Sanna-Maria Käkönen

Strong Prediction of Fractures Among Older Adults by the Ratio of Carboxylated to Total Serum Osteocalcin

We examined serum total osteocalcin (TOC), carboxylated osteocalcin (COC), and their ratio (COC/TOC) by one‐step two‐site immunofluorescent assays in 87% (n = 792) of all home‐dwelling persons of 70 years or older living in a defined area in northern Finland. Other baseline subject‐related risk factors of fractures were assessed by postal questionnaires, interviews, clinical examinations, and tests. During a 5‐year follow‐up period, all falls and fractures (n = 106) were recorded by regular phone calls and by examining all the medical records yearly. Serum TOC and COC concentrations increased with advancing age and were higher in women than in men, but corresponding differences were not found in the case of COC/TOC. The adjusted relative risk of fracture was elevated in association with low (≤−1 SD from the mean) COC; hazard ratio (HR, 95% CI) 2.00 (1.20‐3.36) and low COC/TOC; HR 5.32 (3.26‐8.68), the relative risk being highest in the population older than 80 years; and HR 7.02 (2.42‐20.39). The predictive value of low COC/TOC lasted 3 years. The multivariable‐adjusted relative risk of hip fracture (n = 26) in regard to low COC/TOC ratio was 3.49 (1.12‐10.86), as compared with the persons who did not suffer hip fractures. Our results suggest that serum COC concentrations and, more strongly, COC/TOC, predict the occurrence of fractures in older community‐dwelling adults. The risk of fracture associated with low COC/TOC equals the hip fracture risk previously verified for concomitant high serum undercarboxylated OC concentrations and low bone mineral density.

Characterization of Serum Tartrate-Resistant Acid Phosphatase and Development of a Direct Two-Site Immunoassay†

Osteoclasts secrete tartrate‐resistant acid phosphatase (TRAP) to the circulation, where the amount of TRAP is expected to correlate with the bone resorption rate. We have developed two monoclonal antibodies, O1A and J1B, using purified human bone TRAP as antigen. The antibodies recognized different epitopes, allowing us to develop a two‐site fluoroimmunoassay. The immunoreactivity in fresh serum specimens was less than 10% of the concentrations measured from the same specimens after 24 h of storage at 4°C, or after addition of 5 mM EDTA or EGTA to them. When fresh serum was gel filtrated using Sephacryl S‐200 column, all of the enzyme eluted in the void volume as a complex with a molecular weight of more than 250 kDa. If the serum was treated with EDTA before the gel filtration, the complex was destroyed and the enzyme eluted in fractions corresponding to a molecular weight of 30 kDa, the size of monomeric purified human bone TRAP. The immunoassay was used to measure TRAP concentrations from serum samples that had been stored at 4°C for 24 h. According to the assay, premenopausal women had 13.1 ± 3.1, postmenopausal women 17.6 ± 4.2, and children 32.6 ± 12.2 μg TRAP/l of serum. We conclude that TRAP circulates in the serum as part of a complex, which also contains Ca2+, and that TRAP‐immunoassay is a potentially useful method for determining bone resorption rates, as long as the complex is destroyed before the assay.

Identification of MicroRNAs Inhibiting TGF-β-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 β (TGF-β) 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-β 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-β-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-β 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.

Two‐Site Immunoassays for Osteoclastic Tartrate‐Resistant Acid Phosphatase Based on Characterization of Six Monoclonal Antibodies

Tartrate‐resistant acid phosphatase (TRAP), an enzyme expressed in bone‐resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two‐site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4°C and −20°C or five thawing‐freezing cycles, did not change the TRAP concentration detected using the two‐site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1–5C1 and O1A–J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1–5C1 and O1A–J1B may be useful in determining the bone resorption rate.

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.

Fracture-induced changes in bone turnover: a potential confounder in the use of biochemical markers in osteoporosis

