Accelerated Bone Resorption, Due to Dietary Calcium Deficiency, Promotes Breast Cancer Tumor Growth in Bone

Research Article

Yu Zheng, Hong Zhou, James R.K. Modzelewski, Robert Kalak, Julie M. Blair, Markus J. Seibel, and Colin R. Dunstan

Bone Research Program, ANZAC Research Institute, University of Sydney, Sydney, New South Wales, Australia

Abstract erating tumor cells, increase osteoclastic bone destruction by acting on osteoblasts to increase expression of receptor activator of The skeleton is a major site of breast cancer metastases. High n bone turnover increases risk of disease progression and death. nuclear factor- B ligand. As bone breaks down, growth factors, such as transforming growth factor-h and insulin-like growth factors, are However, there is no direct evidence that high bone turnover is causally associated with the establishment and progression of released from the bone matrix in active forms to stimulate tumor metastases. In this study, we investigate the effects of high growth leading in turn to further bone destruction (4). In animal bone turnover in a model of breast cancer growth in bone. models of breast cancer metastasis, antiresorptive treatment with Female nude mice commenced a diet containing normal (0.6%; either bisphosphonates or osteoprotegerin has shown skeletal ‘Normal-Ca’) or low (0.1%; ‘Low-Ca’) calcium content. Mice protection and reduced tumor growth (5–8). Therefore, bone were concurrently treated with vehicle or osteoprotegerin resorption seems to provide the cellular microenvironment that (1 mg/kg/d s.c; n = 16per group). Three days later (day 0), enhances growth of breast cancer metastases in bone. 50,000 Tx-SA cells (variant of MDA-MB-231 cells) were Several clinical studies showed that in patients with newly implanted by intratibial injection. On day 0, mice receiving diagnosed cancer, high bone turnover is associated with a Low-Ca had increased serum parathyroid hormone (PTH) significantly higher risk of future skeletal-related events, disease and tartrate-resistant acid phosphatase 5b levels, indicating progression, and death (9–15). However, thus far, there is no direct secondary hyperparathyroidism and high bone turnover, which evidence indicating that high bone turnover is causally associated with the establishment and progression of metastatic bone disease. was maintained until day 17. Osteoprotegerin increased serum PTH but profoundly reduced bone resorption. On day 17, in Of note, secondary hyperparathyroidism due to low dietary calcium mice receiving Low-Ca alone, lytic lesion area, tumor area, and intake and/or vitamin D deficiency is frequent in middle-aged and cancer cell proliferation increased by 43%, 24%, and 24%, elderly women in whom breast cancer is also common (16, 17); respectively, compared with mice receiving Normal Ca elevated bone remodeling and progressive bone loss typically occur (P < 0.01). Osteoprotegerin treatment completely inhibited in these individuals (18). Taking these facts into consideration, we lytic lesions, reduced tumor area, decreased cancer cell hypothesized that increased bone turnover may enhance the proliferation, and increased cancer cell apoptosis. Increased growth and destructive potential of breast cancer metastases in bone turnover, due to dietary calcium deficiency, promotes bone. In this study, we therefore evaluated whether induction of tumor growth in bone, independent of the action of PTH. high bone turnover as a consequence of a calcium-deficient diet in mice promotes the establishment and growth of breast cancer cells Breast cancer patients frequently have low dietary calcium intake and high bone turnover. Treatment to correct calcium in bone. insufficiency and/or treatment with antiresorptive agents, such as osteoprotegerin, may be of benefit in the adjuvant as well as Materials and Methods palliative setting. [Cancer Res 2007;67(19):9542–8] Breast cancer cell line. The estrogen-independent bone-seeking human breast cancer cell line, Tx-SA, was derived from the cell line MDA-MB-231 Introduction and was obtained from Dr. Toshi Yoneda (M. D. Anderson Cancer Center, San Antonio, TX; ref. 19). All tissue culture media and supplements were Breast cancer is one of the most common malignancies in supplied by Invitrogen unless otherwise stated. The cell line was cultured in women and affects approximately one million women per year DMEM supplemented with 10% FCS (JRH Biosciences) and 1% penicillin- worldwide. Approximately 70% of patients who develop advanced streptomycin solution. To assess whether osteoprotegerin had direct effects breast cancer will have secondary tumors in the bone (1). These on cell growth in vitro, 10,000 Tx-SA cells were plated in DMEM medium bone metastases frequently produce osteolytic bone lesions by with 2% FCS per well in 12-well tissue culture plates and allowed to adhere activating local osteoclasts to break down existing bone and often overnight. Osteoprotegerin (10, 100, and, 1,000 Ag/mL) or vehicle (PBS) was result in significant morbidity, including pain, fracture, immobility, added to the media on day 0, with replacement every 48 h without washing. and spinal cord compression (2, 3). Cells were trypsinized and cell numbers were counted at days 1, 3, and 5 by trypan blue exclusion. Experiments were done two times. In established bone metastases, a ‘‘vicious cycle’’ seems to exist Mouse model of breast cancer growth in bone. Four-week-old female between tumor and bone cells. Proresorptive factors, such as BALB/c nu/nu mice (Animal Resources Center, Canning Vale, Western parathyroid hormone (PTH)–related protein, released from prolif- Australia, Australia) were used for the experiments. Mice were maintained under specific pathogen-free, temperature-controlled conditions throughout this study at the animal facilities of the ANZAC Research Institute in Requests for reprints: Colin R. Dunstan, Bone Research Program, ANZAC accordance with Institutional Animal Welfare Guidelines and an approved Research Institute, Hospital Road, Concord, New South Wales 2139, Australia. Phone: protocol. All mouse manipulations were done inside a laminar-flow hood 61-2-9767-9163; Fax: 61-2-9767-9101; E-mail: [email protected]. I2007 American Association for Cancer Research. under aseptic conditions while maintaining general anesthesia with i.p. doi:10.1158/0008-5472.CAN-07-1046 injection of ketamine/xylazine (75/10 mg/kg), unless otherwise noted.

