The role of bisphosphonates in breast cancer: Actions of bisphosphonates in animal models of breast cancer

The skeleton is the most common site of breast cancer metastases. These bone metastases are usually osteolytic and cause significant morbidity. Bisphosphonates, potent inhibitors of bone resorption, reduce skeletal morbidity in breast cancer patients with bone metastases. Animal studies with bisphosphonates are crucial to understanding the mechanisms by which these compounds affect bone and tumor cells in vivo. Such animal models of breast cancer that are used to test the efficacy of bisphosphonates are discussed. These studies may offer insight into the treatment of other tumor types that frequently metastasize to bone.


Introduction
Up to one-third of patients with early stage breast cancer will eventually die from the disease, and most of these (~80%) will have bone metastases. Although a majority of these bone metastases are destructive or osteolytic, a significant percentage also causes abnormal bone formation or osteosclerotic lesions. Once tumor has metastasized to bone, the disease is incurable. Because the average survival of breast cancer patients following diagnosis of bone metastases is 24-36 months, the morbidity of bone pain, fracture, hypercalcemia and nerve compression syndromes is longstanding. Therapeutics to treat and prevent these devastating complications of bone metastases are therefore in great demand.

Seed and soil hypothesis
It is well established that the skeleton is the most common site of distant metastases of breast cancer cells. Paget proposed, in 1889, that the affinity of certain cancers to metastasize to bone was due to the fact that the bone provides a 'fertile soil' or environment for the cells to germinate [1]. This seed and soil hypothesis is supported by the fact that bone is a repository for a number of growth factors and that osteoclastic bone resorption releases these growth factors.
Histological sections of breast cancer metastases to bone reveal tumor cells adjacent to osteoclasts that are resorbing bone. These observations, combined with the clinical data demonstrating that bisphosphonate inhibitors of bone resorption reduce skeletal morbidity in breast cancer patients, indicate that bone destruction in breast cancer osteolysis is mediated by the osteoclast.
Our laboratory and other laboratories have provided evidence of a 'vicious cycle' involving breast cancer and bone. In this vicious cycle, metastatic breast cancer cells in bone produce factors (such as parathyroid hormonerelated protein) that stimulate osteoclastic bone resorption. This production results in the release of growth factors, such as transforming growth factor-β, from the bone matrix [2,3]. Growth factors, in turn, stimulate tumor growth and production of more parathyroid hormonerelated protein, resulting in a 'vicious cycle' that further fuels the bone destruction and tumor growth [2,3]. the metastatic cascade before it comfortably settles in bone. Specifically, the tumor cell must detach from the primary site, enter tumor vasculature to reach the circulation, survive host immune response and physical forces in the circulation, arrest in a distant capillary bed, escape the capillary bed, and proliferate in the metastatic site.
The events involved in entering the tumor vasculature are similar to those involved with exiting the vasculature in the bone marrow cavity. These events include attachment of tumor cells to the basement membrane, tumor cell secretion of proteolytic enzymes to disrupt the basement membrane, and migration of tumor cells through the basement membrane. Attachment of tumor cells to basement membranes and to other cells is mediated through cell adhesion molecules such as laminin and E-cadherin. Tumor cell secretion of substances such as metalloproteinases facilitates disruption of the basement membranes and enhances invasion. Inherent tumor cell motility or motility in response to chemotactic stimuli are also important factors for tumor cell invasion to the secondary site.
A number of other tumor types frequently metastasize to bone, including prostate, lung, thyroid, and renal cell carcinomata. Multiple myeloma is a primary hematologic malignancy that causes osteoclast-mediated bone destruction but is not a metastatic tumor per se. However, many of the tumor cell-bone cell interactions that cause the bone destruction are similar to those implicated in breast cancer osteolysis. Although this review focuses on breast cancer metastases to bone, the studies reviewed here offer general mechanisms that can be applied to other tumor types.

