Investigation On The Role Of MED1in The Breast Cancer Therapy

Breast cancer is the most common malignancy and one of the leading causes of cancer-related mortality amongst female worldwide. The incidence of breast cancer in China, especially in most big cities, is dramatically increasing nowadays. Pure anti-estrogen fulvestrant has been shown to be a promising estrogen receptor (ER) antagonist for locally advanced and metastatic breast cancer. Unfortunately, a significant proportion of patients developed resistance to this type of endocrine therapy but the molecular mechanisms governing cellular responsiveness to this agent remain poorly understood. Meanwhile, metastasis is the major cause of death associate with breast cancer, and the molecular mechanisms of metastasis of breast cancer also remain unclear. MED1(Mediator subunit1), as a known ER coactivator, plays a crucial role in ER-dependent gene expression and estrogen-dependent breast cancer cell growth, and is associated with the resistance of tamoxifen, another class of endocrine therapy agent, and poor outcome of breast cancer patients. Here, this study investigated the role of MED1in the processes of fulvestrant resistance and metastasis of breast cancer.1. This study successfully constructed Tet-inducible MED1shRNA lentiviral system using pLKO-Tet-On vector and generated breast cancer cell sublines that stably express firefly luciferase and Tet-inducible MED1shRNA (MCF7-tet-shMED1, BT474-tet-shMED1and ZR75-1-tet-shMEDl). The expressions of MED1in all these Tet-inducible cells were significantly down-regulated after Dox (doxycycline) treatment. Consistent with the previous reports, MED1knockdown induced by Dox strongly inhibited the cell proliferation, anchorage-dependent and anchorage-independent colony formation. In a word, these inducible cells are reliable and useful for further studies.2. This study first investigated the role of MEDl on fulvestrant resistance. It was revealed that knockdown of MED1sensitized fulvestrant resistance breast cancer cells (BT474, ZR75-1, MCF7-F) to fulvestrant treatment in vitro. MED1knockdown further promoted cell cycle arrest induced by fulvestrant. Using an orthotopic BT474xenograft mouse model, it was found that knockdown of MED1further significantly potentiated tumor growth inhibition by fulvestrant. Mechanistic studies indicated that combination of fulvestrant treatment and MED1knockdown was able to cooperatively inhibit the expression of ER target genes. Chromatin immunoprecipitation (ChIP) assays further supported a role for MED1in regulating the recruitment of RNA polymerase II, ER and transcriptional corepressor HDAC1on endogenous ER target gene promoter in the presence of fulvestrant. Furthermore, there is a regulator loop between HER2and MED1in controlling fulvestrant resistance of human breast cancer cells. These results demonstrate a role for MED1in mediating resistance to the pure anti-estrogen fulvestrant both in vitro and in vivo.3. This study first established the relationship between MED1and the aggressiveness of ER-positive breast cancer cells. Using Tet-inducible MED1knockdown cells, it was revealed that silencing MED1efficiently blocks the abilities of cell migration and invasion in vitro, as well as the metastasis of breast carcinoma in vivo using orthotopic or i.v. xenograft mice models. Meanwhile, the expression of matrix metalloproteinase9(MMP9) was down-regulated by silencing MED1. Exogenous restoration of MMP9can rescue cell migration and invasion decreased by MED1knockdown. Whereas, over-expression of MED1significantly enhances the migration and invasion of MCF7and BT474cells through increasing MMP9expression. Mechanistically, promoter reporter assays indicated that the proximal AP-1binding site is required for MED1-modulated transcriptional regulation of MMP9. ChIP assays further supported that MED1regulated the assembly of AP-1complex and chromatin remodeling through modulating the recruitment of AP-1, transcriptional corepressor HDAC1and coactivator CBP on the proximal AP-1binding site of endogenous MMP9promoter. This perhaps is the main mechanism for MED1activating MMP9transcription. Collectively, our results reveal that MED1plays a key role on invasion and metastasis of ER-positive breast carcinoma through transcriptional regulation of MMP9.Taken together, this study has demonstrated the key role of MED1on modulating fulvestrant resistance and metastasis of breast cancer. MED1therefore may offer advantageous therapeutic outcome by overcoming both fulvestrant resistance and metastasis of breast cancer. Targeted intervention of MEDl expression may provide a novel therapeutic strategy to inhibit breast cancer endocrine resistance and distant metastasis.