RESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Receptores ErbB/antagonistas & inhibidores , Humanos , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Colorrectales/metabolismo , Complejos Multiproteicos/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adulto , Anciano , Línea Celular Tumoral , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Persona de Mediana Edad , Complejos Multiproteicos/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Pronóstico , Serina-Treonina Quinasas TOR/genética , Análisis de Matrices TisularesRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Cerebelosas/metabolismo , Meduloblastoma/metabolismo , Complejos Multiproteicos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Adolescente , Adulto , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/mortalidad , Neoplasias Cerebelosas/patología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Lactante , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/mortalidad , Meduloblastoma/patología , Complejos Multiproteicos/antagonistas & inhibidores , Estadificación de Neoplasias , Fosforilación/efectos de los fármacos , Pronóstico , Transducción de Señal/efectos de los fármacos , Tasa de Supervivencia , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Análisis de Matrices Tisulares , Células Tumorales Cultivadas , Adulto JovenRESUMEN
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.
Asunto(s)
Enfermedad de Hodgkin/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Niño , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/patología , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Sirolimus/farmacología , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Adolescente , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular , Línea Celular Tumoral , Niño , Preescolar , Supervivencia sin Enfermedad , Sinergismo Farmacológico , Humanos , Lactante , Estimación de Kaplan-Meier , Análisis Multivariante , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , Curva ROC , Sirolimus/farmacología , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Resultado del TratamientoRESUMEN
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.