Ágnes Márk

Characteristic mTOR activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease--a combined tissue microarray, in vitro and in vivo study.

BackgroundTargeting signaling pathways is an attractive approach in many malignancies. The PI3K/Akt/mTOR pathway is activated in a number of human neoplasms, accompanied by lower overall and/or disease free survival. mTOR kinase inhibitors have been introduced in the therapy of renal cell carcinoma and mantle cell lymphoma, and several trials are currently underway. However, the pathological characterization of mTOR activity in lymphomas is still incomplete.MethodsmTOR activity and the elements of mTOR complexes were investigated by immunohistochemistry on tissue microarrays representing different human non-Hodgkin-lymphomas (81 cases) and Hodgkin-lymphomas (87 cases). The expression of phospho-mTOR, phospho-4EBP1, phospho-p70S6K, phospho-S6, Rictor, Raptor and Bcl-2, Bcl-xL, Survivin and NF-kappaB-p50 were evaluated, and mTOR activity was statistically analyzed along with 5-year survival data. The in vitro and in vivo effect of the mTOR inhibitor rapamycin was also examined in human Hodgkin-lymphoma cell lines.ResultsThe majority (>50%) of mantle cell lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma and Hodgkin-lymphoma cases showed higher mTOR activity compared to normal lymphoid tissues. Hodgkin-lymphoma was characterized by high mTOR activity in 93% of the cases, and Bcl-xL and NF-kappaB expression correlated with this mTOR activity. High mTOR activity was observed in the case of both favorable and unfavorable clinical response. Low mTOR activity was accompanied by complete remission and at least 5-year disease free survival in Hodgkin-lymphoma patients. However, statistical analysis did not identify correlation beetween mTOR activity and different clinical data of HL patients, such as survival. We also found that Rictor (mTORC2) was not overexpressed in Hodgkin-lymphoma biopsies and cell lines. Rapamycin inhibited proliferation and induced apoptosis in Hodgkin-lymphoma cells both in vitro and in vivo, moreover, it increased the apoptotic effect of chemotherapeutic agents.ConclusionsTargeting mTOR activity may be a potential therapeutic tool in lymphomas. The presence of mTOR activity probably indicates that the inclusion of mTOR inhibition in the therapy of Hodgkin-lymphomas may be feasible and beneficial, especially when standard protocols are ineffective, and it may also allow dose reduction in order to decrease late treatment toxicity. Most likely, the combination of mTOR inhibitors with other agents will offer the highest efficiency for achieving the best clinical response.

The activated targets of mTOR signaling pathway are characteristic for PDGFRA mutant and wild-type rather than KIT mutant GISTs.

The therapy for gastrointestinal stromal tumors (GISTs) has been revolutionized by tyrosin kinase inhibitors. Clinicopathologic studies have been conducted to assess therapeutical responses in cases with KIT and platelet-derived growth factor receptor &agr; (PDGFRA) gene mutations. Cell culture data suggest that Akt/mammalian target of rapamycin (mTOR) kinase signaling may be important in GIST. The aim of our study was to determine the activity of the mTOR pathway in a larger series of GISTs (108 different cases) with different exon mutation types. The KIT and/or PDGFRA mutation status of 108 GIST patients was analyzed by direct DNA sequencing. Immunohistochemistry was performed on tissue microarrays using antibodies for phospho-p70S6 kinase, phospho-4EBP1, and phospho-S6, which are downstream target proteins of mTOR. DNA sequencing identified 73 cases with mutations of KIT and 12 cases with PDGFRA mutations. Wild-type receptors were present in 23 cases. KIT exon mutations were accompanied by the activation of the mTOR pathway in 28 of 73 (38.4%) cases, whereas PDGFRA mutant GISTs showed activation in 10 of 12 (83.3%) cases. Wild-type cases were accompanied by mTOR activation in 17 of 23 (73.9%) cases. Our results indicate that the activation of the mTOR pathway is not a general hallmark of GIST with KIT mutations. However, mTOR signaling seems to be activated in PDGFRA mutants and in wild-type cases, which suggests that mTOR or upstream mTOR inhibitors may be therapeutically useful in primary resistant GISTs and confirms earlier data that mTOR is a crucial survival pathway in resistant GISTs.

Mammalian Target of Rapamycin (mTOR) Activity Dependent Phospho-Protein Expression in Childhood Acute Lymphoblastic Leukemia (ALL)

Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25–30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments.

