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Mice were sacrificed 1 week after the last injection

Mice were sacrificed 1 week after the last injection. of sclerostin reduces bone metastatic burden and muscle weakness, with a prolongation of survival time. This might provide novel options for treating musculoskeletal complications in breast cancer patients. and and weaker alizarin red S staining (Physique 1, A and B). Furthermore, using a TOPflash reporter gene assay, we decided that cancer cellCconditioned medium suppressed the activity VU591 of the canonical Wnt signaling pathway in osteoblasts in a dose-dependent manner (Physique 1C). These findings indicate that metastatic breast malignancy cells may secrete factors that inhibit canonical EC-PTP Wnt signaling in osteoblasts VU591 in a paracrine fashion. Open in a separate window Physique 1 Breast cancerCderived sclerostin inhibits Wnt signaling in osteoblasts.(A and B) Calvarial cells were differentiated into osteoblasts in the presence of control medium or cancer-conditioned medium (CM) collected from MDA-MB-231 metastatic breast malignancy cells. Osteoblast differentiation was determined by quantification of and osteocalcin (= 4 impartial experiments). (C) Calvarial osteoblasts were cultured in the presence of the indicated amount of MDA-MB-231Cderived CM. Wnt signaling activity was determined by TOPflash reporter assay (= 4 impartial experiments). (D) Sclerostin mRNA expression was quantified in nonmetastatic MCF-7 and metastatic MDA-MB-231 breast malignancy cells by qRT-PCR (= 6 impartial experiments). (E) mRNA expression was analyzed in breast cancer tissue from 48 patients. Proportion of sclerostin-positive and sclerostin-negative tissue samples is shown for all patients and for triple-negative (ERC, PRC, HERC) and in hormone receptorCnegative (ERC, PRC, HER+) patients. All, = 48; ERC, PRC, HER-, = 9; ERC, HERC, HER+, = 7. (F) Wnt signaling activity in calvarial osteoblasts cultured with control medium or with CM from MDA-MB-231 cells transfected with scrambled control siRNA (si-Ctrl CM) or siRNA against sclerostin (si-Sclerostin CM) (= 6 impartial experiments). (G) Wnt signaling activity in calvarial osteoblasts isolated from mice heterozygous for the mutation G171V (= 4 impartial experiments). Data are presented as mean SEM. Two-tailed Students test was used to compare 2 groups (A and D), and ANOVA followed by Tukeys post hoc analysis was used to compare 3 or more groups (C, F, and G); * 0.05, ** 0.01, *** 0.001. Breast cancer cells have been shown to express Dickkopf 1 (Dkk1), a soluble antagonist of canonical Wnt signaling. However, antagonizing cancer cellCderived Dkk1 did not fully restore the activity of the Wnt pathway (19), suggesting that additional mechanisms might exist. Indeed, expression analysis revealed significantly higher expression of the Wnt inhibitor sclerostin in metastatic MDA-MB-231 breast cancer cells compared with nonmetastatic MCF-7 breast malignancy cells (Physique 1D). To determine whether sclerostin expression VU591 is a general feature of breast malignancy cells, we performed an in silico analysis using the EMBL-EBI Expression Atlas (20). In addition to cells of the MDA-MB-231 cell line, expression of sclerostin was found in cells of the SUM159PT, CAL51, HCC 1187, HCC 1197, HCC 1395, HCC 1806, and KPL-4 breast malignancy cell lines. Interestingly, 6 of these cell lines (SUM159PT, CAL51, HCC 1187, HCC 1197, HCC 1395, and HCC 1806) express neither the estrogen receptor (ER) nor the progesterone receptor (PR) and do not bear an amplification of the HER-2/Neu gene (referred to as triple-negative breast malignancy cells) (21, 22). Furthermore, although KPL-4 cells have a HER-2/Neu amplification, they do not express the ER or the PR (23), suggesting that sclerostin expression is usually a common feature of hormone receptorCnegative breast cancer cells. To address the question of whether sclerostin is usually expressed in primary breast malignancy tissue from patients,.