Using ROI boxes of the same size, data analysis tools were used to calculate the trabecular bone compartment, which was manually delineated from the cortical bone. Since current treatment for bone metastasis has limited efficacy, development of an effective therapeutic strategy is urgently needed. To improve the prognosis of breast cancer bone metastasis (BCBM), molecular mechanisms involved in breast cancer proliferation, invasion, metastasis and osteolytic lesions should be further explored. Sclerostin, Cloprostenol (sodium salt) a key regulator of normal bone remodeling, inhibits bone formation through inhibition of Wnt signaling6, 7. Recent studies have revealed a vital role of sclerostin in multiple myeloma with osteolytic bone lesions8C12. BCBM patients exhibit a high level of circulating sclerostin, which correlates with disease stage and fractures, however, the origin and impact of sclerostin on BCBM remains to be defined. Thus, the purpose of our study was to evaluate the expression level of sclerostin in tumor tissue derived from BCBM patients and explore its association with clinical outcome and tumor characteristics, including the presence of lytic bone disease. Activation of Wnt signaling pathway has been indicated to participate in both initiation and progression of cancer metastasis13, 14. During multiple-steps of metastasis to bone, breast cancer cells successfully induce a sequence of changes, for instance, secreting cytokines to inhibit differentiation and maturation of osteoblasts whereas to enhance the activity of osteoclasts. As such, we hypothesized that decreased sclerostin would suppress osteolytic bone lesions and reduce tumor burden, which may represent a target for inhibiting cancer-induced bone diseases and facilitating restoration of normal bone homeostasis. Results Sclerostin is up-regulated in tumor tissues derived from patients with BCBM To assess the expression of sclerostin in BCBM, paraffin sections of tissues from BCBM, localized breast cancer and benign breast tumor (n?=?15 per group) were evaluated with immunohistochemistry (Fig.?1A). Among the tumor tissues from BCBM, 13 (86.7%) exhibited strongly positive expression of sclerostin. In breast cancer patients, mainly in the cytoplasm of tumor cells, 12 (80%) of samples were weakly positive for sclerostin. In contrast, all tissues from benign breast tumor were negative. We next quantified sclerostin in the plasma of breast cancer patients and matched health individuals by ELISA (Fig.?1B). Significantly upregulated expression of sclerostin was observed in BCBM compared with localized breast cancer and benign breast tumor (Fig.?1C), which was further confirmed by NR4A3 Western blot analysis (Fig.?1D). Open in a separate window Figure 1 Expression of sclerostin (SOST) in tumor tissues. (A) Representative expression of sclerostin by immunohistochemical staining (100??magnification). Left: strongly positive expression of sclerostin in breast cancer bone metastasis (BCBM) tumor cells; middle: weakly positive expression of sclerostin in localized breast cancer (BC); right: negative expression of sclerostin in benign breast tumor (NC) tissue. (B) Quantification of sclerostin in the plasma of NC, BC and BCBM patients by ELISA. Expression of sclerostin mRNA (C), protein (D) and protein (E) were quantified in NC, BCBM and BC tissues. Each bar represents mean??SEM. *P? ?0.05, **P? ?0.01, ***P? ?0.005. Expression of sclerostin in breast cancer cell lines To determine the expression profile of sclerostin in different breast cancer cell lines (MDA-MB-231, BT-549, MCF-7, MDA-MB-453 and SK-BR3), qRT-PCR assay and Western blot analysis were adopted. Sclerostin protein extracted from supernatant Cloprostenol (sodium salt) culture medium was detectable in all cell lines (Fig.?2A). Cloprostenol (sodium salt) A higher expression level was observed in MDA-MB-231 and MCF-7 cells, whereas a Cloprostenol (sodium salt) lower level in SK-BR3, BT-549 and MDA-MB-453 cells. However, concerning expression of sclerostin mRNA, SK-BR3 was higher than MCF-7 (Fig.?2B). Open in a separate window Figure 2 Expression of sclerostin in human breast cancer cell lines. (A)Expression of sclerostin protein. GAPDH was used as an internal control. Sclerostin protein was extracted from supernatant culture medium after centrifugation and concentration. (B) Quantification of sclerostin are normalizing to GAPDH. (C) Expression of sclerostin mRNA (RT-PCR: 45 cycles of amplification). GAPDH mRNA was used as an internal control. Each bar represents mean??SEM. Effect of sclerostin on migration and invasion of MDA-MB-231 and MCF-7 cells To explore the impact of reduced Cloprostenol (sodium salt) sclerostin expression on migration and invasion, breast cancer cell lines with high endogenous sclerostin were treated with blocking antibody against sclerostin. A subsequent MTT assay revealed that reduced.
Using ROI boxes of the same size, data analysis tools were used to calculate the trabecular bone compartment, which was manually delineated from the cortical bone