Evidence is accumulating that there is a vicious cycle between

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1 Skeletal Complications of Malignancy Supplement to Cancer 2979 Actions of Bisphosphonate on Bone Metastasis in Animal Models of Breast Carcinoma Toshiyuki Yoneda, Ph.D., D.D.S. 1 Toshimi Michigami, M.D. 2 Bing Yi, M.D. 1 Paul J. Williams, B.S. 1 Maria Niewolna, M.S. 1 Toru Hiraga, Ph.D., D.D.S. 3 1 Division of Endocrinology and Metabolism, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas. 2 Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka, Japan. 3 Department of Biochemistry, Osaka University Faculty of Dentistry, Osaka, Japan. Presented at the Second North American Symposium on Skeletal Complications of Malignancy, Montreal, Québec, Canada, October 15 16, Supported by National Institutes of Health Grants PO1-CA40035, RO1-AR28149, and RO1-DK The authors are grateful to Dr. Fred Miller for generously providing them with the 4T1 murine mammary tumor cells. The authors thank Roche, Novartis, and Taiho Pharmaceutical Company for providing them with ibandronate, zoledronate, and UFT, respectively. The authors also thank Miss Mie Masuda and Ms. Christa Martinez for their excellent secretarial assistance. Address for reprints: Toshiyuki Yoneda, Division of Endocrinology and Metabolism, Department of Medicine, UTHSCSA, 7703 Floyd Curl Drive, San Antonio, TX Received March 2, 2000; accepted March 10, BACKGROUND. Bone, which abundantly stores a variety of growth factors, provides a fertile soil for cancer cells to develop metastases by supplying these growth factors as a consequence of osteoclastic bone resorption. Accordingly, suppression of osteoclast activity is a primary approach to inhibit bone metastasis, and bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used for the treatment of bone metastases in cancer patients. To obtain further insights into the therapeutic usefulness of BP, the authors studied the effects of BP on bone and visceral metastases in animal models of metastasis. METHODS. The authors used two animal models of breast carcinoma metastasis that they had developed in their laboratory over the last several years. One model uses female young nude mice in which inoculation of the MDA-MB-231 or MCF-7 human breast carcinoma cells into the left cardiac ventricle selectively develops osteolytic or osteosclerotic bone metastases, respectively. Another model uses syngeneic female mice (Balb/c) in which orthotopic inoculation of the 4T1 murine mammary carcinoma cells develops metastases in bone and visceral organs including lung, liver, and kidney. RESULTS. BP inhibited the development and progression of osteolytic bone metastases of MDA-MB-231 breast carcinoma through increased apoptosis in osteoclasts and breast carcinoma cells colonized in bone. In a preventative administration, however, BP alone increased the metastases to visceral organs with profound inhibition of bone metastases. However, combination of BP with anticancer agents such as uracil and tegafur or doxorubicin suppressed the metastases not only in bone but also visceral organs and prolonged the survival in 4Tl mammary tumor-bearing animals. Of interest, inhibition of early osteolysis by BP inhibited the subsequent development of osteosclerotic bone metastases of MCF-7 breast carcinoma. CONCLUSIONS. These results suggest that BP has beneficial effects on bone metastasis of breast carcinoma and is more effective when combined with anticancer agents. They also suggest that the animal models of bone metastasis described here allow us to design optimized regimen of BP administration for the treatment of breast carcinoma patients with bone and visceral metastases. Cancer 2000;88: American Cancer Society. KEYWORDS: osteolytic bone metastasis, osteoclasts, bisphosphonate, osteosclerotic bone metastasis, breast carcinoma. Evidence is accumulating that there is a vicious cycle between osteoclasts, osteoblasts, and cancer cells during the development and progression of bone metastases. As depicted in Figure 1, osteoclastic bone resorption releases bone-stored growth factors including insulin-like growth factor (IGF) and transforming growth factor (TGF ), which stimulate the growth and production of parathyroid hormone-related protein (PTH-rP) in metastatic cancer cells, 1 respec American Cancer Society

