SMO expression level correlates with overall survival in patients with malignant pleural mesothelioma
- Yi Zhang1Email author,
- Jianxing He2Email author,
- Fang Zhang3Email author,
- Hui Li4,
- Dongsheng Yue4, 5,
- Changli Wang5,
- David M Jablons4,
- Biao He4Email author and
- Natalie Lui4Email author
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 24 October 2012
Accepted: 1 February 2013
Published: 5 February 2013
Malignant mesothelioma is an aggressive, treatment-resistant tumor arising from mesothelium of pleura, peritoneum and pericardium. Despite current combined regimen, its prognosis remains dismal, calling for more effective targeted therapies. We investigated whether aberrant Hh activation may play a role in mesothelioma.
SMO and SHH expression levels were analyzed in 46 mesothelioma tissue specimens with real-time RT-PCR, and correlation with survival was analyzed with univariate and multivariate Cox proportional hazards models, Kaplan-Meier survival curves, and the log-rank test. We also examined multiple mesothelioma cell lines for SMO expression and the effect of Hh inhibition by a specific SMO antagonist on cell proliferation by MTS assay.
We observed strong correlation between higher SMO and SHH expression levels with poorer overall survival. Remarkably, Hh inhibition by a specific SMO inhibitor significantly suppressed cell proliferation in the mesothelioma cell lines examined.
Our data strongly support that Hh signaling deregulation plays critical roles in proliferation of mesothelioma, and consistently exerts significant impact on prognosis of the disease. Therefore our findings revealed the hitherto unappreciated role of Hh activation in mesothelioma, and pinpointed Hh signaling antagonist as a potential new therapy against this devastating disease.
KeywordsMesothelioma Hedgehog signaling Prognosis Proliferation suppression
Malignant mesothelioma is an aggressive, treatment-resistant tumor, arising from transformed mesothelial cells lining the pleura, peritoneum and pericardium. Athough relatively a rare disease, its incidence rate is increasing throughout the world [1, 2]. Its major risk factor is asbestos exposure, besides it can also be caused by ionizing radiation, erionite exposure, chest injuries, and presumably SV40 virus . Patients with malignant pleural mesothelioma (MPM) usually present with shortness of breath and chest pain with pleural effusions. Patients are diagnosed with cytopathology of mesothelioma effusions or fine-needle aspirations, and histopathology is often required to establish the diagnosis . Despite the current regimen of surgical resection, chemotherapy, and radiation therapy for treating MPM, the prognosis remains dismal, with median survival being 9–12 months from diagnosis . Therefore developing new molecular targeted therapies may pose promise for this devastating illness.
The pathogenic mechanisms underlying mesothelioma involve deregulation of multiple signaling pathways, including activation of multiple receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) family and MET, and subsequent deregulations of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)-AKT signaling cascades, the TNF-α / NF-κB survival pathway, Wnt signaling, and loss of tumor suppressors such as Neurofibromatosis type 2(NF2), p16INK4A, and p14ARF–. Understanding mechanisms of the dysregulated signaling pathways allows strategies for development of targeted new therapies against this devastating disease.
It has been recently reported that sonic hedgehog (Hh) signaling, another important pathway during development and tumorigenesis, is aberrantly activated in MPM, and inhibition of hedgehog signaling suppresses tumor growth . Deregulated Hedgehog (Hh) pathway activation has been implicated in several human cancers including glioma, basal cell carcinoma, medulloblastoma, lung, breast, pancreatic and gastric cancers –. The Hh family of proteins controls multiple fundamental cellular functions, including cell proliferation and survival, body patterning and organ morphogenesis during embryonic development , –. Hh signaling is orchestrated by two trans-membrane receptors, Patched (Ptch1) and Smoothened (SMO). In the absence of the Hh ligand, PTCH1 inhibits SMO, causing cleavage of GLI1 to the N-terminal repressor form. Once Hh binds to PTCH1, the inhibitory effect on SMO is released, causing active full-length GLI1 to transport into the nucleus and activate transcription of the Hh target genes in a context- and cell-type specific manner, including GLI1, PTCH1, HHIP and C-MYC –. Targeted inhibition of aberrant Hh signaling leads to suppression of cancer stem cells awakened and propelled by inappropriate Hh signaling [10, 11, 16].
We propose that the Hh signaling pathway may play an essential role during pathogenesis of MPM. To test this hypothesis, we measured SMO and SHH expression levels in MPM tissue specimens, and studied the relation of those expression levels with regard to overall survival. We also examined multiple mesothelioma cell lines for SMO expression and their cell proliferation responses to a specific SMO inhibitor. We therefore aim to better elucidate the role of Hh signaling in the tumorigenesis of MPM, and such finding may lead to development of improved molecular targeted therapies against this fatal disease.
