Osteolytic Breast Cancer Research Articles

Wnt signaling induces gene expression of factors associated with bone destruction in lung and breast cancer.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

Abstract 4858: A novel strategy for the selective and tissue specific inhibition of MMPs in active breast cancer to bone metastases

Abstract Bone metastasis is a common event during breast cancer progression: The delicate balance between bone matrix synthesizing osteoblasts and bone resorbing osteoclasts activities is typically subverted by the presence of tumor cells at the bone site. The resulting incurable lesions cause extensive osteolysis and severely impact the patient's quality of life. Understanding how active breast-to-bone metastases manipulate the bone microenvironment is critical for the development of new therapies to treat the disease. We have recently shown in preclinical animal models of breast to bone metastases that MMP-2 proteolytic activity is critical for the growth of the tumor and the associated osteolysis. We therefore hypothesized that the selective inhibition of MMP-2 at the tumor site would be a potent means through which to prevent the progression of the malignancy. Given that previous broad-spectrum MMP inhibitor (MMPI) trials were unsuccessful mainly because of consistent side effects, we took a novel chemical approach to generate a selective MMPI that would preferentially target the skeleton. To this end, we utilized a bisphosphonate backbone (tiludronate) to synthesize an MMPI (BMMPI) with high specificity for MMP-2 (IC50 0.14± 0.04μM). To test the efficacy of the BMMPI in vivo, we inoculated 6-week old immunocompromized female mice with a luciferase expressing osteolytic breast cancer cell line, PyMT-Luc (105 cells). Following tumor injection, mice (n=6/group) received saline, tiludronate (25mg/kg, to control for bisphosphonate effects) or BMMPI (25mg/kg) sub-cutaneously, twice weekly. Tumor growth was measured by quantitating luminescence over time. Our results show that application of the BMMPI significantly reduced the breast cancer growth over a two-week period (p<0.05). Furthermore, using Faxitron analysis to determine the extent of tumor induced bone destruction, we observed significantly less osteolysis in the tibia images of BMMPI treated mice compared to the control group. Our initial studies also suggest that BMMPIs perform better than clinically used bisphosphonates such as tiludronate. We are currently following up our in vivo/ex vivo analyses with histomorphometrical and histology approaches. In conclusion, our data thus far demonstrate that an MMP-2 specific BMMPI may be an effective means to prevent the progression of breast to bone metastases while at the same time eliminating some of the noted off-target effects of broad-spectrum MMP inhibitors. Since bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be rapidly translated to the patient. Citation Format: Marilena Tauro, Antonio Laghezza, Paolo Tortorella, Conor C. Lynch. A novel strategy for the selective and tissue specific inhibition of MMPs in active breast cancer to bone metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4858. doi:10.1158/1538-7445.AM2014-4858

Abstract 1114: Combination of a novel CXCR4 antagonist with chemotherapy reduces breast cancer bone metastatic tumor burden

Abstract Background: Bone is the most common site of metastasis for patients with breast cancer. Tumor cells migrate to and reside in the protective bone marrow microenvironment niche through adhesive interaction between tumor CXCR4 and stromal CXCL12 (SDF1). CXCL12 is produced by activated osteoblasts, bone marrow and lung stromal cells, and endothelial cells. Nearly 60% of breast cancers express CXCR4 and this is associated with decreased survival. We hypothesized that a Protein Epitope Mimetic (PEM) POL5551, a novel CXCR4 antagonist, will limit the extent of tumor metastasis by disrupting stromal-mediated protection from cytotoxic chemotherapy and in turn may prolong survival. Approach: In vitro, POL5551 had no direct cytotoxic activity and did not reduce proliferation of CXCR4+ MDA-MB-231 or 4T1 osteolytic breast cancer cell lines. However, in an in vitro scratch-wound assay POL5551 inhibited migration of MDA-MB-231 cells. In a Gaussia luciferase (GLuc) complementation model in MDA-MB-231 cells, the interaction of CXCL12 with CXCR4, but not CXCR7, was blocked by low nanomolar concentrations of POL5551. At 20 mg/kg administered from day 10 post inoculation, POL5551 displayed no single agent activity on primary tumor xenografts, and in combination with eribulin there was no synergistic effect on primary tumor xenografts. However, continuation of treatment with POL5551 after surgical tumor removal decreased tumor metastasis and prolonged survival to 58 days compared to control at 45 days and eribulin alone at 51 days. Also, POL5551 showed effects on immune infiltration to the primary tumor. To test the hypothesis that CXCR4 antagonism disrupts the protective bone marrow niche in which tumor cells reside and sensitizes them to cytotoxic chemotherapy, we employed a “framing dosing strategy”. In a second model of metastasis produced by intracardiac injection of MDA-MB-231 cells, POL5551 (20 mg/kg, s.c.) was administered to mice with bone metastases 4 hours before and 4 & 18 hours after eribulin chemotherapy (0.2 mg/kg, i.v.). While bone is the predominant site of metastasis with these cell lines, lung and liver metastasis also occurs. POL5551 in combination with eribulin decreased bone tumor burden more than eribulin alone (reduction in leg bone & liver tumor burden, versus control respectively: eribulin alone 58% & 75%, n.s.; POL5551+eribulin: 89% & 86%, P<0.05; n=4-5 mice per group). Conclusion: These preclinical data support a synergism of CXCR4 antagonism during chemotherapy for the treatment of metastatic breast cancer. These and additional data support initiation of a Phase I clinical trial to evaluate a related PEM CXCR4 antagonist in combination with eribulin chemotherapy in patients with metastatic breast cancer who have failed at least 2 lines of therapy. Note: This abstract was not presented at the meeting. Citation Format: Jingyu Xiang, Michelle A. Hurchla, Kathryn Luker, Garry Douglas, Barbara Romagnoli, Eric Chevalier, Michael Bauer, Johann Zimmermann, Klaus Dembowsky, Gary Luker, Katherine N. Weilbaecher. Combination of a novel CXCR4 antagonist with chemotherapy reduces breast cancer bone metastatic tumor burden. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1114. doi:10.1158/1538-7445.AM2014-1114

