Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-β (TGF-β), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-β inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-β responses, any role of TGF-β in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-β promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant-negative mutant (TβRII∆cyt) of the TGF-β type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-β. In a murine model of bone metastases, expression of TβRI-I∆cyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant-negative signaling blockade by expression of a constitutively active TGF-β type I receptor in the breast cancer cells increased tumor production of parathyroid hormone-related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant-negative TβRII∆− cyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-β in the development of breast cancer metastasis to bone, via the TGF-β receptor-mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.

Breast cancer almost invariably metastasizes to bone in patients with advanced disease and causes local osteolysis. Much of the morbidity of advanced breast cancer is a consequence of this process. Despite the importance of the problem, little is known of the pathophysiology of local osteolysis in the skeleton or its prevention and treatment. Observations in patients with bone metastases suggest that breast cancer cells in bone express parathyroid hormone-related protein (PTHrP) more frequently than in soft tissue sites of metastasis or in the primary tumor. Thus, the role of PTHrP in the causation of breast cancer metastases in bone was examined using human breast cancer cell lines.

O steoblastic metastases occur in advanced cases of prostate cancer and frequently in breast cancer (1). Many factors have been proposed to cause disorganized new bone formation at sites of metastases, including insulin-like growth factors 1 and 2, transforming growth factor (TGF)-␤, prostate-specific antigen, urokinase-type plasminogen activator, fibroblast growth factors (FGF)-1 and -2, bone morphogenic proteins (BMPs), and, in particular, endothelin-1 (ET-1) (2-7).ET-1 is a potent vasoconstrictor that binds to ET A and ET B receptors with the latter functioning in ligand clearance (8,9). ET-1 is produced by and affects bone cells (10-12). It stimulates mitogenesis in osteoblasts, which express both ET A and ET B receptors (13-15). ET-1 can decrease osteoclast activity and motility (16).The prostate expresses ET-1 ligand and receptors (5-7). Primary and metastatic prostate cancers make 6,17,18), which can stimulate autocrine proliferation and potentiate effects of insulin-like growth factors, platelet-derived growth factor, epidermal growth factor, and FGF-2 (5). ET B receptor expression is decreased in prostate cancer (5). Nelson et al. (6) found that plasma ET-1 concentrations were higher in men with advanced prostate cancer with bone metastases compared with men with organ-confined disease (6). ET-1 concentrations did not correlate to tumor burden in bone. Five human prostate cancer cell lines expressed ET-1 messenger RNA, and ET-1 increased BMP-initiated bone formation (6).Breast cancers also express ET-1 and are the next most common cause of osteoblastic metastases. Breast cancer cells can convert preproET-1 to 20). Thus, substantial data implicate ET-1 in the pathogenesis of osteoblastic metastases, but a causal role for ET-1 in bone metastasis has not been directly tested. Questions remain about the importance of ET-1 on bone formation in vivo and whether ET-1 receptor blockade would decrease osteoblastic metastases.We found three human breast cancer cell lines that produce ET-1 and cause osteoblastic bone metastases. We used nude mice inoculated with ZR-75-1 cells to demonstrate a causal role for ET-1 in osteoblastic metastasis. Endothelin A receptor blockade in this model dramatically decreased metastases and tumor burden in bone. Materials and MethodsCells. ZR-75-1 and T47D were from American Type Culture Collection. C. Kent Osborne (San Antonio, TX) provided MCF-7 and MDA-MB-231. ZR-75-1 and T47D cells were grown in RPMI medium 1640; MDA-MB-231 in DMEM; MCF-7 in Iscove's modified Eagle's medium (IMEM); and BT483, BT549, MDA-MB435s, HS578T, MDA-MB-436, MDA-MB-361 PC-3, DU145, LNCaP, and TSU-Pr1 in 1:1 mixture of F12͞DMEM. All media contained 10% FCS, 1% penicillin͞streptomycin, and 1% nonessential amino acids in a 37°C atmosphere of 5% CO 2 ͞95% air. T47D and MCF-7 culture media were supplemented with insulin. At 80% confluence, 250 l of serum-free media was conditioned in 48-well plates for 48 h, and cells were counted. ET-1 and BQ-123 were from American Peptide (Sunnyvale, CA). New Bone Formation ...

