Zoledronic Acid Prevents Bone Resorption Caused by the Combination of Radium-223, Abiraterone Acetate, and Prednisone in an Intratibial Prostate Cancer Mouse Model.

An increased risk of non-pathological fractures in patients with prostate cancer and bone metastases has been associated with combination treatment with radium-223, abiraterone, and prednisone/prednisolone in the absence of bone-protecting agents. Here, we investigated possible mechanisms leading to this outcome using an intratibial LNCaP model mimicking prostate cancer bone metastases. Male NOD.scid mice were inoculated intratibially with LNCaP prostate cancer cells and treated with vehicle, radium-223, abiraterone, prednisone, zoledronic acid, or their combinations for 28 days. Serum TRACP 5b and PSA levels were measured. Bone structure, quality, and formation rate of non-tumor-bearing and tumor-bearing tibiae were analyzed by microCT, 3-point bending assay, and dynamic histomorphometry, respectively. Radium-223 incorporation into bone was also measured. Radium-223/abiraterone/prednisone combination treatment induced a transient increase in bone resorption indicated by elevated TRACP 5b levels, which was inhibited by concurrent treatment with zoledronic acid. Furthermore, radium-223/abiraterone/prednisone combination reduced periosteal and trabecular new bone formation and the number of osteoblasts, but bone structure or biomechanical quality were not affected. The abiraterone/prednisone treatment decreased radium-223 incorporation into tumor-bearing bone, possibly explaining the lack of additional antitumor efficacy. In conclusion, radium-223/abiraterone/prednisone combination increased bone resorption, which may have been one of the mechanisms leading to an increased fracture risk in patients with mCRPC.

Enhanced Antitumor Efficacy of Radium-223 and Enzalutamide in the Intratibial LNCaP Prostate Cancer Model.

Radium-223 dichloride and enzalutamide are indicated for metastatic castration-resistant prostate cancer and their combination is currently being investigated in a large phase 3 clinical trial. Here, we evaluated the antitumor efficacy of radium-223, enzalutamide, and their combination in the intratibial LNCaP model mimicking prostate cancer metastasized to bone. In vitro experiments revealed that the combination of radium-223 and enzalutamide inhibited LNCaP cell proliferation and showed synergistic efficacy. The combination of radium-223 and enzalutamide also demonstrated enhanced in vivo antitumor efficacy, as determined by measuring serum PSA levels in the intratibial LNCaP model. A decreasing trend in the total area of tumor-induced abnormal bone was associated with the combination treatment. The serum levels of the bone formation marker PINP and the bone resorption marker CTX-I were lowest in the combination treatment group and markedly decreased compared with vehicle group. Concurrent administration of enzalutamide did not impair radium-223 uptake in tumor-bearing bone or the ability of radium-223 to inhibit tumor-induced abnormal bone formation. In conclusion, combination treatment with radium-223 and enzalutamide demonstrated enhanced antitumor efficacy without compromising the integrity of healthy bone. The results support the ongoing phase 3 trial of this combination.

Additive Benefits of Radium-223 Dichloride and Bortezomib Combination in a Systemic Multiple Myeloma Mouse Model.

Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor-bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease.

The Mode-of-Action of Targeted Alpha Therapy Radium-223 as an Enabler for Novel Combinations to Treat Patients with Bone Metastasis.

Bone metastasis is a common clinical complication in several cancer types, and it causes a severe reduction in quality of life as well as lowering survival time. Bone metastases proceed through a vicious self-reinforcing cycle that can be osteolytic or osteoblastic in nature. The vicious cycle is characterized by cancer cells residing in bone releasing signal molecules that promote the differentiation of osteoclasts and osteoblasts either directly or indirectly. The increased activity of osteoclasts and osteoblasts then increases bone turnover, which releases growth factors that benefit metastatic cancer cells. In order to improve the prognosis of patients with bone metastases this cycle must be broken. Radium-223 dichloride (radium-223), the first targeted alpha therapy (TAT) approved, is an osteomimetic radionuclide that is incorporated into bone metastases where its high-linear energy transfer alpha radiation disrupts both the activity of bone cells and cancer cells. Therefore, radium-223 treatment has been shown preclinically to directly affect cancer cells in both osteolytic breast cancer and osteoblastic prostate cancer bone metastases as well as to inhibit the differentiation of osteoblasts and osteoclasts. Clinical studies have demonstrated an increase in survival in patients with metastatic castration-resistant prostate cancer. Due to the effectiveness and low toxicity of radium-223, several novel combination treatment strategies are currently eliciting considerable research interest.

