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Bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used as a beneficial agent for the treatment of bone metastases in patients with breast cancer. It is well recognized that BP reduces osteolysis by promoting apoptosis in osteoclasts. However, recent animal and human data suggest that BPs not only reduce osteolysis associated with metastatic breast cancer, but also decrease tumor burden in bone. The mechanisms by which tumor burden is decreased following BP administration are unknown. Here we examined the effects of the BP ibandronate on MDA-231 human breast cancer cells in bone metastases in a well-characterized animal model of bone metastasis. Ibandronate, which was administered (s.c. daily; 4 microg/mouse/day) after bone metastases were established, inhibited the progression of established osteolytic bone metastases as assessed by radiographic analysis. Histological and histomorphometrical examination revealed that ibandronate reduced osteoclastic bone resorption, with increased apoptosis in osteoclasts. Furthermore, ibandronate also significantly decreased the MDA-231 tumor burden, with increased apoptosis in MDA-231 breast cancer cells in bone metastases. In contrast, ibandronate failed to inhibit MDA-231 tumor formation with no effects on apoptosis in MDA-231 breast cancer cells in the orthotopic mammary fat pads. These data suggest that the effects of ibandronate on apoptosis in MDA-231 breast cancer cells are restricted in bone in which ibandronate selectively deposits. Consistent with these in vivo results, a relatively high concentration of ibandronate (100 microM) increased caspase-3 activity and induced DNA fragmentation in MDA-231 breast cancer cells in culture. Moreover, a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone, blocked ibandronate-induced DNA fragmentation in MDA-231 cells, suggesting an involvement of caspase-3 in ibandronate-induced apoptosis. Our results suggest that BP suppresses bone metastases through promotion of apoptosis in metastatic cancer cells as well as in osteoclasts. However, it still remains open whether BP has direct anticancer actions in vivo.

Humoral hypercalcemia of malignancy results from the effects of tumor-produced factors on bone, kidney, and intestine that disrupt normal calcium homeostasis. Although parathyroid hormone-related protein (PTHrP) is a major mediator of the syndrome, tumors also produce other hypercalcemic factors, such as tumor necrosis factor (TNF), which may modulate the effects of PTHrP. It has been postulated that TNF may counteract the stimulatory effects of PTHrP on bone formation. To examine the effects of TNF on PTHrP-induced changes in calcium and bone metabolism, a murine tumor model of hypercalcemia was used. Nude mice were inoculated with Chinese hamster ovarian (CHO) cells expressing human TNF (CHO/TNF) or nontransfected CHO cells (CHO/-) and further treated with injections of human PTHrP(1-34) or vehicle. The effects of TNF, PTHrP, and the combination of the two factors on blood ionized calcium, osteoclast recruitment, and bone histomorphometry were evaluated. Mice bearing CHO/TNF tumors that were injected with PTHrP had significantly higher calcium concentrations, increased committed osteoclast progenitors, and mature osteoclasts as well as enhanced bone resorption compared with mice bearing CHO/TNF tumors injected with vehicle or those bearing CHO/- tumors injected with PTHrP or vehicle. A 2-fold increase in new woven bone formed in the calvaria at sites of previous bone resorption was observed in CHO/TNF mice treated with PTHrP. Bone formation rates in the vertebrae were similar in both CHO/- and CHO/TNF mice treated with PTHrP. These data demonstrate that the hypercalcemic effects of PTHrP are enhanced by TNF and that this effect is due to the increased production of committed osteoclast precursors with a subsequent increase in osteoclastic bone resorption. Furthermore, PTHrP caused a coupled increase in osteoclastic bone resorption and new bone formation that was not inhibited by TNF. These findings highlight the complex interactions that may occur between tumor-produced factors on bone that result in malignancy-associated hypercalcemia and suggest that TNF may not be responsible for the decreased bone formation seen in some patients with this condition.

