Jukka P. Rissanen

Properties and expression of human tartrate-resistant acid phosphatase isoform 5a by monocyte-derived cells

Human serum tartrate‐resistant acid phosphatase exists as two enzyme isoforms (TRACP 5a and 5b), derived by differential, post‐translational processing of a common gene product. Serum TRACP 5b is from bone‐resorbing osteoclasts (OC) and becomes elevated in diseases of increased bone resorption. TRACP 5a is secreted by macrophages (MΦ) and dendritic cells (DC) and is increased in many patients with rheumatoid arthritis. Our purpose was to fully characterize the properties of human TRACP isoforms and to produce an antibody specific to TRACP 5a for use as a biomarker in chronic inflammatory diseases. Partially purified, natural serum TRACP isoforms and recombinant TRACP 5a (rTRACP 5a) were compared with respect to specific activity and subunit structure and presence of sialic acid. Mice were immunized with rTRACP 5a, and resulting hybridomas were screened for monoclonal antibody to serum TRACP 5a. One antibody, 220, was tested for its epitope specificity and use in various immunological techniques. rTRACP 5a had properties identical to serum TRACP 5a. Antibody 220 was specific for the trypsin‐sensitive epitope in the loop peptide, present only in TRACP 5a. Antibody 220 was effective for specific immunoprecipitation, immunoassay, and immunoblot of TRACP 5a. Intact TRACP was present in MΦ, DC, and OC. TRACP 5a was the predominant isoform secreted by MΦ and DC, whereas TRACP 5b was the predominant isoform secreted by OC. TRACP isoforms 5a and 5b may have different functions inside and outside of monocyte‐derived cells. Antibody 220 is an important resource for studies of the biosynthetic relationship among TRACP isoforms and of the significance of serum TRACP 5a as a marker in diseases of bone metabolism and inflammation.

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.

Effects of bioactive peptide, valyl-prolyl-proline (VPP), and lactobacillus helveticus fermented milk containing VPP on bone loss in ovariectomized rats.

Background: Valyl-prolyl-proline (VPP), a bioactive peptide formed during the fermentation with Lactobacillus helveticus LBK-16H (L. helveticus), has been shown to increase bone formation in vitro. The aim of the study was to determine whether VPP and L. helveticus fermented milk prevent bone loss in ovariectomized (OVX) rats. Methods: During the 12-week intervention study, the OVX rats received VPP in water or L. helveticus fermented milk, containing VPP. Sham-operated rats receiving water acted as controls. The trabecular and cortical bone mineral density were determined by peripheral quantitative computed tomography before the operation and at 4 and 12 weeks. The mechanical testing and ash weight analysis as well as the static and dynamic histomorphometrical parameters were assessed at the end of the intervention. Results: VPP given in water showed no clear effect on bone loss. L. helveticus fermented milk prevented bone loss by decreasing bone turnover and increasing the bone mineral density. Ovariectomycaused a 57% loss in the trabecular bone, which was attenuated by 16% in the L. helveticus group. Conclusions: VPP peptide did not prevent ovariectomy-induced bone loss, which could be due to the poor bioavailability of VPP from water solution. L. helveticus fermented milk prevented bone loss, whether this is due to the VPP peptide cannot be concluded.

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.

Heparin-like Polysaccharides Reduce Osteolytic Bone Destruction and Tumor Growth in a Mouse Model of Breast Cancer Bone Metastasis

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β 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-β–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-β–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-β 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. Mol Cancer Res; 10(5); 597–604. ©2012 AACR.

Improved methods for testing antiresorptive compounds in human osteoclast cultures

We cultured human bone marrow-derived stem cells on bovine bone slices in 96-well plates in the presence of M-CSF and RANKL, allowing them to differentiate into osteoclasts. Secreted TRACP 5b was a useful endpoint measurement to demonstrate effects of inhibitors of osteoclast differentiation in the culture system, reflecting accurately the number of formed osteoclasts. Inhibitors of osteoclast activity were added into the cultures after the differentiation period, and the cultures were continued to allow the formed osteoclasts to resorb bone. CTX values obtained after the resorption period were normalized with TRACP 5b values obtained after the differentiation period, before adding the inhibitors. This normalization prevents false results that could be obtained from the presence of different amounts of osteoclasts in different wells before adding the inhibitors. These results demonstrate that the use of TRACP 5b and CTX allows rapid and reliable testing of antiresorptive compounds in human osteoclast cultures.

Models and screening assays for drug discovery in osteoporosis

Importance of the field: Osteoporosis affects nearly 100 million people in Europe, Japan and the US, and the number is increasing due to aging of the population. Preclinical efficacy studies performed according to regulatory guidelines are large, long and expensive, and there is a need for guidance and recommendations on how to perform preliminary studies prior to the regulatory studies. Areas covered in this review: We review research models that can be used for preclinical efficacy testing of new drug candidates for osteoporosis. Our focus is on testing compounds targeted to directly decrease osteoclastic bone resorption or increase osteoblastic bone formation. What the reader will gain: We provide an overview of in vitro bone cell culture systems and osteoporosis animal models useful for preclinical efficacy studies and a step-by-step approach on how the most interesting compound can be selected from thousands of drug candidates. Different approaches for testing anti-catabolic and anabolic compounds are provided. Take home message: Efficacy of new osteoporosis drug candidates can be first proven conveniently using in vitro bone cell cultures and then confirmed in short-term animal studies, followed by more extensive animal studies, and finally a regulatory study performed according to the guidelines of regulatory authorities.