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At day À3, mice were placed on a diet with normal (0.6%; ‘Normal-Ca’) tumor area was measured in each section and the average tumor area was or low (0.1%; ‘Low-Ca’) calcium content (Specialty Feeds) and then treated used as an index of tumor burden. Cortical bone area was measured in the with vehicle (0.9% sterile saline) or osteoprotegerin (1 mg/kg/d s.c.), using a same sections. Osteoclasts were identified as TRAcP-positive multinucle- recombinant construct of osteoprotegerin containing amino acids 22 to 194 ated cells associated with bone surfaces. The number of osteoclasts at the of human osteoprotegerin fused to the Fc domain of human immunoglobin bone surface (day 0) was counted in each section (Â200 magnification) and G (kindly provided by Amgen, Inc.). The dose of osteoprotegerin was osteoclast number per millimeter of bone surface was calculated. Bone selected to produce rapid and profound inhibition of bone resorption volume and osteoclast surface were also measured at the same time. despite the strongly proresorptive effects of Tx-SA cells in vivo. At day 0, five Osteoblast surface was calculated on H&E-stained sections following mice from each treatment group were sacrificed for biochemical and morphologic identification of osteoblasts associated with bone surfaces histologic assessment. The remaining mice (n = 16 per group) were (Â400 magnification). anesthetized as described above and injected intratibially with 5 Â 104 Biochemical measurements. Serum calcium, PTH, osteocalcin, and Tx-SA cells suspended in 10 AL PBS. The contralateral tibiae were similarly TRAcP5b were measured at days 0 and 17. Serum calcium levels were injected with PBS as a control. The analgesic carprofen (Rimadyl; 5 mg/kg measured by standard autoanalyzer techniques. TRAcP5b, a marker of s.c.) was administered at the time of inoculation to minimize postsurgical bone resorption, was analyzed by ELISA (sensitivity, 0.1 U/L; intra-assay pain. variation, 2.2%; Suomen Bioanalytiikka). Serum levels of PTH and osteocalcin For in vivo follow-up of lytic bone lesions, mice were anesthetized as (a marker of bone formation) were analyzed by immunoradiometric assay above and assessed by digital radiography (MX-50 X-ray cabinet, Faxitron) [PTH: sensitivity, 3 pg/mL; intra-assay variation, 7.9%; osteocalcin: sensitivity, on days 7, 10, and 14 after cell implantation. On day 17, mice were 0.1 ng/mL; intra-assay variation, 3.1%; Immutopics International]. anesthetized and examined again radiologically for osteolytic bone lesions Statistical analysis. All data were presented as the mean F SE and before being sacrificed for tissue harvest. statistics were done using one-way ANOVA, followed with Bonferroni’s Micro–computed tomography imaging. After tissue harvest, repre- adjustment where there were multiple comparisons by means of SPSS 15.0 sentative micro–computed tomography (CT) images of tibiae were obtained for Windows (SPSS, Inc.). Significance was accepted where P < 0.05. using a SkyScan 1172 scanner (SkyScan). Scanning was done at 100 kV, 100 AA using a 1-mm aluminium filter. In total, 1,800 projections were Results collected at a resolution of 6.93 Am/pixel. Reconstruction of sections was in vitro done using a modified Feldkamp cone-beam algorithm with beam Osteoprotegerin does not affect Tx-SA cell growth . hardening correction set to 50%. VGStudio MAX 1.2 software (Volume At doses of 10 to 1,000 Ag/mL, given over 5 days, osteoprotegerin Graphics GmbH) was used to obtain three-dimensional visualization of had no detectable effect on the increase in cell numbers (Fig. 1) or tibiae from reconstructed sections. the morphology of Tx-SA cells in