Model Of Breast Cancer Bone Metastasis Research Articles

1,25(OH)2D3 deficiency increases TM40D tumor growth in bone and accelerates tumor-induced bone destruction in a breast cancer bone metastasis model

Breast cancer is one of the most common malignancies and bone is the commonest site of distant metastases. Evidences indicate that adequate supply of vitamin D will decrease the morbidity and mortality of breast cancer. However, the main role of vitamin D deficiency in breast cancer bone metastases remains unclear. In this study, the relationship between vitamin D and breast cancer bone metastases were evaluated. Results showed that 1,25(OH)2D3 can not only inhibit the proliferation, migration and invasion of breast cancer cell TM40D in vitro, but also attenuate the breast cancer cell TM40D-induced bone destruction in vivo, whose underlying mechanism was at least partially through decreasing the number of the osteoclasts. To our knowledge, this is the first to use 1-alpha-hydroxylase [1α(OH)ase] knockout mice which characterized vitamin D deficiency to establish the breast cancer bone metastases model. Based on this model, we also found that vitamin D deficiency will accelerate the osteolytic lesions, and 1,25(OH)2D3 supplement will restrain osteolytic lesions. Therefore, these findings suggest that vitamin D has the potential capacity to be a therapeutic agent for the breast cancer bone metastases.

Abstract P6-18-01: Site-specific activation of curcuminoids in the breast cancer bone metastases microenvironment

Abstract The majority of women with advanced breast cancer (BCa) develop incurable osteolytic bone metastases. Our laboratory has previously demonstrated that curcuminoids, bioactive compounds isolated from turmeric rhizomes, prevent the development of lytic bone lesions in a murine xenograft model of human BCa bone metastasis and inhibit tumor cell secretion of TGFβ-stimulated parathyroid-related protein (PTHrP), a signaling pathway known to drive bone metastasis progression. In both humans and mice, glucuronidated phase II metabolites of curcumin, which are thought to be biologically inactive, are the primary form detected in circulation after consuming curcuminoid-containing foods or supplements. This has led to an untested postulate that curcuminoids may be deglucuronidated at sites of action to form bioactive, aglycone (free) curcumin. Studies were therefore undertaken to test this hypothesis in the context of the murine BCa bone metastasis model, assessing site-specific deconjugation of curcuminoids in the tumor-bone microenvironment. Effects of curcumin-glucuronide (G-CURC) vs. curcumin (CURC) on TGFβ-stimulated PTHrP secretion by bone-tropic MDA-MB-231 (MDA) cells was determined by immunoradiometric assay. G-CURC and CURC levels were quantified by LC-MS in plasma and bone marrow specimens isolated from curcuminoid-treated female nude mice. Endogenous β-glucuronidase enzyme expression was localized by immunohistochemical (IHC) staining in paraffin-embedded sections of decalcified, MDA metastases-containing hind limbs of nude mice inoculated, via intracardiac injection 21 days prior, with MDA cells. Glucuronide deconjugation activity of bone marrow lysates was determined by colorimetric assay. In contrast to the inhibitory effects of a naturally occurring curcuminoid mixture or pure CURC, G-CURC did not alter TGFβ-stimulated PTHrP secretion, confirming its postulated lack of biologic activity. In mice treated with curcuminoids, the majority of circulating curcumin was conjugated (91%). In contrast, the majority of curcumin in the bone marrow (56%) was unconjugated free (aglycone) curcumin (p <0.01). IHC staining of MDA tumor-bearing hind limbs of nude mice demonstrated expression of β-glucuronidase (GUSB), an enzyme that deconjugates compounds in mice and humans, by bone marrow cells, but not by tumor cells. Consistent with IHC, bone marrow from female nude mice displayed significantly higher (30-fold) deconjugation enzyme activity compared to MDA cells (p < 0.01). Female C3H/HeJ mice, which possess a partial loss-of-function mutation in the GUSB gene, had significantly decreased (66% lower) deconjugation enzyme activity compared to normal female C57BL/6 (p < 0.001) or nude (p < 0.01) mice. In addition, treatment with the β-glucuronidase inhibitor, saccharolactone (SL), also decreased deconjugation enzyme activity in marrow lysates from female nude mice. These results suggest that curcuminoids, in the setting of breast cancer bone metastases, may act as a pro-drug, becoming activated within the tumor-bone microenvironment by glucuronidase-expressing hematopoietic bone marrow cells, to limit the progression of osteolytic lesion formation in a murine model of BCa bone metastasis. Supported by: R01CA174926-01, R01AT006896, and R03CA159382. Citation Format: Kunihiro AG, Frye JB, Brickey JA, Luis PB, Schneider C, Funk JL. Site-specific activation of curcuminoids in the breast cancer bone metastases microenvironment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-18-01.