Molecular Mechanisms of Bone Metastasis and Associated Muscle Weakness

Bone is a preferred site for breast cancer metastasis and leads to pathologic bone loss due to increased osteoclast-induced bone resorption. The homing of tumor cells to the bone depends on the support of the bone microenvironment in which the tumor cells prime the premetastatic niche. The colonization and growth of tumor cells then depend on adaptations in the invading tumor cells to take advantage of normal physiologic responses by mimicking bone marrow cells. This concerted effort by tumor cells leads to uncoupled bone remodeling in which the balance of osteoclast-driven bone resorption and osteoblast-driven bone deposition is lost. Breast cancer bone metastases often lead to osteolytic lesions due to hyperactive bone resorption. Release of growth factors from bone matrix during resorption then feeds a "vicious cycle" of bone destruction leading to many skeletal-related events. In addition to activity in bone, some of the factors released during bone resorption are also known to be involved in skeletal muscle regeneration and contraction. In this review, we discuss the mechanisms that lead to osteolytic breast cancer bone metastases and the potential for cancer-induced bone-muscle cross-talk leading to skeletal muscle weakness.

Pharmacologic inhibition of bone resorption prevents cancer-induced osteolysis but enhances soft tissue metastasis in a mouse model of osteolytic breast cancer.

Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily, which binds to the receptor activator of nuclear factor κB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was previously shown to inhibit tumor growth in bone and to prevent cancer-induced osteolysis. In this study, we examined the effect of OPG, when produced locally by breast cancer cells located within bone, using a mouse model of osteolytic breast cancer. MDA-MB-231-TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human OPG, were transplanted directly into the tibial marrow cavity of nude mice. Overexpression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis and diminished intra-osseous tumor growth but had no effect on extra-skeletal tumor growth. This effect was associated with a significant reduction in the number of osteoclasts that lined the bone surface, resulting in a net increase in bone volume. Despite limiting breast cancer-mediated bone loss, OPG overexpression resulted in a significant increase in the incidence of pulmonary metastasis. Our results demonstrate that inhibition of osteoclastic bone resorption by OPG when secreted locally by tumors in bone may affect the behaviour of cancer cells within the bone microenvironment and their likelihood of spreading and establishing metastasis elsewhere in the body.