Bisphosphonates: what they are and what they do
Bisphosphonates are synthetic analogs of inorganic pyrophosphate. They are taken up preferentially by the skeleton and are strongly bound to hydroxyapatite on the surface of bone. Bisphosphonates are potent inhibitors of osteoclastic bone resorption. The effects of these drugs are primarily on the bone-resorbing osteoclasts but may also target osteoblasts, macrophages and tumor cells [4][5][6][7][8]. The mechanisms by which bisphosphonates inhibit osteoclast activity, and the relative potencies with which they do so, are dependent on the molecular structure of each compound.
The major mechanism of the nitrogen-containing bisphosphonates to decrease osteoclast number, osteoclast activity and bone resorption is by induction of osteoclast apoptosis [9]. As described by Fleisch in this issue [10], bisphosphonates inhibit farnesyl diphosphate synthase in the mevalonate pathway and thereby prevent protein prenylation of small GTPase signaling proteins required for osteoclast function [11,12]. The degree to which nitrogen-containing bisphosphonates inhibit bone resorption correlates with the capacity to cause apoptosis in cells of the osteoclast lineage, as well as with the capacity to inhibit farnesyl diphosphate synthase and protein prenylation in the osteoclast [11]. The non-nitrogen-containing bisphosphonates, such as clodronate, do not inhibit protein prenylation and have a different mode of action that may involve the formation of cytotoxic metabolites in osteoclasts or inhibition of protein tyrosine phosphatases [12].
Bisphosphonates also affect cells other than osteoclasts in the bone microenvironment. Derenne et al. showed that the bisphosphonates pamidronate and zoledronate inhibited interleukin-6-induced production of matrix metalloproteinase-1 by bone marrow stromal cells [13]. In vitro studies have also shown that nitrogen-containing bisphosphonates inhibit the adhesion of breast cancer cells to bone and bone matrix [14].
Even more intriguing are those results indicating that bisphosphonates may have direct effects on the tumor cells themselves, as reviewed in detail by Senaratne and Colston in this issue [15]. Bisphosphonates have been shown to decrease the proliferation and viability of human tumor cells lines, as well as to increase the apoptotic index of the human tumor cell lines [13,16]. Numerous investigators have demonstrated that bisphosphonates significantly reduce tumor invasion and adhesion to bone [13,14,[16][17][18][19].

Bisphosphonates in preclinical animal models of breast cancer
Animal studies with bisphosphonates are essential to understanding the effects of these compounds on both bone and tumor cells in vivo. Four distinct models of osteolytic bone metastases (MDA-MB-231, 4T1, ENU-1564, and Walker carcinosarcoma 256B) and one model of osteoblastic metastases (MCF-7) in breast cancer have been used to test the efficacy and dosing of bisphosphonates. Table 1 summarizes the effects of bisphosphonates on the development and progression of bone metastases in these models.  morphometric analysis of the bones showed a decrease in tumor volume in the bone in mice treated with risedronate [21]. Risedronate also increased the survival of the animals compared with untreated mice [21]. Sasaki et al. tested an experimental bisphosphonate, YH529, for the ability to decrease bone metastases, and observed a dose-dependent decrease in both osteolytic lesion number and area when the drug was given in a preventative manner (from the time of tumor inoculation until the end of the experiment) [22]. When YH529 was administered to treat established bone metastases, however, it had little effect [22].

MDA-MB-231 experimental model
Yoneda et al. also studied the effects of the bisphosphonate ibandronate on a subclone of MDA-MB-231 cells that is reported to more reliably form metastases to the skeleton and adrenal glands [23]. Ibandronate was administered in a 'preventative' protocol and a 'therapeutic' protocol. In groups treated according to the preventative protocol, in which mice received daily injections of ibandronate beginning at the time of tumor inoculation and continued for the duration of the experiment, a decrease in osteolytic skeletal metastases was observed [23]. However, adrenal metastases were increased in mice treated with ibandronate; an observation that is consistent with the data of other studies [21][22][23][24]. In the therapeutic protocol, mice received ibandronate daily only after the development of osteolytic bone metastases. In this case, ibandronate (unlike YH529 in earlier experiments) decreased the progression of the established bone metastases compared with the control group, with no effect on adrenal metastases [23].
Hiraga et al. have more recently provided evidence that ibandronate acts by reducing osteoclastic bone resorption and by increasing the apoptosis of osteoclasts [25]. In addition, ibandronate was shown to increase the apoptosis of tumor cells in the experimental bone metastasis model but not in orthotopic mammary fat pad tumors, indicating that the primary effect is in bone and tumors in bone [25].