Redox and pH dual sensitive bone targeting nanoparticles to treat breast cancer bone metastases and inhibit bone resorption.

Bone is an especially prone metastatic site for breast cancer, and to block the vicious cycle between bone resorption and tumor growth is an important strategy for the treatment of breast cancer bone metastasis. In this paper, pH- and redox-sensitive as well as breast cancer bone metastasis-targeting nanoparticles (DOX@ALN-(HA-PASP)CL) were prepared, and also their anti-tumor activity and anti-bone resorption effect were investigated in detail. The in vitro experimental results indicated that DOX released from DOX@ALN-(HA-PASP)CL exhibited a GSH-, DTT- and pH-dependent manner. Moreover, in an in vitro 3D breast cancer bone metastasis model, DOX@ALN-(HA-PASP)CL decreased bone resorption through inhibiting the proliferation of human breast cancer cells (MDA-MB-231 cells) and reducing the activity of osteoclasts. The in vivo experimental results indicated that a large amount of DOX was delivered to a breast cancer bone metastasis site after tumor-bearing mice were treated with DOX@ALN-(HA-PASP)CL; meanwhile, DOX@ALN-(HA-PASP)CL significantly decreased the tumor volume and bone resorption in tumor-bearing mice without causing obvious systemic toxicity. In conclusion, the in vitro and in vivo experimental results indicate that DOX@ALN-(HA-PASP)CL has great potential in the treatment of breast cancer bone metastasis.

Doxorubicin-Mediated Bone Loss in Breast Cancer Bone Metastases Is Driven by an Interplay between Oxidative Stress and Induction of TGFβ

Breast cancer patients, who are already at increased risk of developing bone metastases and osteolytic bone damage, are often treated with doxorubicin. Unfortunately, doxorubicin has been reported to induce damage to bone. Moreover, we have previously reported that doxorubicin treatment increases circulating levels of TGFβ in murine pre-clinical models. TGFβ has been implicated in promoting osteolytic bone damage, a consequence of increased osteoclast-mediated resorption and suppression of osteoblast differentiation. Therefore, we hypothesized that in a preclinical breast cancer bone metastasis model, administration of doxorubicin would accelerate bone loss in a TGFβ-mediated manner. Administration of doxorubicin to 4T1 tumor-bearing mice produced an eightfold increase in osteolytic lesion areas compared untreated tumor-bearing mice (P = 0.002) and an almost 50% decrease in trabecular bone volume expressed in BV/TV (P = 0.0005), both of which were rescued by anti-TGFβ antibody (1D11). Doxorubicin, which is a known inducer of oxidative stress, decreased osteoblast survival and differentiation, which was rescued by N-acetyl cysteine (NAC). Furthermore, doxorubicin treatment decreased Cu-ZnSOD (SOD1) expression and enzyme activity in vitro, and treatment with anti-TGFβ antibody was able to rescue both. In conclusion, a combination therapy using doxorubicin and anti-TGFβ antibody might be beneficial for preventing therapy-related bone loss in cancer patients.

Abstract 2050: BAY 1125976, a highly selective and potent allosteric AKT1/2 inhibitor, for the treatment of cancers with aberrations in the PI3K-AKT-mTOR pathway.