vitro (data not shown). Measurement of lytic lesions. Lytic bone areas of injected and sham- Dietary calcium deficiency induces hyperparathyroidism. At injected tibiae were measured on digitally recorded radiographs using the time of tumor inoculation (day 0), animals receiving Low-Ca for interactive image analysis software (ImageJ, NIH). At each time point (days 3 days were found to have decreased serum calcium and increased 10, 14, and 17), changes in the size of osteolytic lesions were calculated and serum PTH, osteocalcin, and TRAcP5b levels, indicative of growth of lytic lesions was compared between Normal-Ca and Low-Ca secondary hyperparathyroidism and accelerated bone turnover. group of mice from day 10 to day 17. Histologic assessment. Harvested tibiae were fixed for 36 h in 4% Treatment with osteoprotegerin decreased serum calcium and paraformaldehyde buffered with 0.1 mol/L phosphate buffer (pH 7.4) and increased serum PTH levels in mice on both diets; these effects decalcified in 10% EDTA at 4jC for 2 weeks. The tissues were then were most pronounced in mice receiving the Low-Ca diet. In processed and embedded in paraffin. Five-micron sections were cut from contrast with vehicle-treated mice, osteoprotegerin treatment each specimen and stained with H&E for routine histologic examination. profoundly reduced serum osteocalcin and TRAcP5b levels in mice Histochemical examination for tartrate-resistant acid phosphatase (TRAcP), on either diet, signifying reduced bone remodeling in these animals as a marker for osteoclasts, was done by using naphthol AS-BI phosphate (Table 1). At day 17, when mice were sacrificed, animals on Low-Ca (Sigma Chemical Co.) as a substrate and fast red violet Luria-Bertani salt (Sigma Chemical) as a stain for the reaction product; incubation was done at room temperature for 30 min (8). Immunohistochemistry and terminal deoxynucleotidyl transferase– mediated dUTP nick end labeling staining. As an indicator of proliferation, 5-Am sections were examined immunohistochemically for expression of human Ki67 using a polyclonal rabbit antibody to human Ki67 (Santa Cruz Biotechnology, Inc.) and a secondary goat anti-rabbit antibody (Vector Laboratories). Tumor cell apoptosis rates were assessed by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL), which was done on 5-Am sections with the In situ Cell Death Detection kit, POD (Roche Diagnostics), according to the manufacturer’s protocol. The proportion of Ki67-positive cells and apoptotic TUNEL-stained cells was determined by counting positive and negative cells in five random fields of nonnecrotic areas of tumors in a representative section in each bone specimen (Â400 magnification). Bone histomorphometry. Histomorphometric analysis of the proximal tibial metaphysis was conducted at days 0 and 17 to evaluate bone volume, bone remodeling, and tumor burden (day 17). Measurements were done in longitudinal 5-Am sections stained with H&E or reacted to show TRAcP Figure 1. Representative growth curves of Tx-SA cells grown in vitro in the activity (Â12.5 magnification) with measurement made interactively using presence of osteoprotegerin. Tx-SA cells were grown under conditions as described in Materials and Methods. At concentrations of 10 to 1,000 Ag/mL, the OsteoMeasure System (Osteometrics). To determine tumor area, three osteoprotegerin (OPG) had no effect on the growth rate over 5 days of treatment. sagittal sections through the tibia were taken at f200-Am intervals, These experiments were done two times. Points, mean (n = 6 at each time representing upper, mid, and lower regions of the proximal tibia. Total point); bars, SD. www.aacrjournals.org 9543 Cancer Res 2007; 67: (19). October 1, 2007

