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Skeletal Complications of Malignancy

Supplement to Cancer

Molecular Mechanisms of Osteolytic Bone Metastases

Theresa A. Guise,
M.D.

BACKGROUND. Breast carcinoma commonly metastasizes to the skeleton in patients with advanced disease to cause bone destruction and the associated pain, hypercalcemia, fracture, and nerve-compression syndromes. In this scenario, the bone destruction is mediated by the osteoclast. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process. Transforming growth factor (TGF ), which is abundant in bone matrix and is released as a consequence of osteoclastic bone resorption, may promote breast carcinoma osteolysis by stimulating PTHrP production by tumor cells. METHODS. Stable breast carcinoma MDA-MB-231 cell lines were constructed that expressed mutant TGF receptors, Smad proteins, or estrogen receptor (ER)- and were used to determine the role of TGF in modulating tumor production of PTHrP. These stable cell lines were applied to a mouse model of human breast carcinoma metastases to the bone to dissect the molecular mechanisms responsible for osteolytic bone metastases. RESULTS. TGF promoted the development and progression of osteolytic bone metastases by inducing tumor production of PTHrP, the effect of which was mediated through the Smad signaling pathway. PTHrP stimulated osteoclastic bone resorption by increasing osteoblast production of the receptor activator of nuclear factor B (RANK) ligand and decreasing osteoblast production of osteoprotegerin (OPG). A constitutively active ER- mutation (Tyr537Asn), identied from a human bone metastases, when it was expressed in human breast carcinoma cells, caused increased production of PTHrP. TGF signicantly enhanced the ER- -mediated transcriptional activity induced by ER- (Tyr537Asn), and this resulted in further stimulation of PTHrP production. CONCLUSIONS. These data indicate a central role for TGF in the pathogenesis of osteolytic bone metastases from breast carcinoma by 1) the induction of PTHrP through the Smad signaling pathway and 2) the potentiation of ER- -mediated transcription induced by a constitutively active ER- . Understanding the mechanisms of osteolysis at a molecular level will generate more effective therapeutic agents for patients with this devastating complication of cancer. Cancer 2000;88: 2892 8. 2000 American Cancer Society.

Division of Endocrinology, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas.

Presented at the Second North American Symposium on Skeletal Complications of Malignancy, Montreal, Quebec, Canada, October 1516, 1999. Supported by grants from the National Institutes of Health (AR01899 and CA69158) and from the Department of Defense-United States Army (DAMD17-94-J-4213). Address for reprints: Theresa A. Guise, M.D., Division of Endocrinology, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. Received March 2, 2000; accepted March 10, 2000. 2000 American Cancer Society

KEYWORDS: parathyroid hormone-related protein, transforming growth factor osteolysis, bone metastasis, breast carcinoma.

arcinoma that is metastatic to bone often causes bone destruction or osteolysis, which results in the complications of bone pain, fracture, hypercalcemia, and nerve-compression syndromes. Although several tumor types, such as prostate, lung, renal, and thyroid, are associated with osteolytic lesions, breast carcinoma is the most common. Sixty-nine percent of patients dying of breast carcinoma have bone metastases, and bone is the most common site of rst distant recurrence.1 In those patients with disease conned to the skeleton, the median survival was 24 months compared with 3

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months in those patients whose rst recurrence was located in the liver. Because patients with breast carcinoma may survive for several years with their bone metastases, it is important to understand the pathophysiology of this process so that therapy and prevention can be improved. This overview will highlight recent developments in our understanding of the molecular mechanisms of osteolytic bone metastases. The fact that breast carcinoma is associated with signicant morbidity in the skeleton was noted in 1889, when Stephen Paget observed that in a cancer of the breast the bones suffer in a special way, which cannot be explained by any theory of embolism alone.2 Indeed, breast carcinoma is one of a limited number of primary neoplasms that display osteotropism, an extraordinary afnity to grow in bone. The mechanisms underlying this osteotropism are complex and involve unique characteristics of both the breast carcinoma cells and the bone to which these tumors metastasize. Paget proposed the seed and soil hypothesis to explain this phenomenon: When a plant goes to seed, its seeds are carried in all directions; but they can only grow if they fall on congenial soil.2 In essence, the microenvironment of the organ to which the tumor cells metastasize may serve as a fertile soil on which the tumor cells (or seeds) may grow. Although this concept was proposed over a century ago, it remains a basic principle in the eld of metastasis at the present time. Thus, breast carcinoma cells possess certain properties that enable them to grow in bone, and the bone microenvironment provides a fertile soil on which to grow.

