Tumorigenic role of tacrolimus through mTORC1/C2 activation in post-transplant renal cell carcinomas.

BACKGROUND: Kidney transplant recipients (KTRs) face an increased risk of renal cell carcinoma (RCC), in which the immunosuppressive regimen plays an important role. This study aimed to identify intracellular signalling alterations associated with post-transplant (post-tx) tumour formation. METHODS: Expression of mTOR-related proteins were analysed in kidneys obtained from end-stage renal disease (ESRD) patients and RCCs developed in KTRs or non-transplant patients. The effects of tacrolimus (TAC) and rapamycin (RAPA) on mTOR activity, proliferation, and tumour growth were investigated through different in vitro and in vivo experiments. RESULTS: Elevated mTORC1/C2 activity was observed in post-tx RCCs and in kidneys of TAC-treated ESRD patients. In vitro experiments demonstrated that TAC increases mTOR activity in a normal tubular epithelial cell line and in the investigated RCC cell lines, moreover, promotes the proliferation of some RCC cell line. In vivo, TAC elevated mTORC1/C2 activity in ischaemic kidneys of mice and enhanced tumour growth in xenograft model. CONCLUSIONS: We observed significantly increased mTOR activity in ischaemic kidneys and post-tx RCCs, which highlights involvement of mTOR pathway both in the healing or fibrotic processes of kidney and in tumorigenesis. TAC-treatment further augmented the already elevated mTOR activity of injured kidney, potentially contributing to tumorigenesis during immunosuppression.

Revealing a Phenotypical Appearance of Ibrutinib Resistance in Patients With Chronic Lymphocytic Leukaemia by Flow Cytometry.

Background: Ibrutinib is widely known as an effective and well-tolerated therapeutical choice of the chronic lymphocytic leukaemia (CLL). However, acquired resistance may occur during the treatment, causing relapse. Early detection of ibrutinib resistance is an important issue, therefore we aimed to find phenotypic markers on CLL cells the expression of which may correlate with the appearance of ibrutinib resistance. Methods: We examined 28 patients' peripheral blood (PB) samples (treatment naïve, ibrutinib sensitive, clinically ibrutinib resistant). The surface markers' expression (CD27, CD69, CD86, CD184, CD185) were measured by flow cytometry. Furthermore, the BTKC481S resistance mutation was assessed by digital droplet PCR. Moreover, the CLL cells' phenotype of a patient with acquired ibrutinib resistance was observed during the ibrutinib treatment. Results: The expression of CD27 (p = 0.030) and CD86 (p = 0.031) became higher in the clinically resistant cohort than in the ibrutinib sensitive cohort. Besides, we found that high CD86 and CD27 expressions were accompanied by BTKC481S mutation. Our prospective study showed that the increase of the expression of CD27, CD69 and CD86 was noticed ahead of the clinical resistance with 3 months. Conclusion: Our study suggests that the changes of the expression of these markers could indicate ibrutinib resistance and the examination of these phenotypic changes may become a part of the patients' follow-up in the future.

Limitations of VS38c labeling in the detection of plasma cell myeloma by flow cytometry.

Plasma cell myeloma (multiple myeloma [MM]) is a malignant neoplasm originating from the plasma cells. Besides other methods, flow cytometric analysis of the patient's bone marrow aspirate has an important role in the diagnosis and also in the response assessment. Since the cell surface markers, used for identifying abnormal plasma cells, are expressed diversely and the treatment can also alter the phenotype of the plasma cells, there is an increasing demand for new plasma cell markers. VS38c is a monoclonal antibody that recognizes the CLIMP-63 protein in the membrane of the endoplasmic reticulum. CLIMP-63 is known to be expressed at high levels in normal and pathologic plasma cells in the bone marrow, thus VS38c antibody can be used to identify them. Although VS38c staining of plasma cells is reported to be constant and strong even in myeloma, we were wondering whether sample preparation can affect the staining. We have investigated the effect of different permeabilization agents and washing of the cells on the quality of the VS38c staining and found that in many cases the staining is inadequate to identify the plasma cells. We measured the VS38c staining of the bone marrow aspirates of 196 MM patients and observed that almost all cases showed bright staining with VS38c. However, permeabilization with mild detergent resulted in the appearance of a significant VS38cdim subpopulation, which showed increased sensitivity to mechanical stress (centrifugation). Our results indicate that VS38cdim MM cells can appear due to the improper permeabilization of the endoplasmic reticulum and this finding raises the possibility of the existence of a plasma cell subpopulation with different membrane properties. The significance of this population is unclear yet, but these cells can be easily missed with VS38c staining and can be lost due to centrifugation-induced lysis during sample preparation.

