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The role of parathyroid hormone-related protein in giant cell tumour of bone
S48
Abstracts / Bone 48 (2011) S22–S55
the count of CD31-positive microvessels. Whereas Twist-1 expression did not affect MDA-BO2 proliferation in vitro, it significantly shortened their entry in cell cycle. Finally, Twist-1 activity did not interfere with the gene expression and/or subcellular localisation of proteins involved in bone remodelling and osteomimicry such as RUNX2, DKK-1, VEGF, MMP-9 and YB-1, suggesting that Twist-1 promotes the bone metastasis formation by increasing the tumor burden in bone rather than modifying the osteomimetic properties of MDA-BO2. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.134
P-79 The role of parathyroid hormone-related protein in giant cell tumour of bone R.W. Cowana,c,*, G. Singha,c, M. Ghertb,c a Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada b Department of Surgery, McMaster University, Hamilton, ON, Canada c Juravinski Cancer Centre, Hamilton, ON, Canada The etiology of giant cell tumour of bone (GCT), an aggressive primary osteolytic bone tumour, remains unclear. GCTs present with similar clinical features as brown tumours of hyperparathyroidism, which are caused by a systemic increase in parathyroid hormone (PTH) concentration. Multiple tumours secrete PTH-related protein (PTHrP), which similarly cause an increase in systemic PTHrP concentrations and lead to increased bone resorption and hypercalcemia. Both PTH and PTHrP are known to stimulate the same receptor, the PTH type 1 receptor (PTH1R). We investigated whether PTHrP could play a role in the pathogenesis of GCT. Immunohistochemistry analyses of paraffin-embedded GCT tissues revealed a positive expression of both PTHrP and PTH1R in the stromal cells of GCT. Moreover, the giant cells showed consistently strong expression of PTH1R, whereas PTHrP was found in a small percentage of giant cells. Western blot analyses of primary cell cultures from patients presenting with GCT confirmed the PTHrP expression by the neoplastic stromal cell population. To assess whether PTHrP could contribute to the abundance of giant cells present in the tumour, we treated GCT stromal cells with PTHrP (1–34) and examined the expression of the receptor activator of nuclear factor-kappaB ligand (RANKL) by real-time PCR. Results revealed increased expression of RANKL following administration of the peptide. We further assessed whether such treatment would result in increased stromal cell-mediated formation of multinucleated cells from murine myeloid RAW 264.7 cells. Results suggest that PTHrP may contribute to the formation of giant cells in GCT. (This work is supported by a grant from the Canadian Institutes of Health Research.) Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.135
P-80 Osteocyte-derived FGF23 activates EGR-1 signaling in prostate cancer cells: A new vicious cycle in bone metastases P. Fourniera, D. Edwardsa, K. Whiteb, J.M. Chirgwina,* a Medicine, Indiana University, Indianapolis, USA b Medical Genetics, Indiana University, Indianapolis, USA Prostate cancer metastasizes to bone more frequently than other solid tumors, often causing osteoblastic responses. The unique features of skeletal metastases are due to specific, local interactions between tumor cells and the bone microenvironment. Bone is the sole source of fibroblast growth factor 23, an endocrine hormone that acts on cells expressing its receptor (FGFR1c +klotho) to increase the transcription factor EGR-1 and
alter metabolism of vitamin D. This pathway controls kidney handling of phosphate. Expression of klotho by breast cancers has been reported. Human prostate cancer lines all expressed mRNAs for klotho and the FGF receptor 1iiic isoform. DU145 human prostate cancer cells, which cause bone lesions in mice, were treated with 100 ng/ml recombinant human FGF23 and analyzed by real-time PCR. EGR-1 mRNA increased 3× at 1 h (n= 3; p < 0.0001), returning to baseline by 2 h. The vitamin Dinactivating 24-hydroxylase, CYP24A1, increased 2× at 2 h (p< 0.0001), returning to baseline by 4 h. There was little change in 1-alpha hydroxylase, CYP27B1. Treatment of C4-2B prostate cells with 100 nM 1,25-dihydroxy-vitamin D3 for 6 days caused 80% inhibition of growth by MTT assay (p< 0.05). Addition of 100 ng/ml FGF23 caused a 40% increase in cell number (p< 0.05), despite the presence of 100 nM 1,25dihydroxy-vitamin D3. This response was not seen with MDA-MB-231 breast cancer cells, which express low klotho (<5% of mRNA made by DU145 or C4-2B cells by PCR). Prostate tumor lines which cause osteoblastic lesions had very high klotho expression, suggesting that FGF23/klotho signaling may contribute to osteoblastic responses via genes downstream of EGR1, such as VEGF and TRPV6. PTHrP and FGF8, made by prostate cancers, increase FGF23; hypoxia and TGFbeta activate EGR-1. The FGF23/klotho/EGR-1 signaling axis may sustain a vicious cycle of bone metastases and be a novel target for therapy. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.136