Pathogenesis of Osteolytic Breast Carcinoma Metastases to Bone Role of tumor-produced parathyroid hormone-related protein
Histologic analysis as well as scanning electron microscopy of osteolytic bone metastases indicate that the bone destruction is mediated by the osteoclast.3,4 Bisphosphonates, which are potent inhibitors of bone resorption, signicantly reduce skeletal morbidity in patients with advanced breast carcinoma.5 Evidence in humans and mice indicates that tumor-produced factors, such as parathyroid hormone-related protein (PTHrP), often are responsible for the osteoclast-stimulating activity and the bone destruction associated with breast carcinoma.6 11 PTHrP was puried from human lung carcinoma,12 breast carcinoma,13 and renal cell carcinoma14 as a hypercalcemic factor simultaneously by several independent groups in 1987 and was cloned shortly thereafter.15 PTHrP has 70% homology to the rst 13 amino acids of parathyroid hormone (PTH),15 binds to the

common PTH/PTHrP receptors,16 and shares biologic activity similar to PTH.17,18 Hypercalcemia of malignancy was the rst identied consequence of PTHrP in cancer. In this syndrome, tumor-produced PTHrP interacts with the common PTH/PTHrP receptors in bone and kidney to cause hypercalcemia, osteoclastmediated bone resorption, increased nephrogenous cyclic AMP, and phosphate excretion. The PTH-like properties of PTHrP and, specically, its ability to increase osteoclastic bone resorption and renal tubular calcium reabsorption are responsible for the hypercalcemia. Approximately 80% of hypercalcemic patients with solid tumors have detectable or increased plasma PTHrP concentrations.19 In humans, 50 60% of primary breast carcinoma tumors express PTHrP.6,7,20 However, PTHrP expression by breast carcinoma bone metastases is greater than at the primary site or that of nonbone metastases.8,9,20 Taken together, these observations suggest that PTHrP, as a bone-resorbing factor, favors tumor growth in bone or that the bone microenvironment enhances the production of PTHrP. Neutralizing antibodies to PTHrP-(1-34) inhibited the development of breast carcinoma metastases to bone by the human breast carcinoma cell line MDA-MB-231, which produces moderate amounts of PTHrP, in a mouse model of bone metastases.11 Histomorphometric analysis of long bones from tumor-bearing mice revealed significantly fewer osteoclasts at the tumor-bone interface and less tumor in mice that were treated with the PTHrP antibody compared with control animals. Thus, neutralizing the effects of PTHrP decreases not only osteoclastic bone resorption but also tumor burden in bone. Conversely, experimental overexpression of PTHrP by MCF-7 breast carcinoma cells, which do not express PTHrP and do not cause osteolytic metastases, induced marked bone destruction associated with increased osteoclast formation compared with empty vector or parental controls, which caused no bone metastases in the mouse model.21 Collectively, these data suggest that tumor production of PTHrP is important for the establishment and progression of osteolytic bone metastases. However, these data do not address the possibility that the bone microenvironment enhances the production of PTHrP.

Bone microenvironment: The role of transforming growth factor in osteolysis

Bone matrix is a repository for growth regulatory factors.22 Transforming growth factor (TGF ), which is one of the most abundant, is released in active form into the bone microenvironment as a consequence of osteoclastic bone resorption23 and enhances produc-

