- Email: [email protected]
An exopolysaccharide produced by alteromonas infernus reduces lung metastasis and prolongs survival rate of osteosarcoma-bearing mice
S42
Abstracts / Bone 48 (2011) S22–S55
whether CpG-ODN influenced BMP-induced osteoblast differentiation or BMP-induced apoptosis of malignant plasma cells. We found that PTOCpG-ODN inhibited BMP-2-induced osteoblast differentiation from human mesenchymal stem cells (hMSCs). Further, PTO-CpG-ODN counteracted BMP-2- and BMP-6-induced apoptosis of the human myeloma cell lines IH-1 and INA-6, respectively. In contrast, PTO-CpGODN did not antagonize the anti-proliferative effect of BMP-2 on hMSC or IH-1 cells. Inhibition of Smad-signaling and p38 MAPK-signaling indicated that apoptosis of IH-1 cells is dependent on Smad-signaling downstream of BMP, whereas the anti-proliferative effect of BMP-2 on IH-1 cells also involves p38 MAPK-signaling. Together, the data suggested a specific inhibition by PTO-CpG-ODN on BMP-Smad-signaling. Supporting this we found that PTO-CpG-ODN inhibited BMP-induced phosphorylation of receptor-Smads in hMSC and myeloma cell lines. This effect appeared to be independent of TLR9 since GpC-ODN and other ODNs with the ability to form multimeric structures inhibited Smad-signaling as efficiently as PTO-CpG-ODNs, and since knockdown of TLR9 by siRNA in INA-6 cells did not blunt the effect of PTO-CpG-ODN. In conclusion, our results demonstrate that PTO-CpG-ODN inhibits BMP-signaling, and thus might provoke unwanted TLR9-independent side effects in patients. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.117
P-62 First evidence for anti-tumour effects of doxorubicin and zoledronic acid from an immunocompetent mouse model P.D. Ottewella,*, H. Browna, C.A. Evansa, N.S. Winda, N.J. Brownb, R.E. Colemana, I. Holena a Academic Unit of Clinical Oncology, University of Sheffield, Sheffield, UK b Academic Unit of Surgical Oncology, University of Sheffield, Sheffield, UK Studies in immunocompromised mice have shown that weekly administration of doxorubicin followed 24 h later by zoledronic acid for 6 weeks inhibits growth of subcutaneous MDA-MB-436 breast tumours. In addition, tumour growth does not re-occur when treatment is stopped (Ottewell, 2009). These studies have been carried out in athymic mice bearing human tumour xenografts at non-orthotopic sites. To more closely mimic human breast cancer development and response to treatment we are now using an immunocompetent transgenic mouse model (PyMT) to investigate the effects of doxorubicin and zoledronic acid on spontaneous mammary tumour growth and metastasis. 7-week-old PyMT/FVB mice were randomly divided into 4 treatment groups (n= 15/group). Mice were treated 1× per week for 6 weeks with 100 μl PBS (control), 2 mg/kg doxorubicin, 100 μg/kg zoledronic acid, or doxorubicin followed 24 h later by zoledronic acid. Mice were sacrificed 24 h after last treatment or when primary tumour volume reached 1.5 cm3. Primary tumour mass was recorded on sacrifice. Lungs were analysed for metastatic tumour nodules and hind limbs were assessed by X-ray and μ-CT for lytic lesions and bone metastasis. Mammary tumours were non-palpable in mice treated sequentially with doxorubicin followed by zoledronic acid and tumours could only be detected on resection. Administration of 2 mg/kg doxorubicin followed by 100 μg/kg zoledronic acid for 6 weeks significantly reduced mammary tumour growth compared with control. Mean mammary tumour mass was 1.34 ± 0.38 g following sequential treatment and 3.03 ± 0.21 g in control mice (p= 0.007). All mice receiving combination treatment survived the entire experiment, whereas, 40% of control animals were sacrificed early due to excessive tumour volume. Mammary tumour metastases were not detected in lungs or hind limbs of any animals. Sequential administration of doxorubicin followed 24 h later by zoledronic acid significantly reduces spontaneous mammary tumour growth and increases survival in immunocompetent PyMT mice.
