Over‐expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma

Abstract

Osteosarcoma is the most common primary bone malignancy in children and is associated with rapid bone growth. Parathyroid hormone-related peptide (PTHrP) signaling via parathyroid hormone Type 1 receptor (PTHR1) is important for skeletal development and is involved in bone metastases in other tumors. The aim of this study was to investigate the status of PTHrP/PTHR1 and its possible role in osteosarcoma. In a preliminary screening, a higher level of PTHR1 mRNA, but not PTHrP, was found in 4 osteosarcoma xenografts as compared with 4 standard cell lines, or 5 patient derived cell lines (p < 0.05) using quantitative RT-PCR. It was therefore extended to 55 patient specimens, in which a significantly higher level of PTHR1 mRNA was detected in metastatic or relapsed samples than those from primary sites (p < 0.01). Cell behavior caused by PTHR1 overexpression was further studied in vitro using PTHR1 transfected HOS cell line as a model. Over-expression of PHTR1 resulted in increased proliferation, motility and Matrigel invasion without addition of exogenous PTHrP suggesting an autocrine effect. Importantly, the aggressiveness in PTHR1-expressing cells was completely reversed by RNAi mediated gene knockdown. In addition, PTHR1 overexpression led to delayed osteoblastic differentiation and upregulation of genes involved in extracellular matrix production, such as TGF-b1 and connective tissue growth factor. When cocultured with bone marrow derived monocytes, PTHR1 transfected HOS cells induced a greater number of osteoclasts. This study suggests that PTHR1 over-expression may promote osteosarcoma progression by conferring a more aggressive phenotype, and forming a more favorable microenvironment. ' 2007 Wiley-Liss, Inc.Key words: parathyroid hormone Type 1 receptor; metastasis; osteosarcoma Osteosarcoma is typically a high-grade malignancy that has a peak incidence in children and young adults. More than 75% of osteosarcoma in adolescents occurs near growing joints, such as the distal femur, proximal tibia and proximal humerus, implying an association with rapid bone growth.1 Osteosarcoma has a tendency to metastasize early. The 5-year event free survival of osteosarcoma has been improved dramatically from 10% to nearly 70% for patients without detectable metastases at initial diagnosis with the adoption of neo-adjuvant and adjuvant chemotherapy, which is aimed at eliminating microscopic metastatic disease and making primary tumor more amenable for limb salvage procedures. 2,3However, for patients with radiographically detectable metastases or relapse, cure is extremely difficult to achieve even using the regimens of intensified chemotherapy that have being explored during the past 2 decades.4,5 Therefore, identification of factors contributing to tumor progression and metastatic behavior will be essential to improve the outcome for patients with this disease.Parathyroid hormone Type 1 receptor (PTHR1) is the common receptor for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). PTH and PTHrP sha...

Transcriptional Profiling Identifies the Signaling Axes of IGF and Transforming Growth Factor-β as Involved in the Pathogenesis of Osteosarcoma

“…Tumor cells from patient-derived cell lines OS160CL and OS63CL were used to establish xenografts in mice as previously described according to a protocol approved by the Memorial Hospital Institutional Animal Care and Use Committee [37]. Xenografts were resected when the size was approximately 1.5 cm in diameter.…”

“…The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for template loading. Thermal cycling was performed with a 7500-fast real-time PCR system (Applied Biosystems 1 ) using a 2 9 TaqMan 1 Fast Universal PCR Master Mix provided by the manufacturer [37]. The relative quantity was calculated based on standard 2…”

Immunohistochemical identification of the PTHR1 parathyroid hormone receptor in normal and neoplastic human tissues

“…In parallel to the occurrence of PTHrP, PTHR1 also seems to be present in other non-malignant and malignant tissues and is thought to mediate PTHrP-dependent effects on cell proliferation and differentiation. Most previous studies have detected the PTHR1 receptor on the mRNA level or via ligand-binding assays (7)(8)(9)(21)(22)(23)(24). Consequently, only limited information is currently available concerning the cellular and subcellular localisation of the PTHR1 receptor in human tissues.…”

Parathyroid Hormone–Related Protein Promotes Epithelial–Mesenchymal Transition

“…26,27 Moreover, a recent report has shown that PTHR1 overexpression in a human osteosarcoma cell line resulted in its increased invasive capacity, associated with upregulation of TGF-␤1. 28 Also of interest in the present context, in both human embryonic kidney cells HEK-293 and osteoblasts, stimulation of PTHR1 may lead to EGFR transactivation. 29,30 Collectively, these findings suggest that PTHrP might interact with TGF-␤ and/or EGFR to modulate EMT.…”