Dickkopf-1 is regulated by the mevalonate pathway in breast cancer

IntroductionAmino-bisphosphonates and statins inhibit the mevalonate pathway, and may exert anti-tumor effects. The Wnt inhibitor dickkopf-1 (DKK-1) promotes osteolytic bone lesions by inhibiting osteoblast functions and has been implicated as an adverse marker in multiple cancers. We assessed the effects of mevalonate pathway inhibition on DKK-1 expression in osteotropic breast cancer.MethodsRegulation of DKK-1 by bisphosphonates and statins was assessed in human breast cancer cell lines, and the role of the mevalonate pathway and downstream targets was analyzed. Moreover, the potential of breast cancer cells to modulate osteoblastogenesis via DKK-1 was studied in mC2C12 cells. Clinical relevance was validated by analyzing DKK-1 expression in the tissue and serum of women with breast cancer exposed to bisphosphonates.ResultsDKK-1 was highly expressed in receptor-negative breast cancer cell lines. Patients with receptor-negative tumors displayed elevated levels of DKK-1 at the tissue and serum level compared to healthy controls. Zoledronic acid and atorvastatin potently suppressed DKK-1 in vitro by inhibiting geranylgeranylation of CDC42 and Rho. Regulation of DKK-1 was strongest in osteolytic breast cancer cell lines with abundant DKK-1 expression. Suppression of DKK-1 inhibited the ability of breast cancer cells to block WNT3A-induced production of alkaline phosphates and bone-protective osteoprotegerin in preosteoblastic C2C12 cells. In line with the in vitro data, treatment of breast cancer patients with zoledronic acid decreased DKK-1 levels by a mean of 60% after 12 months of treatment.ConclusionDKK-1 is a novel target of the mevalonate pathway that is suppressed by zoledronic acid and atorvastatin in breast cancer.

Abstract 2143: Anti-TGFβ antibody treatment rescues doxorubicin- mediated bone loss in preclinical osteolytic breast cancer metastasis models.

Abstract Anthracyclines (doxorubicin) are one of the most effective and widely used chemotherapeutic agent for the treatment of several cancer types, including early stage and metastatic breast cancer. However, doxorubicin has been known to cause long-term bone damage and growth defects in patients with childhood cancers, which was also confirmed by several preclinical studies. This raises concern regarding the usage of doxorubicin for the treatment of patients with metastatic breast cancer who are already at increased risk of osteolytic bone damage leading to severe bone loss due to. A number of clinical and preclinical studies have indicated that doxorubicin causes both long term and short term bone damages by lowering bone mineral density, increasing bone loss and may disrupt bone homeostasis by directly affecting bone marrow stem cells, however, the underlying mechanism is poorly understood. We have previously reported that treatment with doxorubicin increases circulating levels of TGFβ, a cytokine which has been implicated in the increase in osteoclast differentiation and survival, suppression of osteoblast differentiation and has been associated with advancement of breast cancer to bone metastasis. We hypothesize, breast cancer patients undergoing doxorubicin treatment will experience accelerated bone loss due to an increase in serum TGFβ levels and blocking TGFβ will rescue this bone loss, at least partially. We have tested the efficacy of anti-TGFβ antibody 1D11, in blocking doxorubicin-mediated bone damage in cardiac injection and orthotopic models of breast cancer to bone metastasis. X-ray image analysis revealed that doxorubicin treatment resulted in an eightfold increase in osteolytic lesion areas (P=0.002) and microCT analysis has shown almost 50% decrease in BV/TV (P=0.0005), in a preclinical murine model metastatic breast cancer. Doxorubicin treatment has also decreased BV/TV in non-tumor bearing animals, suggesting a direct damaging effect of this agent to bone at the sites which are not affected by cancer infestation. As anticipated, treatment with anti-TGFβ antibody 1D11 was able to rescue doxorubicin-mediated bone loss in all three preclinical models tested. At the cellular level, direct effect of doxorubicin treatment on the bone marrow microenvironment included decreased osteoblast survival and reduced mineralized matrix formation and increased osteoclast formation, thereby affecting both bone formation and bone resorption. Taken together, we conclude that, doxorubicin therapy decreases bone formation and increases bone resorption causing additional bone loss in patients with osteolytic bone disease and an anti-TGFβ therapy may be affective in controlling that bone loss. Citation Format: Tapasi Rana, Anwesa Chakrabarti, Michael Freeman, Swati Biswas. Anti-TGFβ antibody treatment rescues doxorubicin- mediated bone loss in preclinical osteolytic breast cancer metastasis models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2143. doi:10.1158/1538-7445.AM2013-2143

Quantitative Contrast-Enhanced Ultrasound for Imaging Antiangiogenic Treatment Response in Experimental Osteolytic Breast Cancer Bone Metastases