Activity and complexes of mTOR in diffuse large B-cell lymphomas—a tissue microarray study

Diffuse large B-cell lymphoma is a heterogeneous group of diseases with different responses to therapy. Targeting mTOR (mammalian target of rapamycin) offers a new approach to improve the treatment. mTOR inhibitors are being developed and are in clinical use in mantle cell lymphoma therapy and clinical trials are ongoing in other high-grade lymphomas as well. However, there is limited data about mTOR activity and the expression of its different complexes in diffuse large B-cell lymphomas. Tissue microarray blocks were constructed from paraffin-embedded biopsy specimens. More than 700 immunohistochemical stainings (mTOR signaling-related proteins and phosphoproteins, markers for lymphoma classification) were evaluated from 68 diffuse large B-cell lymphoma biopsies from conventionally treated and followed patients. Approximately 30% of cases were characterized as germinal center-derived diffuse large B-cell lymphomas, which showed virtually no mTOR activity, as determined by phospho-ribosomal S6 expression, the most sensitive marker of mTOR activity. In about 80% of non-germinal center-derived diffuse large B-cell lymphoma cases, positivity of mTOR-related phosphoproteins was observed, denoting mTOR activity. Moreover, Rictor (a characteristic protein of the mTOR complex2) was overexpressed in 43% of all diffuse large B-cell lymphomas and in 63% of mTOR-active non-germinal center diffuse large B-cell lymphoma samples. Rictor overexpression with mTOR activity indicated significantly worse survival for patients than mTOR inactivity or mTOR activity with low Rictor expression. These results suggest that mTOR activity is characteristic in most non-germinal center-derived diffuse large B-cell lymphomas with potentially variable mTOR-inhibitor sensitivity. Taken together, mTOR inhibitors may be useful in addition to regular therapy in diffuse large B-cell lymphomas, however, patient and inhibitor selection criteria must be carefully considered.

Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas

TGF-β1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-β1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-β signaling, and through these pathways it is able to enhance TGF-β induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-β1 combination on cell growth and on TGF-β and mTOR signalling events in lymphoma cells. Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-β1 induced apoptosis and restored TGF-β1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-β sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin+TGF-β1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin+TGF-β1 induced apoptosis was not completely eliminated by a caspase inhibitor. These results suggest that high mTOR activity contributes to TGF-β resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-β1.

Revisiting CB1 receptor as drug target in human melanoma

Previous studies have indicated the antitumoral effect of human melanocytes, human melanoma cell lines expressing CB1 receptor (CB1), and of the peritumoral administration of endocannabinoids. In the present study, we systematically screened several human melanoma cell lines for the expression of CNR1 and demonstrated transcription of the authentic gene. The product of CNR1, the CB1 protein, was found localized to the cell membrane as well as to the cytoskeleton. Further, the studied human melanoma cell lines expressed functional CB1 since physiological and synthetic ligands, anandamide (AEA), Met-F-AEA, ACEA and AM251 showed a wide range of biological effects in vitro, for example anti-proliferative, proapoptotic and anti-migratory. More importantly, our studies revealed that systemic administration of a stable CB1 agonist, ACEA, into SCID mice specifically inhibited liver colonization of human melanoma cells. Since therapeutic options for melanoma patients are still very limited, the endocannabinoid-CB1 receptor system may offer a novel target.

mTOR activity and its prognostic significance in human colorectal carcinoma depending on C1 and C2 complex-related protein expression

Aims Tumour heterogeneity and altered activation of signalling pathways play important roles in therapy resistance. The PI3K/Akt/mTOR signalling network is a well-known regulator of several functions that contribute to tumour growth. mTOR exists in two functionally different multiprotein complexes. We aimed to determine mTOR activity-related proteins in clinically followed, conventionally treated colon carcinomas and to analyse the correlation between clinical data and mTORC1 and mTORC2 activity. Methods Immunohistochemistry was performed with different antibodies on tissue microarray blocks from 103 patients with human colorectal adenocarcinoma. mTORC1- and mTORC2-related activity were scored on different stainings including analysis of the expression of Raptor and Rictor—specific elements of mTORC1 and C2 complexes. The staining scores and clinical/survival data were compared and analysed. Results Detailed characterisation showed stage and grade independent high mTOR activity in 74% of cases. High mTOR activity was present in mTORC1 and/or mTORC2 complexes; >60% of cases had mTORC2-related high mTOR activity. Based on our analysis, high mTOR activity and Rictor overexpression could be markers of a bad prognosis. Combined phosphoprotein and Rictor/Raptor expression evaluation revealed even stronger statistical correlation with prognosis. Conclusions The presented staining panel could be appropriate and highly recommended for the accurate specification of mTORC1 and C2 activity of tumour tissues. This could help in the selection of mTOR inhibitors and can provide information about prognosis, which may guide decisions about the intensity of therapy.