2 2980 CANCER Supplement June 15, 2000 / Volume 88 / Number 12 FIGURE 1. The vicious cycle between osteoclasts, metastatic carcinoma cells, and osteoblasts during the development and progression of bone metastases is shown. Growth factors such as IGFs and TGF released as a consequence of osteoclastic bone resorption stimulate metastatic breast carcinoma cells to proliferate and produce PTH-rP. The PTH-rP, in turn, stimulates osteoblasts/stromal cells to express RANKL, which then binds to its cognitive receptors RANK on osteoclasts leading to further activation of osteoclasts. PTH-rP: parathyroid hormone-related protein; RANKL: receptor-activated NF- B ligand; TGF : transforming growth factor ; IGF: insulin-like growth factor; GF: growth factor. tively. Parathyroid hormone-related protein produced by these cancer cells then increases the expression of the receptor-activated nuclear factor (NF)- B ligand (RANKL) in osteoblasts. Receptor-activated NF- B ligand subsequently binds to its cognitive receptor RANK that is expressed in osteoclasts and enhances osteoclast activity. 2 Thus, osteoclasts play a key role in the development of bone metastasis. 3 6 Accordingly, suppression of osteoclasts would be a primary approach to inhibit bone metastases. Bisphosphonate (BP) has characteristic chemical structure that leads to selective accumulation of BP in bone. 7 Bisphosphonate in bone is shown to specifically inhibit osteoclasts through yet-unclear mechanisms. 8 Because of these unique properties, BP has been widely used and effectively suppresses bone metastasis and its complications, particularly bone pain, in patients with breast carcinoma, 9,10 prostate carcinoma, 11 and multiple myeloma Questions Remained Unclear in Clinical Studies It is evident that BP has beneficial effects in cancer patients with bone metastases. 4,15 18 However, there are still many questions about the actions of BP that remain unclear in clinical studies. For example, a recent clinical study reported by Diel et al. 15 suggests that BP may have some effects on metastatic carcinoma cells not only in bone but also visceral organs in breast carcinoma patients and causes controversy as to whether BP has anticancer actions. Furthermore, several in vitro studies demonstrate that several BPs promote apoptosis in certain cancer cells However, in vivo effects of BP on metastatic carcinoma cells that are virtually the principal player in the development of bone metastasis are still unclear. Determination of the effects of BP on cancer cells colonizing bone is critical to increase our understanding of the mode of BP action and, more importantly, therapeutic efficacy of BP for the treatment of bone metastasis. Effects of BP on metastases to visceral organs are also unclear. Cancer patients with bone metastases, in almost all cases, already have developed metastases in visceral organs. 15,22,23 Accordingly, it is important to know what effects BP shows on established metastases in visceral organs. In this context, it is noteworthy that a recent clinical study has reported that the BP clodronate in combination with conventional anticancer therapies enhanced inhibition of visceral organ metastases and prolonged the survival compared with anticancer therapy alone in breast carcinoma patients. 15 The data suggest that the clodronate may possess direct anticancer and/or adjuvant effects on metastases not only in bone but also nonbone organs. However, a subsequent analogous clinical study showed clodronate did not suppress visceral organ metastases nor prolong the survival in breast carcinoma patients. 18 Thus, the effects of BP on visceral organ metastasis are still controversial and need to be elucidated. Limited information is available regarding the effects of BP on osteosclerotic bone metastases. In most cases, prostate carcinoma 11,24 and often breast carcinoma 25 patients develop osteosclerotic bone metastases. Although very little is known about the mechanisms underlying the osteosclerotic bone metastasis, there has been a long-standing notion that bone resorption is a prerequisite for the development of osteosclerotic bone metastases. 26,27 It has been proposed that bone resorption releases growth factors that allow the proliferation and differentiation of cells of osteoblast lineage and calcium that is required for mineralization. Consistent with this notion, blood or urinary levels of biochemical markers of bone resorption frequently are elevated during the advancement of osteosclerotic bone metastases in prostate carcinoma patients. 28 Bisphosphonate therefore could inhibit the development of osteosclerotic bone metastases through inhibiting preceding bone resorption. However, this intriguing possibility has not been extensively explored yet. Effects of BP on the survival of cancer patients are controversial. Most clinical studies described that BP fails to increase the survival of cancer patients, al-