Conflict of interest statement: R.E. Coleman: research funding; Novartis, consultancy: Novartis and Amgen, speaker fees: Novartin, Amgen. I. Holen: funding from Novartis and speaker fee from Novartis. doi:10.1016/j.bone.2010.10.118
P-63 The causal role of the BMP signalling antagonist noggin in suppressing bone formation in osteolytic bone metastasis C. Secondini*, A. Wetterwald, R. Schwaninger, G.N. Thalmann, M.G. Cecchini Department of Urology, University of Bern, Bern, Switzerland Department of Clinical Research, University of Bern, Bern, Switzerland Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. We previously reported that lack of expression of the BMP antagonist noggin by osteoinductive cell lines derived from mammary and prostate cancers is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic cell lines derived from the same cancer types express noggin constitutively. In multiple myeloma, the Wnt antagonist dickkopf-1 has been shown to participate to the osteolytic process by repressing bone formation. Therefore, we hypothesized that noggin may contribute by an equivalent mechanism to the osteolysis caused by solid cancers. An shRNA-mediated silencing strategy was used to investigate whether constitutive noggin expression participates to the osteolytic response induced by intra-osseous xenografts of the prostate cancer cell line PC-3. Tumour growth in vivo was monitored by bioluminescent imaging. Modifications in bone architecture were determined by radiography, micro-computed tomography, peripheral quantitative computed tomography and histomorphometry. The PC-3 cell-induced osteolytic lesions showed not only enhanced osteoclast-mediated bone resorption, but also decreased osteoblast-mediated bone formation. Noggin knock-down in PC-3 cells restored bone formation in the osteolytic lesions, thus reestablishing, at least partially, physiologic bone coupling. Furthermore, intra-osseous tumour growth of noggin-silenced PC-3 cells was limited by the restored osteoblast activity. In the PC-3 model, uncoupling of the bone remodelling process contributes to osteolysis, similarly to the mechanism shown in multiple myeloma. Constitutive noggin secretion by cancer cells mediates the repression of bone formation, at least in the PC-3 model. Cancer cell targeted noggin suppression restores bone formation and, as possible consequence of this, interferes with tumour growth. Therefore, noggin neutralization may be an additional therapeutic option to treat osteolytic bone metastases by prostate cancer. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.119
P-64 An exopolysaccharide produced by alteromonas infernus reduces lung metastasis and prolongs survival rate of osteosarcoma-bearing mice C. Ruiz Velascoa,*, S. Colliec-Jouaultb, C. Sinquinb, D. Heymanna, M. Padrinesa a INSERM U957, Université de Nantes, Nantes, France b IFREMER, Laboratoire de biotechnologie et molécules marines, Nantes, France Heparin, which is traditionally used as an anticoagulant, has a variety of additional biological activities. It has been shown in several
Abstracts / Bone 48 (2011) S22–S55
retrospective and prospective clinical trials to have an effect on cancer patient survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. Here, we investigated the in vivo effects of a “heparin-like”, an oversulfated exopolysaccharide (OS-EPS) produced by a mesophilic strain found in deep-sea hydrothermal vents, on lung metastasis progression and animal survival. We compared this effect with that of a non-oversulfated exopolysaccharide (EPS) and unfractionated heparin. An animal model of lung metastasis was developed in C3H/He mice inoculated intravenously with a spontaneous murine osteosarcoma POS-1 cell line. Lung metastasis was determined at the time of autopsy. Independent experiments (series treated with 6 mg/kg of EPS or OS-EPS or heparin) showed a significant increase of the survival rate in treated animals versus controls. The number of metastatic nodules was