Bone morphogenetic protein-10 (BMP-10) inhibits aggressiveness of breast cancer cells and correlates with poor prognosis in breast cancer

Abstract

Our recent study showed that a novel member of bone morphogenetic protein (BMP) family, BMP-10, was decreased in prostate cancer. In the present study, we investigated the implication of BMP-10 in breast cancer, particularly the relation of its expression with clinical aspects. The expression of BMP-10 was examined in a cohort of human breast cancer specimens (normal, n = 23; cancer, n = 97), using both quantitative real-time PCR and immunohistochemical staining. The full-length human BMP-10 was cloned into a mammalian expression plasmid vector and then transfected into breast cancer cells. The effect on growth, cell matrix adhesion, motility, and invasion of MDA-MB-231 cells by BMP-10 was then investigated using in vitro growth assays. Immunohistochemical staining and quantitative real-time PCR revealed a decreased expression of BMP-10 in breast cancer. Further analysis of BMP-10 transcript level against the clinical aspect demonstrated that the decreased BMP-10 expression correlated with disease progression, bone metastasis, and poor prognosis. The disease-free survival of the patients with a higher level of BMP-10 was 132.8 (95% CI, 122.0-143.5) months, significantly longer compared to 93.7 (95% CI, 60.3-127.2) months for patients with a lower level of BMP-10 expression (P = 0.043). The overexpression of BMP-10 has broad inhibitory effects on the in vitro growth, invasion, and motility of breast cancer cells. Taken together, BMP-10 can inhibit the cell growth of breast cancer cells, and decreased BMP-10 expression correlates to poor prognosis and disease progression, particularly the lymphatic and bone metastasis. Bone morphogenetic protein-10 (BMP-10) may function as a tumor suppressor in breast cancer.

Figure 1

Expression of bone morphogenetic protein‐10 (BMP‐10) in breast cancer. (a) The expression of BMP‐10 mRNA in breast cancer cell lines using RT‐PCR. (b) The BMP‐10 transcript level is decreased in human breast cancer using quantitative PCR. (c) Immunohistochemical staining revealed a decreased staining of BMP‐10 in breast cancer compared to normal background tissue.

Figure 2

Bone morphogenetic protein‐10 (BMP‐10) and histological type, tumor grade, nodal status, and TNM stage. (a) Decreased levels of BMP‐10 transcripts were seen in lobular and other histological types of breast cancer compared to ductal breast cancer. (b) Bone morphogenetic protein‐10 (BMP‐10) transcripts were reduced in moderate‐ and poorly differentiated cancer cells compared to well‐differentiated tumor cells. (c) Decreased BMP‐10 expression was associated with lymphatic metastasis. (d) Lower levels of BMP‐10 transcripts were seen in the advanced breast cancer, including TNM3 and TNM4.

Figure 3

Bone morphogenetic protein‐10 (BMP‐10) transcript level and estrogen receptor (ER) status.

Figure 4

BMP‐10 and clinical outcomes and prognosis. (a) Bone morphogenetic protein‐10 (BMP‐10) and Nottingham prognostic index (NPI). (b) Expression of BMP‐10 is decreased in patients with poor prognosis compared to disease‐free patients. (c) Decreased BMP‐10 expression in primary tumor is associated with bone metastasis. (d) BMP‐10 and clinical outcomes. Expression of BMP‐10 is significantly decreased in patients with local recurrence or death due to breast cancer compared to disease‐free patients. (e) Higher level of BMP‐10 expression in primary breast tumors is correlated with longer disease‐free survival.

Figure 5

Forced expression of bone morphogenetic protein‐10 (BMP‐10) in breast cancer cells. (a) Overexpression of BMP‐10 was seen in MDA‐MB‐231^BMP−10exp cells using RT‐PCR, compared to both MDA‐MB‐231^WT and MDA‐MB‐231^pEF cells. (b) Overexpression of BMP‐10 protein in MDA‐MB‐231^BMP−10exp cells was also verified using western blot analysis, in comparison with both controls.

Figure 6

The effects on biological functions of breast cancer cells by bone morphogenetic protein‐10 (BMP‐10) overexpression. BMP‐10 inhibits in vitro growth breast cancer cells (a); and reduces invasion of breast cancer cells (b). (c) Overexpression of BMP‐10 results in a reduction of cell motility. (d) Cell–matrix adhesion of breast cancer cells was enhanced by BMP‐10. Shown are representative results of three independent experiments of each function assay. **P < 0.01, *P < 0.05.

Figure 7

Influence of bone morphogenetic protein‐10 (BMP‐10) overexpression on in vivo tumor growth.