3 Bisphosphonate Actions on Bone Metastasis/Yoneda et al FIGURE 2. Heart injection model of bone metastasis (experimental bone metastasis model) is shown. One to five hundred thousand human cancer cells, such as MDA-MB-231 human estrogen-independent breast carcinoma cells, are inoculated into the left ventricle of the heart in 4 6-week-old female nude mice (left). Four to five weeks after the inoculation of MDA-231 cells, metastases are selectively developed in bone as seen in the radiograph (arrows, top right). Histologic examination demonstrates that bone marrow cavity is replaced by metastatic MDA-231 breast carcinoma cells with increased numbers of osteoclasts (bottom right, arrows) along the endosteal surface of bone. OCL: osteoclasts. though BP markedly suppresses bone metastases. 9,16,17 Because visceral organ metastasis is one of the direct causes of death in cancer patients, these results suggest that BP has little effect on visceral organ metastasis and indicate that suppression of bone metastases does not improve the survival of cancer patients. However, because cancer patients with distant metastases usually receive varieties of anticancer treatments together with BP, the effects of BP alone on the survival in these patients are difficult to evaluate. These questions regarding BP actions are difficult to study in cancer patients in a systematic well controlled manner. Here, we describe the results of the experiments in which these questions are studied using animal models of bone metastasis we developed over the last several years. Study Model of Osteolytic Bone Metastasis in Breast Carcinoma Heart injection model using human cancer cells (experimental bone metastasis model) Characteristics of this model has been described in detail elsewhere. In this model, human cancer cells are directly introduced into the arterial circulation through the left ventricle of the heart in young female nude mice (Fig. 2). Thus, this model represents an experimental bone metastasis model. The most notable feature of this model is that the development of metastases is preferentially observed in bone through yet-unknown mechanisms. Metastasis to nonbone organs including adrenal glands, ovary, and brain is rarely observed, and few metastases are detected in lung or liver at a histologic level. As such, this model is suitable to specifically study the events involved in bone metastasis. In most of our studies, we used a human estrogen-independent breast carcinoma cell line MDA-MB-231 (MDA-231). These cancer cells develop radiologically distinctive osteolytic bone metastases 3 4 weeks after cell inoculation (Fig. 2). Histologic examination demonstrates numeral numbers of osteoclasts along the endosteal surface of bone and extensive tumor colonization in the bone marrow cavity (Fig. 2). Orthotopic bone metastasis model of mouse mammary tumor 4T1 Experimental metastasis models such as the heart injection model described above lack critical early steps occurring between tumor formation at the primary site and

4 2982 CANCER Supplement June 15, 2000 / Volume 88 / Number 12 FIGURE 3. Orthotopic bone metastasis model (spontaneous bone metastasis model) is shown. Inoculation of 4T1 mouse mammary carcinoma cells in the mammary fat pad in 4 6-week-old female Balb/c mice caused tumor formation (arrow) and metastases to multiple organs including bone and visceral organs as a function of time. entry into the circulation (intravasation). Moreover, the heart injection model described above rarely forms visceral organ metastases, which most cancer patients already have developed at the time of detection of bone metastases. Thus, the results obtained in experimental bone metastasis models are not necessarily applicable to cancer patients who have primary tumor and develop metastases to bone and visceral organs. To overcome these problems, we have established an orthotopic metastasis model of mouse mammary tumor called 4T1. 32,33 These mammary tumor cells form tumors 7 10 days after subcutaneous inoculation into the orthotopic mammary fat pad and subsequently develop bone and visceral organ metastases 3 4 weeks after inoculation in female Balb/c mice (Fig. 3). From technical points of view, this model produces bone metastases at 100%, whereas the heart injection model does not produce 100% success due to its requirement of technical skillfulness. Another advantageous feature is that this model allows us to use immunocompetent syngeneic female mice (Balb/c). Overall, this model more closely resembles the situation in breast carcinoma patients than the heart injection model, except that 4T1 tumor is not of human origin. Osteolytic