The aim of this study was to investigate the feasibility of using contrast-enhanced ultrasound (CEUS) in experimental breast cancer bone metastases and its utilization for assessment of early antiangiogenic treatment response in these lesions. Nude rats bearing bone metastases (n = 20) were treated with the antiangiogenic tyrosine kinase inhibitor sunitinib daily from days 30 to 35 after MDA-MB-231 tumor cell inoculation (n = 10) and compared with sham-treated controls (n = 10). Imaging with ultrasound (US) and magnetic resonance imaging (MRI) was performed on days 30, 32, and 35 after tumor cell inoculation to determine tumor volume and parameters of vascularization in bone metastases. Contrast-enhanced US images were used to calculate wash-in and wash-out values, peak enhancement, and area under the curve from time intensity curves. In addition, a quantitative analysis software was used to determine regional blood volume and flow as well as filling times within bone metastases. For comparison, dynamic contrast-enhanced MRI provided amplitude A and exchange rate constant kep, respectively. Immunohistological analysis of the vasculature in bone metastases followed in vivo imaging experiments. Although no changes in tumor volume were assessed in the observation time, significantly decreased values for peak enhancement, area under the curve, and wash-out were determined by CEUS in animals treated with sunitinib at day 35 after tumor cell inoculation. Analysis of CEUS images with quantitative analysis software showed significantly lower values for regional blood volume and regional blood flow as well as higher values for filling time in treated animals as early as 2 days after therapy onset. Dynamic contrast-enhanced MRI revealed significantly decreased values for parameter A at day 35 and kep at days 32 and 35 after tumor cell inoculation for treated animals. Immunohistology of bone metastases revealed significantly larger vessels and decreased positive area fraction for von Willebrand Factor in animals treated with sunitinib. Contrast-enhanced US is feasible in experimental breast cancer bone metastases and depicts early antiangiogenic treatment response in advanced osteolytic lesions.

Abstract 5313: Sustained hypercalcemia primes non-invasive breast cancer cells for metastasis to high calcium microenvironments

Abstract Breast cancer frequently spreads to calcium-rich organs/metastatic sites such as skeletal tissues but the role of hypercalcemia in breast cancer progression remains poorly understood. While patients with metastatic or end-stage cancer are likely to become hypercalcemic, up to 30% of breast cancer patients develop cancer-induced hypercalcemia (CIH) in the absence of metastases or bone diseases. The resulting high extracellular ionized calcium (Ca2+) activates the calcium sensing receptor (CaSR) that not only promotes the proliferation of breast cancer cells but also the secretion of parathyroid hormone-related protein by the tumor cells. This suggests that CIH may promote both the vicious osteolytic cycle and breast cancer progression. In this study, we examined whether sustained hypercalcemia primes breast cancer cells for metastasis to high Ca2+ microenvironments. We demonstrate that sustained hypercalcemia enables Ca2+-sensitive non invasive breast cancer cells such as MCF-7 to become high Ca2+-adapted and that this adaptation is accompanied by increased motility and more aggressive growth (increased proliferation). Consistent with this observation, invasive and metastatic breast cancer cells are high Ca2+-adapted cells that efficiently grow over a wide range of Ca2+ concentrations (up to 10 mM). Interestingly, we also show that at high Ca2+ (5 mM) breast cancer cells expressing the Q1011E CaSR variant that is common among African Americans proliferate faster than those expressing the A986S CaSR variant that is common among Caucasians. These data suggest that differences in the adaptation of breast cancer cells to hypercalcemia due in part to the expression of distinct CaSR variants underlie the distinct race-related breast cancer outcomes. This work is funded by the following grants made available to JO: DOD-W81XWH-07-1-0254 and 1SC1CA134018-01 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5313. doi:1538-7445.AM2012-5313

Abstract 5712: Alpharadin (Radium-223 chloride) completely prevents tumor growth in bone and increases survival in a mouse model of breast cancer bone metastases in preventive and micro-metastatic settings