Radium-223 Inhibits Osseous Prostate Cancer Growth by Dual Targeting of Cancer Cells and Bone Microenvironment in Mouse Models.

Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models.Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12-17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology.Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects.Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335-46. ©2017 AACR.

Survival benefit with radium-223 dichloride in a mouse model of breast cancer bone metastasis.

BACKGROUND: Bone metastases are associated with increased morbidity and poor prognosis in breast cancer patients. Radium-223 dichloride is a calcium mimetic that localizes to bone, providing targeted therapy for skeletal metastasis. METHODS: We investigated the mode of action of radium-223 dichloride using breast cancer cell, osteoclast, and osteoblast cultures as well as a mouse model of breast cancer bone metastasis. A single dose of radium-223 dichloride was used in three different settings mimicking the prevention or treatment of bone metastasis. Disease progression was monitored using fluorescence and radiographic imaging and histological analyses. The effect of radium-223 dichloride alone and in combination with doxorubicin or zoledronic acid on survival of mice was analyzed by Kaplan-Meier methods. All statistical tests used were two-sided. RESULTS: Radium-223 dichloride incorporated into bone matrix and inhibited proliferation of breast cancer cells and differentiation of osteoblasts and osteoclasts (all P values < .001) in vitro. In an established bone metastasis setting, radium-223 dichloride prevented tumor-induced cachexia (0/14 vs 7/14 control mice) and decreased osteolysis by 56% and tumor growth by 43% (all P values < .05). Radium-223 dichloride induced double-strand DNA breaks in cancer cells in vivo. Finally, radium-223 dichloride extended survival as a monotherapy (29.2 days, 95% confidence interval [CI] = 26.6 to 31.8 days, P = .039) and in combination with zoledronic acid (31.4 days, 95% CI = 28.8 to 34.0 days, P = .004) or doxorubicin (31.5 days, 95% CI = 29.5 to 33.5 days, P < .001) compared to the vehicle group (24.9 days, 95% CI = 23.4 to 26.4 days). Similar but even more pronounced effects were observed when radium-223 dichloride was administered in a preventive or micrometastatic setting. CONCLUSIONS: Our findings strongly support the development of radium-223 dichloride for the treatment of breast cancer patients with or at high risk of developing bone metastases.

Identification of microRNAs inhibiting TGF-ß-induced IL-11 production in bone metastatic breast cancer cells.

Development of bone metastases is dependent on the cancer cell-bone cell interactions in the bone microenvironment. Transforming growth factor ß (TGF-ß) is released from bone during osteoclastic bone resorption and induces production of osteolytic factors, such as interleukin 11 (IL-11), in breast cancer cells. IL-11 in turn increases osteolysis by stimulating osteoclast function, launching a vicious cycle of cancer growth and bone destruction. We aimed to identify and functionally characterize microRNAs (miRNAs) that mediate the bone metastatic process, focusing on miRNAs that regulate the TGF-ß induction of IL-11. First, we profiled the expression of 455 miRNAs in a highly bone metastatic MDA-MB-231(SA) variant as compared to the parental MDA-MB-231 breast cancer cell line and found 16 miRNAs (3.5%) having a >3-fold expression difference between the two cell types. We then applied a cell-based overexpression screen with Pre-miRNA constructs to functionally identify miRNAs regulating TGF-ß-induced IL-11 production. This analysis pinpointed miR-204, miR-211, and miR-379 as such key regulators. These miRNAs were shown to directly target IL11 by binding to its 3' UTR. MiR-379 also inhibited Smad2/3/4-mediated transcriptional activity. Gene expression analysis of miR-204 and miR-379-transfected cells indicated that these miRNAs downregulated the expression of several genes involved in TGF-ß signaling, including prostaglandin-endoperoxide synthase 2 (PTGS2). In addition, there was a significant correlation between the genes downregulated by miR-379 and a set of genes upregulated in basal subtype of breast cancer. Taken together, the functional evidence and clinical correlations imply novel mechanistic links between miRNAs and the key steps in the bone metastatic process in breast cancer, with potential clinical relevance.

