| Background and objective:Bone morphogenetic protein 4 (BMP4) is an extracellular signaling molecule that belongs to the transforming growth factor β(TGF-β) superfamily and is expressed in both breast cancer cell lines and tumor samples as being reported. In vitro, several studies, especially in breast cancer, have implicated BMP4 both in inhibition of cell proliferation and in promotion of cell migration and invasion. BMP4 binds to its specific transmembrane receptors on the surface of breast cancer cells through autocrine mechanism to play a role mainly through the canonical SMAD signaling pathway. The latest research found out that BMP4 can stimulate fibroblasts in the breast cancer microenvironment to promote mammary carcinoma cell invasion. Cancer-associated fibroblasts (CAFs) are one of the most important components of the tumor microenvironment and are shown to be vital in tumor growth and progression, characterized by the expression of α-smooth muscle actin (α-SMA). Recent studies have demonstrated that adipose-derived stem cells (ADSCs) can differentiate into CAF-like cells to promote tumor cell invasion under the influence of breast cancer cells-derived TGF-β.However, TGF-β isn’t the only factor involved in this phenomenon. This study firstly detect the expression of BMP4 in both breast cancer cell lines and tumor samples and investigate the effects of BMP4 expression on the prognosis of breast cancer. Then we focus on exploring whether BMP4 secreted by breast cancer cells can affect ADSCs isolated from breast adipose tissue differentiating into CAF-like cells, in order to help us find new potential mechanisms involved in the effects of BMP4 on breast cancer progression, and offer clues for prevention and treatment of breast cancer.Methods:Real-time PCR was used to detect the expression of BMP4 in two kinds of breast cancer cell lines, MCF-7 and MDA-MB-231. We studied the clinical features in a cohort of 50 patients (25 are ER positive and 25 are ER negative) which were selected randomly among the patients with primary breast cancer who were treated at Shanghai Zhongshan Hospital during 2008～2009. Immunohistochemistry was used to detect the expression of BMP4 in paraffin samples of the 50 cases. The associations of BMP4 expression with clinicopathological parameters and its influence on the prognosis of breast cancer were analyzed. Human ADSCs were isolated from breast adipose tissue. The expression of α-SMA was increased when hADSCs were directly concultured with MCF-7/MDA-MB-231, observed by immunofluorescence assay. Tumor conditioned media of MCF-7/MDA-MB-231 were collected and used to culture hADSCs. Recombinant human BMP4 protein was added to the TCM of MCF-7, while anti-BMP4 neutralizing-antibody was added to the TCM of MDA-MB-231.Real-time PCR and western blot were used to detect the expression of a-SMA.Results:1) The expression of BMP4 was detected in both MCF-7 and MDA-MB-231, and the level was relatively higher in the latter cell line.2) BMP4 expression was determined in 62%(31/50) of the tumors. The samples were divided into two groups: BMP4 positive (n=31) and BMP4 positive (n=19). Except for histological grade, no further statistically significant associations were found between BMP4 expression and other clinicopathological parameters including tumor size, nodal status, TNM stage, surgical treatment, pathology, ER/PR status and ERBB2 status.3) For all the 50 cases, the 3-year and 5-year disease-free survival (DFS) of BMP4 positive cases were 90.3%, 61.5%. And the 3-year and 5-year overall survival (OS) were 90.3%,87.0%. While the 3-year and 5-year DFS of BMP4 negative cases were 94.4%,87.7%. And the 3-year and 5-year OS were 100%、90.0%. There were no differences in DFS and OS of patients whether the tumors expressed BMP4 or not.4) Among 25 cases of ER positive,16 cases were BMP4 positive, and their 3-year and 5-year DFS were 100%, 83.0%, OS were 100%,90.0%. The rest cannot detect the BMP4 expression. During follow-up, one case was missing and no recurrence or metastasis or death occured.5) Among 25 cases of ER negative,15 cases were BMP4 positive, and their 3-year and 5-year DFS were 80.0%,32.7%, OS were 80.0%,73.3%. The rest were BMP4 negative, and the 3-year and 5-year DFS were 90.0%,80.0%, OS were 100.0%, 85.7%. There were no differences in DFS and OS of patients whether the tumors expressed BMP4 or not.6) hADSCs can express significantly higher a-SMA when concultured with MCF-7/MDA-MB-231 directly or indirectly.7) Recombinant human BMP4 protein, added to the TCM of MCF-7, can make hADSCs express higher a-SMA.8) Compared to the hADSCs cultures with the TCM of MDA-MB-231, the level of a-SMA expressed in hADSCs decreased when the activity of BMP4 in the TCM of MDA-MB-231 was neutralized. However, it was still higher than the a-SMA expression when cultured in serum-free medium.Conclusions:In present study, we confirm that BMP4 are expressed in both breast cancer cell lines and tumor samples. We demonstrate that BMP4 expression is statistically significant with histoligical grade, however, no significant associations with the prognosis of breast cancer. This study investigate that BMP4 secreted by breast cancer cells may act on ADSCs in tumor microenvironment through paracrine mechanism, resulting in significantly higher expression of a-SMA, suggesting that ADSCs may differentiate into CAF-like cells. It is expected to discover the important effects of BMP4 on breast cancer microenvironment and reveal new molecular targets for the treatment of breast cancer. |
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