The molecular mechanisms by which human cancer cells spread to bone are largely unexplored. The process likely involves cell adhesion molecules (CAMs) that are responsible for homophilic and heterophilic cell-cell interactions. One relevant CAM may be the calcium-dependent transmembrane glycoprotein E-cadherin. To investigate the involvement of E-cadherin in breast cancer metastasis to bone, we used an in vivo model in which osteolytic bone metastases preferentially occur after injections of cancer cells directly into the arterial circulation through the left ventricle of the hearts of nude mice. We have found that E-cadherin-negative human breast cancer cells MDA-MB-231 (MDA-231) develop radiographically detectable multiple osteolytic bone metastases and cachexia in this model. However, MDA-231 breast cancer cells that were transfected with E-cadherin cDNA showed a dramatically impaired capacity to form osteolytic metastases and induce cachexia. Histological and histomorphometrical analyses of bones of mice bearing mock-transfected MDA-231 revealed aggressive metastatic tumor, whereas metastatic tumor burden was significantly decreased in the bones of mice bearing E-cadherin-expressing MDA-231. Nude mice bearing E-cadherin-transfected MDA-231 breast cancer cells survived longer than mice bearing mock-transfected MDA-231 breast cancer cells. Anchorage-dependent and -independent growth in culture and tumor enlargement in the mammary fat pad of nude mice were unchanged between mock-transfected and E-cadherin-expressing MDA-231, suggesting that these differences in metastatic behavior are not due to an impairment of cell growth and tumor-igenicity. Our results show the suppressive effects of E-cadherin expression on bone metastasis by circulating breast cancer cells and suggest that the modulation of expression of this CAM may reduce the destructive effects of breast cancer cells on bone.

Tumors occasionally stimulate bone formation and cause osteoblastic metastases. Although this occurs most frequently in widespread prostate cancer, human prostate cancer cells are difficult to grow in culture without changing their phenotype, and the few available prostatic cancer lines do not increase bone formation in vivo. To identify tumor-derived osteoblast-stimulatory factors, we studied a long-established human tumor cell line derived from human amnion that has, in the past, been reported to cause bone formation in vivo when inoculated into nude mice. Tumor cells were inoculated into nude mice and induced extensive new bone formation. To characterize osteoblast growth factors produced by these tumor cells, solid tumor was isolated from the mice and extracted at neutral pH. Biological activity, assessed by stimulation of proliferation of MG-63 osteoblast-like cells, was used to monitor purification after heparin-Sepharose column chromatography, Mono-S, and C4 reverse-phase high-performance liquid chromatography. An extend amino-terminal form of basic fibroblast growth factor (FGF) was purified by its capacity to stimulate proliferation in MG-63 cells and partially sequenced. Basic FGF is also known to stimulate proliferation in MG-63 cells and other osteoblasts in vitro and bone formation in vivo. In summary, these human tumor cells stimulate new bone formation in vivo and produce an osteoblast stimulating activity in vitro, which has been identified as a form of basic FGF.

Human breast cancer frequently metastasizes to the skeleton to cause osteolysis and subsequent pain, pathological fracture, and hypercalcemia. Because bone continuously releases growth factors stored in bone matrix by bone resorption during physiological remodeling and, thus, possibly provides a favorable microenvironment for metastatic breast cancer cells to proliferate, inhibitors of bone resorption used either prophylactically or in patients with established disease, therefore, would seem likely to be useful adjuvant therapy in patients with breast cancer. However, the parameters for monitoring progressive osteolytic bone disease in humans are imprecise. We examined the effects of the third generation bisphosphonate, risedronate, which is a specific inhibitor of osteoclastic bone resorption, in a bone metastasis model in nude mice in which intracardiac injection of the human breast cancer cell line MDA-231 leads to osteolytic bone metastases. Risedronate (4 micrograms/animal/day) was given s.c. to animals (a) after radiologically small but defined osteolytic metastases were observed; (b) simultaneously with MDA-231 cell inoculation through the entire experimental period; or (c) by short-term prophylactic administration before inoculation of MDA-231 cells. In all experiments, risedronate either slowed progression or inhibited the development of bone metastases assessed radiographically. Furthermore, mice treated continuously with risedronate showed significantly longer survival than did control mice. Histomorphometrical analysis revealed that osteoclast numbers were diminished at metastatic tumor sites. Unexpectedly, there was also a marked decrease in tumor burden in bone in risedronate-treated animals. In contrast, the growth of metastatic breast cancer in soft tissues surrounding bones was not affected by risedronate. Moreover, risedronate had no effects on the local growth of s.c. implanted MDA-231 breast cancers in nude mice or on MDA-231 cell proliferation in culture. These data demonstrate that risedronate decreases metastatic MDA-231 breast cancer burden selectively in bone, as well as suppresses progression of established osteolytic lesions and prevents the development of new osteolytic lesions; thus, the data suggest that inhibition of osteoclastic bone resorption may be a useful adjunctive therapy for the treatment of cancers that have colonized in bone.