Heparin-like polysaccharides reduce osteolytic bone destruction and tumor growth in a mouse model of breast cancer bone metastasis.

TGF-ß regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-ß is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-ß-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-ß-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-ß induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.

Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis.

Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, an understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidences for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L: -serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L: -serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L: -serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L: -serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions.

D-18F-fluoromethyl tyrosine imaging of bone metastases in a mouse model.

UNLABELLED: The presence and localization of metastatic bone lesions is important for the staging of the disease and subsequent treatment decisions. Detecting tumor cells would have additional value over the current indirect bone scintigraphy method for detecting areas of elevated skeletal metabolic activity. d-(18)F-fluoromethyl tyrosine (d-(18)F-FMT) has recently shown good uptake and fast elimination, resulting in good tumor-to-background ratios. The potential of d-(18)F-FMT for imaging bone metastases has been investigated. METHODS: 786-O/luciferase human renal adenocarcinoma cells were injected intracardially, resulting in the formation of bone metastases in mice. Small-animal PET was performed 51 and 65 d after tumor cell inoculation. RESULTS: d-(18)F-FMT showed specific uptake in the bone metastases, giving excellent images with a little background in the pancreas. All imaged metastases were histologically confirmed. A bone scan with (18)F-fluoride showed elevated skeletal metabolic activity in the areas of osteolytic lesions. CONCLUSION: d-(18)F-FMT is a useful PET tracer for the detection of bone metastases and should be evaluated in the clinical setting.

Characterization of a new renal cell carcinoma bone metastasis mouse model.

Metastatic bone disease caused by renal cell carcinoma (RCC) occurs frequently and becomes more and more prevalent presumably because survival times among patients with disseminated cancers are increasing. Patients with bone metastases from renal cell carcinoma suffer from severe pain, nerve compression syndromes and pathologic fractures. Very little is known about the mechanisms of skeletal metastases of RCC. Thus, to better understand the molecular mechanism of renal cell cancer (RCC) bone metastasis, it is crucial to develop new animal models. We have established a new animal model of RCC metastasis to bone by inoculation of human 786-O/luciferase cells into the left cardiac ventricle of athymic nude mice. The animals developed aggressive osteolytic bone destruction as monitored by radiography and micro-CT-scans with the mean endpoint at 62 +/- 8 days. The extensive bone destruction observed was comparable to the clinical setting and mainly occurred in hind limbs, forelimbs and the spine. The tumors were primarily located within the bone and resulted in destruction of cortical bone. No soft tissue metastases were detected by BLI or histomorphometry. To increase the bone-metastatic potential of the 786-O cell line, an in vivo selection was done yielding a subpopulation causing osteolytic lesions with the mean endpoint of 47 +/- 3 days. The selected subline secreted more proangiogenic factors VEGF and bFGF in vitro compared to the parental cell line suggesting that these tumors are highly vascular. This model provides a reliable reproduction of the clinical situation and therefore, is suitable for designing and evaluating more effective treatments for RCC bone metastasis.

Luminescent imaging technology as an opportunity to reduce and refine animal experiments: Light at the end of the tunnel?

In vivo luminescent imaging technology was introduced in experimental life science research several years ago and has rapidly gained wide acceptance. By making use of this technology substantially more information can be gained from animal experiments than was previously possible. The concept of the 3Rs describes the aim to Refine, Reduce and Replace animal models in research. The goal of the present paper is to systematically investigate the impact of luminescent imaging on the 3Rs. In particular, three examples of applications are explained in detail so as to be accessible to the reader unfamiliar with the procedure. The examples are subsequently analysed for and categorised according to their concrete effect on animal welfare as defined by the 3Rs.

Sagopilone inhibits breast cancer bone metastasis and bone destruction due to simultaneous inhibition of both tumor growth and bone resorption.

