Induction of osteoclastogenesis in co-cultures of human osteoclast precursors and prostate cancer cell lines

Abstracts / Bone 48 (2011) S251–S260 Osteoclastic precursor cells (PBMC) were isolated from human peripheral blood. PBMC (5 × 105 cells/cm2) were co-cultured with three different cell densities (1 × 102–1 × 104 cells/ 2 cm ) of a Ewing sarcoma cell line (91304), in the absence of any exogenous osteoclastogenic stimuli. The involvement of the signaling pathways MEK, NFkB, PKC, MAPK, JNK and p38 and the synthesis of PGE2 was addressed. Cell cultures were analysed at days 7, 14 and 21 for tartarate-resistant acid phosphatase activity and histochemical staining, presence of multinucleated cells with actin rings and expressing vitronectin and calcitonin receptors, expression of several osteoclast-related genes and ability to resorb bone. It was observed that the presence of the 91304 cell line significantly increased the osteoclastogenic behaviour of PBMC, in all tested cell densities. However, the response was higher on co-cultures performed with the two lower 91304 cell densities, which suggests that Ewing sarcoma cells have a high ability to induce osteoclast development. The different osteoclastogenic-related signaling pathways analysed revealed important differences in their relative contribution for the observed responses. In conclusion, this work demonstrated that cell-cell communications between Ewing sarcoma cells and osteoclast precursors are sufficient to promote a high degree of osteoclast differentiation and activation. Data regarding the intracellular mechanisms involved can reveal new insights about the bone metabolic processes affected in patients suffering from Ewing sarcoma. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

doi:10.1016/j.bone.2011.03.629

PP499-S Induction of osteoclastogenesis in co-cultures of human osteoclast precursors and prostate cancer cell lines J. Costa-Rodrigues a,⁎, R. Fernandes a,b, M.H. Fernandes a a Laboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Portugal b Faculdade de Ciências, Universidade do Porto, Porto, Portugal Abstract: Cellular crosstalks among prostate cancer cells and bone cells have an important role in the development of bone metastasis. With this study, one normal prostate cell line (PNT2) and two prostate cancer cell lines with different bone metastatic characteristics (PC3 — osteolytic metastasis; LNCaP — osteoblastic/mixed metastasis) were co-cultured with human osteoclast precursor cells, and the osteoclastogenic response was assessed. Osteoclastic precursors were isolated from peripheral blood and were co-cultured (5×105 cells/cm2) with three different cell densities (102–104 cells/cm2) of the prostate cell lines for 21 days in the absence of any exogenous stimuli. When indicated, cell cultures were treated with inhibitors of MEK, NFkB, PKC, MAPK, JNK and p38 signaling pathways, and a PGE2 production blocker. Co-cultures were analysed (days 7, 14 and 21) for tartarate-resistant acid phosphatase activity and histochemical staining, presence of multinucleated cells with actin rings and expressing vitronectin and calcitonin receptors, expression of osteoclast-related genes and ability to resorb bone. It was observed that PNT2 exhibited a low osteoclastogenic-inducing ability that decreased with the increase on its cellular density. On the other hand, PC3 and LnCaP displayed a high ability to induce osteoclast differentiation, especially at low cell densities. However, the osteoclastogenic induction was higher for PC3 cell line. The characterization of the intracellular mechanisms involved in the observed behaviors revealed important differences among the different cell lines tested. In conclusion, it was demonstrated that prostate cancer cell lines, unlike the nontumorigenic cell line, have the ability to induce osteoclastogenic through mechanisms that might involve, at least partially, direct cell–cell contacts with osteoclast precursor cells. The characterization of the intracellular processes involved in the different cellular behaviors can open new insights on the study of bone metastasis induced by prostate cancer cells. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