Table 1. Group-specific and longitudinal changes in serum calcium, PTH, and bone markers

Serum assay Normal-Ca Low-Ca Normal-Ca OPG Low-Ca OPG

Day 0 (n =5) cb Calcium (mmol/L) 2.22 F 0.04 2.11 F 0.02* 2.12 F 0.05* 1.68 F 0.05 , c,b PTH (pg/mL) 31.01 F 4.50 63.13 F 16.99* 73.18 F 27.01* 177.53 F 37.29 c c mTRAcP5b (units/L) 8.66 F 0.61 11.78 F 1.17* 1.60 F 0.14 2.94 F 0.22 Osteocalcin (ng/mL) 179.24 F 13.92 267.81 F 16.91* 132.39 F 12.55* 95.71 F 8.39*

Day 17 (n =8) cb Calcium (mmol/L) 2.28 F 0.03 2.16 F 0.02* 2.17 F 0.04* 2.11 F 0.01 , cb PTH (pg/mL) 32.76 F 4.90 82.31 F 13.14* 64.02 F 12.36* 173.60 F 18.00 , c c mTRAcP5b (units/L) 5.74 F 0.46 6.22 F 0.60 0.89 F 0.05 1.25 F 0.11 Osteocalcin (ng/mL) 177.69 F 12.19 224.45 F 15.73* 111.18 F 6.85* 101.43 F 7.81*

NOTE: Data are expressed as mean F SE. Abbreviations: mTRAcP5b, serum mouse TRAcP5b; OPG, osteoprotegerin. * P < 0.05. cP < 0.01 versus Normal-Ca. bP < 0.05 versus Normal-Ca osteoprotegerin.

still had significantly decreased serum calcium and increased PTH diet induced hyperparathyroidism and both accelerated bone and osteocalcin levels, but TRAcP5b levels were similar to animals resorption and formation; in contrast, treatment with osteoprote- on Normal-Ca. At day 17, mice receiving osteoprotegerin on either gerin increased PTH levels but inhibited both bone resorption and diet continued to show low serum calcium, osteocalcin, and formation. TRAcP5b levels and increased PTH levels, consistent with the Effects of low dietary calcium and/or antiresorptive changes observed at day 0 (Table 1). treatment on intratibial tumor growth and bone destruction. Histomorphometric analysis of mice sacrificed at day 0 showed Seventeen days after tumor cell implantation, lytic bone lesions that mice on a Low-Ca diet showed significantly increased were established in 100% of the vehicle-treated animals in both the numbers of osteoclasts per millimeter bone surface compared Normal-Ca and Low-Ca groups, as determined by plain X-rays and with animals receiving Normal-Ca. At the same time, relative to micro-CT (Fig. 2A). In animals receiving Low-Ca alone, lytic lesion values in mice on Normal-Ca, bone volume was decreased in mice area was significantly increased by 42% on day 17 compared with receiving Low-Ca. A Low-Ca diet was also associated with animals on Normal-Ca diet (Fig. 2B). In contrast, no lytic lesions increased osteoblast surface (Table 2), indicating a general increase were detected in any of the mice treated with osteoprotegerin. No in bone remodeling, even if the net effect of changes was to induce bony lesions were seen in any of the sham-injected legs. bone loss. Treatment with osteoprotegerin increased bone volume Compared with mice on Normal-Ca, animals fed the Low-Ca diet and decreased osteoclast numbers and osteoclast- and osteoblast- showed significantly lower cortical bone area (9% decrease) and lined bone surfaces regardless of diet. This indicates that significantly higher tumor area (24% increase; Fig. 3A–C) 17 days osteoprotegerin provided effective inhibition of bone resorption after tumor implantation. For both Low-Ca and Normal-Ca groups, and coupled bone formation, even in the presence of elevated PTH treatment with osteoprotegerin significantly increased cortical levels. Taken together, these data show that 3 days of a low-calcium bone area (Fig. 3A–C). Osteoclast numbers at the tumor bone

Table 2. Day 0 histomorphometric data

Normal-Ca Low-Ca Normal-Ca OPG Low-Ca OPG

c c BV/TV (%) 11.67 F 0.47 9.03 F 0.62* 21.22 F 1.20 20.56 F 0.70 c c NOc/BS 7.19 F 0.23 9.33 F 0.35* 3.06 F 0.24 2.96 F 0.38 c c Oc.S/BS (%) 40.41 F 1.46 52.68 F 1.53* 17.92 F 1.38 19.29 F 0.71 c c Ob.S/BS (%) 24.60 F 1.37 38.03 F 2.31* 11.96 F 1.23 12.59 F 0.50