PURPOSE: Bone metastases have a considerable impact on quality of life in patients with breast and other cancers. Tumors produce osteoclast-activating factors, whereas bone resorption promotes the growth of tumor cells, thus leading to a "vicious cycle" of bone metastasis. Sagopilone, a novel, fully synthetic epothilone, inhibits the growth of breast cancer cells in vitro and in vivo, and here we report its activity in the MDA-MB-231(SA) breast cancer bone metastasis mouse model. EXPERIMENTAL DESIGN: The potency of sagopilone was determined in treatment models simulating the adjuvant (preventive) and metastatic (therapeutic) settings in the clinic. RESULTS: We showed that sagopilone inhibited tumor burden and bone destruction, in addition to reducing tumor-induced cachexia and paraplegia. The reduction in osteolytic lesions, tumor growth in bone, and weight loss was statistically significant in the preventive model compared with the vehicle group. In the therapeutic model, sagopilone treatment significantly lowered the number of activated osteoclasts and significantly reduced the osteolytic lesion area, bone volume loss, and bone resorption compared with vehicle treatment while simultaneously inhibiting tumor burden. An in vitro assay confirmed that sagopilone inhibited osteoclast activation without cytotoxic effects, whereas paclitaxel resulted in lower inhibition and high levels of cytotoxicity. CONCLUSIONS: Sagopilone seems to inhibit the vicious cycle at both the tumor growth and bone resorption stages, suggesting the possibility for substantial benefit in the treatment of patients with breast cancer at risk from bone metastases or with bone lesions already present. Phase II clinical trials with sagopilone in patients with breast cancer are ongoing.

Mechanisms of osteolytic bone metastases in breast carcinoma.

Osteolytic and osteoblastic metastases are often the cause of considerable morbidity in patients with advanced prostate and breast carcinoma. Breast carcinoma metastasis to bone occurs because bone provides a favorable site for aggressive behavior of metastatic cancer cells. A vicious cycle arises between cancer cells and the bone microenvironment, which is mediated by the production of growth factors such as transforming growth factor beta and insulin growth factor from bone and parathyroid hormone-related protein (PTHrP) produced by tumor cells. Osteolysis and tumor cell accumulation can be interrupted by inhibiting any of these limbs of the vicious cycle. For example, bisphosphonates (e.g., pamidronate, ibandronate, risedronate, clodronate, and zoledronate) inhibit both bone lesions and tumor cell burden in bone in experimental models of breast carcinomametastasis. Neutralizing antibodies to PTHrP, which inhibit PTHrP effects on osteoclastic bone resorption, also reduce osteolytic bone lesions and tumor burden in bone. Other pharmacologic approaches to inhibit PTHrP produced by breast carcinoma cells in the bone microenvironment also produce similar beneficial effects. Identification of the molecular mechanisms responsible for osteolytic metastases is crucial in designing effective therapy for this devastating complication.

Transforming growth factor-beta stimulates parathyroid hormone-related protein and osteolytic metastases via Smad and mitogen-activated protein kinase signaling pathways.

Transforming growth factor (TGF)-beta promotes breast cancer metastasis to bone. To determine whether the osteolytic factor parathyroid hormone-related protein (PTHrP) is the primary mediator of the tumor response to TGF-beta, mice were inoculated with MDA-MB-231 breast cancer cells expressing a constitutively active TGF-beta type I receptor. Treatment of the mice with a PTHrP-neutralizing antibody greatly decreased osteolytic bone metastases. There were fewer osteoclasts and significantly decreased tumor area in the antibody-treated mice. TGF-beta can signal through both Smad and mitogen-activated protein (MAP) kinase pathways. Stable transfection of wild-type Smad2, Smad3, or Smad4 increased TGF-beta-stimulated PTHrP secretion, whereas dominant-negative Smad2, Smad3, or Smad4 only partially reduced TGF-beta-stimulated PTHrP secretion. When the cells were treated with a variety of protein kinases inhibitors, only specific inhibitors of the p38 MAP kinase pathway significantly reduced both basal and TGF-beta-stimulated PTHrP production. The combination of Smad dominant-negative blockade and p38 MAP kinase inhibition resulted in complete inhibition of TGF-beta-stimulated PTHrP production. Furthermore, TGF-beta treatment of MDA-MB-231 cells resulted in a rapid phosphorylation of p38 MAP kinase. Thus, the p38 MAP kinase pathway appears to be a major component of Smad-independent signaling by TGF-beta and may provide a new molecular target for anti-osteolytic therapy.