Breast Cancer-derived Factors Stimulate Osteoclastogenesis through the Ca2+/Protein Kinase C and Transforming Growth Factor-β/MAPK Signaling Pathways

Abstract

Breast cancer commonly metastasizes to bone where its growth depends on the action of bone-resorbing osteoclasts. We have previously shown that breast cancer cells secrete factors able to directly stimulate osteoclastogenesis from receptor activator of nuclear factor B ligand (RANKL)-primed precursors and that transforming growth factor-␤ (TGF␤) plays a permissive role in this process. Now, we evaluate the signaling events triggered in osteoclast precursors by soluble factors produced by MDA-MB-231 human breast carcinoma cells. In mouse bone marrow cultures and RAW 264.7 murine monocytic cells, MDA-MB-231-derived factors increased osteoclast number, size, and nucleation. These factors failed to induce Smad2 phosphorylation, and short interfering RNAs against Smad4 did not affect their ability to induce osteoclastogenesis. In contrast, MDA-MB-231 factors induced phosphorylation of p38 and ERK1/2, and pharmacological inhibitors against p38 (SB203580) and MEK1/2 (PD98059) impeded the osteoclastogenic effects of cancer-derived factors. Neutralizing antibodies against TGF␤ attenuated p38 activation, whereas activation of ERK1/2 was shortened in duration, but not decreased in amplitude. ERK1/2 phosphorylation induced by cancer-derived factors was blocked by MEK1/2 inhibitor, but not by Ras (manumycin A) or Raf (GW5074) inhibitors. Inhibition of protein kinase C␣ using Gö6976 prevented both ERK1/2 phosphorylation and osteoclast formation in response to MDA-MB-231-derived factors. Using microspectrofluorimetry of fura-2-AM-loaded osteoclast precursors, we have found that cancer-derived factors, similar to RANKL, induced sustained oscillations in cytosolic free calcium. The calcium chelator BAPTA prevented calcium elevations and osteoclast formation in response to MDA-MB-231-derived factors. Thus, we have shown that breast cancer-derived factors induce osteoclastogenesis through the activation of calcium/protein kinase C␣ and TGF␤-dependent ERK1/2 and p38 signaling pathways.Breast cancer exhibits a high propensity to metastasize to bone causing bone pain, pathological fractures, hypercalcemia, spinal cord compression, and immobility (1, 2). Breast cancer cells do not resorb bone; instead they rely on stimulation of osteoclasts, cells physiologically responsible for bone destruction (1-4). Breast cancer cells can stimulate osteoclasts indirectly, by producing factors, such as parathyroid hormone-related peptide, interleukin-1, -6, and -11, which act on bone-forming osteoblasts to increase the production of an essential osteoclast stimulator, receptor activator of nuclear factor B (RANK) 4 ligand (RANKL) (1, 5-11). We have found that soluble factors produced by human or mouse breast cancer cells can directly stimulate osteoclast formation from late human or mouse osteoclast precursors (12). These effects depended on the permissive action of TGF␤, and we observed that TGF␤ type I receptor expression (T␤RI) was up-regulated in late osteoclast precursors (12). The expression of TGF␤ and T␤RI increases at the interface between...

CCN3 Impairs Osteoblast and Stimulates Osteoclast Differentiation to Favor Breast Cancer Metastasis to Bone

“…However, we did not observe osteoclast formation in cultures treated only with rCCN3 (see Supplemental Figure S3, CC, at http://ajp.amjpathol.org), which resembled the undifferentiated monocytes present in RAW 264.7 cells cultured in DMEM alone (see Supplemental Figure S3, DD, at http://ajp.amjpathol.org). We previously showed that soluble factors produced by breast cancer cells can induce osteoclast formation from monocytes primed for a short time with rRANKL 30,32 ; therefore, we considered whether rCCN3 could be effective in inducing osteoclastogenesis from rRANKL-primed precursors. RAW 264.7 cells were primed for 2 days with rRANKL, after which the media was replaced by DMEM alone or DMEM containing rCCN3 (600 ng/mL).…”

“…In these studies, activation of the p38, ERK, and PKC pathways were observed after treatment with conditioned media from breast cancer cells. 30,32 Thus, CCN3 seems to engage different signaling pathways to promote osteoclastogenesis of rRANKL-primed precursors. The present results are consistent with previous observations indicating that CCN3 can impair ERK phosphorylation in chronic myeloid leukemia.…”

“…30 Thus, we sought evidence of whether CCN3 played a role in activating these signaling pathways in rRANKL-primed RAW 264.7 cells. We observed that CCN3 treatment resulted in an increase in phosphorylation of JNK and PKC but not p38 (data not shown) or ERK1/2 compared with the control treatment (DMEM) (see Supplemental Figure S5 at http://ajp.amjpathol.org).…”

“…30 Briefly, 5 ϫ 10 3 RAW 264.7 cells were plated in each well of a 48-well plate. On day 1 and day 3, fresh media containing the specified factors was applied to the cultures (rRANKL, 50 ng/mL; rCCN3, 600 ng/mL).…”

“…30 Raw 264.7 cells were plated on glass-bottom 35-mm dishes (MatTek Corp., Ashland, MA). rRANKL-primed cells were washed twice and loaded at room temperature for 40 minutes with fura-2-AM (catalog number F1221; Invitrogen).…”