ABSTRACT
Malignant pleural mesothelioma (MPM) is a relatively chemoresistant malignancy. Diverse biological targets are under investigation to develop new therapeutic options. One of these targets, namely the phosphoinositide-3-kinase (PI3K) pathway, has been shown to be a regulator of the side population (SP) phenotype in different cancers. The SP phenotype is due to drug efflux abilities providing drug-resistant properties. The presence of a SP fraction in MPM was recently observed in our laboratory. The aim of this study was to investigate the role of the PI3K pathway in the regulation of the SP phenotype in MPM. Treatment of overnight serum-starved cells with IGF increased phosphorylation of downstream target AKT, S6 and 4EBP1 and SP fraction in ZL55, ZL34 and SDM103T2 MPM cell lines. The PI3K/mTOR inhibitor NVP-BEZ235 and PI3K inhibitor wortmannin reduced the phosphorylation of downstream target AKT, S6 and 4EBP1 and decreased the SP fraction. Chemotherapy resistance mediated by drug efflux was tested by treating the cells with mitoxantrone. NVP-BEZ235 increased mitoxantrone cytotoxicity and this effect was mimicked by fumitremorgin C, a specific ABCG2 inhibitor, although not to the same extent, indicating that ABCG2-mediated drug efflux participates to chemoresistance. The involvement of ABCG2 in drug efflux was confirmed by determination of ABCG2-mediated decrease of intracellular mitoxantrone accumulation and silencing experiments. NVP-BEZ235-mediated decrease in drug efflux was associated with a significant decrease of ABCG2 present at the cell surface in ZL55 and SDM103T2 cells. In conclusion, the PI3K pathway is playing an important role in regulating the SP phenotype in MPM cells and inhibition of this activity may contribute to a more efficient cancer treatment.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Mesothelioma/metabolism , Neoplasm Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Pleural Neoplasms/metabolism , Signal Transduction/drug effects , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Androstadienes/pharmacology , Androstadienes/therapeutic use , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Membrane/metabolism , Drug Resistance, Neoplasm , Enzyme Inhibitors/therapeutic use , Glutamates/pharmacology , Guanine/analogs & derivatives , Guanine/pharmacology , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Mesothelioma/drug therapy , Mitoxantrone/pharmacology , Pemetrexed , Phenotype , Phosphoinositide-3 Kinase Inhibitors , Pleural Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Quinolines/pharmacology , Quinolines/therapeutic use , Side-Population Cells/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , WortmanninABSTRACT
PURPOSE: The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM). EXPERIMENTAL DESIGN: The expression of hedgehog signaling components was assessed by quantitative PCR and in situ hybridization in 45 clinical samples. Primary MPM cultures were developed in serum-free condition in 3% oxygen and were used to investigate the effects of smoothened (SMO) inhibitors or GLI1 silencing on cell growth and hedgehog signaling. In vivo effects of SMO antagonists were determined in an MPM xenograft growing in nude mice. RESULTS: A significant increase in GLI1, sonic hedgehog, and human hedgehog interacting protein gene expression was observed in MPM tumors compared with nontumoral pleural tissue. SMO antagonists inhibited GLI1 expression and cell growth in sensitive primary cultures. This effect was mimicked by GLI1 silencing. Reduced survivin and YAP protein levels were also observed. Survivin protein levels were rescued by overexpression of GLI1 or constitutively active YAP1. Treatment of tumor-bearing mice with the SMO inhibitor HhAntag led to a significant inhibition of tumor growth in vivo accompanied by decreased Ki-67 and nuclear YAP immunostaining and a significant difference in selected gene expression profile in tumors. CONCLUSIONS: An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach.
Subject(s)
Anilides/administration & dosage , Lung Neoplasms , Mesothelioma , Pleural Effusion, Malignant , Pyridines/administration & dosage , Receptors, G-Protein-Coupled , Transcription Factors , Adaptor Proteins, Signal Transducing/metabolism , Adult , Aged , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitor of Apoptosis Proteins/blood , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mesothelioma/drug therapy , Mesothelioma/metabolism , Mesothelioma/pathology , Mice , Middle Aged , NIH 3T3 Cells , Phosphoproteins/metabolism , Pleural Effusion, Malignant/drug therapy , Pleural Effusion, Malignant/metabolism , Pleural Effusion, Malignant/pathology , RNA, Small Interfering , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Smoothened Receptor , Survivin , Tomatine/administration & dosage , Tomatine/analogs & derivatives , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Transplantation, Heterologous , Veratrum Alkaloids/administration & dosage , YAP-Signaling Proteins , Zinc Finger Protein GLI1ABSTRACT
DyeCycleViolet was used to set up the side population (SP) functional assay aimed at identifying subpopulations of malignant pleural mesothelioma (MPM) tumor cells with chemoresistance phenotype associated with ABCG2 transporter activity. Self-renewal, chemoresistance and tumorigenicity were tested for SP and non-side population (NSP) cells. Tumors were characterized by mesothelin, calretinin, N-cadherin, D2-40 and Wilms tumor 1 (WT1) immunohistochemistry. Surface expression of mesenchymal stem cell markers CD90, CD73 and CD105 was investigated in SP and NSP cells. We identified SP cells with self-renewal properties and increased chemoresistance in MPM cell lines and tumor-derived primary cell cultures. Compared with the non-SP fraction (NSP), the SP fraction led to the development of tumors including cells with mesothelium precursor phenotype characterized by mesenchymal morphology, being WT1 negative but cytoplasmic D2-40 positive and having a tendency of increased tumorigenicity. The same phenotypic shift was observed in patients with relapsing tumors after chemotherapy. Furthermore, the SP cells were enriched in CD105(-)(/low) expressing cells, which were small sized and had increased tumorigenicity compared with CD105(high) cells. Taken together, our results support the hypothesis that MPM CD105(-)(/low), chemoresistant small sized SP cells may constitute the cellular pool out of which recurrence develops. Further characterization of mechanisms of chemoresistance and self-renewal should lead to targets specific for this subpopulation in MPM patients.