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

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Abstract 1803: A new spectrum-selective cathepsin inhibitor, VBY-825, inhibits bone destruction in a syngeneic 5TGM1 multiple myeloma mouse model

Multiple myeloma (MM) is the second most common blood cancer after non Hodgkin lymphoma. It is a monoclonal B-cell neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, fractures and hypercalcemia. Chemo- or radiotherapy may induce remissions, but MM is generally thought to be incurable. Our aim was to observe the effects of a cathepsin inhibitor VBY-825 on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model using immunocompetent C57BL/KaLwRij mice. VBY-825 is a potent inhibitor of cathepsins K, L, B, V, and S. 5TGM1 cells were inoculated via tail vein in 7 weeks old female C57BL/KaLwRij mice, which were divided to 4 groups: Control group received vehicle of VBY-825 (5% dextrose 10 ml/kg daily), Control group received bortezomib vehicle (3 ml/kg twice a week), Reference group received bortezomib (0.5 mg/kg twice a week) and Study group received VBY-825 (100 mg/kg daily). Administration of all compounds began one day before tumor cell inoculation and continued until day 34. Disease progression was followed by measuring the serum levels of paraprotein (IgG2b) and TRACP 5b, radiography, and body weight. The animals were sacrificed 5 weeks after inoculation, examined macroscopically, and their bones were collected for histomorphometric analysis. The reference compound bortezomib had no effects on body weight but it delayed the disease progression based on IgG2b measurements. It also decreased the number and total area of osteolytic lesions, but not mean osteolytic lesion area (MOLA). VBY-825 had no effect on body weight or IgG2b level, frequency of soft tissue tumors or intraosseous tumor area. VBY-825 decreased total and MOLA, consistent with inhibited resorption. There was also a trend of increased relative trabecular bone area. Serum TRACP 5b activity in the VBY-825 treated group did not differ from the respective vehicle group, whereas the number of osteoclasts at tumor-bone interface was increased in VBY-825 treated animals. These findings suggest that VBY-825 decreased osteoclast function and resorption activity without decreasing the number of osteoclasts In conclusion, VBY-825 had no effects on tumor growth but it inhibited bone destruction in this mouse model of MM, which is consistent with its potency on cathepsin S and K, which are known to be important in osteoclast-mediated bone resorption. VBY-825 is a promising candidate for the treatment of tumor-associated bone disease. Citation Format: Mari I. Suominen, Johanna Tuomela, Esa Alhoniemi, Katja M. Fagerlund, Jukka P. Rissanen, Jussi M. Halleen, Leslie J. Holsinger. A new spectrum-selective cathepsin inhibitor, VBY-825, inhibits bone destruction in a syngeneic 5TGM1 multiple myeloma mouse model. [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 1803. doi:10.1158/1538-7445.AM2014-1803

Abstract 5202: Additive benefits of radium-223 dichloride and bortezomib combination in a syngeneic 5TGM1 multiple myeloma mouse model

Radium-223 dichloride (radium-223, Xofigo®), a targeted alpha-therapy, improves overall survival in prostate cancer patients with bone metastases. It inhibits disease progression by reducing tumor growth and tumor-induced pathological bone reaction in breast and prostate cancer mouse models. Radium-223 is actively incorporated into the bone matrix by osteoblasts. Multiple myeloma (MM) is characterized by increased osteoclast and reduced or no osteoblast activity. Bortezomib (Velcade®), a treatment for MM, restores the impaired osteoblast activity in MM. Here, we report the effects of radium-223, bortezomib and their combination on myeloma cell proliferation in vitro and on myeloma bone disease model in mice. Proliferation assays were performed with human plasma cell leukemia (JJN-3, L-363), human MM (LP-1, MOLP-8, RPMI-8226 and OPM-2), and mouse MM (5TGM1) cells. Corresponding in vivo effects were studied in a syngeneic 5TGM1 mouse MM model. Female C57BL/KaLwRij mice (7 weeks old, n=15/group) were inoculated with 5TGM1 cells via tail vein and 26 days later, radium-223 (300 kBq/kg, single iv injection) and/or bortezomib (1 mg/kg ip, twice a week; total of 3 doses) or vehicle control were administered. The development of osteolytic lesions was detected by radiography. Hind limbs were used for histological analyses and total activity measurement was performed by a gamma-counter. TRAP-stained osteoclasts were counted at tumor-bone interface. Bortezomib inhibited proliferation of all cancer cell lines tested at 25 nM (JJN3 and OPM-2 at 2.5 nM) and radium-223 at 0.8 kBq/ml (L-363 and MOLP-8 at 0.2 kBq/ml) concentrations. Additive effects were observed with combination treatment in vitro. The 5TGM1 in vivo model demonstrated that both bortezomib and radium-223 decreased osteolytic lesion area as monotherapy (p In conclusion, radium-223 dichloride (Xofigo®) therapy in combination with bortezomib decreased osteolytic lesion area and almost completely eradicated tumor-associated osteoclasts in a mouse model of myeloma bone disease. Incorporation of radium-223 to bone matrix was improved, possibly via induction of osteoblast activity by bortezomib. These data suggest that combination of radium-223 and bortezomib could be a new effective therapy in MM, which is currently being investigated in a Phase Ib/II trial in patients with early relapsed MM (NCT02928029). Citation Format: Mari I. Suominen, Jukka P. Rissanen, Anniina Luostarinen, Katja M. Fagerlund, Birgitta Sjoholm, Esa Alhoniemi, Sanna-Maria Kakonen, Dominik Mumberg, Jussi M. Halleen, Karl Ziegelbauer, Arne Scholz. Additive benefits of radium-223 dichloride and bortezomib combination in a syngeneic 5TGM1 multiple myeloma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5202. doi:10.1158/1538-7445.AM2017-5202