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Abstract: Breast cancers commonly metastasis to the bone microenvironment, disrupting the bone remodelling cycle causing predominantly osteolytic metastases. Insulin like growth factor 1 (IGF-1) is known to play functional roles in both bone cell and breast cancer cell biology. Here, we investigate the effects of PQIP, a novel, selective dual inhibitor of IGF-1 receptor (IGF-1R) and IR kinases, on bone cell differentiation, activity and interaction with breast cancer cell lines in vitro. Treatment of calvarial osteoblasts with PQIP resulted in a significant reduction in alkaline phosphatase activity (56%, p< 0.05), and bone nodule formation (40%, p< 0.05), in the presence and absence of IGF-1, without affecting cell viability. Moreover, PQIP suppressed IGF-1 induced migration of the osteoblast-like MC3T3-E1 cells (24%, p< 0.05) as measured by the wound healing assay. Interestingly, PQIP also inhibited RANKL production in osteoblasts (89%, p< 0.05), indicating a possible indirect inhibitory effect on osteoblast support for osteoclastagenesis. Studies in osteoclast cultures demonstrated that IGF-1 enhanced RANKL induced osteoclast formation (31%, p< 0.05), and that this effect was completely abolished by the presence of PQIP. Functional studies showed that PQIP treatment prevented IGF-1 induced AKT phosphorylation in osteoblast, osteoclast, and MDA-MB-231 cultures, confirming inhibition of IGF-1 receptor kinase signalling. Conditioned media from human MDA-MB-231, human MCF7, and mouse 4T1 breast cancer cells stimulated osteoclast formation in bone marrow cultures treated with RANKL and M-CSF, and this was significantly suppressed in the presence of PQIP (40–50% reduction, p< 0.05). Further mechanistic studies showed that PQIP also inhibited AKT phosphorylation in mature osteoclasts when induced by condition media from MDA-MB-231 cells, indicating an inhibitory effect on tumour cell support for osteoclastogenesis. In conclusion, the IGF-1R kinase inhibitor PQIP suppresses differentiation of bone cells and for the first time we showed that IGF-1R inhibition suppresses both osteoblast and breast cancer cell support for osteoclastogenesis in vitro. Therefore, derivatives of PQIP, which are now in advanced clinical development, may be of clinical value for the treatment of bone loss and metastases associated with breast cancer. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

doi:10.1016/j.bone.2011.03.631

PP501-T Utility of quantitative bone ultrasound to assess anastrozole effect on bone mass in patients with breast cancer J.F. Calderon-Garcia a,⁎, S. Gonzalez-Santiago b, J.M. Lavado-Garcia a, M.H. Lopez-Ceballos b, P. Rey-Sanchez a, J.D. Pedrera-Zamorano a a Metabolic Bone Diseases Research Group, University of Extremadura, Spain b Oncology Unit, Hospital San Pedro de Alcantara, Caceres, Spain Abstract: Background: Anastrozole, a third generation aromatase inhibitor, block estrogen production in peripheral tissues and reduce circulating estrogen levels, leading to accelerated bone loss and an increased risk of fracture. Quantitative ultrasound (QUS) is widely used for the assessment of the skeletal status; however, the usefulness of QUS in long-term monitoring has yet to be defined. The aim of this study was to evaluate the utility of quantitative bone ultrasound measurements to monitoring bone mass in women with breast cancer in treatment with anastrozole. Patients and Methods: The study was a longitudinal analysis of 101 postmenopausal women (62.05±9.91 years old; 13.81±10.71 years since menopause) with breast cancer (stages I, III and III) in treatment with anastrozole (1 mg/24 h). Bone mass was measured at 0, 6 and 12 months using peripheral quantitative computed tomography (pQCT) of the non-dominant distal forearm, phalangeal bone ultrasound, as the amplitude-dependent speed of bone ultrasound (Ad-SoS), and dual energy X-ray absorptiometry (DXA) at lumbar spine and hip. Results: Bone mass and bone mineral density (by means of QUS, DXA and pQCT) reduce significatively after 12 months (p< 0.05 in all cases). QUS measurements have showed a significatively decrease in bone mass after 6 months of treatment (p< 0.05). Conclusion: These data suggest that QUS may have an important role in the monitoring of bone mass in patients in treatment with anastrozole. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

doi:10.1016/j.bone.2011.03.630 doi:10.1016/j.bone.2011.03.632

PP500-M/NIOP07 The novel and selective insulin-like growth factor-1 receptor kinase inhibitor pqip suppresses bone cell function and osteoclast- breast cancer cell cross-talk J.G. Logan a,b,⁎, A. Sophocleous a, E. Landao-Bassonga a, V.G. Brunton b, A.I. Idris a,b a Centre for Molecular Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK b Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK

PP502-S Effect of strontium ranelate in women with breast cancer after combined therapy. K.F. Vartanyan. Department of Radiology, Russian Academy of Advanced Medical Studies, Moscow, Russian Federation. Background new therapeutic agents with radiobiological EFFE K.F. Vartanyan ⁎ Department of radiology, Russian Academy of Advanced Medical Studies, Moscow, Russian Federation