Rapamycin Plus Doxycycline Combination Affects Growth Arrest and Selective Autophagy-Dependent Cell Death in Breast Cancer Cells.

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.

Lenalidomide abrogates the survival effect of bone marrow stromal cells in chronic lymphocytic leukemia.

In the pathogenesis of chronic lymphocytic leukemia (CLL) the microenvironment plays an important role, as it produces survival signals and mediates drug resistance. Lenalidomide, which has immunomodulatory effect, can enhance the activation of T-, NK-cells and endothelial cells, however there are no data available whether it can modulate bone marrow stromal cells (BMSCs). In our study, we investigated the effects of lenalidomide on BMSCs and CLL cells. CLL cells were cultured alone or with BMSCs and were treated with lenalidomide. Apoptosis, immunophenotype, and cytokine secretion of BMSCs and CLL cells were determined by flow cytometry. Lenalidomide slightly increased the apoptosis of CLL cells and abrogated the anti-apoptotic effect of BMSCs on CLL cells. Lenalidomide treatment decreased the expression of antigens on CLL cells, which mediate the interactions with the microenvironment. Interestingly, lenalidomide enhanced the expression of IRF4 and the co-stimulatory molecule CD86. The secretion of several cytokines was not changed significantly by lenalidomide. CD49d-negative CLL cases were more sensitive to lenalidomide treatment. Our results suggest that lenalidomide has a limited effect on BMSCs, but it renders CLL cells more immunogenic and unresponsive to survival signals provided by BMSCs.

In contrast to high CD49d, low CXCR4 expression indicates the dependency of chronic lymphocytic leukemia (CLL) cells on the microenvironment.

CD49d and CXCR4 are key determinants of interactions between chronic lymphocytic leukemia (CLL) tumor cells and their microenvironment. In this study, we investigated the effect of CD49d and CXCR4 expressions on survival of CLL cells. Primary CLL cells were cultured with CD49d ligand, VCAM-1, or bone marrow stromal cells (BMSCs); then, apoptosis and immunophenotype analyses were performed. VCAM-1 treatment could not induce direct apoptosis protection or immunophenotype change on the CD49d-expressing CLL cells, but resulted in actin reorganization. The BMSC-induced apoptosis protection was independent from the presence of CD49d expression of CLL cells, but showed an inverse correlation with their CXCR4 expression level. We suppose that CD49d contributes to enhanced survival of leukemic cells by mediating migration to the protective microenvironment, not by direct prevention of apoptosis. Moreover, CLL cells with low CXCR4 expression represent a subpopulation that is more dependent on the microenvironmental stimuli for survival, and show increased "death by neglect" when separated from the supportive niche.

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.

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.

The effect of microenvironmental factors on the development of myeloma cells.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of monoclonal plasma cells (PCs) in the bone marrow and other tissues. Although there are several new therapies, MM remains fatal. The interaction between MM cells and the bone marrow microenvironment promotes drug resistance and cancer cells survival. In our present work, we compared the antigen expression pattern of normal and pathological PCs and investigated the possible connections between various surface receptors, adhesion molecules, and recurrent genetic aberrations. We showed that the expression of CD29, CD27, and CD81 is lower in MM cells than in normal PCs. We found correlation of chromosome 11 hyperdiploidity and the decrease of CD27 expression. We demonstrated that MM cells with CD20 positivity also have CD28 expression. Multiple myeloma patients with active CD29 showed better response to treatment. Our results suggest that these changes may result in an alteration of the interaction between stromal cell and MM cell facilitating cell survival and the development of a more aggressive and resistant phenotype.

Growth inhibitory effect of rapamycin in Hodgkin-lymphoma cell lines characterized by constitutive NOTCH1 activation.

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.

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

TGF-ß1 (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-ß1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-ß signaling, and through these pathways it is able to enhance TGF-ß induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-ß1 combination on cell growth and on TGF-ß 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-ß1 induced apoptosis and restored TGF-ß1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-ß 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-ß1, 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-ß1 induced apoptosis was not completely eliminated by a caspase inhibitor. These results suggest that high mTOR activity contributes to TGF-ß 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-ß1.

