gdc-0449 and Mesothelioma

An autocrine-driven upregulation of the Hedgehog (Hh) signaling pathway has been described in malignant pleural mesothelioma (MPM), in which the ligand, desert Hh (DHH), was produced from tumor cells. However, our investigation revealed that the Hh pathway is activated in both tumor and stroma of MPM tumor specimens and an orthotopic immunocompetent rat MPM model. This was demonstrated by positive immunohistochemical staining of Glioma-associated oncogene 1 (GLI1) and Patched1 (PTCH1) in both tumor and stromal fractions. DHH was predominantly expressed in the tumor fractions. To further investigate the role of the Hh pathway in MPM stroma, we antagonized Hh signaling in the rat model of MPM using a Hh antagonist, vismodegib, (100 mg/kg orally). Daily treatment with vismodegib efficiently downregulated Hh target genes Gli1, Hedgehog Interacting Protein (Hhip), and Ptch1, and caused a significant reduction of tumor volume and tumor growth delay. Immunohistochemical analyses revealed that vismodegib treatment primarily downregulated GLI1 and HHIP in the stromal compartment along with a reduced expression of previously described fibroblast Hh-responsive genes such as Fibronectin (Fn1) and Vegfa Primary cells isolated from the rat model cultured in 3% O2 continued to express Dhh but did not respond to vismodegib in vitro However, culture supernatant from these cells stimulated Gli1, Ptch1, and Fn1 expression in mouse embryonic fibroblasts, which was suppressed by vismodegib. Our study provides new evidence regarding the role of Hh signaling in MPM stroma in the maintenance of tumor growth, emphasizing Hh signaling as a treatment target for MPM. Mol Cancer Ther; 15(5); 1095-105. ©2016 AACR.

Topics: Anilides; Animals; Biomarkers; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Lung Neoplasms; Magnetic Resonance Imaging; Mesothelioma; Mesothelioma, Malignant; Mice; NIH 3T3 Cells; Pleural Neoplasms; Pyridines; Rats; Signal Transduction; Stromal Cells; Tumor Burden; Xenograft Model Antitumor Assays

The present study sought to determine whether the Hedgehog (Hh) pathway is active and regulates the cell growth of cultured malignant pleural mesothelioma (MPM) cells and to evaluate the efficacy of pathway blockade using smoothened (SMO) antagonists (SMO inhibitor GDC-0449 or the antifungal drug itraconazole [ITRA]) or Gli inhibitors (GANT61 or the antileukemia drug arsenic trioxide [ATO]) in suppressing MPM viability.. Selective knockdown of SMO to inhibit Hh signaling was achieved by small interfering RNA in 3 representative MPM cells. The growth inhibitory effect of GDC-0449, ITRA, GANT61, and ATO was evaluated in 8 MPM lines, with cell viability quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was determined by annexinV/propidium iodide staining and flow cytometry.. SMO small interfering RNA mediated a two- to more than fivefold reduction of SMO and Gli1 gene expression as determined by real-time quantitative reverse-transcriptase polymerase chain reaction, indicating significant Hh pathway blockade. This was associated with significantly reduced cell viability (34% ± 7% to 61% ± 14% of nontarget small interfering RNA controls; P = .0024 to P = .043). Treating MPM cells with Hh inhibitors resulted in a 1.5- to 4-fold reduction of Gli1 expression. These 4 Hh antagonists strongly suppressed MPM cell viability. More importantly, ITRA, ATO, GANT61 induced significant apoptosis in the representative MPM cells.. Hh signaling is active in MPM and regulates cell viability. ATO and ITRA were as effective as the prototypic SMO inhibitor GDC-0449 and the Gli inhibitor GANT61 in suppressing Hh signaling in MPM cells. Pharmaceutical agents Food and Drug Administration-approved for other indications but recently found to have anti-Hh activity, such as ATO or ITRA, could be repurposed to treat MPM.

Topics: Anilides; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Itraconazole; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Molecular Targeted Therapy; Oxides; Pleural Neoplasms; Pyridines; Pyrimidines; Receptors, G-Protein-Coupled; RNA Interference; Signal Transduction; Smoothened Receptor; Time Factors; Transcription Factors; Transfection; Zinc Finger Protein GLI1

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis. Current treatment is rarely curative, thus novel meaningful therapies are urgently needed. Inhibition of Hedgehog (Hh) signaling at the cell membrane level in several cancers has shown anti-cancer activity in recent clinical studies. Evidence of Hh-independent Gli activation suggests Gli as a more potent therapeutic target. The current study is aimed to evaluate the potential of Gli as a therapeutic target to treat MPM. The expression profiles of Gli factors and other Hh signaling components were characterized in 46 MPM patient tissue samples by RT-PCR and immunohistochemistry. Cultured cell lines were employed to investigate the requirement of Gli activation in tumor cell growth by inhibiting Gli through siRNA or a novel small molecule Gli inhibitor (Gli-I). A xenograft model was used to evaluate Gli-I in vivo. In addition, a side by side comparison between Gli and Smoothened (Smo) inhibition was conducted in vitro using siRNA and small molecule inhibitors. Our study reported aberrant Gli1 and Gli2 activation in a large majority of tissues. Inhibition of Gli by siRNAs or Gli-I suppressed cell growth dramatically both in vitro and in vivo. Inhibition of Gli exhibited better cytotoxicity than that of Smo by siRNA and small molecule inhibitors vismodegib and cyclopamine. Combination of Gli-I and pemetrexed, as well as Gli-I and vismodegib demonstrated synergistic effects in suppression of MPM proliferation in vitro. In summary, Gli activation plays a critical role in MPM. Inhibition of Gli function holds strong potential to become a novel, clinically effective approach to treat MPM.

Topics: Aged; Anilides; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Glutamates; Guanine; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Male; Mesothelioma; Mice; Mice, Nude; Molecular Targeted Therapy; Nuclear Proteins; Pemetrexed; Pleural Neoplasms; Pyridines; Receptors, G-Protein-Coupled; RNA, Small Interfering; Signal Transduction; Smoothened Receptor; Transcription Factors; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2

The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM).. 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.. 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.. An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Anilides; Animals; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Male; Mesothelioma; Mice; Middle Aged; NIH 3T3 Cells; Phosphoproteins; Pleural Effusion, Malignant; Pyridines; Receptors, G-Protein-Coupled; RNA, Small Interfering; Signal Transduction; Smoothened Receptor; Survivin; Tomatine; Transcription Factors; Transplantation, Heterologous; Veratrum Alkaloids; YAP-Signaling Proteins; Zinc Finger Protein GLI1