bone metastases in 4T1 tumorbearing mice are less distinctive compared with those in MDA-231 tumor-bearing mice (Fig. 4). However, histologic examination reveals the occurrence of profound osteoclastic bone resorption (Fig. 4). Effects of BP on Osteoclasts and Breast Carcinoma Cells Metastasized to Bone We studied the effects of the BP risedronate, ibandronate, and zoledronate on bone metastasis of MDA- FIGURE 4. Bone metastases of 4T1 mammary carcinoma are shown. Note osteolytic bone metastases in the distal femur and proximal tibia (left, arrows). Histologic examination reveals that 4T1 carcinoma cells almost completely occupy the bone marrow cavity with osteoclastic bone resorption along endosteal surface (right). MB-231 human breast carcinoma by using the heart injection model. These BPs were given either from the time of cell inoculation (preventative) or after osteolytic bone metastases were established (therapeutic). Development and progression of osteolytic bone metastases were monitored weekly by radiographs. Bones with radiologically evident lesions were processed for histologic and histomorphometric examination. Radiographic examination showed that BP significantly inhibited the development of new bone metastases and progression of established bone metastases. 31,34 Histomorphometric analysis of these

5 Bisphosphonate Actions on Bone Metastasis/Yoneda et al In support of this interpretation, it has been shown that considerably high concentrations ( 10 4 M) of BP are required to induce apoptosis in cancer cells in culture Furthermore, BP has been found to induce apoptosis in myeloma cells in culture through inhibiting mevalonate pathway as is the case in osteoclasts. 20 Thus, whether BP increases apoptosis in metastatic breast carcinoma cells in bone through direct anticancer actions or secondary effects of inhibition of bone resorption is still an open question (Fig. 6). FIGURE 5. Apoptosis in osteoclast and metastatic carcinoma cells in bone in ibandronate-treated mice in the heart injection model is shown. A tartrateresistant acid phosphatase -positive osteoclast (thick arrow) exhibits representative apoptosis with nuclear condensation and is detached from bone surface. In addition, metastatic MDA-231 human breast carcinoma cells (thin arrows) also show histologically representative apoptotic morphology. osteolytic lesions demonstrated that BP decreased osteoclast number and metastatic tumor burden in bone. Moreover, histologic examination revealed that BP markedly increased apoptosis in osteoclasts (Fig. 5). 35 As a mechanism of BP-induced apoptosis in osteoclasts, inhibition of mevalonate pathway that is critical to the prenylation of small GTP proteins such as Ras, Rho, and Rab has been reported. 36,37 Subsequently, we also determined the effects of BP on apoptosis and mitosis in MDA-231 breast carcinoma cells metastasized to bone. We found that apoptosis in these cancer cells also was increased by the treatment with BP (Fig. 5), whereas mitosis was not changed, suggesting that increased apoptosis is not due to cytotoxic effects of BP. To determine whether BP-increased apoptosis in MDA-231 breast carcinoma cells is specific for bone, we next examined effect of BP on apoptosis in MDA-231 tumor at the mammary fat pad. Our data showed that BP did not increase apoptosis in MDA-231 tumor at the orthotopic site. The data suggest that BP specifically enhances apoptosis in MDA- 231 breast carcinoma cells colonized in bone. The results also suggest that BP may not have direct anticancer effects and may increase apoptosis in bonemetastatic breast carcinoma cells through restriction of the supply of bone-derived growth factors resulting from inhibition of bone resorption. There could be, however, an alternative interpretation of these in vivo results. Because BP is known to predominantly accumulate in bone after systemic administration, the local concentration of BP at the mammary fat pad may not be high enough to increase apoptosis in cancer cells. Effects of BP on Visceral Organ Metastasis Heart injection model of bone metastasis Single use of BP. MDA-231 human breast carcinoma cells occasionally spread to adrenal glands after intracardiac inoculation. By repeated passages in the metastases in adrenal glands and in culture of metastatic MDA-231 cells, we established a subclone that selectively and consistently metastasizes to both bone and adrenal glands. We then stably transfected the reporter gene luciferase into this subclone (MDA- 231F9AD/Luc). Using this subclone, we determined the effects of the BP ibandronate on metastases in bone and adrenal glands. In the first set