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.

Toll-like receptor 9 (TLR9) recognizes microbial DNA. We show here that TLR9 protein is expressed in human breast cancer cells and clinical breast cancer samples. Stimulation of TLR9-expressing breast cancer cells with the TLR9 agonistic CpG oligonucleotides (1-10 Mmol/L) dramatically increased their in vitro invasion in both Matrigel assays and three-dimensional collagen cultures. Similar effects on invasion were seen in TLR9-expressing astrocytoma and glioblastoma cells and in the immortalized human breast epithelial cell line MCF-10A. This effect was not, however, dependent on the CpG content of the TLR9 ligands because the non-CpG oligonucleotides induced invasion of TLR9-expressing cells. CpG or non-CpG oligonucleotide-induced invasion in MDA-MB-231 cells was blunted by chloroquine and they did not induce invasion of TLR9 À breast cancer cells.Treatment of MDA-MB-231 cells with CpG or non-CpG oligonucleotides induced the formation of f50-kDa gelatinolytic band in zymograms. This band and the increased invasion were abolished by a matrix metalloproteinase (MMP) inhibitor GM6001 but not by a serine proteinase inhibitor aprotinin. Furthermore, CpG oligonucleotide treatment decreased tissue inhibitor of metalloproteinase-3 expression and increased levels of active MMP-13 in TLR9-expressing but not TLR9 À breast cancer cells without affecting MMP-8. Neutralizing anti-MMP-13 antibodies inhibited the CpG oligonucleotide-induced invasion. These findings suggest that infections may promote cancer progression through a novel TLR9-mediated mechanism. They also propose a new molecular target for cancer therapy, because TLR9 has not been associated with cancer invasiveness previously.

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.

TLR9-mediated invasion may represent a novel mechanism through which infections promote prostate cancer.

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13 -mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasioninducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotideinduced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer. (Mol Cancer Res 2008;6(10):1534 -43)