We identified patients who underwent surgical resection for malignant pleural mesothelioma at our institution from April 2000 to January 2010 and had a tissue specimen available in our tissue bank. Clinical and histological data were obtained by review of electronic medical records and entered prospectively into our tissue bank database. Vital status was obtained through the Social Security Death Master File. Overall survival was calculated from the date of surgery. Our institutional review board approved this study.
RNA extraction and real-time RT-PCR
Total RNA was isolated from MPM tissue samples using the RNeasy kit (Qiagen). Genomic DNA contamination was eliminated by DNase I treatment. 250 ng of RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). The resulting cDNAs were analyzed with real-time RT-PCR using Gene Expression Assays in a 7900 Real-Time PCR System (Applied Biosystems) for 40 cycles (96°C for 15 seconds and 60°C for 1 minute). Gene expressions were normalized to 18S rRNA expression.
Peroxidase-based immunohistochemistry using paraffin-sections was performed per standard protocol. Smo antibody (abcam, ab72130) and Shh antibody (abcam, ab19897) were employed following the manufacturer's instructions. These slides were then mounted in Citifluor.
Cell lines and cell culture
Mesothelioma cell lines NCI-H28, REN, and H290 were cultured in RPMI 1640 (Life Technology, Carlsbad, CA) supplemented with 10% FBS and penicillin (100 IU/ml) and streptomycin (100 ug/ml), at 37°C in a humid incubator with 5% CO2. Cells were seeded one day before treatment with cyclopamine (Selleckchem) at 10 uM and 20 uM or vehicle (DMSO) for 72 hours. Cells were subjected to proliferation assays at 0, 24, 48 and 72 hours after drug treatment.
Cell proliferation assay
Cells will be treated with Cyclopamine at indicated doses in 96-well plates for 6–7 days. Cell proliferation was assayed by MTS assay (Promega) according to the manufacturer’s protocol and as described previously . The quantity of formazan product as measured by the absorbance at 490 nm is directly proportional to the number of living cells in culture. Data are representative of at least 3 independent experiments with similar results.
Whole cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with the indicated antibodies: α-human SMO mouse monoclonal antibody (Sigma), α-ß-actin mouse monoclonal antibody (Sigma) as described previously . Data represent three independent experiments with consistent results.
Survival and statistical analyses
Survival analysis was performed using univariate and multivariate Cox proportional hazards models, Kaplan-Meier survival curves, and the log-rank test. For the Cox proportional hazards models, age and sex were included in the multivariate model a priori. Race, histological type, stage, smoking status were included in the multivariate model only if the p-value was less than 0.10 in the univariate analysis. For all statistical tests, a two-sided alpha level less than 0.05 was considered statistically significant. Analyses were performed using Stata version 11.
Results and discussion
Patient baseline characteristics
All patients (N = 46)
Age, mean ± SD—yr.
67.2 ± 10.7
Smoking status—no. (%)
Histologic type—no. (%)
SMO and SHH expression analysis
Univariate and multivariate Cox proportional hazards model
Age (10 years)
SMO expression level
In the multivariate Cox proportional hazards model, SMO expression level remained associated with worse survival (Table 2). However, sex was no longer associated with overall survival (p = 0.50) and histological type was less strongly associated with overall survival (p = 0.08). After adjusting for age, sex, and histological type, the hazard ratio and significance of SMO expression level increased compared to the univariate model (p = 0.03).
SHH expression level was analyzed separately because data was only available for 26 patients. In the univariate model, SHH expression level was significantly associated with overall survival. Increase in SHH expression level strongly correlates with elevated risk of death (95% CI, 1-28%; p = 0.04; data not shown). When SHH expression level was dichotomized at the median, log-rank test was not significant (p = 0.45), although the Kaplan-Meier survival curve showed separation after 12 months (Figure 3B).
After including SHH expression level in the multivariate model above, SHH expression level remained significant and even increased the significance of SMO expression level. After adjusting for age, sex, and histological type, increase in SMO expression level strongly correlates with increase in risk of death (95% CI, 8-72%; p = 0.009; data not shown); and so does increase in SHH expression level (95% CI, 1-26%; p = 0.04; data not shown). Histological type was no longer associated with overall survival (p = 0.87).
SMO Inhibition suppresses mesothelioma cell proliferation
Role of Hh activation in mesothelioma
As deregulated Hh signaling pathway has been implicated in many different types of cancer, and inhibition of Hh signaling leads to suppression of tumor growth [10, 11], we addressed whether Hh signaling plays critical roles in proliferation of mesothelioma cells. Remarkably, we observed elevated endogenous SMO expression in 3 mesothelioma cell lines tested (Figure 5A). Furthermore, utilizing a specific Hh inhibitor cycloplamine, which significantly suppressed expression of Gli downstream targets (Figure 4), we observed significant inhibition of cell proliferation in all 3 mesothelioma cell lines examined (Figure 5B-D). These data indicate that aberrant Hh activation plays critical roles in tumor cell proliferation in mesothelioma, consistent with recent data by Shi Y et al. .