Abstract Radium-223 chloride is an alpha-emitting radiopharmaceutical that has been shown to improve overall survival in the phase III clinical study (ALSYMPCA) in the treatment of castration resistant prostate cancer with bone metastases (Parker et al. ECCO/ESMO 2011, abstract LBA1). As a calcium mimetic, radium-223 localizes to bone, where the emission of alpha-particles provide an efficient and localized radiation treatment to metastatic skeletal tumor lesions. As previously reported, radium-223 decreases osteolysis and also tumor burden in established model of breast cancer bone metastasis in nude mice (Suominen et al. AACR 2011, abstract 2664). In this study, we investigated the effects of radium-223 on the development of breast cancer bone metastases when administered in the preventive and micro-metastastic settings. The effects of radium-223 were studied in a breast cancer bone metastatic model using intra-cardiac injection of human MDA-MB-231SA/GFP cells in nude mice at day 0. In the first study, the animals were randomized into four groups (n=7) and dosed with either vehicle or a single dose of radium-223 (300 kBq/kg) at days -1, 2 or 15. Three mice were also sacrificed at day 2 for immunohistochemical (pan-cytokeratin and GFP) detection of tumor cells in the bone marrow. Radiography and fluorescence imaging were performed at sacrifice (day 25). Tumor burden was also analysed from mid-sagittal sections of both hind limbs. The second study was a survival study, in which the animals were randomized to three groups (n=12) and dosed with either vehicle or a single dose of radium-223 (300 kBq/kg) at days -1 or 2. Radiography and fluorescence imaging were performed only on animals surviving to day 50. In the first study there were disseminated tumor cells in the bone marrow of all three animals (altogether 18/18 positive sections) sacrificed at day 2, as detected by IHC staining. Radium-223 decreased whole body tumor burden by 81, 84 and 55% and osteolysis by 98, 99.6 and 82%, when administered at days -1, 2 or 15, respectively. No tumor foci were detected in the bone sections of mice treated at days -1 and 2, and tumor area was decreased by 65% in animals treated at day 15. In the survival study, median survival was 24.5 days in the control group, 39.5 days (p <0.001 vs ctrl) in the group administered at day -1, and 35.5 days (p<0.001 vs ctrl) in the group administered at day 2. Three of twelve animals, all administered at day -1, survived until day 50 and two of them had only minimal residual disease. In summary, Alpharadin (radium-223) administered in a preventive or micro-metastatic setting completely prevented progression of osteolytic breast cancer bone metastases and increased survival in this preclinical model. These findings strongly support the clinical development of radium-223 for patients at risk of developing bone metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5712. doi:1538-7445.AM2012-5712

Sustained hypercalcemia primes non‐invasive breast cancer cells for metastasis to high calcium microenvironments

Breast cancer frequently spreads to calcium‐rich organs/metastatic sites such as skeletal tissues. While patients with metastatic or end‐stage cancer are likely to become hypercalcemic, up to 30% of breast cancer patients develop cancer‐induced hypercalcemia (CIH) in the absence of metastases or bone diseases. The resulting high extracellular ionized calcium (Ca2+) activates the calcium sensing receptor (CaSR) that promotes cell proliferation and the secretion of parathyroid hormone‐related protein by tumor cells. This suggests that CIH promotes both the vicious osteolytic cycle and breast cancer progression. In this study, we examined whether sustained hypercalcemia primes breast cancer cells for metastasis to high Ca2+ microenvironments. We demonstrate that sustained hypercalcemia enables Ca2+‐sensitive non invasive breast cancer cells such as MCF‐7 to become high Ca2+ adapted and that this adaptation is accompanied by increased motility and more aggressive growth. Moreover, breast cancer cells expressing the Q1011E variant (common among African Americans)grow faster at high Ca2+ than those expressing either the wild type or the A986S variant (common among Caucasians)of the CaSR. These data suggest that differences in the adaptation of breast cancer cells to hypercalcemia due in part to the expression of distinct CaSR variants underlie the distinct race‐related breast cancer outcomes.

CCN3 Impairs Osteoblast and Stimulates Osteoclast Differentiation to Favor Breast Cancer Metastasis to Bone

Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.

Abstract 2664: Alpharadin inhibits osteoclast differentiation in vitro and progression of established breast cancer bone metastases in vivo

Abstract Alpharadin is an alpha-emitting radium isotope currently in phase III clinical study for the treatment of castration resistant prostate cancer bone metastases. As a calcium mimetic Alphapharmaceutical, radium localizes to bone, where the emitted alpha-particles provide an efficient and localized radiation treatment to the metastatic skeletal tumor lesion. While promising clinical data in predominately osteoblastic bone metastases such as prostate cancer have already been obtained (Nilsson, Lancet Oncol 2007), robust clinical evidence for the activity of Alpharadin in osteolytic bone metastases associated with other cancer entities is lacking. As previously reported (Henriksen, Cancer Res 2002), Alpharadin increases symptom-free survival in an osteolytic breast cancer bone metastasis model in nude rats. Besides this study on the therapeutic efficacy in a preclinical skeletal metastasis tumor model it is still unclear how Alpharadin affects the vicious cycle of tumor growth and osteolysis. We report here the effects of Alpharadin on differentiation and activity of human osteoclasts in vitro and on treatment of established breast cancer bone metastases in vivo. The in vitro effects of Alpharadin (50-1600 Bq/ml, n=8/group) on osteoclasts were studied using human osteoclast precursor cells cultured on bovine bone slices. Alpharadin was added in the osteoclast differentiation assay at day 0, and in the activity assay at day 7. Secreted tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) was measured at day 7 as a marker of osteoclast differentiation and secreted C-terminal cross-linked telopeptides of type I collagen (CTX) at day 10 as a marker of resorption during days 7-10. The in vivo effects were studied using a model where human MDA-MB-231SA/GFP cells were inoculated intracardially in nude mice. The animals were randomized to two groups (n=15) based on body weight and presence of osteolytic lesions at day 14. Either vehicle or Alpharadin (300 kBq/kg) were administered iv at day 15. Radiography and fluorescence imaging were performed at day 14 and at sacrifice (day 25). Alpharadin inhibited dose dependently differentiation of osteoclasts in vitro at all tested concentrations, but had no effect on resorption activity of mature osteoclasts. In vivo, Alpharadin decreased whole body tumor burden by 43% as quantified by fluorescence imaging and osteolysis by 56% compared to the vehicle control group. Furthermore, Alpharadin treatment completely prevented tumor associated cachexia observed in the control group. In summary, Alpharadin inhibits differentiation of osteoclasts and progression of established, osteolytic breast cancer bone metastases. These preclinical findings strongly support the clinical development of Alpharadin in additional cancer indications which form predominantely osteolytic bone metastasis, e.g. breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2664. doi:10.1158/1538-7445.AM2011-2664

