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

Abstract

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. Immunohistochemical analysis showed that osteosarcoma is expressed in ∼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) Osteosarcoma is the most common primary malignant tumor of bone characterized by production of osteoid and a high propensity to metastasize (1). It most frequently occurs in the second decade of life with ∼60% of patients ages <25 years, whereas 30% of osteosarcoma occurs in patients ages >40 years.At present, the standard treatment for high-grade osteosarcoma includes neoadjuvant chemotherapy followed by surgical resection and postoperative chemotherapy. Several clinical factors have been related to survival, including the presence of metastatic disease and the histologic response to preoperative

Cortactin (CTTN) overexpression in osteosarcoma correlates with advanced stage and reduced survival

“…All samples were obtained with written informed consent from patients and/or their parents, and the protocol was approved by the local ethics committee (CEIC-CUN). Human osteoblasts were isolated and characterized according to procedures we have published previously [4,8]. Normal osteoblastic cells were passaged 1 to 5 times, whereas tumoral cells were passaged between 20 and 40 times; in all cases, cells were 90% confluent and in logarithmic growth phase prior to molecular analysis.…”

“…RNA was isolated, labelled and hybridized on HGU133A chips (Affymetrix) by Progenika Biopharma as previously described [4]. The test set comprised 14 samples: osteosarcoma cell lines and the paired osteoblastic control cell lines derived from 7 patients (numbers 473, 475, 486, 491, 500, 524 and 531 in Table 1).…”

“…We have previously reported the transcriptomic profiling [4] of pediatric osteosarcoma based on a paired analysis of tumoral and normal cell lines derived from high-grade conventional osteosarcomas. This approach enabled assessment of the changes associated with oncogenesis and determination of molecular alterations influencing differential clinical behaviour and/or response to treatment.…”

Identification of polymorphisms in ultraconserved elements associated with clinical outcomes in locally advanced colorectal adenocarcinoma

“…Several studies have shown that EBF inactivation due to genomic deletion, epigenetic silencing, or somatic point mutations exists in several types of cancer, including leukemia, glioblastoma, and pancreatic cancer, supporting the emerging roles of the EBF family in tumor suppression 303132 . Another study showed that silencing EBF2 led to a reduced resistance to apoptosis in chemo-naive tumor-derived cell populations from patients diagnosed with sporadic osteosarcoma 33 . However, EBF2 has not been investigated within the context of a fluoropyrimidine regimen.…”

EPCR promotes breast cancer progression by altering SPOCK1/testican 1-mediated 3D growth

“…Low expression probes were removed by filtering those that did not exceed a level of expression of 32 in at least one of the samples for each condition. Differentially expressed genes were identified using LIMMA (linear models for microarray data) method [27]. …”