P-81 Inhibition of discoidin domain receptor-1 (DDR1) impairs tumor-induced osteoclastogenesis preventing bone metastatic homing and colonization K. Valencia*, C. Zandueta, C. Ormazábal, D. Luis-Ravelo, I. Antón, S. Martínez, F. Lecanda Division of Oncology, University of Navarra, Center for Applied Medical Research, Pamplona, Spain Bone microenvironment and tumor–stromal interactions are critical for the initiation and development of metastasis. The aim of this study was to delineate the role of DDR1 (discoidin domain receptor 1) in this process. DDR1 was found in several lung tumor cells lines through qPCR. Lentiviral transduction of a lung tumor cell line with shRNA using different DDR1 targets led to 80–90% inhibition. Knock-down cells (shDDR1) displayed a stunted decrease of invasiveness in collagen type I and type IV as compared to control cells (p<0.001). In contrast adhesiveness of shDDR1 cells to collagen type I or IV was unaltered. To delineate the functional contribution of DDR1 in metastatic activity, tumor burden and osteolytic lesions were assessed by bioluminescence imaging, X-Ray and μCT image analysis, after intracardiac inoculation (i.c.) in nude mice. Interestingly, shDDR1 inoculated animals displayed an overt increase in metastasis-free survival as compared to scramble and control mice (p < 0.001). Bioluminescence imaging showed a dramatic decrease in skeletal tumor burden in shDDR1 treated mice. More importantly, a marked reduction in osteolytic lesions assessed by X-ray imaging and μCT scans was also detected in shDDR1 inoculated mice (p<0.001). These results were correlated with a marked decrease in osteoclastogenic and metalloproteolytic activities in conditioned media derived from shDDR1 cells. To evaluate whether these effects were the result of impaired bone homing, we performed i.c. inoculation of shDDR1 cells and we assessed tumor burden at day 5 post-inoculation. Quantification of metastatic cell number after bone marrow “flushing” revealed a marked decrease in shDDR1 inoculated mice (p<0.01) indicating that DDR1 mediates tumor–bone engagement in early events of bone homing. These data suggest that disruption of DDR1 alters heterotypic tumor–stromal interactions during bone homing and abrogates osteoclatogenic activity, critically impairing osseous colonization. We suggest that DDR1 represents a novel therapeutic target involved in lung cancer bone metastasis.
Abstracts / Bone 48 (2011) S22–S55
the count of CD31-positive microvessels. Whereas Twist-1 expression did not affect MDA-BO2 proliferation in vitro, it significantly shortened their entry in cell cycle. Finally, Twist-1 activity did not interfere with the gene expression and/or subcellular localisation of proteins involved in bone remodelling and osteomimicry such as RUNX2, DKK-1, VEGF, MMP-9 and YB-1, suggesting that Twist-1 promotes the bone metastasis formation by increasing the tumor burden in bone rather than modifying the osteomimetic properties of MDA-BO2. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.134
P-79 The role of parathyroid hormone-related protein in giant cell tumour of bone R.W. Cowana,c,*, G. Singha,c, M. Ghertb,c a Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada b Department of Surgery, McMaster University, Hamilton, ON, Canada c Juravinski Cancer Centre, Hamilton, ON, Canada The etiology of giant cell tumour of bone (GCT), an aggressive primary osteolytic bone tumour, remains unclear. GCTs present with similar clinical features as brown tumours of hyperparathyroidism, which are caused by a systemic increase in parathyroid hormone (PTH) concentration. Multiple tumours secrete PTH-related protein (PTHrP), which similarly cause an increase in systemic PTHrP concentrations and lead to increased bone resorption and hypercalcemia. Both PTH and PTHrP are known to stimulate the same receptor, the PTH type 1 receptor (PTH1R). We investigated whether PTHrP could play a role in the pathogenesis of GCT. Immunohistochemistry analyses of paraffin-embedded GCT tissues revealed a positive expression of both PTHrP and PTH1R in the stromal cells of GCT. Moreover, the giant cells showed consistently strong expression of PTH1R, whereas PTHrP was found in a small percentage of giant cells. Western blot analyses of primary cell cultures from patients presenting with GCT confirmed the PTHrP expression by the neoplastic stromal cell population. To assess whether PTHrP could contribute to the abundance of giant cells present in the tumour, we treated GCT stromal cells with PTHrP (1–34) and examined the expression of the receptor activator of nuclear factor-kappaB ligand (RANKL) by real-time PCR. Results revealed increased expression of RANKL following administration of the peptide. We further assessed whether such treatment would result in increased stromal cell-mediated formation of multinucleated cells from murine myeloid RAW 264.7 cells. Results suggest that PTHrP may contribute to the formation of giant cells in GCT. (This work is supported by a grant from the Canadian Institutes of Health Research.) Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.135