4T1 experimental model
The second model, also used by Yoneda and colleagues, involves a mouse mammary tumor cell line, 4T1. This model is clinically relevant because syngeneic, immunocompetent mice are inoculated orthotopically into the mammary fat pad, and metastases occur in bone and soft tissue. This is one of the few models in which the cells must go through the multiple steps of the metastatic cascade to develop bone metastases. A primary tumor is usually evident in these mice about 1 week post tumor inoculation. Metastases are identified in the lungs and liver around 2 weeks post tumor inoculation. By week 3 the mice have distant metastases to the skeleton, the kidney, the adrenal glands, the heart and the spleen, and they do not usually survive beyond week 4 or 5 post tumor inoculation. The pattern of metastases and the histologic appearance are similar to those seen in human patients. This model allows for the simultaneous study of the effects of bisphosphonates on bone and soft tissue metastases.
This model was used to examine the therapeutic value of zoledronate, the most potent of the approved bisphosphonates. Administration of the bisphosphonate was begun as soon as the orthotopic tumor was apparent (approximately 7 days post tumor inoculation) [23]. Analysis of Xrays of both the treated and the untreated mice revealed a decrease in lesion area in the long bones of the mice receiving zoledronate. In addition, zoledronate-treated mice exhibited a decrease in osteolytic tumor lesion area in the lumbar spine by histomorphometric analysis [26]. The compound prevented the marked bone destruction seen in the trabecular bone of the tibial growth plates of control mice [26]. Daily treatment of mice bearing 4T1 tumors with zoledronate increased both osteoclast and tumor cell apoptosis within the bone metastases [26]. Finally, zoledronate also resulted in a decrease in the lesion area by X-ray analysis of existing bone metastases by 4T1 cells, while ibandronate and alendronate had no effect on established bone metastases [26]. No effect was observed on visceral metastases or on the primary tumor, however, indicating that the actions of zoledronate as used in this study are limited to bone.
The 4T1 bone metastases model has also been utilized to look at combinations of bisphosphonates with anticancer agents, a situation that more closely resembles the clinical scenario. Yoneda et al. [23] examined the effects of incadronate and zoledronate with the anticancer agent, UTF. The combination therapy inhibited metastasis to bone, the liver and the lung. UTF alone resulted in a decrease in tumor burden in the mammary fat pad, as well as in decreased metastases to the lung, the liver and the skeleton [23]. The decrease in bone metastases by UTF alone is probably due to the initial decrease in the size of the primary tumor.

ENU-1564 experimental model
A third model used to study the effects of bisphosphonates is a rat model using the ENU1564 mammary adenocarcinoma cell line. Rats are administered the mammary adenocarcinoma cells via intracardiac inoculation and are monitored for tumor development and the subsequent development of metastases.
This model was used to examine the effects of risedronate on bone metastases. Consistent with the results observed with the MDA-MB-231 model, risedronate resulted in a reduction in the number of skeletal metastases and in the size of the lesions in the skeleton [27]. No difference in visceral metastases was observed [27].

Walker carcinosarcoma 256B experimental model
The fourth model used to assess the use of bisphosphonates in the treatment of skeletal metastases secondary to breast cancer is the Walker carcinosarcoma 256B model. This is a rat model of skeletal metastases in which the cells are implanted directly into the bone. The growth of the Walker carcinosarcoma 256B cells in bone also leads to hypercalcemia, a common complication of bone metastases. Krempien et al. [28] have also found that intra-aortic administration of Walker carcinosarcoma 256B cells results in both bone and adrenal metastases in rats. Prophylactic treatment with clodronate, both short-term (5-day treatment) and long-term (21-day treatment), inhibited osteolysis is this model [28]. However, Krempien and Manegold also found that the longer the treatment-free intervals after short-term therapy, the less effective the inhibition [29]. The Walker carcinosarcoma 256B models have also been used to examine the effect of pamidronate on skeletal metastases [24,30]. Krempien et al. reported a reduction in skeletal metastases in rats treated with pamidronate [30]. Surprisingly, a second group reported that the tumor burden in bone increased following treatment with pamidronate [30]. The latter result, however, is not consistent with the clinical data that pamidronate reduced skeletal complications in patients with multiple myeloma and breast cancer [31][32][33].
Kurth et al. more recently examined the effects of daily treatment with ibandronate on the bone quality of rats inoculated with Walker carcinosarcoma 256B cells [34]. Treatment with ibandronate was shown to increase the bone density and the bone stability compared with controls [34].