Abstract The PI3K/AKT/mTOR pathway is frequently activated in human cancer. AKT, a central element in the pathway, is essential for tumor growth, proliferation, survival, invasion and metastasis. Activation of AKT is a key mechanism in resistance to chemo-, radio- and targeted therapies. Thus, AKT is considered an attractive drug target. Herein, we report on the preclinical profile and combinability of BAY 1125976, a potent, highly selective, allosteric AKT1/2 inhibitor, which is particularly effective in models with PI3K-AKT pathway aberrations. In biochemical assays, BAY 1125976 demonstrates equal potency against AKT1 and AKT2 in the low nanomolar range (IC50 ∼ 10 nM) while it displays weaker activity against AKT3 (IC50 ∼ 500 nM) and is inactive against ∼230 other protein/ lipid kinases (IC50 > 1 μM). Mechanistically, BAY 1125976 blocks AKT signalling by inhibiting the phosphorylation of AKT at both Thr308 and Ser473 (IC50 < 1 nM), as well as downstream phosphorylation of 4E-BP1 (IC50 < 50 nM). The strong inhibition of cellular p-AKT and downstream signalling translates to a broad inhibition of tumor cell proliferation in vitro. In particular, tumor cell lines carrying defects in the tumor suppressor PTEN, or oncogenic mutations in PIK3CA are most sensitive to BAY 1125976 treatment. Daily oral dosing of BAY 1125976 in human xenograft tumor models induces strong pharmacodynamic inhibition of AKT phosphorylation that correlates with drug exposure. In vivo, BAY 1125976 demonstrates dose-dependent anti-tumor efficacy in multiple xenograft tumor models of different histological types with PIK3CA mutations or PTEN deletions while being well tolerated. BAY 1125976 can be effectively combined with various anti-cancer therapies. In vitro combination profiling shows synergistic anti-proliferative effects with anti-hormonal therapeutics in breast and prostate cancer cell lines, which translates to enhanced anti-tumor efficacy with durable tumor regressions in vivo. Furthermore, in vivo combination of BAY 1125976 with external beam radiation results in strong additive to synergistic efficacy and significant tumor growth delay. Moreover, the combination of BAY 1125976 with the bone-targeting agent Radium 223 in a breast cancer bone metastasis model results in reduced tumor and metastases burden and increased necrotic and fibrotic bone area. In conclusion, BAY 1125976 is a highly selective, potent allosteric AKT1/2 inhibitor with strong in vitro and in vivo activity in tumor models with activated AKT signalling and strong synergistic activity in combination. Targeting AKT might also provide a promising strategy for overcoming chemo/radio-resistance and increasing radio-sensitization and radio-potentiation. Citation Format: Oliver Politz, Lars Baerfacker, Stuart Ince, William J. Scott, Roland Neuhaus, Ulf Boemer, Martin Michels, Dominik Mumberg, Franz von Nussbaum, Karl Ziegelbauer, Andrea Haegebarth. BAY 1125976, a highly selective and potent allosteric AKT1/2 inhibitor, for the treatment of cancers with aberrations in the PI3K-AKT-mTOR pathway. [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 2050. doi:10.1158/1538-7445.AM2013-2050

Abstract 3947: AMG 161, a fully human monoclonal antibody to human RANKL, inhibits tumor-induced osteoclastogenesis and reduces skeletal tumor burden in mice that express chimeric (murine / human) RANKL.

Abstract Tumor cells can induce RANKL expression in the bone stroma, causing activation of osteoclasts which consequently mediate bone breakdown and subsequent tumor proliferation. RANKL is an essential mediator of osteoclast function and survival. RANKL inhibition by OPG-Fc can prevent tumor-induced osteolysis, decrease skeletal tumor burden, and increase survival in preclinical models. Denosumab is an anti-huRANKL antibody approved for the prevention of skeletal-related events due to bone metastases from solid tumors. Selective anti-huRANKL antibodies do not bind murine RANKL, therefore efficacy cannot be tested in typical mouse models. We developed a breast cancer bone metastasis model in mice in which exon 5 from murine RANKL was replaced with its human ortholog (“mu/huRANKL”). We tested the effect of AMG 161, an antibody which binds huRANKL and chimeric mu/huRANKL, on tumor burden and associated osteoclasts. After intra-cardiac injection of MCF-7Luc breast carcinoma cells, hind limb metastases formed and tumor burden was tracked by bioluminescence imaging (BLI). AMG 161 (0.3 or 3.0 mg/kg, 2x/week), OPG-Fc (3.0 mg/kg, 2x/week) or vehicle were administered beginning 10 days post tumor injection to treat established bone metastases until study termination at day 31. AMG 161 resulted in a dose-dependent decrease in skeletal tumor burden (3.0 mg/kg: 84% decrease vs. control p=0.0052) similar to that observed with OPG-Fc (85% decrease vs. control, p=0.0038). Histological analysis at the end of the study showed reductions in skeletal tumor burden consistent with the BLI. AMG 161 treatment resulted in a significant decrease in tumor-associated osteoclasts (TRAP staining) and inhibition of osteolytic lesions (X-ray). The inhibition of tumor burden by the anti-RANKL antibody was indirect via osteoclast inhibition since MCF-7Luc cells do not express RANK, and RANKL inhibition does not affect subcutaneous growth of MCF-7Luc cells. These data support the efficacy of anti-RANKL antibody treatment to inhibit tumor-induced osteoclastogenesis and to block the growth of bone metastases. Citation Format: Jude R. Canon, Rebecca Bryant, Martine Roudier, William C. Dougall. AMG 161, a fully human monoclonal antibody to human RANKL, inhibits tumor-induced osteoclastogenesis and reduces skeletal tumor burden in mice that express chimeric (murine / human) RANKL. [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 3947. doi:10.1158/1538-7445.AM2013-3947