Substantial data support major roles for bone-derived TGF-␤ 1 and tumor-derived parathyroid hormone-related protein (PTHrP) in the vicious cycle of local bone destruction that characterizes osteolytic metastases. Tumor-produced PTHrP stimulates osteoclastic bone resorption to result in the bone destruction associated with breast cancer metastases (1, 2). Neutralizing antibodies to PTHrP not only decreased osteoclastic bone resorption but also inhibited the development of metastases to bone by the human breast cancer cell line, MDA-MB-231 (3). TGF-␤, stored in bone matrix (4) and released locally in active form during osteoclastic resorption (5), stimulates PTHrP production by tumor cells (6 -8). A dominantnegative TGF-␤ type II receptor (T␤RII⌬cyt) stably expressed in the MDA-MB-231 breast cancer line rendered the cells unresponsive to TGF-␤ and inhibited TGF-␤-induced PTHrP secretion and the development of bone metastases in a mouse model. This dominant-negative type II blockade was reversed by a constitutively active TGF-␤ type I receptor (T␤RI(T204D)). Furthermore, transfection of the cDNA for PTHrP into the dominant-negative MDA-MB-231 line also increased PTHrP production and accelerated bone metastases (9). These published data establish that TGF-␤ in bone can promote osteolysis by increasing PTHrP secretion from breast cancer cells. They do not, however, exclude contributions from other TGF-␤-responsive tumor factors. Here we demonstrate that PTHrP is the central mediator of TGF-␤-induced osteolytic metastasis. We also show that TGF-␤ increases PTHrP secretion from MDA-MB-231 cells by signaling through both Smad and p38 MAP kinase pathways.

Some of the most common human cancers, including breast cancer, prostate cancer, and lung cancer, metastasize with avidity to bone. What is the basis for their preferential growth within the bone microenvironment? Bidirectional interactions between tumor cells and cells that make up bone result in a selective advantage for tumor growth and can lead to bone destruction or new bone matrix deposition. This review discusses our current understanding of the molecular components and mechanisms that are responsible for those interactions.

Epithelial-mesenchymal transition (EMT) is a developmental program of signaling pathways that determine commitment to epithelial and mesenchymal phenotypes. In the prostate, EMT processes have been implicated in benign prostatic hyperplasia and prostate cancer progression. In a model of Pten- and TP53-null prostate adenocarcinoma that progresses via transforming growth factor β-induced EMT, mesenchymal transformation is characterized by plasticity, leading to various mesenchymal lineages and the production of bone. Here we show that SLUG is a major regulator of mesenchymal differentiation. As microRNAs (miRs) are pleiotropic regulators of differentiation and tumorigenesis, we evaluated miR expression associated with tumorigenesis and EMT. Mir-1 and miR-200 were reduced with progression of prostate adenocarcinoma, and we identify Slug as one of the phylogenetically conserved targets of these miRs. We demonstrate that SLUG is a direct repressor of miR-1 and miR-200 transcription. Thus, SLUG and miR-1/miR-200 act in a self-reinforcing regulatory loop, leading to amplification of EMT. Depletion of Slug inhibited EMT during tumorigenesis, whereas forced expression of miR-1 or miR-200 inhibited both EMT and tumorigenesis in human and mouse model systems. Various miR targets were analyzed, and our findings suggest that miR-1 has roles in regulating EMT and mesenchymal differentiation through Slug and functions in tumor-suppressive programs by regulating additional targets.

CD97, an adhesion-linked G-protein-coupled receptor (GPCR), is induced in multiple epithelial cancer lineages. We address here the signaling properties and the functional significance of CD97 expression in prostate cancer. Our findings show that CD97 signals through Ga12/13 to increase RHO-GTP levels. CD97 functioned to mediate invasion in prostate cancer cells, at least in part, by associating with lysophosphatidic acid receptor 1 (LPAR1), leading to enhanced LPA-dependent RHO and extracellular signal-regulated kinase activation. Consistent with its role in invasion, depletion of CD97 in PC3 cells resulted in decreased bone metastasis without affecting subcutaneous tumor growth. Furthermore, CD97 heterodimerized and functionally synergized with LPAR1, a GPCR implicated in cancer progression. We also found that CD97 and LPAR expression were significantly correlated in clinical prostate cancer specimens. Taken together, these findings support the investigation of CD97 as a potential therapeutic cancer target. Cancer Res; 71(23); 7301-11. Ó2011 AACR.

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