RESUMO
Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is still increasing in Europe and the prognosis remains poor. We investigated the oncogenic function of signal transducer and activator of transcription 1 (STAT1) in MPM in more detail. A miRNA profiling was performed on 52 MPM tissue samples. Upregulated miRNAs (targeting SOCS1/3) were knocked-down using miRNA inhibitors. mRNA expression levels of STAT1/3, SOCS1/3 were detected in MPM cell lines. STAT1 has been knocked-down using siRNA and qPCR was used to detect mRNA expression levels of all JAK/STAT family members and genes that regulate them. An immunohistochemical staining was performed to detect the expression of caspases. STAT1 was upregulated and STAT3 was downregulated, SOCS1/3 protein was not detected but it was possible to detect SOCS1/3 mRNA in MPM cell lines. The upregulated miRNAs were successfully knocked-down, however the expected effect on SOCS1 expression was not detected. STAT1 knock-down had different effects on STAT3/5 expression. Caspase 3a and 8 expression was found to be increased after STAT1 knock-down. The physiologic regulation of STAT1 via SOCS1 is completely lost in MPM and it does not seem that the miRNAs identified by now, do inhibit the expression of SOCS1. MPM cell lines compensate STAT1 knock-down by increasing the expression of STAT3 or STAT5a, two genes which are generally considered to be oncogenes. And much more important, STAT1 knock-down induces apoptosis in MPM cell lines and STAT1 might therefore be a target for therapeutic intervention.
Assuntos
Apoptose/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , Neoplasias Pleurais/genética , Fator de Transcrição STAT1/genética , Transdução de Sinais/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Mesotelioma Maligno , MicroRNAs/genética , Prognóstico , RNA Mensageiro/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Regulação para Cima/genéticaRESUMO
Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is increasing in Europe and the prognosis remains poor. We compared epithelioid MPM in short and long survivors, and identified signal transducer and activator of transcription 1 (STAT1) as probably being responsible for antiapoptotic signaling and chemoresistance. Six mesothelioma cell lines were evaluated by Western Blot. We also analyzed 16 epithelioid MPM tissue samples for the phosphorylation status of STAT1 and the expression of its negative regulator, the suppressor of cytokine signaling 1 (SOCS1). Formalin-fixed and paraffin-embedded tissue specimens were evaluated by protein-lysate microarray and immunohistochemistry. We found STAT1 to be highly expressed and STAT3 downregulated in MPM cell lines. The expression of STAT1 phosphorylated on tyrosine 701 (Y701) was increased by interferon-gamma (IFN-γ) treatment, whereas SOCS1 was not expressed. The expression of STAT1 phosphorylated on serine 727 (S727) was not detected in mesothelioma cell lines and was not stimulated by IFN-γ. STAT1 was phosphorylated on tyrosine 701 and serine 727 in MPM tissue samples. The expression of pSTAT1-Y701 was increased compared to pSTAT1-S727. SOCS1 was again not detectable. STAT1 is upregulated in MPM, and its action may be prolonged by a loss of the negative regulator SOCS1. STAT1 might, therefore, be a target for therapeutic intervention, with the intention to restore apoptotic mechanisms and sensitivity to chemotherapy. However, other regulatory mechanisms need to be investigated to clarify if lack of expression of SOCS1 is the only reason for sustained STAT1 expression in MPM.
Assuntos
Neoplasias Pulmonares/fisiopatologia , Mesotelioma/fisiopatologia , Oncogenes , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Interferon gama/uso terapêutico , Neoplasias Pulmonares/etiologia , Mesotelioma/etiologia , Mesotelioma Maligno , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/deficiência , Regulação para CimaRESUMO
Human malignant pleural mesothelioma (MPM) is an aggressive cancer due to former asbestos exposure with little knowledge about prognostic factors of outcome and resistance to conventional therapy. BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene that is frequently lost in MPM. Germline mutations of BAP1 predispose to several different tumors including malignant mesothelioma. Our study aimed to clarify if asbestos exposure has an influence on BAP1 expression and if BAP1 expression could be used as a prognostic factor of outcome. An immunohistochemical staining for BAP1 was performed on 123 MPM tissue samples and the expression levels have been correlated with asbestos exposure and overall survival time. BAP1 expression was not associated with asbestos exposure but we detected a significant effect of BAP1 expression on overall survival time--the higher the BAP1 expression (non-mutated BAP1), the shorter the overall survival. BAP1 mutation has been linked to non-asbestos induced familial mesotheliomas, which usually belong to the long survivor group and BAP1 is most probably functioning differently than in sporadic cases. Further investigations need to be performed to characterize the BAP1 mutations and to identify the BAP1 downstream targets in MPM.
Assuntos
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesotelioma/genética , Mesotelioma/patologia , Neoplasias Pleurais/genética , Neoplasias Pleurais/patologia , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Amianto/efeitos adversos , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Mesotelioma Maligno , Mutação , PrognósticoRESUMO
FineFix, RCL-2 and HOPE, three formalin-free fixatives, were compared to the common used formalin fixed tissue samples of lung cancer and were evaluated for their effects on quality, quantity and integrity of RNA and microRNA. Two commercially available RNA extraction Kits (RNeasy FFPE by Qiagen and RecoverAll™ Nucleic Acid Isolation by Ambion) were tested and optimized in order to determine an extraction protocol for RNA as well as miRNA independent of the fixative. Two selected miRNAs were quantified via TaqMan MicroRNA assays. The optimized RNA extraction protocol for Qiagen's Kit leads to similar results for RNA quality and integrity for all fixatives. Highest RNA yield was obtained for formalin and the highest average miRNA ratio was found for FineFix. RNA fragments smaller than 500 bases were detected in FineFix, formalin and RCL2 fixed tissues; HOPE was the only fixative showing long fragments in one third of the samples. Our findings demonstrate that formalin-free fixatives are in general not superior for RNA studies. With our optimized RNA extraction protocol, there is no difficulty in extracting great amounts of RNA with high quality. According to the quality obtained, quantitative real-time PCR analysis can be performed without any negative impact. Similar results can be achieved for the tested fixatives and therefore no fixative seems to represent a new "gold-standard" for tissue fixation.