Porcine pancreatic extracts (PXs) have previously been shown to decrease blood ionized calcium in BALB/c mice (T. Yoneda, Y. Takaoka, and G. R. Mundy. FEBS Lett., 278: 171-174, 1991). In the present study, we show that the PX is effective in preventing progression of hypercalcemia and decreasing osteoclastic bone resorption associated with a human squamous carcinoma in nude mice. PX inhibited osteoclast-like cell formation in mouse bone marrow cultures and bone resorption in organ cultures of fetal rat long bones which had been stimulated by serum-free culture supernatants of this cancer. In addition, PX increased food intake, decreased weight loss, and prevented development of cachexia. In parallel with these effects, PX prolonged survival of tumor-bearing animals. PX might have therapeutic potential for management of hypercalcemia and cachexia associated with malignancy.

Bone resorbing cytokines may be associated with abnormalities in bone cell function and calcium homeostasis in a number of pathological conditions. One of these cytokines is interleukin 6 (IL-6), which is a multifunctional cytokine which has been shown to be associated with increased formation of bone resorbing osteoclasts in vitro. In this report, we demonstrate that neutralizing antibodies to human IL-6 lower the blood calcium in nude mice carrying a human tumor associated with increased IL-6 production. There was no effect on blood calcium in hypercalcemic nude mice carrying tumors not associated with increased IL-6 production and normocalcemic tumor-bearing nude mice. These results suggest that increased production of IL-6 may contribute to disturbances in calcium homeostasis in some malignancies, and suggest that neutralization of IL-6 effects can lower the serum calcium in these tumors.

The mechanisms by which tumor cells metastasize to bone are not well understood. We have investigated the role of the basement membrane glycoprotein, laminin, in bone metastasis, since antagonists to laminin have been shown to inhibit the formation of lung metastases. We studied the formation of osteolytic metastases caused by a human tumor which is known to cause osteolysis and hypercalcemia in nude mice. We found that tumor-bearing nude mice developed hypercalcemia, cachexia, and characteristic osteolytic lesions throughout the skeleton after injection of this human melanoma cell line (A375) into the left ventricle. When we gave injections to nude mice with A375 cells which had been exposed to C(YIGSR)3-NH2, a laminin-derived synthetic peptide containing three linear sequences of YIGSR with an amino-terminal cysteine which competes with laminin for its receptor, we found a decrease in the formation of detectable osteolytic bone metastases. The tumor cells were incubated with the antagonist and then inoculated into nude mice which were administered the antagonist i.p. Hypercalcemia and cachexia were also decreased in tumor-bearing mice treated with the laminin antagonist. In contrast, laminin itself increased the number of osteolytic bone metastases, as has been shown for other tumor cells. These data suggest that laminin plays a role in the formation of osteolytic bone metastases in this model and that laminin antagonists may be useful in the prevention of bone metastases in some human tumors.