Longitudinal live animal micro-CT allows for quantitative analysis of tumor-induced bone destruction

The majority of breast cancer and prostate cancer patients with metastatic disease will go on to develop bone metastases, which contribute largely to the patient's morbidity and mortality. Numerous small animal models of cancer metastasis to bone have been developed to study tumor-induced bone destruction, but the advancement of imaging modalities utilized for these models has lagged significantly behind clinical imaging. Therefore, there is a significant need for improvements to live small animal imaging, particularly when obtaining high-resolution images for longitudinal quantitative analyses. Recently, live animal micro-computed tomography (μCT) has gained popularity due to its ability to obtain high-resolution 3-dimensional images. However, the utility of μCT in bone metastasis models has been limited to end-point analyses due to off-target radiation effects on tumor cells. We hypothesized that live animal in vivo μCT can be utilized to perform reproducible and quantitative longitudinal analyses of bone volume in tumor-bearing mice, particularly in a drug treatment model of breast cancer metastasis to bone. To test this hypothesis, we utilized the MDA-MB-231 osteolytic breast cancer model in which the tumor cells are inoculated directly into the tibia of athymic nude mice and imaged mice weekly by Faxitron (radiography), Imtek μCT ( in vivo), and Maestro (GFP-imaging). Ex vivo μCT and histology were performed at end point for validation. After establishing a high-resolution scanning protocol for the Imtek CT, we determined whether clear, measurable differences in bone volume were detectable in mice undergoing bisphosphonate drug treatments. We found that in vivo μCT could be used to obtain quantifiable and longitudinal images of the progression of bone destruction over time without altering tumor cell growth. In addition, we found that we could detect lesions as early as week 1 and that this approach could be used to monitor the effect of drug treatment on bone. Taken together, these data indicate that in vivo μCT is an effective and reproducible method for longitudinal monitoring of tumor-associated bone destruction in mouse models of tumor-induced bone disease.

Matrix Metalloproteinase (MMP)-13 Regulates Mammary Tumor–Induced Osteolysis by Activating MMP9 and Transforming Growth Factor-β Signaling at the Tumor-Bone Interface

Abstract The tropism of breast cancer cells for bone and their tendency to induce an osteolytic phenotype are a result of interactions between breast cancer cells and stromal cells and are of paramount importance for bone metastasis. However, the underlying molecular mechanisms remain poorly understood. We hypothesize that tumor-stromal interaction alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic breast cancer bone metastasis and that inhibition of such interactions can be developed as targeted therapeutics. Microarray analysis was performed to investigate gene expression profiling at the tumor-bone (TB) interface versus the tumor alone area from syngenic mice injected with three different syngenic mammary tumor cell lines that differ in their metastatic potential. We identified matrix metalloproteinase 13 (MMP13), receptor activator of NF-κB ligand (RANKL), and integrins binding sialoprotein to be genes upregulated at the TB interface and validated. To determine the functional role of MMP13 in tumor-induced osteolysis, mice with Cl66 mammary tumors were treated with MMP13 antisense oligonucleotides (MMP13-ASO) or control scrambled oligonucleotides (control-ASO). Knockdown of MMP13 expression at the TB interface leads to significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. Further analysis to evaluate the mechanism of MMP13-dependent osteolytic bone metastasis revealed that MMP13-ASO treatment decreased active MMP9, RANKL levels, and transforming growth factor-β signaling at the TB interface. Together, our data indicate that upregulation of MMP13 at the TB interface is important in tumor-induced osteolysis and suggest that MMP13 is a potential therapeutic target for breast cancer bone metastasis. Cancer Res; 70(9); 3494–504. ©2010 AACR.

