The Mechanism Of Metadherin Modulating Angiogenesis In Breast Cancer

BACKGROUND AND AIMSBreast cancer is one of the most common malignant tumors in women, which is a serious affect on women’s physical and mental health and even is life-threatening. According to statistics, the incidence ratio is about 7% to 10% of all malignant tumor diseases。Moreover, the global incidence of breast cancer has been on the rise since the end of the 20th century. The incidence is also the highest of various female malignant tumors in China. The development of breast cancer as well as other malignant tumor is a complex biological process, including the proliferation of tumor cells invasion, metastasis, etc.Research has been shown that tumor angiogenesis was the significant role in the process of the local growth and metastasis of tumor. Tumor angiogenesis can supply enough nutrition for the continuous proliferation of tumor cells and provide a pathway for distant metastasis of tumor cells. Preclinical models of studies confirmed that angiogenesis had happened before malignant transformation of mammary gland hyperplasia. The breast cancer cells were transfected with angiogenic polypeptide also showed stronger ability of proliferation, invasion and metastasis. Since angiogenesis is closely associated with the development and metastasis of breast cancer, inhibition of tumor angiogenesis by some means may become the effective way of treatment of breast cancer. Angiogenesis relies on a variety of molecular pathways and is regulated by multiple genes and microRNAs. The research on mechanism of angiogenesis can not only helps to further understand the oncogenetic mechanism of breast cancer, also provide a basis for anti-angiogenesis therapy of breast cancer.Metadherin (MTDH) is a novel multifunctional oncogene. Previous studies have found MTDH were high expression in breast cancer, prostate cancer, liver cancer, esophageal cancer and other malignant tumors, and play important role in progression, metastasis and chemotherapy drug resistance of malignant tumor, and is associated with poor clinical outcome. Our laboratory studies have found that MTDH expression was closely related with the upregulated expression of some tumor angiogenesis markers, such as MMP-2, CD31 in breast cancer cells. Therefore, we suspect that MTDH may regulate the angiogenesis of breast cancer, so the purpose of our study is to investigate the relationship between the MTDH and angiogenesis in breast cancer and explore the molecular mechanism.METHODSDue to the high expression level of MTDH in MDA-MB-231 cells, we choose MDA-MB-231 cell line which has characteristics of triple-negative breast cancer for further study. We designed interference sequences target MTDH and connect them into the interfering vector pSUPER.retro.pure. The interference vector pSUPER.retro.pure MTDH shRNA was applied to establish the MTDH knockdown cell line and the empty vector was used to establish the control cell line. Cells were selected with puromycin to generate stable cell lines. The expression of MTDH was detected by qRT-PCR and western blot. And then the angiogenesis is researched through tube formation assay, mouse aortic ring assay and chick embryo chorioallantoic membrane (CAM) assay. In addition, We also collect 84 human triple-negative breast cancer tissue sample and detect the expression of MTDH and microvessel density (MVD) by immunohistochemical techniques to verify the clinical significance of MTDH in angiogenesis.To elucidate the potential molecular mechanism of MTDH in angiogenesis, we screening the pathway by western blot and selected ERK1/2 pathway for further study. We also detect the molecular markers of angiogenesis as VEGF and MMP2. To further investigate the function of microRNAs in the pathway of angiogenesis, miR-21 mimics were transfected into MDA-MB-231-prpM cell lines to up-regulate the level of miRNA-21 and then verify the change of angiogenesis through tube formation assay, mouse aortic ring assay and detect the level of VEGF and MMP2 by qRT-PCR and Western blot. Since PTEN is one of the target genes of miR-21 and regulates various biological behavior of tumor, so we tested the expression of PTEN after up-regulate the level of miRNA-21 to explore the mechanism of miRNA-21.RESULTSThe MTDH knockdown plasmids were constructed and were verified, then were transfected into MDA-MB-231 cells. The shRNA interference vector was applied to establish the MDA-MB-231-prpM cell line and the empty vector was used to establish the MDA-MB-231-prpn cell line. Cells were selected with 0.5 ug/ml puromycin to generate stable cell lines. MTDH expression levels were detected by qRT-PCR and western blot analysis. MTDH expression levels were obviously lower in MDA-MB-231-prpM cells than that in control MDA-MB-231-prpn cells (p<0.01).To investigate whether the MTDH could regulate angiogenesis of breast cancer cells, tube formation assay, mouse aortic ring assay and chick embryo chorioallantoic membrane (CAM) assay were performed. The results confirm that the knockdown of MTDH significantly inhibited the angiogenesis(P=0.006, P=0.016, P=0.024). We stained 84 triple-negative breast cancer tissue samples with the MTDH and CD31 antibody.33 cases were CD31-positive in the 48 MTDH-positive cases in total. Of these,14 cases were CD31-positive in the total 36 MTDH-negative staining cases. The data were evaluated with the t test (P=0.017).ERK1/2 molecule was widely regarded as a signal pathway activator of angiogenesis. Therefore, the protein levels of ERK1/2 and p-ERK1/2 were monitored in our test to explore a potential mechanism of action. As shown in our results, the expression of p-ERK1/2 was decreased. We measured the mRNA levels and protein levels of MMP-2 and VEGF in MTDH knockdown cells. The levels of the two markers were significantly reduced in MDA-MB-231-prpM cells.The miRNA arrays were adopted to detect the changes of miRNAs after MTDH knockdown. From the miRNA array data, we found MTDH regulated miRNA expression in MDA-MB-231-prpM cell. Among them, miRNA-21 level was significantly decreased. The level of miRNA-21 obviously increased after miR-21mimics transfection (p<0.01). Then, we applied tube formation assay and mouse aortic ring assay to explore the role of miRNA-21 in angiogenesis. The results showed that upregulated expression of miRNA-21 could partially reverse the inhibition of angiogenesis in MDA-MB-231-prpM cells (P=0.008, P=0.032) Previous studies indicated that miRNA-21 regulated expression of PTEN. Therefore, we detected PTEN and p-PTEN in miRNA-21 mimic-transfected MDA-MB-231-prpM cells. Up-regulation of miRNA-21 increased the protein level of p-ERKl/2 via suppressing PTEN, and then increased the levels of MMP2 and VEGF.CONCLUSION1. MTDH knockdown inhibits angiogenesis in breast cancer2. MTDH regulates angiogenesis via MTDH/miR-21/PTEN/ERKl/2 pathway.SIGNIFICANCE1. Confirmed that MTDH regulates angiogenesis of breast cancer2. Clarify the detailed molecular mechanisms of MTDH in angiogenesis.3. MTDH can be used as a novel target for anti-angiogenesis treatment of breast cancer in the future.