of experiments, BP was administered in a preventative manner in which mice received daily subcutaneous injections of BP from the time of intracardiac inoculation of cells to the end of experiments. Radiographic and histologic examination demonstrate that BP administered according to this protocol profoundly decreased osteolytic bone metastases. In contrast, metastasis to adrenal glands determined by luciferase activity was increased significantly in BP-treated animals. Similar experimental observations that BP increases soft organ metastases have been reported using various BPs. 34,38,39 The mechanism of increased soft organ metastasis by BP administration is not known. Because tumor-bearing mice received only repeated subcutaneous injections of BP daily for 4 weeks in our experiments, it is likely that this observation is an extreme example in experimental nude mouse model of bone metastasis, which evidently never simulates the situation in cancer patients. In the second set of experiments, BP was administered in a therapeutic fashion in which mice received daily subcutaneous injections of BP after bone metastases were established until the end of experiments. Thus, in these experiments, total amounts of BP administered was less, and the period of administration was shorter than the preventative experiments. Our results demonstrate that the BP ibandronate significantly impaired the progression of bone metastases.

6 2984 CANCER Supplement June 15, 2000 / Volume 88 / Number 12 FIGURE 6. Apoptotic effects of BP on osteoclasts and metastatic carcinoma cells in bone are shown. Bisphosphonate preferentially accumulates in bone, directly enters osteoclasts through yet-unknown mechanisms, and inhibits the mevalonate pathway. Inhibition of the mevalonate pathway prevents the prenylation of small GTP proteins such as Ras, Rho, and Rab, leading to induction of apoptosis and inhibition of bone resorption. Due to inhibition of osteoclastic bone resorption, supply of bone-derived growth factors such as TGF and IGFs to metastatic carcinoma cells is largely restricted, which, in turn, may cause apoptosis in these cancer cells. Moreover, BP released into the marrow cavity as a consequence of osteoclastic bone resorption might directly induce apoptosis in metastatic carcinoma cells through inhibiting the mevalonate pathway, although this is yet to be proved. BP: bisphosphonate; GF: growth factor. Of note, BP administered according to this protocol did not increase adrenal metastases. Therefore, therapeutic administration of BP to breast carcinoma patients with established bone metastases probably does not cause an increase in soft organ metastases. Combined use of BP with anticancer agents. In the experiments described above, tumor-bearing mice were treated with BP only, and no other anticancer therapies were given. This therapeutic regimen is far different from that in breast carcinoma patients with bone metastases who are primarily treated with anticancer agents and given BP as an adjuvant agent only when necessary. In fact, most clinical studies have been performed using BP in combination with conventional anticancer therapies. 9,15 17 We therefore determined the effects of the BP ibandronate combined with an anticancer agent doxorubicin. We found that combined treatment with BP and doxorubicin in the preventative manner more effectively suppressed both bone and adrenal metastases than BP or doxorubicin alone. Doxorubicin alone failed to inhibit bone metastases in these experiments. Thus, combination of BP and an anticancer agent doxorubicin produced synergistic inhibitory effects on both bone and soft organ metastases of MDA-231F9AD/Luc breast carcinoma in the heart injection model. Orthotopic model of bone metastasis As described above, 4T1 mammary tumor cells begin to show tumor formation at the orthotopic site 1 week after cell inoculation and develop both bone and visceral organ metastases 3 4 weeks after the inoculation. Thus, this model is suitable to determine the therapeutic effects of BP and also compare the effects of BP on metastases between bone and nonbone organs. To facilitate quantitative analysis, 4T1 cells were stably transfected with the reporter gene luciferase (4T1/Luc). In addition, metastases in bone and nonbone organs were analyzed by histology and histomorphometry. Single use of BP. We tested the newest and most potent BP zoledronate in this model. Bisphosphonate was administered in a therapeutic manner in which single bolus intravenous injection from the tail vein was given when tumor formation at the orthotopic inoculation site became visible 7 10 days after cell inoculation. Similar administration protocol of BP to this has been most frequently used for breast carcinoma patients with bone metastases. Histomorphometric and histologic examination showed that BP suppressed bone metastases in a dose-dependent fashion. Moreover, BP decreased osteoclast number, increased apoptosis in osteoclasts and metastatic 4T1 cancer cells,

