BMPs were originally discovered because they can induce bone and cartilage formation ectopically18–20, and later studies revealed that BMPs are widely distributed in multiple tissues and cells of the human body21,22 and can be involved in processes such as embryogenesis, embryonic development, histiocyte differentiation and proliferation23. Reports in recent years have shown that BMPs and their signaling pathways are closely related to human tumors24–26. BMPs have been found to be expressed in gastric cancer, prostate cancer, breast cancer, colorectal cancer, and other cell lines27–29. The purpose of this study was to investigate the effects of BMP-2 on the proliferation and invasion of breast cancer cells and the possible mechanism of action.
We selected several representative breast cancer cell lines to detect the expression of BMP-2, and the results showed that BMP-2 was highly expressed in MBA-MD-231 and T47D cell lines, while the expression levels were relatively low in MCF-7 and SKBr-3 cell lines. The morphology of MCF-7 cells treated with rhBMP-2 was changed into longer, narrower and polyantennary shapes, the cell-cell adhesion weakened or even disappeared, and the cells became discrete, which make MCF-7 cells more migratory and metastatic. Given that clonal proliferation is one of the malignant phenotypes of tumor cells, we validated the effects of BMP-2 on the proliferation and colony formation ability of breast cancer cells by MTT assay and plate cloning experiment. The results showed that the proliferation and colony formation ability of MCF-7 cells was significantly increased by the addition of rhBMP-2. However, when Noggin was added to MBA-MD-231 cells, the cell proliferation and colony formation ability was significantly attenuated in a significant time-concentration-dependent manner. Park et al30. found that serum BMP-2 levels in gastric cancer patients were significantly higher than normal and positively correlated with tumor progression, and tissue microarray results showed that BMP-2 expression was higher in gastric cancer than in normal gastric mucosa. Pouliot, F et al. found that BMP-2 can promote the proliferation and survival of breast cancer31.
Immortalization by reduced tumor cell apoptosis is one of the malignant phenotypes of tumor cells, and the effects of BMP-2 on tumor cell apoptosis are still controversial. Hardwick et al.32 found that BMP-2 can promote the apoptosis and differentiation of colon cancer cells, while Arnold, SF et al. found that rhBMP-2 inhibited apoptosis in the breast cancer cell line MBA-MD-23133. To understand the effects of BMP-2 on apoptosis, flow cytometry was used to detect apoptosis of cells treated with BMP-2. The results showed that the apoptotic ratio of breast cancer MCF-7 cells decreased by the addition of BMP-2 (p < 0.05) in a concentration-dependent manner. However, after the addition of Noggin to MBA-MD-231 cells, the level of apoptosis gradually increased with the concentration (p < 0.05) This shows that BMP-2 can reduce apoptosis and enhance immortalization characteristics in breast cancer cells. The above inconsistent findings may be caused by the fact that the purification and concentration of BMP-2 protein used in each study were not consistent, thus providing different conclusions. In addition, the reactivity of different types of tumor cells to BMP-2 also showed different results, and the exact mechanism is not fully clear34–37.
Katsuno, Y found that BMP-2 could promote breast cancer cell migration and bone metastasis38. Clement, JH et al. reported that BMP-2 could improve the proliferation and invasion abilities of breast cancer cells39. Some studies have shown that BMP-2 can enhance the invasion ability of lung cancer cells, and BMP-2 expression is closely related to the prognosis of non-small cell lung cancer (NSCLC) 40. Feeley et al. 41 reported the effects of BMP-2 on prostate cancer cells and found that it promoted the migration and metastasis of prostate tumor cells in a concentration-dependent manner. In digestive tract tumors, Jin et al. found that BMP-2 could cause migration and invasion of colorectal and gastric cancer cells42. The opposite has also been reported, with Buijs JT suggesting that BMP-2 and BMP-7 would significantly inhibit the activity of breast cancer cells43. An analysis of the clinicopathological profiles of BMP-2 published by us in 2019 found that BMP-2 was highly expressed in breast cancer tissues and closely related to poor prognosis and bone metastasis5. A scratch test was performed in this study to validate the effects of BMP-2 on the migration, motility and cytothesis of breast cancer cell lines, and the results showed that the scratch healing rate was significantly increased after adding different concentrations of rhBMP-2 (p < 0.05), indicating that BMP-2 could significantly enhance the migration and motility of MCF-7 cells. Transwell assay showed that the number of cells passing through the membrane was significantly increased by the addition of BMP-2 (p < 0.05), which indicated that BMP-2 could significantly enhance the invasion ability of MCF-7 cells. Then we conducted a deeper exploration of its possible mechanism of action.
BMP-2 is a member of the TGF-β superfamily that can activate the receptor by binding to TGF-β receptors on the cell surface, and it has been reported to be involved in the signaling of multiple signaling pathways, such as the Smad pathway, PI3K/Akt signaling pathway, Ras/MAPK signaling pathway and Rho-kinase signaling pathway 44–47. Moreover, TGF-β is one of the more intensively studied inducers in the field of EMT48–50. It is speculated that the promotion of breast cancer invasion and metastasis by BMP-2, a member of the TGF-β family, may be achieved by inducing EMT. In order to validate this speculation, the expression of BMP-2 in MCF-7 cells was promoted by rhBMP-2 in this study. The changes in mRNA and protein levels indicated that BMP-2 significantly regulated the changes in EMT-related indicators. After treatment with rhBMP-2, the RNA expression levels of E-cadherin and Cx43 in MCF-7 cells were significantly decreased (p < 0.01), while those of N-cadherin, FN and slug were significantly up-regulated (p < 0.05), which indicated the promotion of the transition of MCF-7 cells from epithelial to mesenchymal. To further validate whether BMP-2 induces EMT in MCF-7 cells via the PI3K/Akt/mTOR signaling pathway, we quantitatively detected the changes in the expression of major signaling molecules (p-PI3K, p-Akt, p-mTOR) of the PI3K/Akt/mTOR signaling pathway during BMP-2 induced EMT in MCF-7 cells by Western blot. The changes of signaling molecules in the PI3K/Akt pathway blocked by LY294002 in response to BMP-2 were also analyzed. The results showed that BMP-2 could activate the phosphorylation of the PI3K/Akt signaling pathway, and this activation effect was significantly attenuated after the addition of pathway inhibitors, thus validating the speculation that BMP-2 promotes the EMT process in tumors via PI3K/Akt/mTOR pathway.
By subcutaneous injection of breast cancer MCF-7 cells treated with BMP-2 into nude mice, we successfully established xenograft models with high BMP-2 expression. Through measurement and observation, we found that the growth rate of xenograft tumors in the BMP-2 group was significantly faster than that in the control group. The proportion of lymph node metastasis and bone metastasis in the BMP-2 group was significantly higher than that in the control group, moreover, the expression of E-cadherin was attenuated, while the expression of vimentin was enhanced, showing a significant EMT. The in vivo experiments confirmed that BMP-2 could promote the EMT process and promote the metastasis of breast tumors.