Abstract 484: Treatment strategy to reverse osteolytic breast cancer metastasis using osteoblasts in a murine xenograft model

Abstract The most common site of breast cancer metastasis is bone. The majority of these metastases are osteolytic, characterized by an increase in bone degradation via an upregulation of osteoclast activity. These effects lead to complications including fracture, contractile pain and hypercalcemia, significantly reducing the quality of life in these patients. The role of bone forming osteoblasts at sites of osteolytic lesions is less understood. We have recently shown that breast cancer cells are capable of inducing apoptosis of osteoblasts in vitro as well as in vivo using murine xenograft models. This effect would further exacerbate osteolytic disease, and may explain why treatment strategies capable of inhibiting osteolysis (bisphosphonates) are not capable of repairing lost bone. We hypothesize that modulating the number of osteoblasts at sites of osteolytic metastases, may aid in the repair of resorbed bone. To provide proof-of-principle evidence, we have engineered the murine pre-osteoblast cell line, MC3T3-E1, to overexpress Bcl2, conferring resistance to apoptosis. These cells will be used with an in vivo model of osteolysis using GFP-labeled breast cancer cells previously reported by our laboratory. Apoptosis resistant MC3T3-E1 will be injected into bone both before, and after induction of osteolysis and rates of bone resorption and repair will be measured. The results of this project will provide the first in vivo evidence that restoration of osteoblasts at sites of osteolytic metastases can aid in the repair of lost bone and will test for the first time, experimental manipulation of osteoblast number and function in osteolytic breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 484.

Doxorubicin Therapy Causes Additional Bone Loss in Preclinical Osteolytic Breast Cancer Model

Introduction: Treatment of myeloma (MY) bone disease with bisphosphonates (BP) is standard of care. But renal toxicity due to BP treatment tends to be a major problem in MY patients, especially. Patients with reduced renal function (RF) and patients who had switched BP drugs are at highest risk. Toxicity appears typically after few month of treatment. We evaluated safety data about RF from MY patients, who were treated with ibandronate (IBD). The patients were stratified according to pretreatment and RF.Methods: In a prospective noninterventional study (NIS) about safety and efficacy of IBD in breast cancer patients it turned out that unintentionally MY patients were also included. Out of 3528 documented patients 93 MY patients were identified. We evaluated the data from these MY patients separately. The patients were subdivided in 4 groups according to their RF: GFR>90 (1), 60-90 (2), 30-59 (3) and <30 (4) ml/min. Since pretreatment with or without BP could be of influence, patients were analysed according to former pretreatment: no BP (I), IBD (II), and other BP (III). The kidney function was calculated every month over a period of six month. Results: 93 patients were available for evaluation. In 87 patients RF was documented over a minimum of 5 months. The initial RF was (1) n=11, (2) n=34, (3) n=36 and (4) n=12, respectively. There were no differences in renal function according to their pre-treatment. A total number of 620 infusions were documented. The IBD dosage was 6 mg in 89% of all infusions, 4 mg in 4%, 3 mg in 2% and 2 mg in 5%, respectively. In the subgroups (1-4) RF was stable over time (mean GFR+//0-SD ; difference over the time [ml/min]: (1): 116,8+//0-20,9; -3,38; (2): 74,7+//0-8.4; 2,04; (3): 46,1+//0-8,1; 3,7; (4): 20,4+//0-7; 13,37; all changes n.s.), but there was a trend for improved RF in patients with GFR<30 ml/min. The mean changes were similar in all groups irrespectively of the pretreatment. In 8 patients the treatment was terminated prematurely, due to disease progression (n=3), death due to MY during study period (n=2). Two patients were lost by follow up and one patient declined further treatment. Discussion: The data of this NIS suggest, that there is no evidence for renal toxicity of IBD in MY patients in all stages of renal function. Patients with worse kidney function (stage 4) had a trend of improvement of RF over the study period, however, the changes were not significant due to the limited number of patients.