RANKL inhibition combined with tamoxifen treatment increases anti-tumor efficacy and prevents tumor-induced bone destruction in an estrogen receptor-positive breast cancer bone metastasis model

Tumor cells in bone can induce the activation of osteoclasts, which mediate bone resorption and release of growth factors and calcium from the bone matrix, resulting in a cycle of tumor growth and bone breakdown. Targeting the bone microenvironment by the inhibition of RANKL, an essential mediator of osteoclast function, not only prevents tumor-induced osteolysis but also decreases skeletal tumor burden in preclinical models. The inhibition of skeletal tumor progression after the inhibition of osteoclasts is via interruption of the "vicious cycle" of tumor/bone interactions. The majority of breast cancer patients at risk for bone metastases harbor estrogen receptor-positive (ER+) tumors. We developed a mouse model for ER+ breast cancer bone metastasis and evaluated the effect of RANKL inhibition on tumor-induced osteolysis and skeletal tumor growth both alone and in combination with tamoxifen. Luciferase-labeled MCF-7 cells (MCF-7Luc) formed metastatic foci in the hind limbs following intracardiac injection and caused mixed osteolytic/osteoblastic lesions. RANKL inhibition by OPG-Fc treatment blocked osteoclast activity and prevented tumor-induced osteolysis, as well as caused a marked decrease in skeletal tumor burden. Tamoxifen as a single agent reduced MCF-7Luc tumor growth in the hind limbs. In a combination experiment, OPG-Fc plus tamoxifen resulted in significantly greater tumor growth inhibition than either single agent alone. Histologic analysis revealed a decrease in the proliferation of tumor cells by both single agents, which was enhanced in the combination treatment. Upon treatment with OPG-Fc alone or in combination with tamoxifen, there was a complete absence of osteolytic lesions, demonstrating the ability of RANKL inhibition to prevent skeletal related morbidity in an ER+ model. The combination approach of targeting osteoclasts and the bone microenvironment by RANKL inhibition and the tumor directly via hormonal therapy may provide additional benefit to reducing skeletal tumor progression in ER+ breast cancer patients.

Oncolytic Adenovirus Expressing Soluble TGFβ Receptor II-Fc-mediated Inhibition of Established Bone Metastases: A Safe and Effective Systemic Therapeutic Approach for Breast Cancer

In recent years, oncolytic adenoviruses have shown some promise as a novel class of antitumor agents. However, their utility in targeting bone metastases is relatively less studied. We have examined whether the systemic therapy of oncolytic adenoviruses expressing the soluble form of transforming growth factor-β (TGFβ) receptor II fused with human immunoglobulin G1 can be developed for the treatment of established breast cancer bone metastases. MDA-MB-231-luc2 human breast cancer cells were injected in the left heart ventricle of nude mice to establish bone metastasis. Mice with hind limb tumors were administered (on days 8 and 11) oncolytic adenoviruses-Ad.sTβRFc or mhTERTAd.sTβRFc. Skeletal tumor growth was monitored weekly by bioluminescence imaging (BLI) and radiography. At the termination time on day 28, hind limb bones were analyzed for tumor burden, synchrotron micro-computed tomography, and osteoclast activation. Intravenous delivery of Ad.sTβRFc and mhTERTAd.sTβRFc induced significant inhibition of tumor growth, reduction of tumor burden, osteoclast activation, and increased animals' survival. Oncolytic adenoviruses were safer than dl309, a wild-type virus. A slight elevation of liver enzyme activity was observed after Ad.sTβRFc administration; this subsided with time. Based on these studies, we believe that Ad.sTβRFc and mhTERTAd.sTβRFc can be developed as a safe and effective approach for the treatment of established bone metastasis.