NOTE: Data are expressed as mean F SE. n = 5 per group. Abbreviations: BV/TV, bone volume % tissue volume; NOc/BS, osteoclast number per millimeter bone surface; Oc.S/BS, osteoclast surface % bone surface; Ob.S/BS, osteoblast surface % bone surface. * P < 0.05. cP < 0.01 versus Normal-Ca.

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Figure 2. Effects of low-calcium diet and/or osteoprotegerin treatment on the size of osteolytic lesions in Tx-SA–injected tibiae of nude mice. A, representative X-ray (a–d) and micro-CT (e–g) images of the tibiae of mice inoculated by intratibial injection with Tx-SA cells. Images were taken 17 days after tumor cell inoculation [normal calcium diet (Normal-Ca) with vehicle (a and e), low-calciumdiet (Low-Ca) with vehicle ( b and f), normal diet plus osteoprotegerin (Normal-Ca OPG; c and g), and low-calciumdiet plus osteoprotegerin (Low-Ca OPG; d and h)]. Osteolytic lesions on X-rays are darkened areas within the bone (arrows). B, mean area of osteolytic lesions fromday 10 to day 17. Osteolytic areas were measured fromX-ray imagesas described in Materials and Methods. Mice on Low-Ca had a 42 % larger lesion area (P < 0.01) compared with Normal-Ca by day 17. Treatment with osteoprotegerin inhibited the formation of osteolytic bone lesions completely. Points, mean (n = 16 in each group); bars, SE. *, P < 0.01 versus Normal-Ca. OPG, osteoprotegerin. interface were similar in Normal Ca and Low-Ca groups (14.83 F are useful for the prediction of skeletal-related events in cancer 2.34/mm versus 15.84 F 2.30/mm, respectively). Osteoclast patients remained a controversial question. differentiation and activity were almost completely inhibited by The present study is the first to show in a murine model of osteoprotegerin treatment, as no TRAcP-positive osteoclasts were breast cancer growth in bone that accelerated bone turnover at the detected in sections derived from these groups. time of tumor implantation strongly promotes cancer cell Tissues harvested from animals receiving a Low-Ca diet showed proliferation and tumor growth in bone. Our results further a 26% increase in the numbers of Ki67-positive, proliferating cancer suggest that increased bone turnover, rather than the low-calcium cells, whereas both groups showed similar numbers of TUNEL- diet, hypocalcemia or secondary hyperparathyroidism per se, positive apoptotic cells. For both animals on Low-Ca and on produced the observed effect as osteoprotegerin treatment reduced Normal-Ca, treatment with osteoprotegerin decreased the number tumor growth in mice on a low-calcium diet while enhancing of Ki67-positive, proliferating cells by 45% and 47% and increased hypocalcemia and secondary hyperparathyroidism. These results numbers of TUNEL-positive cells by 170% and 179%, respectively support the concept that high bone turnover enhances breast (Fig. 4A–D). cancer tumor growth in bone. A diet low in calcium has been shown to increase bone remodeling in different animal models (23–26). In the present study, a Discussion low-calcium diet fed for 3 days resulted in mild hypocalcemia, Several clinical studies in cancer patients suggest that the level secondary hyperparathyroidism, and increased bone remodeling of bone resorption at the time of diagnosis is a strong predictor as indicated by both increased numbers of osteoclasts and of skeletal-related events. In particular, high bone resorption rates osteoblasts and higher circulating levels of TRAcP5b and osteocalcin. seem to be associated with rapid disease progression (e.g., bone Hypocalcemia, secondary hyperparathyroidism, and elevated osteo- metastases), poor survival, and even death (10–14, 20–22). For calcin levels persisted throughout the study period until day 17. By example, Coleman et al. (14) found that cancer patients with high then, however, osteoclast number and bone resorption rates urinary N-telopeptide (NTX-I) levels (i.e., high bone resorption (assessed by serum TRAcP5b) in the low-calcium diet group were rates) had a 2-fold increased risk of skeletal complications and similar to those in the group receiving a normal calcium diet. This disease progression and a 4- to 6-fold increased risk of death on discrepancy could be due to the adaptation of mice to a low-calcium study compared with patients with low NTX-I levels. Although diet or, alternatively, to proresorptive tumor effects dominating the these data are intriguing, most if not all studies on the association effects of elevated systemic PTH levels. In contrast, osteoclast between bone turnover and tumor outcome have been observa- number was significantly reduced after 3 days of treatment with tional and provide no direct evidence that high bone turnover is osteoprotegerin, and at day 17, almost no osteoclasts could be seen causally associated with the establishment and progression of histologically, consistent with the known ability of osteoprotegerin to metastatic bone disease. Hence, whether bone turnover markers block osteoclast differentiation (27). The reduced TRAcP5b and www.aacrjournals.org 9545 Cancer Res 2007; 67: (19). October 1, 2007

