We investigated the molecular basis for osteolytic bone metastasis by selecting human breast cancer cell line subpopulations with elevated metastatic activity and functionally validating genes that are overexpressed in these cells. These genes act cooperatively to cause osteolytic metastasis, and most of them encode secreted and cell surface proteins. Two of these genes, interleukin-11 and CTGF, encode osteolytic and angiogenic factors whose expression is further increased by the prometastatic cytokine TGF beta. Overexpression of this bone metastasis gene set is superimposed on a poor-prognosis gene expression signature already present in the parental breast cancer population, suggesting that metastasis requires a set of functions beyond those underlying the emergence of the primary tumor.

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.

Since bone metastatic breast cancer is an incurable disease, causing significant morb idity and mortality, understanding of the underlying molecu lar mechanisms would be h ighly valuable. Here, we describe in v itro and in v ivo evidence for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Geno me -wide gene exp ression profiling of th is isogenic cell line pair revealed that all the three genes involved in the L-serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L-serine in mammalian t issues. Consistently, we observed that the proliferat ion of MDA -MB-231(SA) cells in serine-free conditions was dependent on PSAT1 exp ression. In addition, we observed that L-serine is essential for the fo rmation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L-serine on osteoclastogenesis and cancer cell pro liferat ion indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions.

Our findings strongly support the development of radium-223 dichloride for the treatment of breast cancer patients with or at high risk of developing bone metastases.