To examine the short- and long-term bone metabolic effects of fracture assessed by biochemical markers, we utilized a clinical fracture model—proximal tibial osteotomy—and prospectively followed 14 patients. This model of an induced fracture of a major bone gives the advantage of assessing baseline levels prior to fracture. Follow-up occurred at 6–9 weeks, 4–7 months, 9–13 months, and 14–17 months after fracture. Serum was assayed for type 1 procollagen peptide (PICP), total alkaline phosphatase (ALP), and carboxy-terminal-telopeptide of type I collagen (ICTP), while deoxypyridinoline (Dpyr) was measured in urine. Serum osteocalcin (OC) was measured using two recently developed two-site immunofluorometric assays, which both measure full-length and fragmented forms of OC (OCtot), with one of the assays specifically detecting only the carboxylated form of OC (OCcxy). In addition, OC was measured in urine using the same assays as those used for serum. Serum OCtot increased to a peak at 4–7 months after fracture (P < 0.001) and a similar increase was seen for OCcxy (P < 0.05) and ALP (P < 0.01). Bone formation had returned to baseline after a year. Dpyr increased significantly, with a doubling at 6 weeks, while serum (S)-ICTP increased by 73% (P < 0.01 and P < 0.001). Urine OC increased to a maximum of 84% at 6 weeks. The initial percentage increase of bone resorption was greater than that of bone formation. We conclude that: (1) bone turnover as measured by biochemical markers is altered soon after fracture, (2) the major changes occur within 6 weeks to 6 months, but may persist for up to a year. (3) The initial increase in bone resorption exceeds the increase in bone formation, which may contribute to the enhanced bone loss after fracture. (4) The two novel urine OC assays show a similar pattern of change as established marks of bone resorption, which may indicate that they measure bone resorption. (5) Fracture-induced effects on bone turnover are significant and, thus, are potential confounders in the assessment of osteoporosis.

The Proportion of Carboxylated to Total or Intact Osteocalcin in Serum Discriminates Warfarin-Treated Patients from Control Subjects

We assessed the serum concentration of γ‐carboxylated osteocalcin (OC), total OC, and full‐length OC in a clinical setting of 37 patients on continuous warfarin treatment (international normalized ratio 2.0–3.8). A comparison was done with the results from 30 untreated age‐matched controls. Four monoclonal antibodies, previously generated and characterized as to their ability to recognize different human OC forms and fragments, were used in three two‐site immunofluorometric assays. The warfarin‐treated patients had significantly lower levels of carboxylated OC 4.9 ± 3.8 (± 1 SD) ng/ml compared with the controls 13.1 ± 9.7 (p < 0.0001). There was no difference in the levels of total OC or full‐length OC between the two groups of patients. A strong correlation was found between the serum concentration of carboxylated OC and total OC, both for the warfarin‐treated patients (r = 0.98) and for the controls (r = 0.99). There was a distinct cut‐off level at 0.80, in the quotient carboxylated OC/total OC, at which all warfarin‐treated patients fell below and all controls above this level. Hence, the concentration or ratio of serum γ‐carboxylated OC in clinical settings such as warfarin‐treated patients could be measured using two‐site immunoassays.

Identification of novel proteolytic forms of osteocalcin in human urine

In this study, we report the isolation and characterization of osteocalcin in human urine using mass spectrometry and N-terminal sequencing. Multiple proteolytic forms of osteocalcin were found, which consisted of 16-27 residues from the middle region of the molecule. Several fragments had residue Gly7 at the N-terminus and the most predominant was fragment 7-31. Additional fragments starting from residue Asp14 were detected in the samples of children and young adults. Immunochemical detection of urine osteocalcin fragments had a statistically significant negative correlation to bone mineral density in evaluation of urine samples from 75-year-old women. Thus, the measurement of osteocalcin fragments in urine may have potential applications in diagnostics related to disorders of bone metabolism.

Serum osteocalcin in relation to calcaneal bone mineral density in elderly men and women: a 5-year follow-up

Abstract. A 5-year follow-up study investigated serum concentrations of total (tOC) and intact (iOC) osteocalcin in relation to calcaneal bone mineral density (BMD). The study comprised two cohorts, 75- and 80-year-olds, both resident in the city of Jyväskylä, Finland. Baseline OC and BMD were obtained for 161 men and 233 women, of whom 83 men and 189 women participated in follow-up bone measurements. The mean concentration of tOC increased from 9.6 ± 4.3 to 13.2 ± 8.5 μg/l (P = 0.001) in men and from 11.2 ± 4.9 to 14.0 ± 6.1 μg/l (P < 0.001) in women, whereas mean iOC decreased from 6.4 ± 3.0 to 5.9 ± 3.0 μg/l (P = 0.273) and from 7.7 ± 3.7 to 6.9 ± 3.4 μg/l (P = 0.021) in men and women, respectively. TOC and iOC levels correlated inversely with BMD and change in BMD in both sexes (r ranged from −0.223 to −0.422 and P = 0.048 ≤ 0.001). When we divided the baseline tOC and iOC values into four quartiles, the decrease in BMD was significantly greater in the third tOC quartiles in women and in the fourth tOC and iOC quartiles in men when compared with the lower quartiles. During the 5-year period, 19 men and 59 women sustained at least one fracture. These individuals with fractures had significantly higher iOC values and tended to have higher tOC values compared with the nonfracture group at baseline (P = 0.038 and 0.087, respectively). Our results indicate that baseline serum tOC and iOC were associated with bone loss and predicted fracture in the two cohorts of independently living elderly men and women.

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.