strongly decreased by sulfated polysaccharide (OS-EPS and heparin) treated mice as revealed by macroscopic analysis. In vitro, a 48-hour incubation with 25 μg/mL EPS or OS-EPS or heparin inhibited POS-1 cell line migration. Besides, the efficacy of polysaccharides was tested on primary bone tumor growth using a mouse-transplantable model of osteosarcoma. The preliminary results show that polysaccharides-treatment delayed local tumor growth, as compared to the untreated mice. In conclusion, we demonstrate that these sulfated polysaccharides significantly diminish osteosarcoma-induced lung metastasis in vivo, thereby prolonging survival of POS-1-inoculated animals. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.120
P-65 MNNG-HOS osteosarcoma growth is enhanced by human fat and adipose-derived mesenchymal stem cells: Implication in reconstructive surgery following tumor treatment L. Bourdaisa,b, P. Perrota,b, D. Duteillea,b, S. Hervoueta,b, J. Chesneaua,b, J. Amiauda,b, G. Picardaa,b, G. De Pigneuxc, F. Redinia,b, D. Heymanna,b,*, V. Tricheta,b a U957, INSERM, Nantes, France b EA3822, University, Nantes, France c EA3855, University, Tours, France Growth of osteosarcoma, the most frequent primary bone tumor, is supported by its microenvironment. We recently reported an unexpected local osteosarcoma relapse which occurred at the exact site of autologous fat grafts in a female patient who did not present any predictive factor of local recurrence (Perrot et al., PLoS ONE 5(6): e10999. doi:10.1371/journal.pone.0010999). Using SaOS2 human osteosarcoma induced in mice, we have shown that tumor growth was promoted by human fat injection. One hypothesis for this protumor effect may lie to the presence of mesenchymal stromal/stem cells (MSCs) in the injected fat. In this study, we tested human osteosarcoma MNNG-HOS cells rather than human SaOS2 cells because they led to a greater tumor incidence in mice (100% versus 50%) either by intra-muscular cell injection or by tumor fragment transplantation in close contact with bone. Fresh harvested human fat was injected in progressive tumors and was used to isolate MCSs. These human adipose-derived MSCs (hAD-MSCs) either unmodified or modified to express the firefly luciferase were co-injected with MNNG-HOS cells in mice. Tumor development and bone architecture were analyzed by tumor volume measurement and lung metastasis detection and by micro-computed tomography for small animal imaging, respectively. Histology and in vivo bioluminescence imaging were used to characterize MNNG-HOS tumors and to identify hAD-MSCs. MNNG-HOS is an aggressive osteosarcoma cell lines which induced a high tumor incidence, lung metastases and a strong osteolysis in both
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induction processes. Human fat injections and hAD-MSC co-injections increased significantly the tumor growth but did not increase metastasis and bone remodelling as compared to MNNG-HOS control tumors. This study presents a complete characterization of MNNG-HOS osteosarcoma in vivo. Moreover we confirm that modifications of the tumor microenvironment by fat graft may favour osteosarcoma growth and that this effect could be partially mediated by MSCs contained in fat graft. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.121
P-66 The novel and selective insulin-like growth factor-1 receptor kinase inhibitor PQIP suppresses bone cell function and osteoclast-breast cancer cell cross-talk in vitro J.G. Logana,*, E. Landao-Bassongaa, V.G. Bruntonb, A.I. Idrisa a Bone Research Group, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, Edinburgh, UK b Division of Cancer Biology, Edinburgh Cancer Research Centre, Institute of Genetics & Molecular Medicine, Edinburgh, UK Breast cancers commonly metastasise to the bone microenvironment, disrupting the bone remodelling cycle causing predominantly osteosclerotic metastases. Insulin like growth factor 1 (IGF-1) receptor blockers inhibit bone cell proliferation and