7 Bisphosphonate Actions on Bone Metastasis/Yoneda et al and diminished metastatic tumor burden in bone. Metastases in the lung and liver determined by luciferase activity were not increased under this administration protocol. Combined use of BP with anticancer agents. We tested the BP incadronate or zoledronate in combination with an anticancer agent UFT. UFT is an oral anticancer agent consisting of tegafur, a prodrug of fluorouracil, and uracil (with a fluorouracil degradationinhibitory effect) 40 and has been shown to have therapeutic effects in breast carcinoma patients. 41 In these experiments, BP was administered by single bolus intravenous injection, and UFT was given orally once a day from 7 days after tumor inoculation to the end of experiments. Bisphosphonate combined with UFT inhibited not only bone metastases but also lung or liver metastases in an additive fashion. These results are consistent with those of a previous clinical study reported by Diel et al.. 15 UFT alone marginally but significantly suppressed tumor formation at the orthotopic inoculation site and moderately decreased lung and liver metastases. UFT alone also inhibited bone metastases probably by inhibiting the primary tumor in this orthotopic model. Summary of BP Actions on Osteolytic Bone Metastasis in Breast Carcinoma Our results obtained using animal models of bone metastasis demonstrate that BP is a potent and beneficial agent for the treatment of osteolytic bone metastasis in breast carcinoma. Effectiveness of BP on bone metastasis is further increased by a combination with anticancer agents. Moreover, the combined treatment is more effective at inhibiting visceral organ metastases than single treatment with anticancer agents alone. Bisphosphonate might influence visceral organ metastases in some extreme situations. However, our results also suggest that BP alone has no effects on visceral organ metastases in breast carcinoma patients, as long as BP is administered according to the current therapeutic regimens. FIGURE 7. Osteosclerotic bone metastasis caused by MCF-7 cells is shown. MCF-7 human estrogen-dependent breast carcinoma cells form osteosclerotic bone metastases weeks after heart inoculation in female nude mice. Note the presence of numeral osteoblasts surrounding new bone and colonization of metastatic MCF-7 cells. Effects of BP on Osteosclerotic Bone Metastasis Study model Prostate carcinoma is the most relevant model to osteosclerotic bone metastasis. Unfortunately, however, there is currently no animal model of prostate carcinoma that reproducibly develops osteosclerotic bone metastases. Breast carcinomas ( 30%), especially estrogen receptor positive breast carcinomas, have been known to develop osteosclerotic or mixed type (osteolytic and osteosclerotic) bone metastases. 25 We recently have found that MCF-7 human estrogen-dependent breast carcinoma cells form osteosclerotic bone metastases after heart inoculation into female nude mice (Fig. 7). Of note, histologic examination demonstrates that osteoclastic bone resorption is predominant at the early stages of bone metastases and that these osteolytic lesions are progressively replaced by osteosclerotic lesions as a function of time. Thus, this model appears to be suitable to examine the notion that precedence of osteolysis is necessary for the development of osteosclerotic bone metastases and also to test the effects of BP on osteosclerotic bone metastases. Effects of BP on osteosclerotic bone metastases Using this newly established model of osteosclerotic bone metastasis of breast carcinoma, we examined the effects of the BP ibandronate. One group of mice received daily subcutaneous BP injections from 7 days before cell inoculation to 4 weeks after the inoculation to inhibit initial osteolysis, left untreated thereafter, and killed 10 weeks after inoculation (early treatment). Another group of mice received BP from 6 to 10 weeks after cell inoculation during which period osteosclerosis predominantly takes place and killed at Week 10 (late treatment). Both group of mice received the same total amount of BP. Our data showed that early treatment inhibited the development of the osteosclerotic bone metastases, whereas late treatment failed to inhibit them. These results demonstrate that inhibition of initial osteoclastic bone resorption by the administration of BP inhibits the following development of osteosclerotic bone metastases and thus suggest that