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2007 American Association for Cancer Research. Cancer Research osteocalcin levels and reduced osteoclast and osteoblast surfaces at development of osteolytic lesions, whereas treatment with day 0 indicate that, in mice treated with osteoprotegerin, tumor cells pamidronate inhibited bone resorption but had little effect on were injected into an environment of profoundly reduced bone tumor growth. In contrast, previous in vivo studies have shown that remodeling. inhibition of bone turnover via osteoprotegerin or by bisphosph- When breast cancer cells were injected intratibially in mice with onate (ibandronate and zoledronate) treatment reduces the ability high bone turnover, cancer cell growth was enhanced, as indicated of breast cancer cells to grow in the bone environment in xenograft by increased numbers of proliferating cells. These mice also had models in rats and mice (5–7, 30). It is assumed that these effects larger lytic lesions evident on radiographs and decreased bone are due to the inhibition of a ‘‘vicious cycle’’ where bone resorption volume compared with mice fed a normal diet. In contrast, is driven by the tumor cells, as proposed by Mundy, Guise, and osteoprotegerin-treated mice on either low calcium or normal diet Yoneda (4, 31, 32). exhibited significantly reduced tumor growth with increased Our findings clearly show that physiologically accelerated bone cancer cell apoptosis and reduced cancer cell proliferation. Bone remodeling rates can provide a bone microenvironment conducive in tumor-bearing tibiae was well preserved in osteoprotegerin- to tumor growth. Low-calcium diet increased indices of both bone treated mice on either diet. Interestingly, treatment with osteopro- resorption and bone formation and simultaneously promoted tegerin had similar inhibitory effects on tumor growth on both tumor growth. Conversely, osteoprotegerin treatment suppressed diets despite the presence of more severe hyperparathyroidism in indices of bone resorption and bone formation while also mice on a low-calcium diet. Taken together, these findings provide suppressing tumor growth. The parallel changes in bone resorption support for the hypothesis that preexisting high levels of bone and bone formation reflect the close coupling of these processes remodeling advance tumor growth by creating a more fertile soil (33). However, it is likely that bone resorption, rather than bone that supports and promotes tumor proliferation. formation, is the dominant effector on tumor growth as bone Consistent with the findings of this study, enhanced cancer cell resorption can potentially support tumor growth through the growth in bone has occasionally been reported in association with release of growth factors from bone matrix and remove the treatments that increase bone resorption rates. Price et al. (28) physical constraints on tumor growth (31, 32). conducted a study in which MDA-MB-231 cells were injected into Calcium deficiency is common and its prevalence increases with nude mice by the intracardiac route, after which mice were treated age (34, 35). Vitamin D deficiency is also widespread and promotes with the heat shock protein 90 inhibitor, 17-allylamino-17- calcium deficiency as it results in impaired intestinal calcium demethoxygeldanamycin (17-AAG). 17-AAG treatment enhanced absorption (16, 17, 36–38). In both conditions, PTH secretion may tumor growth in bone while inhibiting tumor growth at orthotopic be increased to compensate for inadequate serum calcium concen- sites. 17-AAG treatment can increase the rate of bone resorption, trations, leading to secondary hyperparathyroidism, which in turn which may have contributed to the observed enhancement of induces an acceleration in bone turnover (16, 17, 36–38). Furthermore, tumor growth in bone. Libouban et al. (29) showed in a mouse the menopausal decrease in endogenous estrogen levels is associa- model of multiple myeloma that increased bone remodeling, ted not only with a significant increase in bone turnover but also with induced by ovariectomy, accelerated tumor growth, and the a substantial decrease in intestinal calcium absorption (39), which