Human breast cancer cell proliferation is regulated by growth factors that bind to receptors with intrinsic tyrosine kinase (TK) activity, including the epidermal growth factor (EGF) receptor. To determine whether inhibition of receptor TK activity inhibits tumor growth, we studied the effects of a tyrosine kinase inhibitor, RG-13022, on cultured human breast cancer cells. RG-13022 represents a class of compounds which have been shown to inhibit preferentially the TK activity of the EGF receptor in a cell-free system and also to inhibit EGF-stimulated growth of cultured cells. RG-13022 significantly inhibited EGF-stimulated autophosphorylation of its receptor in two breast cancer cell lines that have abundant, although not amplified, EGF receptor content (MDA-231 and T47D). RG-13022 also inhibited EGF-stimulated DNA synthesis and proliferation of T47D and MCF-7 breast cancer cells in a reversible and dose-dependent manner. Inhibition was observed at 0.1 microM, and it was maximal at 10 microM. The effect was rapid (within 3 h), persisted for 18 h, and was partially reversed by 24 h at 1 microM. At 5 microM, inhibition persisted for more than 50 h. Inhibitory effects were also observed in a panel of estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RG-13022 inhibited not only EGF-induced growth but also growth stimulated by insulin, insulin-like growth factor I, insulin-like growth factor II, or transforming growth factor alpha. RG-13022 also totally blocked estrogen-stimulated phosphorylation of the EGF receptor, as well as estrogen-induced cell proliferation, suggesting that functioning TK pathways are required for estrogen action. The TK inhibitor RG-13022 is a potent inhibitor of hormonally regulated growth of human breast cancer. Tyrosine kinase inhibitors have the potential of providing a new strategy for the "endocrine therapy" of breast cancer.

Many human tumors of epithelial origin contain cells overexpressing the epidermal growth factor (EGF) receptor, and there is convincing evidence that cancer cell growth is correlated with the loss of the normal regulation of the EGF receptor signal transduction pathway. Some cancers are clearly dependent on activation of the EGF receptor for their proliferation. Recently, a class of compounds, tyrphostins, which inhibit the protein tyrosine kinase activity of the growth factor receptor, have been described. In this report, we have examined the antiproliferative effects of potent new tyrphostins on a well-characterized human squamous cell carcinoma in vitro and in vivo. We found that two of these compounds (RG-13022 and RG-14620) suppressed not only EGF-stimulated cancer cell proliferation in vitro but also tumor growth in nude mice. RG-13022 also increased the life span of these tumor-bearing nude mice. When administered to tumor-bearing nude mice together with monoclonal antibodies to the EGF receptor at a suboptimal dose which had no effect alone, inhibition of tumor growth was markedly enhanced. These data suggest that tyrphostins have potential as anticancer agents.

Increased levels of epidermal growth factor receptor (EGFR) have been shown on squamous cell carcinomas. Recently, we described a squamous cell carcinoma (MH-85) derived from the oral cavity which was associated with several paraneoplastic syndromes including hypercalcemia and cachexia. This tumor induced the same paraneoplastic syndromes in nude mice (BALB/c, nu/nu, male, 4-6 weeks old). Scatchard analysis revealed that there are two classes of EGFR in MH-85. The dissociation constant and number of binding sites for the high affinity receptors were 38 pM and 5 x 10(4)/cell, respectively, and 2.2 nM and 6 x 10(5) cell, respectively, for the low affinity receptors. Growth of MH-85 in culture was stimulated by epidermal growth factor (EGF) and inhibited by monoclonal antibody 108 to human EGFR, which recognizes the extracellular domain of the EGF receptor. Surgical removal of submandibular glands from male nude mice resulted in a dramatic decrease in plasma EGF levels and a significant reduction of tumor growth, hypercalcemia, and cachexia. When EGF (5 micrograms/mouse, every 2 days for 6 weeks, i.p.) was administered to these sialoadenectomized animals, tumor growth increased, with a parallel increase in hypercalcemia. When monoclonal antibody 108 (1 mg/mouse, i.p.) was given 1, 5, and 10 days after MH-85 tumor implantation, tumor formation was retarded, which resulted in delayed onset of hypercalcemia and cachexia. Moreover, when the antibody was injected 6 times in nude mice exhibiting large tumors and profound hypercalcemia and cachexia, there was a striking decrease in tumor growth, which was accompanied with a reversal of hypercalcemia and cachexia. These results indicate that growth of the human squamous cell carcinoma MH-85 is dependent on the EGFR pathway and that subsequent development of hypercalcemia and cachexia is dependent on tumor growth. They also suggest that agents which interfere with the EGFR pathway may have therapeutic potential as anticancer agents in some human tumors.