suppress metastasis of breast cancer cells, but their effects on bone and tumour cell interaction are unknown. Here, we investigate the effects of PQIP, a novel, selective, IGF-1 receptor kinase inhibitor, on bone cell differentiation, proliferation and migration of the human breast cancer cell line MDA-MB-231 and bone–tumour cell interaction in vitro. Treatment of primary osteoblasts with IGF-1 (100 ng/ml) stimulated bone nodule formation. PQIP (200 nM) treatment resulted in a significant reduction in osteoblast differentiation (56%, p <0.05) and bone nodule formation (40%, p <0.05) in the presence and absence of IGF-1, without affecting cell viability. PQIP (200 nM) also suppressed both basal and IGF-1 induced migration of the osteoblast-like MC3T3-E1 and MDA-MB-231 cells. PQIP (200 nM) inhibited the increase in osteoclast number, size and multinuclearity in the presence and absence of IGF-1 or following treatment with MDA-MB-231 conditioned media, indicating a significant inhibitory effect on osteoclast–tumour cell interaction. Further mechanistic work showed that PQIP (200 nM) treatment completely abolished IGF-1 induced phosphorylation of AKT in osteoblast, osteoclast, and MDA-MB-231 cultures, confirming inhibition of IGF-1 signalling. Tellingly, treatment of osteoclast cultures with PQIP (200 nM) also significantly reduced AKT phosphorylation stimulated by treatment with MDA-MB-231 conditioned media (62%, p <0.05). In conclusion, the IGF-1 kinase inhibitor PQIP suppresses differentiation of bone and tumours cells and for the first time showed that IGF-1 receptor inhibition suppresses breast cancer cell support for osteoclastogenesis in vitro. This identifies PQIP as a novel agent, which along with other IGF-1 and insulin receptor inhibitors, may be of clinical value for the treatment for bone loss and metastases associated with breast cancer. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.122
P-67 Inhibition of Dickkopf-1 (DKK-1) delays prostate cancer growth in vivo through induction of P21CIP-1 C.L. Halla,*, H. Zhanga, S. Bailea, S. Kuhstossb, E.T. Kellera
Abstracts / Bone 48 (2011) S22–S55
whether CpG-ODN influenced BMP-induced osteoblast differentiation or BMP-induced apoptosis of malignant plasma cells. We found that PTOCpG-ODN inhibited BMP-2-induced osteoblast differentiation from human mesenchymal stem cells (hMSCs). Further, PTO-CpG-ODN counteracted BMP-2- and BMP-6-induced apoptosis of the human myeloma cell lines IH-1 and INA-6, respectively. In contrast, PTO-CpGODN did not antagonize the anti-proliferative effect of BMP-2 on hMSC or IH-1 cells. Inhibition of Smad-signaling and p38 MAPK-signaling indicated that apoptosis of IH-1 cells is dependent on Smad-signaling downstream of BMP, whereas the anti-proliferative effect of BMP-2 on IH-1 cells also involves p38 MAPK-signaling. Together, the data suggested a specific inhibition by PTO-CpG-ODN on BMP-Smad-signaling. Supporting this we found that PTO-CpG-ODN inhibited BMP-induced phosphorylation of receptor-Smads in hMSC and myeloma cell lines. This effect appeared to be independent of TLR9 since GpC-ODN and other ODNs with the ability to form multimeric structures inhibited Smad-signaling as efficiently as PTO-CpG-ODNs, and since knockdown of TLR9 by siRNA in INA-6 cells did not blunt the effect of PTO-CpG-ODN. In conclusion, our results demonstrate that PTO-CpG-ODN inhibits BMP-signaling, and thus might provoke unwanted TLR9-independent side effects in patients. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.117
