Endothelin-1 Single Nucleotide Polymorphisms and Risk of Pulmonary Metastatic Osteosarcoma

Zhang C (2013) Endothelin-1 Single Nucleotide Polymorphisms and Risk of Pulmonary Metastatic Osteosarcoma. PLoS ONE 8(9): e73349. doi:10.1371/journal.pone.0073349 Endothelin-1 Single Nucleotide Polymorphisms and Risk of Pulmonary Metastatic Osteosarcoma Xiaofang Zang 0 Yong Zhou 0 Zufa Huang 0 Chaoyue Zhang 0 Francesc Calafell, Universitat Pompeu Fabra, Spain 0 Department of Orthopaedics, the Third Xiangya Hospital, Central South University , Changsha, Hunan , China Pulmonary metastases are the major cause of death of osteosarcoma (OS) patients. Endothelin-1 (ET-1) reportedly plays an important role in OS metastasis. In the present study, we for the first time explored the association of ET-1 SNPs with the risk of pulmonary metastatic OS. We genotyped three SNPs (rs1800541, rs2070699 and rs5370) in the ET-1 gene in a case-control study, using 260 pairs of age-, sex-, residence area- and tumor location-matched subjects. Patients with pulmonary metastatic OS and patients with localized high-grade (stage IIB) OS were enrolled as cases and controls, respectively. The G allele at rs1800541 was found associated with reduced risk of pulmonary metastatic OS after adjustment for body mass index, systolic blood pressure, diastolic blood pressure and the plasma ET-1 level (P=10-4; adjusted OR, 0.55; 95% CI, 0.42-0.70), while the G allele at rs2070699 was not significantly associated with the risk of pulmonary metastatic OS (P=0.15; adjusted OR, 1.15; 95% CI, 0.87-1.50). The mRNA and the secreted protein levels of ET-1 in primary OS cell cultures (POCCs) established from surgically resected primary OS in the rs1800541 TT homozygotes were higher than those from the TG heterozygotes (P<0.05), who in turn showed higher ET-1 mRNA and secreted ET-1 levels than the GG homozygotes (P<0.05). In the control subjects, the rs1800541 TT homozygotes showed an 18.4% relapse rate, significantly higher than that of the GG homozygotes (0%) (P<0.01). On the other hand, the GG homozygotes showed a 71.4% complete recovery rate, significantly higher than that of the TG heterozygotes (7.3%) and the TT homozygotes (0%) (P<0.01). This study provides the first evidence of an association between the ET-1 gene SNPs and the risk of pulmonary metastatic OS. - Funding: This work was supported by Hunan Provincial Natural Science Foundation (grants #07B2697 and #10C6833), Hunan, P.R. China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Osteosarcoma (OS) is the most frequent malignant bone tumor in children and adolescents [1]. OS is a devastating disease, characterized by high local aggressiveness and a tendency to metastasize to the lungs and distant bones. In improvement in surgical technology that have increased the survival rate to 65-75%, pulmonary metastasis occurs in approximately 40%-50% of OS patients and remains a major cause of fatal outcome [2-4]. The cure rate of OS is approximately 65% for patients with localized diseases. When presenting with metastases at the time of diagnosis, the survival rate is 25% [5,6]. Thus, it is important to uncover the particularly, pulmonary metastasis. Although there have been many studies on its genetics, biology, pathology and clinical aspects, the etiology of osteosarcoma is not well understood. Previous studies suggest a genetic predisposition of osteosarcoma [7]. Endothelin-1 (ET-1) is a potent vasoconstrictor initially isolated from endothelial cells [8]. ET-1 signaling reportedly is involved in a wide range of cancer-relevant processes, such as inhibition of apoptosis, matrix remodeling, bone deposition, and metastases [8]. ET-1 and ET A receptor (ETAR) are expressed in OS tissue and cells [8,9]. Previous studies suggest that ET-1 is important for OS progression and metastasis [8-10]. Zhao et al. reported that ET-1 could promote OS cell invasion and survival [8]. Felx et al. reported that ET-1 could promote metalloproteinase induction in human OS [9]. Li et al. showed that ETAR, the major target for ET-1, was critical for OS pulmonary metastasis in an orthotopic xenograft OS model [10]. Single nucleotide polymorphisms (SNPs) of the ET-1 gene have been reportedly associated with pulmonary and cardiovascular diseases [11-14]. Despite the important role of ET-1 signaling in OS progression, no study has investigated the association of ET-1 gene polymorphisms with OS. In the Table 1. Characteristics of study subjects. Cases (n=260) Controls (n=260) P Age (years) 16.5 8.3 16.8 7.9 0.67 Age Range (years) 4-37 5-34 N/A Age Group n(%) 20 years 190 (73.1) 190 (73.1) >20 years 70 (26.9) 70 (26.9) 1.00 Gender n(%) Male 155 (59.6) 155 (59.6) female 105 (40.4) 105 (40.4) 1.00 Body Mass Index (kg/m2) 17.8 3.2 18.3 3.5 0.09 Tumor Location Long Tubular bones 196 (75.4) 196 (75.4) Axial skeleton 64 (24.6) 64 (24.6) 1.00 Systolic Blood Pressure (mmHg) 112.5 14.3 110.7 16.5 0.18 Diastolic Blood Pressure (mmHg) 76.7 5.2 76.1 5.9 0.21 Plasma ET-1 Level (pg/mL) 14.3 1.9 14.1 1.5 0.19 Note: For continuous variables, all values were expressed as MeanSD. Independent student t tests were performed to compare means between the groups. For categorical variables, all values were expressed as n(%) and comparisons were performed with Chi-square tests. present study, we for the first time explored the association of ET-1 SNPs with the risk of pulmonary metastatic OS in a casecontrol study, using 260 pairs of age-, sex-, residence areaand tumor location-matched subjects. Materials and Methods Ethics Statement This study was approved by the Ethics Committee of the Third Xiangya Hospital, Central South University. Written informed consent was obtained from adult participants or the parent or guardian of minor participants before the start of the study. Subjects From January 2007 to July 2012, blood samples were collected from 260 Han Chinese patients with pulmonary metastatic (stage III) OS at the Third Xiangya Hospital of Central South University. 260 age-, sex-, residence area- and tumor location-matched Han Chinese patients diagnosed with stage IIB OS (localized high-grade OS with extracompartmental lesions) were recruited as controls [15]. All diagnoses were based on biopsy. The inclusion criteria were as follows: (1) metastatic pulmonary OS (for cases) or stage IIB OS (for controls) at diagnosis; (2) had not received any treatment; (3) without a family history of osteosarcoma or any other cancers. Patients with any other malignancies were excluded. Baseline characteristics of all subjects are summarized in Table 1. After blood sample collection, all subjects received neoadjuvant chemotherapy followed by surgical resection of the primary tumor. SNP Selection and Genotyping Three SNPs in the ET-1 gene, including rs1800541 in the promoter region, rs2070699 in intron, and rs5370 in the coding region were sel (...truncated)