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tion of PTHrP by tumor cells.24 27 Because previous clinical and experimental studies demonstrated increased PTHrP expression by breast carcinoma cells in the bone microenvironment, the effect of factors known to be present in bone matrix were tested on PTHrP production by human MDA-MB-231 breast carcinoma cells in vitro. Only TGF signicantly increased PTHrP production by these cells in a dose dependent manner.27 Other growth factors that are abundant in bone, such as broblast growth factor 1 (FGF-1) and FGF-2, IGF-1 and IGF-2, BMP-2 and platelet-derived growth factor, had no effect on PTHrP secretion over a wide range of concentrations. Therefore, the role of TGF as a pathogenic factor in osteolytic bone metastases was investigated further. In the normal state, TGF stimulates mesenchymal cell proliferation and synthesis of extracellular matrix proteins, and it inhibits the growth of epithelial cells.28 These effects are mediated through interactions with transmembrane serine threonine kinase receptors. TGF binds to the type II receptor, and this complex recruits and phosphorylates the type I receptor, which, in turn, initiates signal transduction mediated by members of the Smad protein family.28,29 Smad proteins contain a characteristic C-terminal SerSer-X-Ser motif in which the two most terminal serines become phosphorylated in response to TGF .28,29 TGF appears to have a dual role in malignancy. Although TGF inhibits the growth of epithelial cells and of some tumor cells, and although certain carcinomas are associated with inactivations or deletions of TGF receptors or Smad proteins,30 TGF also can promote tumorigenesis and invasion.3133 We hypothesized that TGF plays unique role in the development and progression of metastases to bone by stimulating tumor production of PTHrP. This was investigated using a mouse model of bone metastases. Expression of a dominant-negative mutant (T RII cyt) of the TGF type II receptor,34 which lacks the kinase domain and does not phosphorylate the type IR, rendered MDA-MB-231 cells unresponsive to the effects of TGF compared with controls. The TGF stimulation of PTHrP production was blocked. In vivo, MDA/ T RII cyt cells caused signicantly less bone destruction, stimulated fewer osteoclasts, and formed less tumor in bone compared with controls. Survival was longer in MDA/T RII cyt-bearing mice. Reversal of the dominant negative blockade by expression of a constitutively active TGF type I receptor [T RI(T204D)],35 caused increased PTHrP production and marked enhancement of osteolytic bone metastasis and decreased survival. Furthermore, transfection of the cDNA for PTHrP into the MDA-MB-231 clonal line,

which expressed the dominant negative type II TGF receptor, also resulted in increased PTHrP production and accelerated bone metastases.27 Thus, the dominant negative blockade of TGF on MDA-MB-231 cells was reversed either by a constitutively active, TGF type I receptor or by PTHrP. These data demonstrate that both TGF receptor activation and PTHrP are important for the development and progression of bone metastases.

PTHrP is the effector of TGF in the pathogenesis of osteolytic bone metastases

The data suggest that the effects of TGF in promoting breast carcinoma metastases to bone are mediated by tumor production of PTHrP but do not exclude other possible effects of TGF . To determine whether the effects of TGF in promoting breast carcinoma metastases to bone are mediated by tumor production of PTHrP, female nude mice were inoculated with the MDA-MB-231 clonal line, which expressed the constitutively active type I TGF receptor, T RI(T204D), and the mice were treated with a neutralizing monoclonal antibody directed against PTHrP(1-34) (PTHrP-Ab). Osteolytic lesion area and lesion numbers were significantly lower in mice treated with PTHrP-Ab compared with those treated with control immunoglobulin G. Bone histomorphometry demonstrated signicantly smaller tumor area and fewer osteoclasts in tumor-bearing mice treated with PTHrP antibody. These results provided further evidence that the effector of TGF on the development and progression of bone metastases was PTHrP.36

Role of Smad signaling pathway in osteolytic metastases as a mediator of TGF to stimulate tumor production of PTHrP
Although many of the effects of TGF are mediated by the Smad signaling pathway, it is clear that this pathway is not responsible for all actions of TGF .37 To determine whether the effects of TGF in stimulating PTHrP production were mediated through the Smad signaling pathway, MDA-MB-231 cells were transfected with a cDNA that encodes a dominant negative Smad2 [Smad2(3S-A)] in which three C-terminal serines are replaced by alanine. This Smad2 mutant has been shown by Macias-Silva et al.38 to abolish TGF receptor dependent phosphorylation. Stable clones were unresponsive to TGF , as assessed by transient transfection with the TGF -responsive plasminogen activator inhibitor promoter linked to luciferase, 3TPlux. PTHrP production by MDA/ Smad2(3S-A) did not increase in response to TGF , in contrast to parental MDA-MB-231 cells or stable

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MDA-MB-231 clones expressing wild-type Smad2 or empty vector.36