Mitotic lymphoma cells are characterized by high expression of phosphorylated ribosomal S6 protein

Growth factors and mitogens influence signaling pathways and often induce the activity of p70S6 kinase (p70S6K), which in turn phosphorylates the ribosomal S6 protein (S6). Although recent data are rather conflicting, the overall view suggests that phosphorylated S6 is a regulator of global protein synthesis, cell proliferation, cell size and glucose homeostasis. In the present work, emphasis was given to cell cycle-dependent activation of S6 focusing mainly on human lymphoid and lymphoma cells. Paraffin-embedded human tissue blocks from lymph node and different tumor biopsies as well as in vitro cell lines were investigated by immunohistochemistry, immunocytochemistry, flow cytometry and Western blotting using antibodies directed against phospho-S6, phospho-mTOR, phospho-p70S6K and phospho-Histone H3. To enrich the cell number in different phases of the cell cycle, nocodazole, staurosporine or rapamycin were used in cell cultures. We observed strong phospho-S6 positivity by immunostainings in the dividing lymphoid cells of reactive lymph nodes and in lymphoma cells cultured in vitro. Phospho-S6 protein levels were shown to be elevated throughout mitosis in lymphoma cells; however, the high expression of phospho-S6 in mitotic cells was not a general hallmark of tumor cell types studied so far: phospho-S6-negative mitotic cells were detected in several carcinoma and sarcoma biopsies. These observations may have practical implications as they raise the possibility to consider p70S6K and/or S6 as a potential therapeutic target—besides mTOR—in certain lymphomas and perhaps in clinical immunosuppression.

Expression of mTORC1/2-related proteins in primary and brain metastatic lung adenocarcinoma

Brain metastases (BMs) are common complications of adenocarcinomas (ADCs) of the lung and are associated with a poor prognosis. Although an increasing amount of data indicates that dysregulated activity of mammalian target of rapamycin (mTOR) can influence the metastatic potential of various tumors, the role of mTOR complexes in the development of BMs from ADCs of the lung is largely unknown. To estimate mTOR activity, we studied the expression of mTOR-related proteins (mTORC1: p-mTOR, p-S6; mTORC2: p-mTOR, Rictor) in primary (n=67) and brain metastatic (n=67) lung ADCs, including 15 paired tissue samples, using immunohistochemistry and tissue microarrays. Correlation with clinicopathological parameters was also analyzed. Increased p-mTOR, p-S6, and Rictor expressions were observed in 34%, 33%, and 37% of primary ADCs and in 79%, 70%, and 66% of BMs, respectively. Expression of these markers was significantly higher in BMs as compared with primary carcinomas (P<.0001, P<.0001, P<.001). Rictor expression was significantly higher in primary ADCs of the paired cases with BMs as compared with primary ADCs without BMs (67% versus 28%; P<.01). No other statistically significant correlations were found between mTOR activity and clinicopathological parameters. The increased mTORC1/C2 activity in a subset of pulmonary ADCs and the higher incidence of increased mTORC1/C2 activity in BMs suggest that the immunohistochemistry panel for characterizing mTOR activity and its potential predictive and prognostic role warrants further investigations.

Characterisation of bioenergetic pathways and related regulators by multiple assays in human tumour cells

BackgroundAlterations in cellular metabolism are considered as hallmarks of cancers, however, to recognize these alterations and understand their mechanisms appropriate techniques are required. Our hypothesis was to determine whether dominant bioenergetic mechanism may be estimated by comparing the substrate utilisation with different methods to detect the labelled carbon incorporation and their application in tumour cells.MethodsTo define the bioenergetic pathways different metabolic tests were applied: (a) measuring CO2 production from [1-14C]-glucose and [1-14C]-acetate; (b) studying the effect of glucose and acetate on adenylate energy charge; (c) analysing glycolytic and TCA cycle metabolites and the number of incorporated 13C atoms after [U-13C]-glucose/[2-13C]-acetate labelling. Based on [1-14C]-substrate oxidation two selected cell lines out of seven were analysed in details, in which the highest difference was detected at their substrate utilization. To elucidate the relevance of metabolic characterisation the expression of certain regulatory factors, bioenergetic enzymes, mammalian target of rapamycin (mTOR) complexes (C1/C2) and related targets as important elements at the crossroad of cellular signalling network were also investigated.ResultsBoth [U-13C]-glucose and [1-14C]-substrate labelling indicated high glycolytic capacity of tumour cells. However, the ratio of certain 13C-labelled metabolites showed detailed metabolic differences in the two selected cell lines in further characterisation. The detected differences of GAPDH, β-F1-ATP-ase expression and adenylate energy charge in HT-1080 and ZR-75.1 tumour cells also confirmed the altered metabolism. Moreover, the highly limited labelling of citrate by [2-13C]-acetate—representing a novel functional test in malignant cells—confirmed the defect of TCA cycle of HT-1080 in contrast to ZR-75.1 cells. Noteworthy, the impaired TCA cycle in HT-1080 cells were associated with high mTORC1 activity, negligible protein level and activity of mTORC2, high expression of interleukin-1β, interleukin-6 and heme oxygenase-1 which may contribute to the compensatory mechanism of TCA deficiency.ConclusionsThe applied methods of energy substrate utilisation and other measurements represent simple assay system using 13C-acetate and glucose to recognize dominant bioenergetic pathways in tumour cells. These may offer a possibility to characterise metabolic subtypes of human tumours and provide guidelines to find biomarkers for prediction and development of new metabolism related targets in personalized therapy.