BMP-2 Promotes Metastasis Of Breast Cancer With Mircrocalcification By Regulating EMT

Objective: Microcalcification show on mammography could be the only sign of the tumor. Bone morphogenetic proteins(BMPs) is one of the important members of the TGF-β superfamily. The biological functions of BMPs are very extensive, including promoting bone and cartilage formation and regulate the proliferation and differentiation of cells and regulating growth and development in various organs. At present, it is found that BMPs is closely related to ectopic calcification. And BMP-2 expression could be found in a variety of tumors tissues. In some recent studies, The BMPs was also considered to induced EMT and promote the invasion of the tumor. The purpose of our study was to detect the expression of BMP-2 in breast cancer with microcalcification and its relationship with the clinicopathological features and prognosis and to explore its influence on breast cancer and its mechanism.Method: 1.Collected 529 cases breast cancer patients with microcalcifications in Tianjin Cancer Hospital. And analyse the relationship between microcalcification and the clinical pathological characteristics and prognosis. 100 patients were selected from these paraffin blocks, and analysis of expression of BMP-2, Runx2, OPN by immunohistochemistry. 2.To verify the function of BMP-2 on breast cancer cells, we selected MCF-7 and MBA-MD-231 cell lines. We detected proliferation of tumor cells by MTT, detected the clone forming ability by plate cloning test, detected the apoptosis by flow cytometry, detected the invasion ability by transwell. 3. To dectect the invasion and metastasis mechanism regulated by BMP-2on breast cancer cell, first using real-time PCR and Western blot analysis of relationship between BMP-2 and EMT correlative index. We hypothesized that PI3 K / Akt pathway is one of the signaling pathway, then we used real-time PCR and Western blot techniques to detecte the relationship of BMP-2 and PI3 K / Akt proteins. And then, we add PI3K/Akt inhibitor LY294002 to verify its mechanism of action.At last we built breast cancer xenograft model to observe the effects of BMP-2 on transplanted tumor growth and metastasis, the expression of EMT related protein. Real-time PCR technique and immunohistochemical method were used on transplanted tumors.Results: 1.Patients with microcalcification of breast cancer has a higher rate of lymph node metastasis and a lager tumor size, and free progression survival is relatively poorer. Microcalcification is one of the independent prognostic factors. The expression of BMP-2, Runx2 and OPN was significantly higher in the breast cancer tissues with microcalcification, which may be involved in the formation of microcalcification. The high expression of BMP-2 was significantly associated with progression free survival, and it was one of the independent prognostic factors. 2. rh BMP-2 can promote the expression of BMP-2 in human breast cancer cell line MCF-7. BMP-2 inhibitor Noggin can inhibit the expression of BMP-2 in MBA-MD-231 cells. Vitro experiment showed that rh BMP-2 could promote the proliferation, clone formation and migration and invasion of breast cancer cells, and decrease the ability of apoptosis. And Noggin can inhibit the function of BMP-2, so as to inhibit tumor proliferation, clone formation, inhibit invasion and migration of breast cancer cells, and can promote apoptosis. These suggested that BMP-2 can promote the malignant degree of breast cancer cells. 3. In breast cancer MCF-7 cells, the expression of BMP-2 was significantly correlated with some indexes of EMT. With the increase of BMP-2 concentration, the expression of E-cadherin, Cx43 was significantly down regulated, and the expression level of N-cadherin, FN and Vimentin m RNA was significantly increased. BMP-2 can promote the expression of PI3K/Akt signaling pathway p-PI3 K, p-Akt and p-m TOR, and promote the process of EMT. After adding this signal pathway inhibitor LY294002, p-PI3 K, p-Akt, p-m TOR did not appear significantly increased, EMT related indicators have no significant change. BMP-2 can activate PI3K/Akt signaling pathway to promote MCF-7 cell EMT, and promote the ability of invasion and metastasis. 4. Application of MCF-7 breast cancer cells treated with BMP-2 successfully established the high expression of BMP-2 transplanted tumor model. The growth rate of tumor in group BMP-2 was significantly faster than the control group. BMP-2 group of lymph node metastasis and bone metastasis were higher than that of control group. Compared with the control group, BMP-2 group decreased the expression of E-cadherin, and N-cadherin and Slug were increased. The BMP-2 can promote the EMT process to regulate breast cancer metastasis.Conclusion: 1. Microcalcification in breast cancer patients is closely related with malignancy. 2. The BMP-2 expression in patients with microcalcification is higher, and closely related to metastasis and poor prognosis. 3. The proliferation and metastasis ability of breast cancer cells were enhanced after BMP-2 stimulation. 4. BMP-2 can stimulate the PI3K/Akt pathway protein phosphorylation, and regulate the expression of E-cadherin, Cx43, N-cadherin proteins, to promote tumor metastasis. 5. In vivo experiments confirmed that BMP-2 can promote tumor growth and metastasis, and BMP-2 may be a new target to control breast cancer in the future.