It has long been known that complex interactions occur between tumors and normal host immune cells. The human melanoma cell line A375 has been used previously as an indicator cell for tumor cell cytotoxicity mediated by monocytes. During other studies on this tumor cell line, we noted that the conditioned media harvested from A375 cultures induced both the human monocytoid cell line U937 and human blood monocytes to release the cytokine tumor necrosis factor (TNF). We characterized this tumor factor which induced TNF release by monocytic cells. Purification was performed using ammonium sulfate precipitation, ion exchange (DEAE) chromatography, gel filtration, and reversed-phase high performance liquid chromatography. The factor copurified with granulocyte-macrophage colony-stimulating factor (GM-CSF). The purified material caused the release of TNF by U937 cells and stimulated formation of granulocyte-macrophage colonies in methyl cellulose. TNF release by U937 cells in response to A375-conditioned medium was inhibited by neutralizing antibodies to GM-CSF. The TNF-inducing activity in A375-conditioned medium was completely removed by an anti-GM-CSF affinity column. Western blotting using antibodies to GM-CSF confirmed a single Mr27,000 band in A375-conditioned medium. We found that recombinant human GM-CSF stimulated TNF production by the same cells as the tumor-conditioned medium. These data show that A375 human melanoma cells produce GM-CSF, which in turn causes TNF production by cells in the monocyte lineage. The combination of GM-CSF production by the tumor and TNF production by immune cells may influence not only tumor growth but also some of the paraneoplastic syndromes associated with malignancy such as hypercalcemia, cachexia and leukocytosis.

Bone marrow biopsy specimens from patients with myeloma were cultured in either 1 of 2 thin-film culture systems, a controlled environment steady state system or a rocker tube configuration of the system, for periods up to 42 days. Both functional and morphological characteristics of the myeloma cells were well-maintained in these systems. Cytocentrifuge preparations of the culture media disclosed hematopoietic cells that included from 5% to almost 100% plasma cells. Histological examination of the cultured specimens disclosed infiltration of the marrow with myeloma cells. Myeloma proteins were released at a steady rate throughout the period of culture after the 1st 4 days. Bone-resorbing activity was demonstrated in the culture media in 7 of 9 myeloma culture media and was well maintained, particularly during the 1st week of culture. This activity was associated with severe osteolytic lesions in the donor patient and marked infiltration of the cultured specimen by myeloma cells. The potential use of these organ culture systems for the further definitive identification of the factor responsible for bone destruction in myeloma is discussed.

The established human breast cancer cell line MCF-7 resorbs bone directly in vitro independently of viable endogenous bone cells, and this resorption of bone is closely correlated with the release of hydrolytic enzymes by the cultured tumor cells. In this study we have examined the effects of a number of drugs which increase the intracellular cyclic adenosine 3':5'-monophosphate (cyclic AMP) content on the release of hydrolytic enzymes by the tumor cells and their capacity to resorb bone. When MCF-7 cultures were treated with cholera toxin (0.05 to 5 micrograms/ml), a potent adenylate cyclase inducer, mineral-releasing, lysosomal enzyme, and collagenolytic activities increased more than 2-fold in the cell culture medium. Prostaglandin E1 (0.1 microM), 8-bromocyclic AMP (10 mM), and isobutylmethylxanthine (30 microM) caused similar effects. These data suggest that adenylate cyclase activation and increases in cyclic AMP content in the tumor cells caused the release of lysosomal enzymes and collagenolytic activity and caused the resorption of bone. We have previously found that colchicine, a drug which inhibits microtubule assembly, also increased hydrolytic enzyme release and release of bone-resorbing activity. Direct measurements of cyclic AMP were made in MCF-7 cells 3 hr after treatment with colchicine (10 microM) as well as MCF-7 cells treated with prostaglandin E1, cholera toxin, and isobutylmethylxanthine. MCF-7 cells showed a 2-fold increase in cyclic AMP content after treatment with all of these agents, although there was no similar increase in mouse 3T3 cells which do not produce bone-resorbing activity under these conditions. These data suggest that increases in cyclic AMP concentrations in human breast cancer cells lead to release of hydrolytic enzymes and resorption of bone.