Taken together, our results demonstrated a strong association between higher SMO and SHH expression levels with poorer overall survival. Furthermore, we showed inhibition of Hh signaling blocked cell proliferation in multiple mesothelioma cell lines, strongly supporting that aberrant Hh signaling is essential for tumor growth in mesothelioma. Therefore our findings revealed the hitherto unappreciated roles of Hh activation in MPM, and pinpointed Hh signaling antagonist as a potential new therapy against this devastating disease.
This work was supported by NIH/NCI grants R01CA125030 and R01CA132566, the Eileen D. Ludwig Endowed for Thoracic Oncology Research, the Kazan, McClain, Abrams, Fernandez, Lyons, Greenwood, Harley & Oberman Foundation, Paul and Michelle Zygielbaum, and the Jeffrey and Karen Peterson Family Foundation, and by a Zhejiang Provincial Natural Science Foundation grant to F. Zhang (Y2110030).
- Bianchi C, Bianchi T: Malignant mesothelioma: global incidence and relationship with asbestos. Ind Health 2007,45(3):379–387.PubMedView Article
- Robinson BW, Musk AW, Lake RA: Malignant mesothelioma. Lancet 2005,366(9483):397–408. ReviewPubMedView Article
- Mott FE: Mesothelioma: a review. Ochsner J. 2012,12(1):70–79.PubMed
- Rusch VW: A proposed new international TNM staging system for malignant pleural mesothelioma. Chest 1995,108(4):1122–1128.PubMedView Article
- Heintz NH, Janssen-Heininger YM, Mossman BT: Asbestos, lung cancers, and mesotheliomas: from molecular approaches to targeting tumor survival pathways. Am J Respir Cell Mol Biol 2010,42(2):133–139. ReviewPubMedView Article
- Sekido Y: Genomic abnormalities and signal transduction dysregulation in malignant mesothelioma cells. Cancer Sci 2010,101(1):1–6.PubMedView Article
- Uematsu K, Seki N, Seto T, Isoe C, Tsukamoto H, Mikami I, You L, He B, Xu Z, Jablons DM, Eguchi K: Targeting the Wnt signaling pathway with dishevelled and cisplatin synergistically suppresses mesothelioma cell growth. Anticancer Res 2007,27(6B):4239–4242.PubMed
- Shi Y, Moura U, Opitz I, Soltermann A, Rehrauer H, Thies S, Weder W, Stahel RA, Felley-Bosco E: Role of hedgehog signaling in malignant pleural mesothelioma. Clin Cancer Res 2012,18(17):4646–4656.PubMedView Article
- Varjosalo M, Taipale J: Hedgehog: functions and mechanisms. Genes Dev 2008, 22:2454–2472.PubMedView Article
- Yang L, Xie G, Fan Q, Xie J: Activation of the hedgehog-signaling pathway in human cancer and the clinical implications. Oncogene 2010, 29:469–481.PubMedView Article
- Scales SJ, de Sauvage FJ: Mechanisms of Hedgehog pathway activation in cancer and implications for therapy. Trends Pharmacol Sci 2009, 30:303–312.PubMedView Article
- Ma X, Chen K, Huang S: Frequent activation of the hedgehog pathway in advanced gastric adenocarcinomas. Carcinogenesis 2005, 26:1698–1705.PubMedView Article
- Hooper JE, Scott MP: Communicating with Hedgehogs. Nat Rev Mol Cell Biol 2005, 6:306–317.PubMedView Article
- Huangfu D, Anderson KV: Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates. Development 2006, 133:3–14.PubMedView Article
- Osterlund T, Kogerman P: Hedgehog signalling: how to go from Smo to Ci and Gli. Trends Cell Biol 2006, 16:176–180.PubMedView Article
- Ruiz i Altaba A, Sanchez P, Dahmane N: Gli and hedgehog in cancer: tumors, embryos and stem cells. Nat Rev Cancer 2002, 2:361–372.PubMedView Article
- Bi X, Han X, Zhang F, He M, Zhang Y, Zhi XY, Zhao H: Triparanol suppresses human tumor growth in vitro and in vivo. Biochem Biophys Res Commun 2012,425(3):613–618.PubMedView Article
- Zhang F, Phiel CJ, Spece L, Gurvich N, Klein PS: Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3. J Biol Chem 2003,278(35):33067–33077.PubMedView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.