Profiling of Chemonaive Osteosarcoma and Paired-Normal Cells Identifies EBF2 as a Mediator of Osteoprotegerin Inhibition to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis
Profiling of Chemonaive Osteosarcoma and Paired-Normal Cells Identifies EBF2 as a Mediator of Osteoprotegerin Inhibition to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis
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DOI：
10.1158/1078-0432.ccr-09-0300
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发表时间：
2009-08-15
影响因子：
11.5
通讯作者：
Lecanda, Fernando
中科院分区：
文献类型：
--
作者：
Patino-Garcia, Ana;Zalacain, Marta;Lecanda, Fernando
目的：骨肉瘤是儿童和青少年中最常见的骨肿瘤。目前，对其发生、维持和转移的机制了解甚少。本研究的目的是确定骨肉瘤发病机制中的相关分子靶点。实验设计：从7例骨肉瘤患者中分离出肿瘤化成骨细胞群和配对对照正常成骨细胞，并对其进行表型表征。通过稳健的微阵列分析分析全球转录组分析。候选基因通过实时荧光定量PCR确认，并按分子途径组织。实时荧光定量PCR检测EBF2和骨保护素（OPG）水平，ELISA检测OPG蛋白水平。对46例骨肉瘤样本进行免疫组化分析。EBF2的沉默是通过慢病毒转导短发夹RNA实现的。caspase-3/7活性测定细胞凋亡。结果：在骨肉瘤中获得了一个强大的聚集转录组特征。转录因子EBF2是一种已知的功能性骨调节因子，是最显著的过度表达基因之一。免疫组织化学分析显示骨肉瘤在70%的肿瘤中表达相似。由于EBF2先前被证明是OPG的转录激活剂，因此与正常成骨细胞相比，骨肉瘤样本中EBF2水平升高与OPG蛋白水平升高相关。EBF2的敲低导致OPG水平的抑制和对肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导的细胞凋亡的敏感性增加。结论：这些发现表明EBF2是一种新的骨肉瘤标志物。EBF2上调可能是骨肉瘤中OPG水平升高的机制之一，有助于减少TRAIL诱导的细胞凋亡，导致TRAIL耐药。（临床癌症杂志2009；15(16):5082-91）
Purpose: Osteosarcoma is the most prevalent bone tumor in children and adolescents. At present, the mechanisms of initiation, maintenance, and metastasis are poorly understood. The purpose of this study was to identify relevant molecular targets in the pathogenesis of osteosarcoma.Experimental Design: Tumor chemonaive osteoblastic populations and paired control normal osteoblasts were isolated and characterized phenotypically from seven osteosarcoma patients. Global transcriptomic profiling was analyzed by robust microarray analysis. Candidate genes were confirmed by real-time PCR and organized in molecular pathways. EBF2 and osteoprotegerin (OPG) levels were determined by real-time PCR and OPG protein levels were assessed by ELISA. Immunohistochemical analysis was done in a panel of 46 osteosarcoma samples. Silencing of EBF2 was achieved by lentiviral transduction of short hairpin RNA. Apoptosis was determined by caspase-3/7 activity.Results: A robust clustered transcriptomic signature was obtained in osteosarcoma. Transcription factor EBF2, a known functional bone regulator, was among the most significantly overexpressed genes. Immunohistochernical analysis showed that osteosarcoma is expressed in similar to 70% of tumors analyzed. Because EBF2 was shown previously to act as a transcriptional activator of OPG, elevated levels of EBF2 were associated with high OPG protein levels in osteosarcoma samples compared with normal osteoblastic cells. Knockdown of EBF2 led to stunted abrogation of OPG levels and increased sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.Conclusions: These findings suggest that EBF2 represents a novel marker of osteosarcoma. EBF2 up-regulation may be one of the mechanisms involved in the high levels of OPG in osteosarcoma, contributing to decrease TRAIL-induced apoptosis and leading to TRAIL resistance. (Clin Cancer Res 2009;15(16):5082-91)