Systemic Delivery of a Novel Liver-Detargeted Oncolytic Adenovirus Causes Reduced Liver Toxicity but Maintains the Antitumor Response in a Breast Cancer Bone Metastasis Model

We are interested in developing oncolytic adenoviruses for the treatment of bone metastasis of cancer. A key limitation of systemic delivery of oncolytic adenovirus type 5 (Ad5) is that the majority of the virus is taken up by the liver, causing liver damage and systemic toxicity. Given that Ad5 hexon binding with blood coagulation factor X is a key factor in liver sequestration, and that a rare serotype, Ad48, has a diminished capacity to bind with factor X, we have generated mHAd.luc2, a novel hexon-chimeric oncolytic adenovirus. To create mHAd.luc2, seven hypervariable regions of Ad5 hexon were substituted with the corresponding regions from Ad48. Compared with Ad5-based oncolytic virus Ad.luc2, intravenous injection of mHAd.luc2 into nude mice resulted in significantly reduced liver uptake. A single high dose (1.0×10(11) viral particles/mouse) of Ad.luc2 resulted in 100% animal death by day 3; whereas none of the mice died in the mHAd.luc2 group. Liver enzyme and liver pathology studies indicated that mHAd.luc2 induced significantly less liver toxicity compared with Ad.luc2. Both mHAd.luc2 and Ad.luc2 exhibited similar binding with breast tumor cells, whereas in the presence of factor X, mHAd.luc2 binding was reduced. Both mHAd.luc2 and Ad.luc2 had nearly equal replication potential in breast cancer cells in vitro. Intravenous injection of mHAd.luc2 and Ad.luc2 into nude mice bearing bone metastases resulted in uptake of the viruses into skeletal tumors, and induced significant inhibition of established bone metastases. Thus, liver-detargeted oncolytic adenovirus can be developed for the treatment of breast cancer bone metastasis.

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

SU11248 inhibits tumor growth and CSF-1R-dependent osteolysis in an experimental breast cancer bone metastasis model.

The aim of the study was to investigate inhibitory effects of the receptor tyrosine kinase (RTK) inhibitor SU11248 against CSF-1R and osteoclast (OC) formation. We developed an in vivo model of breast cancer metastasis to evaluate efficacy of SU11248 against tumor growth and tumor-induced osteolysis in bone. The in vitro effects of SU11248 on CSF-1R phosphorylation, OC formation and function were evaluated. Effects on 435/HAL-Luc tumor growth in bone were monitored by in vivo bioluminescence imaging (BLI), and inhibition of osteolysis was evaluated by measurement of serum pyridinoline (PYD) concentration and histology. Phosphorylation of the receptor for M-CSF (CSF-1R) expressed by NIH3T3 cells was inhibited by SU11248 with an IC50 of 50-100 nM, consistent with CSF-1R belonging to the class III split kinase domain RTK family. The early M-CSF-dependent phase of in vitro murine OC development and function were inhibited by SU11248 at 10-100 nM. In vivo inhibition of osteolysis was confirmed by significant lowering of serum PYD levels following SU11248 treatment of tumor-bearing mice (P = 0.047). Using BLI, SU11248 treatment at 40 mg/kg/day for 21 days showed 64% inhibition of tumor growth in bone (P = 0.006), and at 80 mg/kg/day showed 89% inhibition (P = 0.001). Collectively, these data suggest that SU11248 may be an effective and tolerated therapy to inhibit growth of breast cancer bone metastases, with the additional advantage of inhibiting tumor-associated osteolysis.