RANK ligand mediates the effects of PTHrP to stimulate osteoclastic bone resorption
Although it is clear that PTHrP is a potent stimulator of bone resorption, the molecular mechanisms by which tumor-produced PTHrP stimulates osteoclastic bone resorption have been unclear. Thomas et al.21 have provided evidence that a recently identied osteoclast differentiation factor mediates the effects PTHrP on osteoclastic bone resorption. The stromal cell-derived RANK (receptor activator of NF B) ligand, also known as osteoclast differentiation factor, osteoprotegerin ligand, and TRANCE, has been identied, and a soluble form of the molecule in combination with macrophage colony-stimulating factor can generate osteoclasts from hematopoietic cells in the absence of osteoblastic stromal cells.39 42 It also was identied by its ability to induce NF B and apoptosis of T-cells as RANK ligand and TRANCE, respectively.43 45 For consistency, the nomenclature of RANK ligand, as indicated by a recent review and consensus,46 will be used. RANK ligand is a member of the tumor necrosis factor (TNF) family and is a membrane-bound molecule. Two receptors for RANK ligand exist. The rst, which aided the identication of RANK ligand, was osteoprotegerin (OPG), also reported as osteoclastogenesis inhibitory factor.47,48 OPG is a secreted TNF receptor family member and has a relatively wide distribution. The overexpression of OPG in mice resulted in osteopetrosis.47 Conversely, mice decient in OPG demonstrate osteoporosis and calcication of the aorta. In agreement with the phenotypes of mice with altered OPG production, recombinant OPG inhibited osteoclast formation in cocultures of mouse osteoblastic cells and hematopoietic cells.40,41 The ability of OPG to bind to RANK ligand and to limit the biologic actions of RANK ligand suggested that OPG functions as a decoy receptor.40,41,48 The receptor responsible for signaling RANK ligand biologic actions appears to be receptor activator of NF B (RANK).43 Thomas et al.21 determined that the breast carcinoma cell lines MDA-MB-231, MCF-7, and T47D as well as primary breast tumors did not express RANK ligand but did express OPG and RANK. MCF-7, MDAMB-231, and T47D cells did not act as surrogate osteoblasts to support osteoclast formation in coculture experiments, a result consistent with the fact that they do not express RANK ligand. When MCF-7 cells that overexpressed PTHrP were added to cocultures of murine osteoblasts and hematopoietic cells, osteoclast formation resulted without the addition of any osteotropic agents. Cocultures with MCF-7 or MCF-7 cells

transfected with empty vector required exogenous agents for osteoclast formation. When MCF-7 cells that overexpressed PTHrP were cultured with murine osteoblasts, osteoblastic RANK ligand mRNA levels were enhanced, and osteoblastic OPG mRNA levels diminished. MCF-7 parental cells had no effect on RANK ligand or OPG mRNA levels when cultured with osteoblastic cells. By using a murine model of breast carcinoma metastasis to bone, MCF-7 cells that overexpressed PTHrP caused signicantly more bone metastases, which was associated with increased osteoclast formation, plasma PTHrP concentrations, and hypercalcemia compared with parental or empty vector controls.21

Proposed mechanisms for osteolytic bone metastases

Based on the above data, a proposed mechanism for osteolytic metastases is illustrated in Figure 1. Tumor cells in bone that secrete PTHrP stimulate osteoclastic bone resorption through osteoblast production of RANK ligand. Bone-derived TGF released as a consequence of osteoclastic bone resorption acts on the tumor cells through a TGF receptor-Smad signaling pathway to stimulate further tumor production of PTHrP. This vicious cycle involving PTHrP and TGF results in the bone destruction associated with breast carcinoma and denes specic molecular targets for therapy.

Estrogen receptor, PTHrP, and bone metastases

Estrogen is a mitogen for breast carcinoma cells that express estrogen receptor (ER- ). However, the role of ER- in the pathophysiology of bone metastases has been unclear. Women with ER positive primary tumors are more likely to develop bone metastases.1,49 It has been shown recently that ER- and PTHrP are coexpressed in primary breast carcinoma.50 However, no clear correlation between ER and PTHrP in breast carcinoma bone metastases has been demonstrated. Estrogen increased PTHrP expression in the uterus51 as well as in some human breast carcinoma cell lines.52 The sparse clinical data available on ER expression in breast carcinoma bone metastases indicate that 60 75% are ER negative,49 despite the fact that women with ER positive primary tumors are more likely to develop bone metastases. Furthermore, bone metastases were frequently ER negative in those patients in whom the primary tumors were ER positive.49 Recently, three missense mutations were identied in gene from metastatic breast tumors: the ERSer47Thr, Lys531Glu, and Tyr537Asn59. The rst two ER mutants had activity silimar to that seen in wildtype (wt) ER, whereas the Tyr537Asn ER mutant demonstrated a potent, estradiol independent transcrip-

