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.

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.

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<p>PDF file, 96K, 1A. Expression of HH pathway components in non-tumoral pleural tissue and mesothelioma tumors. Quantitative real-time PCR analysis of SMO, GLI2, IHH, DHH gene expression in non-tumoral pleural tissue (NT) and tumor (T). 1B. Overall survival is inversely correlated with high GLI1 expression (delta Ct below the median) (p=0.042, low GLI1: median survival 22.9 months, 95% confidence interval 0.4- 45.4 months; high GLI1: median survival 17.0 months, 95% confidence interval 9.0- 25.1 months ).</p>

<p>PDF file, 50K, GLI1 transfection (GLI1) leads to increased expression of GLI1 target HHIP compared to control vector (V) transfected ZL55SPT cells.</p>

<p>PDF file, 60K, MPM primary cultures maintained without serum in 3% oxygen show a three-fold increase in expression of ABCG2, GLI1 and PATCH1 compared to cultures without serum in 20% oxygen.</p>

<p>PDF file, 66K, Western blot analysis of NF2 protein in SDM103T, ZL55SPT cells. SDM71 cells were used as positive control (1). 1. Thurneysen C, Opitz I, Kurtz S, Weder W, Stahel RA, Felley-Bosco E. Functional inactivation of NF2/merlin in human mesothelioma. Lung Cancer. 2009;64:140-7.</p>

<p>PDF file, 36K, Table S1: Primers used for RT-PCR.</p>