P-62 First evidence for anti-tumour effects of doxorubicin and zoledronic acid from an immunocompetent mouse model P.D. Ottewella,*, H. Browna, C.A. Evansa, N.S. Winda, N.J. Brownb, R.E. Colemana, I. Holena a Academic Unit of Clinical Oncology, University of Sheffield, Sheffield, UK b Academic Unit of Surgical Oncology, University of Sheffield, Sheffield, UK Studies in immunocompromised mice have shown that weekly administration of doxorubicin followed 24 h later by zoledronic acid for 6 weeks inhibits growth of subcutaneous MDA-MB-436 breast tumours. In addition, tumour growth does not re-occur when treatment is stopped (Ottewell, 2009). These studies have been carried out in athymic mice bearing human tumour xenografts at non-orthotopic sites. To more closely mimic human breast cancer development and response to treatment we are now using an immunocompetent transgenic mouse model (PyMT) to investigate the effects of doxorubicin and zoledronic acid on spontaneous mammary tumour growth and metastasis. 7-week-old PyMT/FVB mice were randomly divided into 4 treatment groups (n= 15/group). Mice were treated 1× per week for 6 weeks with 100 μl PBS (control), 2 mg/kg doxorubicin, 100 μg/kg zoledronic acid, or doxorubicin followed 24 h later by zoledronic acid. Mice were sacrificed 24 h after last treatment or when primary tumour volume reached 1.5 cm3. Primary tumour mass was recorded on sacrifice. Lungs were analysed for metastatic tumour nodules and hind limbs were assessed by X-ray and μ-CT for lytic lesions and bone metastasis. Mammary tumours were non-palpable in mice treated sequentially with doxorubicin followed by zoledronic acid and tumours could only be detected on resection. Administration of 2 mg/kg doxorubicin followed by 100 μg/kg zoledronic acid for 6 weeks significantly reduced mammary tumour growth compared with control. Mean mammary tumour mass was 1.34 ± 0.38 g following sequential treatment and 3.03 ± 0.21 g in control mice (p= 0.007). All mice receiving combination treatment survived the entire experiment, whereas, 40% of control animals were sacrificed early due to excessive tumour volume. Mammary tumour metastases were not detected in lungs or hind limbs of any animals. Sequential administration of doxorubicin followed 24 h later by zoledronic acid significantly reduces spontaneous mammary tumour growth and increases survival in immunocompetent PyMT mice.
Conflict of interest statement: R.E. Coleman: research funding; Novartis, consultancy: Novartis and Amgen, speaker fees: Novartin, Amgen. I. Holen: funding from Novartis and speaker fee from Novartis. doi:10.1016/j.bone.2010.10.118
P-63 The causal role of the BMP signalling antagonist noggin in suppressing bone formation in osteolytic bone metastasis C. Secondini*, A. Wetterwald, R. Schwaninger, G.N. Thalmann, M.G. Cecchini Department of Urology, University of Bern, Bern, Switzerland Department of Clinical Research, University of Bern, Bern, Switzerland Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. We previously reported that lack of expression of the BMP antagonist noggin by osteoinductive cell lines derived from mammary and prostate cancers is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic cell lines derived from the same cancer types express noggin constitutively. In multiple myeloma, the Wnt antagonist dickkopf-1 has been shown to participate to the osteolytic process by repressing bone formation. Therefore, we hypothesized that noggin may contribute by an equivalent mechanism to the osteolysis caused by solid cancers. An shRNA-mediated silencing strategy was used to investigate whether constitutive noggin expression participates to the osteolytic response induced by intra-osseous xenografts of the prostate cancer cell line PC-3. Tumour growth in vivo was monitored by bioluminescent imaging. Modifications in bone architecture were determined by radiography, micro-computed tomography, peripheral quantitative computed tomography and histomorphometry. The PC-3 cell-induced osteolytic lesions showed not only enhanced osteoclast-mediated bone resorption, but also decreased osteoblast-mediated bone formation. Noggin knock-down in PC-3 cells restored bone formation in the osteolytic lesions, thus reestablishing, at least partially, physiologic bone coupling. Furthermore, intra-osseous tumour growth of noggin-silenced PC-3 cells was limited by the restored osteoblast activity. In the PC-3 model, uncoupling of the bone remodelling process contributes to osteolysis, similarly to the mechanism shown in multiple myeloma. Constitutive noggin secretion by cancer cells mediates the repression of bone formation, at least in the PC-3 model. Cancer cell targeted noggin suppression restores bone formation and, as possible consequence of this, interferes with tumour growth. Therefore, noggin neutralization may be an additional therapeutic option to treat osteolytic bone metastases by prostate cancer. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.119