8 2986 CANCER Supplement June 15, 2000 / Volume 88 / Number 12 FIGURE 8. Mechanism of osteosclerotic bone metastasis (hypothesis) is shown. Osteoclastic bone resorption may supply space and bone-stored growth factors that are required for osteoblast proliferation and calcium necessary for calcification. If this is the case, administration of BP at a stage of bone resorption should be able to inhibit the subsequent development of osteoblastic bone metastases. However, it still remains possible that cancer cells directly stimulate osteoblasts by producing osteoblastogenic factors to form osteosclerotic bone metastases. If this is the case, BP may be unable to show inhibitory effects. BP: bisphosphonate; GF: growth factor. bone resorption is necessary for the subsequent development of osteosclerotic bone metastases in this model (Fig. 8). They also suggest that BP may have therapeutic effects on osteosclerotic bone metastases in prostate carcinoma when administered at appropriate stages of the disease. However, our results do not exclude the possibility that prostate carcinoma cells directly stimulate osteoblasts by producing osteoblastogenic factors to cause osteosclerotic bone metastases. Further studies using prostate carcinoma models that consistently develop osteosclerotic bone metastases are required. Once such a model becomes available, BP will be a useful tool to elucidate the mechanism of osteosclerotic bone metastasis. More importantly, such a model should allow us to design effective therapeutic regimen using BP and other agents for the bone diseases associated with prostate carcinoma. Effects of BP on the Survival of Tumor-Bearing Animals In the experiments in which animals were treated with daily subcutaneous injections of BP for 4 weeks in a preventative manner, BP alone significantly increased the survival of MDA-231 breast carcinoma-bearing animals. Conversely, single bolus intravenous injection of BP failed to prolong the survival of 4T1 tumorbearing mice. However, combination of BP with UFT markedly increased the survival of 4T1 tumor-bearing animals. Thus, the effects of BP on survival may depend on the administration protocol. Nevertheless, because bone metastases profoundly limit physical and mental activity in breast carcinoma patients, inhibition of bone metastases by the treatment with BP may increase the survival of these patients. CONCLUSIONS AND FUTURE STUDIES BP has been successfully used for the treatment of cancer patients with bone metastases for almost 3 decades. Nonetheless, numbers of important questions still remain unsolved. In this article, three different types (experimental and orthotopic osteolytic bone metastasis and osteosclerotic bone metastasis) of animal models of bone metastasis of breast carcinoma are introduced. We have shown that these animal models are very useful for the study of BP actions that is not readily conducted in clinical studies. Of particular importance, these animal models allow us to design the optimized regimen of BP administration for the treatment of breast carcinoma patients with bone and visceral organ metastases. However, note that there are still unsolved questions regarding BP actions on cancer metastasis to bone. Is BP effective on bone diseases associated with prostate carcinoma, lung carcinoma, neuroblastoma, and myeloma? Is there a relation between chemical structure of BP and potency or diversity of its action?

9 Bisphosphonate Actions on Bone Metastasis/Yoneda et al Does BP have direct anticancer effects? Combination with what agents or therapies is most effective and beneficial? Does BP have any effects on nonbone tissues and cells? Are there novel molecular mechanisms of BP action other than mevalonate pathway? We believe that these questions can be answered by optimally using our in vivo models and that these studies of BP also will lead us to determine the cellular and molecular mechanisms underlying cancer metastasis to the skeletons. REFERENCES 1. Yin JJ, Selander K, Chirgwin JM, Dallas M, Grubbs BG, Wieser R, et al. TGF signaling blockade inhibits PTH-rP secretion by breast cancer cells and bone metastasis development. J Clin Invest 1999;103: Thomas RJ, Guise TA, Yin JJ, Elliott J, Horwood NJ, Martin TJ, et al. Breast cancer cells interact with osteoblasts to support osteoclasts formation. Endocrinology 1999;140: Mundy GR, Yoneda T. Facilitation and suppression of bone metastasis. 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