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FIGURE 1. Proposed mechanisms for

osteolytic bone metastases. Tumor cells in bone that secrete parathyroid hormone-related protein (PTHrP) stimulate osteoclastic bone resorption through osteoblast production of receptor activator of nuclear factor B ligand (RANK L). PTHrP also reduces the osteoblast production of osteoprotegerin to favor osteoclastic bone resorption. Bone-derived transforming growth factor (TGF ), released as a consequence of osteoclastic bone resorption, acts on the tumor cells through the TGF -receptor-Smad signaling pathway to stimulate further tumor production of PTHrP. This vicious cycle involving PTHrP and TGF results in the bone destruction associated with breast carcinoma.

tional activity compared with wt ER. This constitutive activity of Tyr537Asn was unaffected by estradiol, tamoxifen, or the pure antiestrogen ICI 164,384. This Tyr537Asn mutant was derived from a bone metastases that was ER negative according to ligand-binding analysis. The mutation is located in exon 8, which encodes the carboxy-terminal portion of the hormone-binding domain of the ER- , a potential phosphorylation site implicated in hormone binding, dimerization, and hormone dependent transcriptional activity. Such a mutation may be responsible for the development and progression of breast carcinoma metastases to bone and, because it does not bind ligand, would be classied as an ER negative tumor. Because bone metastases are sampled infrequently, the prevalence of this ER- mutation is unknown. However, the same point mutation also has been identied in an endometrial carcinoma.54 To determine the role of ER- in bone metastases, the cDNA for this ER- (Tyr537Asn) mutant was expressed stably in MDA-MB-231 cells, which do not normally express ER- . Several clonal MDA-MB-231/ER- (Tyr537Asn) lines demonstrated increased transcriptional activity in the absence of estradiol, as assess by transient transfection with the estrogen response element linked to luciferase (ERE-luc) compared with empty vector controls. Transcriptional activity of the stable clones was not affected by estradiol treatment, but exogenous TGF 1 increased ERE-luciferase activity in all stable clones. Furthermore, basal as well as TGF stimulated PTHrP secretion by the Tyr537Asn ER mutant clones was increased compared with the empty vector controls. These data suggest that ER- -medi-

ated transcription is associated with increased tumor production of PTHrP. This, in combination with the effects of TGF to enhance ER- -mediated transcription and potential growth, may be a mechanism for the propensity of breast carcinoma to metastasize to the skeleton. To determine whether the effects of TGF on ERmediated transcription were specic to the ER(Tyr537Asn) mutant, other stable MDA-MB-231 cell lines that expressed wt ER- or ER- mutants, which were identied in soft tissue metastases, Ser47Thr, and Lys531Glu, were constructed. ER-mediated transcription was increased in response to 17 -estradiol in clones, which expressed wt or Ser47Thr and Lys531Glu mutants, but there was no additional effect of TGF . Furthermore, the combination of 17 -estradiol and TGF did not increase PTHrP production over TGF alone. There was no signicant difference between wt ER- and the Ser47Thr or Lys531Glu mutants. In summary, these results suggest that the Tyr537Asn substitution induces conformational changes in the ER that mimic hormone binding and confers a constitutive transactivation function to the receptor. TGF further increases the constitutive ERmediated transcription. The presence of this constitutively active mutant ER in combination with TGF enhances tumor cell production of PTHrP. The molecular mechanisms responsible for these effects appear to be specic to the Tyr537Asn mutant only and suggest cross-talk between the TGF signaling pathway and this mutant ER- . Consistent with this, Yanagasawa et al.55 have demonstrated the convergence of

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TGF and vitamin D signaling pathways on Smad transcriptional coactivators. The clinical relevance of these observations merit further investigation and may have important implications in the pathogenesis of breast carcinoma osteolysis. In conclusion, the molecular mechanisms responsible for osteolytic metastases are complex and involve bidirectional interactions between tumor cells and bone. The data presented in this overview indicate a central role for TGF in the pathogenesis of osteolytic bone metastases from breast carcinoma by several mechanisms: 1) induction of PTHrP through the Smad signaling pathway and 2) potentiation of ER- -mediated transcription induced by a constitutively active ER- . A better understanding of the osteolytic mechanisms at the molecular level will result in more effective therapy for patients with this devastating complication of carcinoma.

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