P-64 An exopolysaccharide produced by alteromonas infernus reduces lung metastasis and prolongs survival rate of osteosarcoma-bearing mice C. Ruiz Velascoa,*, S. Colliec-Jouaultb, C. Sinquinb, D. Heymanna, M. Padrinesa a INSERM U957, Université de Nantes, Nantes, France b IFREMER, Laboratoire de biotechnologie et molécules marines, Nantes, France Heparin, which is traditionally used as an anticoagulant, has a variety of additional biological activities. It has been shown in several
Abstracts / Bone 48 (2011) S22–S55
retrospective and prospective clinical trials to have an effect on cancer patient survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. Here, we investigated the in vivo effects of a “heparin-like”, an oversulfated exopolysaccharide (OS-EPS) produced by a mesophilic strain found in deep-sea hydrothermal vents, on lung metastasis progression and animal survival. We compared this effect with that of a non-oversulfated exopolysaccharide (EPS) and unfractionated heparin. An animal model of lung metastasis was developed in C3H/He mice inoculated intravenously with a spontaneous murine osteosarcoma POS-1 cell line. Lung metastasis was determined at the time of autopsy. Independent experiments (series treated with 6 mg/kg of EPS or OS-EPS or heparin) showed a significant increase of the survival rate in treated animals versus controls. The number of metastatic nodules was strongly decreased by sulfated polysaccharide (OS-EPS and heparin) treated mice as revealed by macroscopic analysis. In vitro, a 48-hour incubation with 25 μg/mL EPS or OS-EPS or heparin inhibited POS-1 cell line migration. Besides, the efficacy of polysaccharides was tested on primary bone tumor growth using a mouse-transplantable model of osteosarcoma. The preliminary results show that polysaccharides-treatment delayed local tumor growth, as compared to the untreated mice. In conclusion, we demonstrate that these sulfated polysaccharides significantly diminish osteosarcoma-induced lung metastasis in vivo, thereby prolonging survival of POS-1-inoculated animals. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.120
P-65 MNNG-HOS osteosarcoma growth is enhanced by human fat and adipose-derived mesenchymal stem cells: Implication in reconstructive surgery following tumor treatment L. Bourdaisa,b, P. Perrota,b, D. Duteillea,b, S. Hervoueta,b, J. Chesneaua,b, J. Amiauda,b, G. Picardaa,b, G. De Pigneuxc, F. Redinia,b, D. Heymanna,b,*, V. Tricheta,b a U957, INSERM, Nantes, France b EA3822, University, Nantes, France c EA3855, University, Tours, France Growth of osteosarcoma, the most frequent primary bone tumor, is supported by its microenvironment. We recently reported an unexpected local osteosarcoma relapse which occurred at the exact site of autologous fat grafts in a female patient who did not present any predictive factor of local recurrence (Perrot et al., PLoS ONE 5(6): e10999. doi:10.1371/journal.pone.0010999). Using SaOS2 human osteosarcoma induced in mice, we have shown that tumor growth was promoted by human fat injection. One hypothesis for this protumor effect may lie to the presence of mesenchymal stromal/stem cells (MSCs) in the injected fat. In this study, we tested human osteosarcoma MNNG-HOS cells rather than human SaOS2 cells because they led to a greater tumor incidence in mice (100% versus 50%) either by intra-muscular cell injection or by tumor fragment transplantation in close contact with bone. Fresh