Osteoblasts suppress high bone turnover caused by osteolytic breast cancer in-vitro

The skeleton is the most common site of breast cancer metastasis, which can occur in up to 85% of patients during their lifetime. The morbidity associated with bone metastases in patients with breast cancer includes pathological fractures, bone pain, hypercalcaemia, and spinal cord compression. When breast cancer metastasizes to bone, the balance of bone resorption (mediated by osteoclasts) and bone formation (mediated by osteoblasts) favors bone resorption, which leads to net bone destruction ( i.e., osteolysis). Anti-resorptive agents such as bisphosphonates are commonly used to treat bone resorption in osteoporosis or osteolytic cancer patients. However, bisphosphonates by themselves are unable to rebuild lost bone tissue, and can cause severe side effects. In this study, we developed a bovine bone explant culture system and have observed that murine osteoblasts can modulate the activity of osteotropic human breast cancer cells on this substrate. Using markers of bone metabolism, we observe diminished bone turnover in organ culture following the addition of exogenous osteoblasts. The data presented in this study supports further investigation into the use of cytotherapies to limit breast cancer mediated osteolysis.

Imaging anti-angiogenic treatment response with DCE-VCT, DCE-MRI and DWI in an animal model of breast cancer bone metastasis

As current classification systems for the assessment of treatment response in bone metastasis do not meet the needs of oncologists, new imaging biomarkers are desirable. Therefore, the diagnostic impact of dynamic contrast enhanced (DCE)-volumetric computed tomography (VCT) (descriptive analysis), DCE-MRI (two-compartment model) and diffusion weighted imaging (DWI) for monitoring anti-angiogenic therapy effects of the VEGF antibody bevacizumab in breast cancer bone metastases in rats was studied. Nude rats ( n = 8 animals treated with bevacizumab and n = 9 untreated control rats) with site-specific osteolytic bone metastasis of the hind leg were imaged with a 1.5 T clinical MRI-scanner in an animal coil as well as in a volumetric CT-scanner at days 30, 40, 50 and 60 after inoculation of MDA-MB-231 human breast cancer cells. From these data, osteolytic lesion size (OLS), peak enhancement (PE), area under the curve (AUC), amplitude ( A), exchange rate constant ( k ep) and apparent diffusion coefficient (ADC) were determined in bone metastases. Prior to changes in OLS ( p ≤ 0.05 at days 50 and 60) there was already a significant decrease in PE, AUC and A ( p ≤ 0.05 at days 40–60) in treated animals compared to controls. However, for k ep and ADC there were no significant differences between the groups at any time point ( p > 0.05 at days 40–60). In conclusion, anti-angiogenic treatment response in osteolytic breast cancer bone metastases can be assessed early with surrogate markers of vascularization, while DWI appears to be insensitive.

CT Fluoroscopy–guided Percutaneous Vertebroplasty for the Treatment of Osteolytic Breast Cancer Metastases: Results in 62 Sessions with 86 Vertebrae Treated

This retrospective study aimed to assess the results of computed tomographic (CT) fluoroscopy-guided percutaneous vertebroplasty (PV) of painful osteolytic spinal metastases from breast cancer, focusing on the frequency and clinical impact of polymethylmethacrylate (PMMA) leaks. Within 48 months, 53 patients (52 women; mean age of 62 y +/- 13) with painful osteolytic breast cancer metastases underwent vertebroplasty. Eighty-six vertebrae were treated in 62 sessions under CT fluoroscopy guidance (single slice and four- and 16-row CT). In the planning CT scan, osteolytic destruction (ie, none, < or =25%, < or =50%, < or =75%, or < or =100%) was assessed regarding the vertebral cross-sectional area, the cortical border of the spinal canal, and the outer circumference. CT performed after vertebroplasty was used to detect local PMMA leaks (intradiscal, intraspinal, paravertebral, intercostovertebral/posterolateral, and vascular). Patient charts were reviewed with respect to adverse events. Clinical outcome was assessed on a visual analog scale (VAS) 24 hours before, immediately after, and 6 months after PV. Overall, 37.2%, 12.8%, and 1.2% of vertebrae (N = 86) showed at least a 50% osteolytic involvement of the cross-sectional area, spinal canal, and outer vertebral cortex, respectively. Intradiscal, intraspinal, paravertebral, and intercostovertebral/posterolateral leaks were seen in 31.3%, 26.9%, 26.9%, and 14.9% of vertebrae, respectively. The ratio of basivertebral to segmental venous leaks was 22.4%/23.9%. No major complications (eg, radiculopathy or pulmonary embolism) occurred. VAS scores decreased significantly (P < .05) from 6.4 at 24 hours before PV to 3.4 at a mean follow-up of 9.2 months. PV of osteolytic breast cancer metastases can be performed safely under CT fluoroscopic guidance even with substantial involvement of the vertebral cross-sectional area or cortical bone. A high clinical success rate was achieved and cortical and vascular PMMA leaks had no impact.