P-80 Osteocyte-derived FGF23 activates EGR-1 signaling in prostate cancer cells: A new vicious cycle in bone metastases P. Fourniera, D. Edwardsa, K. Whiteb, J.M. Chirgwina,* a Medicine, Indiana University, Indianapolis, USA b Medical Genetics, Indiana University, Indianapolis, USA Prostate cancer metastasizes to bone more frequently than other solid tumors, often causing osteoblastic responses. The unique features of skeletal metastases are due to specific, local interactions between tumor cells and the bone microenvironment. Bone is the sole source of fibroblast growth factor 23, an endocrine hormone that acts on cells expressing its receptor (FGFR1c +klotho) to increase the transcription factor EGR-1 and
alter metabolism of vitamin D. This pathway controls kidney handling of phosphate. Expression of klotho by breast cancers has been reported. Human prostate cancer lines all expressed mRNAs for klotho and the FGF receptor 1iiic isoform. DU145 human prostate cancer cells, which cause bone lesions in mice, were treated with 100 ng/ml recombinant human FGF23 and analyzed by real-time PCR. EGR-1 mRNA increased 3× at 1 h (n= 3; p < 0.0001), returning to baseline by 2 h. The vitamin Dinactivating 24-hydroxylase, CYP24A1, increased 2× at 2 h (p< 0.0001), returning to baseline by 4 h. There was little change in 1-alpha hydroxylase, CYP27B1. Treatment of C4-2B prostate cells with 100 nM 1,25-dihydroxy-vitamin D3 for 6 days caused 80% inhibition of growth by MTT assay (p< 0.05). Addition of 100 ng/ml FGF23 caused a 40% increase in cell number (p< 0.05), despite the presence of 100 nM 1,25dihydroxy-vitamin D3. This response was not seen with MDA-MB-231 breast cancer cells, which express low klotho (<5% of mRNA made by DU145 or C4-2B cells by PCR). Prostate tumor lines which cause osteoblastic lesions had very high klotho expression, suggesting that FGF23/klotho signaling may contribute to osteoblastic responses via genes downstream of EGR1, such as VEGF and TRPV6. PTHrP and FGF8, made by prostate cancers, increase FGF23; hypoxia and TGFbeta activate EGR-1. The FGF23/klotho/EGR-1 signaling axis may sustain a vicious cycle of bone metastases and be a novel target for therapy. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.136
P-81 Inhibition of discoidin domain receptor-1 (DDR1) impairs tumor-induced osteoclastogenesis preventing bone metastatic homing and colonization K. Valencia*, C. Zandueta, C. Ormazábal, D. Luis-Ravelo, I. Antón, S. Martínez, F. Lecanda Division of Oncology, University of Navarra, Center for Applied Medical Research, Pamplona, Spain Bone microenvironment and tumor–stromal interactions are critical for the initiation and development of metastasis. The aim of this study was to delineate the role of DDR1 (discoidin domain receptor 1) in this process. DDR1 was found in several lung tumor cells lines through qPCR. Lentiviral transduction of a lung tumor cell line with shRNA using different DDR1 targets led to 80–90% inhibition. Knock-down cells (shDDR1) displayed a stunted decrease of invasiveness in collagen type I and type IV as compared to control cells (p<0.001). In contrast adhesiveness of shDDR1 cells to collagen type I or IV was unaltered. To delineate the functional contribution of DDR1 in metastatic activity, tumor burden and osteolytic lesions were assessed by bioluminescence imaging, X-Ray and μCT image analysis, after intracardiac inoculation (i.c.) in nude mice. Interestingly, shDDR1 inoculated animals displayed an overt increase in metastasis-free survival as compared to scramble and control mice (p < 0.001). Bioluminescence imaging showed a dramatic decrease in skeletal tumor burden in shDDR1 treated mice. More importantly, a marked reduction in osteolytic lesions assessed by X-ray imaging and μCT scans was also detected in shDDR1 inoculated mice (p<0.001). These results were correlated with a marked decrease in osteoclastogenic and metalloproteolytic activities in conditioned media derived from shDDR1 cells. To evaluate whether these effects were the result of impaired bone homing, we performed i.c. inoculation of shDDR1 cells and we assessed tumor burden at day 5 post-inoculation. Quantification of metastatic cell number after bone marrow “flushing” revealed a marked decrease in shDDR1 inoculated mice (p<0.01) indicating that DDR1 mediates tumor–bone engagement in early events of bone homing. These data suggest that disruption of DDR1 alters heterotypic tumor–stromal interactions during bone homing and abrogates osteoclatogenic activity, critically impairing osseous colonization. We suggest that DDR1 represents a novel therapeutic target involved in lung cancer bone metastasis.