harvested human fat was injected in progressive tumors and was used to isolate MCSs. These human adipose-derived MSCs (hAD-MSCs) either unmodified or modified to express the firefly luciferase were co-injected with MNNG-HOS cells in mice. Tumor development and bone architecture were analyzed by tumor volume measurement and lung metastasis detection and by micro-computed tomography for small animal imaging, respectively. Histology and in vivo bioluminescence imaging were used to characterize MNNG-HOS tumors and to identify hAD-MSCs. MNNG-HOS is an aggressive osteosarcoma cell lines which induced a high tumor incidence, lung metastases and a strong osteolysis in both
S43
induction processes. Human fat injections and hAD-MSC co-injections increased significantly the tumor growth but did not increase metastasis and bone remodelling as compared to MNNG-HOS control tumors. This study presents a complete characterization of MNNG-HOS osteosarcoma in vivo. Moreover we confirm that modifications of the tumor microenvironment by fat graft may favour osteosarcoma growth and that this effect could be partially mediated by MSCs contained in fat graft. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.121
P-66 The novel and selective insulin-like growth factor-1 receptor kinase inhibitor PQIP suppresses bone cell function and osteoclast-breast cancer cell cross-talk in vitro J.G. Logana,*, E. Landao-Bassongaa, V.G. Bruntonb, A.I. Idrisa a Bone Research Group, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, Edinburgh, UK b Division of Cancer Biology, Edinburgh Cancer Research Centre, Institute of Genetics & Molecular Medicine, Edinburgh, UK Breast cancers commonly metastasise to the bone microenvironment, disrupting the bone remodelling cycle causing predominantly osteosclerotic metastases. Insulin like growth factor 1 (IGF-1) receptor blockers inhibit bone cell proliferation and suppress metastasis of breast cancer cells, but their effects on bone and tumour cell interaction are unknown. Here, we investigate the effects of PQIP, a novel, selective, IGF-1 receptor kinase inhibitor, on bone cell differentiation, proliferation and migration of the human breast cancer cell line MDA-MB-231 and bone–tumour cell interaction in vitro. Treatment of primary osteoblasts with IGF-1 (100 ng/ml) stimulated bone nodule formation. PQIP (200 nM) treatment resulted in a significant reduction in osteoblast differentiation (56%, p <0.05) and bone nodule formation (40%, p <0.05) in the presence and absence of IGF-1, without affecting cell viability. PQIP (200 nM) also suppressed both basal and IGF-1 induced migration of the osteoblast-like MC3T3-E1 and MDA-MB-231 cells. PQIP (200 nM) inhibited the increase in osteoclast number, size and multinuclearity in the presence and absence of IGF-1 or following treatment with MDA-MB-231 conditioned media, indicating a significant inhibitory effect on osteoclast–tumour cell interaction. Further mechanistic work showed that PQIP (200 nM) treatment completely abolished IGF-1 induced phosphorylation of AKT in osteoblast, osteoclast, and MDA-MB-231 cultures, confirming inhibition of IGF-1 signalling. Tellingly, treatment of osteoclast cultures with PQIP (200 nM) also significantly reduced AKT phosphorylation stimulated by treatment with MDA-MB-231 conditioned media (62%, p <0.05). In conclusion, the IGF-1 kinase inhibitor PQIP suppresses differentiation of bone and tumours cells and for the first time showed that IGF-1 receptor inhibition suppresses breast cancer cell support for osteoclastogenesis in vitro. This identifies PQIP as a novel agent, which along with other IGF-1 and insulin receptor inhibitors, may be of clinical value for the treatment for bone loss and metastases associated with breast cancer. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.122
P-67 Inhibition of Dickkopf-1 (DKK-1) delays prostate cancer growth in vivo through induction of P21CIP-1 C.L. Halla,*, H. Zhanga, S. Bailea, S. Kuhstossb, E.T. Kellera