lymphomas and ALLs. According to these analyses, the activity and expression of mTOR complexes should have potential prognostic value and high importance before mTOR inhibitor treatment, especially in the cases with high mTORC2 related protein expression. The study was supported by OTKA816624, OTKA 84262 projects and Tumor Progression Research Group of Joint Research Organization of the Hungarian Academy of Sciences and Semmelweis University Citation Format: Anna Sebestyén, Noémi Nagy, Ágnes Márk, Anna Molnár, Titanilla Dankó, Melinda Hajdu, Mónika Tóth, Botond Timár, Mónika Csóka, László Kopper. mTOR C1/2 activities - related protein expression and its potential prognostic/therapeutic importance in certain lymphoid malignancies. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B47.