Expression of certain leukemia/lymphoma related microRNAs and its correlation with prognosis in childhood acute lymphoblastic leukemia.

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.

[Significance of mTOR (mammalian target of rapamycin) activity in human lymphomas]. / Az mTOR (mammalian target of rapamycin) aktivitás jelentosége humán lymphomákban.

Neoplastic processes, tumor growth, and tumor cell proliferation and survival are often due to the altered activation of different signaling pathways. The increased activity of PI3K/AKT/mTOR signaling has been shown to be an important regulator of tumor growth in several solid tumors and in mantle cell lymphomas. The active form of mTOR kinase (mammalian target of rapamycin) is a key signaling molecule, and it exists in two different complexes, mTORC1 and mTORC2. In the present work, mTOR activity was investigated in different lymphoma types, in parallel with clinical data. We also examined in Hodgkin lymphomas (HL) the role of mTOR activity in survival mechanisms such as antiapoptotic protein expression and alterations in the microenvironment. We determined which lymphoma types display characteristic high mTOR activity in our TMA (tissue microarray) study. We observed that mTOR activity is increased in mitotic lymphoid cells compared to interphasic cells. The number of diffuse large B cell lymphoma (DLBCL) and HL cases was extended in a further set of TMA. We observed significantly higher mTOR activity in the non-centrum germinativum derived subtype of DLBCL than in the centrum germinativum derived subtype, which was a prognostic marker; 63% of mTOR active cases showed Rictor overexpression, indicating mTORC2 activity. High mTOR activity was also established in 92% of HL cases, which was linked to mTORC1. This finding was not a prognostic marker, however, it can be useful in targeted therapy. We observed the overexpression of the antiapoptotic protein BCL-xL and NFκB-p50 in the majority of mTOR active HLs. HLs showed high numbers of regulatory T cells in the microenvironment and high expression of galectin-1 in tumor cells and in the extracellular matrix, when compared to reactive lymph nodes. We confirmed that mTOR inhibition had significant antiproliferative and antiapoptotic effects in lymphoma cell lines and in lymphoma xenografts (HL, DLBCL, Burkitt lymphoma). We also showed that rapamycin was able to augment the effect of chemotherapeutic agents and TGF-ß. Taken together, mTOR activity may be a potential therapeutic target in different lymphoma types. However, patient and inhibitor selection criteria must be carefully considered. The combination of mTOR inhibitors with other agents will probably offer the highest efficiency for achieving the best clinical response, and may also allow dose reduction in order to decrease late treatment toxicity in these cases.

mTOR pathway as a potential target in a subset of human medulloblastoma.

As mammalian Target of Rapamycin (mTOR) plays role in protein synthesis and metabolism, mTOR pathway activation is involved in the pathogenesis of several types of tumors. Our aim was to elucidate its role in medulloblastoma in terms of prognosis and as a therapeutic target. Members of activated mTOR complex 1 (mTORC1) pathway, phospho-mTOR (p-mTOR) and phospho-S6 (p-S6) were examined by immunohistochemistry in formalin fixed paraffin embedded samples of 40 patients with medulloblastoma, and results were compared to clinical features and survival of patients. In proliferation assays, Daoy and UW228-2 medulloblastoma cell lines were tested by rapamycin, an mTORC1 inhibitor, and NVP-BEZ235, a dual mTOR and phosphatidylinositol 3-kinase (PI3K) inhibitor, each in monotherapy and in combination with cytostatic drugs (cisplatin, etoposide). Components of mTORC1 and mTORC2 complexes were also examined in these cell lines. Neither presence of p-mTOR (32.5 %) nor p-S6 (32.5 %) correlated with age, gender or histological subtype. In 22.5 % of cases simultaneous expression of p-mTOR and p-S6 was shown. Kaplan-Meier analysis showed inferior survival of patients expressing both marker proteins, but it was not statistically significant, probably due to low case number. UW228-2 cells had greater sensitivity to mTOR inhibitors, possibly due to its higher mTORC1 specific protein expression levels, compared to Daoy cells. In both cell lines antiproliferative effect of cytostatic drugs was significantly enhanced by mTOR inhibitors (p < 0.05). Based on our in vitro and clinicopathological studies mTOR inhibitors may have a role in the future treatment of a subset of patients with medulloblastoma.

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.

BACKGROUND: Targeting 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. METHODS: mTOR 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. RESULTS: The 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. CONCLUSIONS: Targeting 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.

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.

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.

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.

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 α (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.