Bisphosphonates and osteoblastic bone metastases
The estrogen receptor positive human breast cancer cell line, MCF-7, has been shown to produce osteoblastic or mixed osteolytic and osteoblastic bone lesions following intracardiac inoculation in a nude mouse model. It has long been hypothesized that bone resorption precedes the new bone formation of osteoblastic metastases, since biochemical markers of osteoclastic bone resorption are markedly increased in patients with osteoblastic metastases. Yoneda et al. tested the hypothesis that blocking bone resorption with bisphosphonates may affect the ability of MCF-7 cells to cause osteoblastic metastases [23]. Using the MCF-7 model, they looked at the effects of both early (prior to tumor inoculation) and late (post tumor inoculation, osteoblastic metastases established) treatment with ibandronate [23]. Only the early treatment with ibandronate resulted in inhibition of osteoblastic metastases. This supports the hypothesis that a bone resorptive phase precedes the development of osteoblastic metastases. The use of bisphosphonates to inhibit this resorptive phase may thus significantly reduce the development of osteoblastic metastases.

Discussion
Studies of bisphosphonates in preclinical models of breast cancer metastases to bone illustrate the importance of the bone microenvironment and osteoclastic bone resorption in the development of skeletal metastases of both osteolytic and osteoblastic nature. We have learned from these studies that the primary action of bisphosphonates is on the bone resorbing osteoclasts, and that bisphosphonates may exert secondary effects on the tumor cells in bone. Zoledronate, the newest and most potent bisphosphonate, showed promise in the preservation of bone in the 4T1 model of breast cancer metastasis to bone [23]. It caused a decrease in the osteoclast number and a decrease in the tumor burden in bone [23].
There are multiple explanations for this decrease in tumor burden in bone. First, and most probable, the decrease in osteoclastic bone resorption makes the skeleton a less hospitable environment for the tumor cells, by reducing the release of bone-derived growth factors that may stimulate tumor growth or production of osteolytic factors. Second, the bisphosphonates may have a direct effect on the tumor cells to induce apoptosis. While there are data in vitro and in animal models to support this, the concentrations of bisphosphonates at which this occurs in vitro are quite high. It is unclear whether such concentrations occur in vivo. Such data have yet to be demonstrated in humans with bone metastases. Finally, it is also possible that the decrease in tumor burden in bone is due to an effect of bisphosphonates on angiogenesis [35]. This potential anti-angiogenic effect on tumors remains to be investigated.
The observation in some of the animal models reported here that clearly needs to be clarified in human studies is the issue of extraskeletal metastases. That is, soft tissue metastases were increased following treatment with bisphosphonates, an observation indicating that if the tumor cells find the bone microenvironment inhospitable then they may seed to other tissues. However, clinical data with regard to this are inconsistent. Many questions remain regarding the use of bisphosphonates in the treatment of metastatic breast disease to the skeleton. More work is needed to determine whether bisphosphonates truly have antitumor activity in humans, or whether the reduction in tumor burden in bone is due to the reduction of bone-derived growth factors released into the local microenvironment as a consequence of inhibiting osteoclastic bone resorption. Second, bisphosphonate use in osteoblastic metastases needs to be further explored to prove definitively that treatment inhibiting bone resorption does indeed reduce osteoblastic disease. In addition, it still remains to be determined whether bisphosphonates increase survival in clinically relevant situations. Patients with metastatic breast cancer are treated with anticancer agents in addition to bisphosphonates, and studies in animal models that mimic this situation would provide more realistic evidence with regard to survival. Finally, the use of bisphosphonates in the treatment of bone metastases of other primary tumors is an area that needs to be explored in both animal models and humans. Many of these questions are currently under investigation, and the answers should provide a strong rationale for the use of bisphosphonates in cancer-induced morbidity of the skeleton.