Figure 3. Effects of low-calciumdiet and/or osteoprotegerin on tumor and bone area in Tx-SA–injected tibiae of nude mice. A, representative H&E-stained sections of tibiae frommiceat day 17 following intratibial inoculation with Tx-SA cells. The cortical bone in Normal-Ca (a) and Low-Ca mice (b) can be seen to be perforated (arrow) adjacent to the tumor mass (T), which fills the medullary cavity. Note the preservation of cortical bone and the accumulation of primary and secondary spongiosa (S) in ‘Normal-Ca OPG’ (c)or ‘Low-Ca OPG’ (d). Magnification, Â40. B, when compared with Normal-Ca, Low-Ca increased tumor area by 24% and osteoprotegerin treatment significantly decreased tumor area by 46% and 45% in mice on Normal-Ca or Low-Ca, respectively. C, when compared with Normal-Ca, Low-Ca decreased cortical bone area and osteoprotegerin increased cortical bone area in combination with normal or low-calcium diet. Columns, mean (n = 16 per group); bars, SE. *, P < 0.05; **, P < 0.01 versus Normal-Ca. OPG, osteoprotegerin.

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Figure 4. Histologic assessment of proliferation and apoptosis of Tx-SA breast cancer cells in bone. Representative sections of tumor from the tibiae of mice inoculated with Tx-SA cells and treated with Normal-Ca, Low-Ca, Normal-Ca OPG, or Low-Ca OPG until day 17. Proliferating cells are indicated by Ki67 staining (positive cells indicated by arrows; A) and apoptotic cells by TUNEL staining (brown stained cells; B). Magnification, Â400. The proportion of proliferating and apoptotic cells was measured by counting five randomfields. Comparedwith Normal-Ca, Low-Ca increased Tx-SA cell proliferation, whereas osteoprotegerin treatment with either calcium diet decreased proliferation (C). Low-Ca alone had no effect on Tx-SA apoptosis in vivo, whereas osteoprotegerin treatment in combination with either diet significantly stimulated apoptosis (D). Columns, mean (n = 16 per group); bars, SE. *, P < 0.05; **, P < 0.01 versus Normal-Ca. OPG, osteoprotegerin.

further aggravates dietary calcium deficiency in older women. Of implications because many older cancer patients, much like the note, the incidence of breast cancer increases after the menopause older population in general, are deficient in calcium and vitamin D and continues to increase with advancing age (40, 41), thus coincid- and may present with accelerated bone turnover. Treatment of ing with a peak in calcium deficiency and bone turnover. In this sense, breast cancer patients to decrease bone turnover through our experimental findings may have clinical implications in that high correction of calcium or vitamin D deficiency may improve patient bone turnover in postmenopausal women with breast cancer may outcomes in the adjuvant as well as palliative settings. Our study increase their risk of disease progression to bone. Our study also also supports the concept that the use of antiresorptives, such as confirms that reversing accelerated bone turnover through anti- bisphosphonates or osteoprotegerin, to suppress bone turnover resorptive treatment can reduce tumor growth. Although human below normal levels, may slow disease progression in addition to studies are required to directly assess the associations between the reducing the risk of skeletal morbidity. incidence of metastatic bone disease and indices of calcium and bone homeostasis, our results do point to a causal relationship between Acknowledgments accelerated bone turnover and skeletal tumor growth. Received 3/20/2007; revised 5/13/2007; accepted 6/12/2007. In conclusion, in mice, a diet low in calcium strongly promotes Grant support: National Health and Medical Research Council of Australia project breast cancer cell growth in bone. Because treatment with grant 352332 and the New South Wales Government (BioFirst Award). The costs of publication of this article were defrayed in part by the payment of page osteoprotegerin reduced tumor growth while at the same time charges. This article must therefore be hereby marked advertisement in accordance enhancing hypocalcemia and secondary hyperparathyroidism, it is with 18 U.S.C. Section 1734 solely to indicate this fact. likely that the rapid tumor growth observed under these conditions We thank Colette Fong-Yee for help in cell culture, Prof. Toshi Yoneda for providing the Tx-SA cell line, Amgen for providing osteoprotegerin, and the staff in the NANO is due to increased bone resorption rather than to hypocalcemia or Major National Research Facility at the Electron Microscope Unit, The University of secondary hyperparathyroidism. Our findings may have clinical Sydney for the facilities as well as scientific and technical assistance.

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