Figure 1.
Knockdown of MED1 sensitizes breast cancer cells to fulvestrant treatment.
(A) BT474-tet-shMED1, ZR75-1-tet-shMED1 and MCF-7-tet-shMED1 cells were treated with vehicle (-Dox) or doxycycline (+Dox) and subjected to western blot analyses using indicated antibodies. (B) (C) (D) MTT assays measuring cell proliferation of cells in (A) after control and fulvestrant treatments for 7 days. Relative cell numbers of both vehicle (-Dox) and doxycycline (+Dox) were normalized to that of their respective fulvestrant untreated control (set as 1). (E) Fulvestrant-resistant MCF7-F cells were infected with lentivirus expressing control scramble or MED1 shRNA and blotted for MED1 expression using β-actin levels as a control. (F) The cells in (E) were subjected to MTT assay after treatment with indicated concentration of fulvestrant. Relative cell numbers of both vehicle (-Dox) and doxycycline (+Dox) were normalized to that of their respective fulvestrant untreated control (set as 1). (n=4. *: P< 0.05; **: P< 0.01).
Figure 2.
MED1 Knockdown promotes fulvestrant-induced cell cycle arrest.
(A) BT474-tet-shMED1 and ZR75-1-tet-shMED1 cells were treated with vehicle (Veh) or doxycycline or/and fulvestrant (+Ful, 5µM) and harvested for flow cytometry analysis. The representative histograms of cell cycle distribution were shown with the percentages of S-phase cells presented. (B) The bar graph of the percentages of S-phase cells in above treatment groups after normalizing to that of the vehicle control treatment. (n=3. *: P< 0.05; **: P< 0.01).
Figure 3.
Silencing MED1 promotes down-regulation of ERα target genes induced by fulvestrant.
(A) and (B) BT474-tet-shMED1 and ZR75-1-tet-shMED1 cells were first treated with vehicle (Veh) or doxycycline or/and fulvestrant (+Ful). Total RNA was then extracted, and the expression of E2-induced ERα target genes TFF1 and Cyclin D1 (A) and EGF-induced ERα target genes ACP6 and LIF (B) was measured by real-time RT-PCR after normalization to that of GAPDH. (n=3. *: P< 0.05; **: P< 0.01).
Figure 4.
MED1 affects the recruitment of RNA pol II and transcriptional corepressor HDAC1 to TFF1 promoter in the presence of fulvestrant.
(A) MCF-7 and BT474 cells were pretreated with fulvestrant (Ful) or vehicle (Veh) for 6h, then followed by treatment with 17β-estradiol (E2, 100nM) or vehicle for 45min. ChIP assays were performed to detect the recruitment of MED1 on TFF1 promoter using antibodies against MED1. (B) BT474-tet-shMED1 cells were pre-incubated with vehicle or doxycycline (Dox) for 7 days, followed by treatments as in (A). ChIP assays were performed to detect the recruitment of MED1, ERα, RPB1 and HDAC1 to TFF1 promoter using respective antibodies. (n=3. *: P< 0.05; **: P< 0.01).
Figure 5.
MED1 knockdown in combination with fulvestrant treatment dramatically inhibits the growth of orthotopic tumor xenografts.
BT474-tet-shMED1 cells were injected into the fat pad of 5-6 weeks old nude mice. After 3 weeks, mice were randomly divided into four groups (4 mice/group): vehicle control (Veh), or doxycycline (+Dox), fulvestrant (+Ful) and the combination treatment (+Dox/+Ful) and examined for the followings: (A) Tumor volumes were measured twice per week by caliper (n=8). Red arrows indicate the time of fulvestrant treatments. (B) Tumors were harvested and the representative images of tumors in each group were shown. (C) Tumor weights were calculated and shown as a box-plot with median and whiskers from minimum to maximum (n=8). (D) Representative tumor bioluminescence images of each group at indicated time point were shown (*: P< 0.05; **: P< 0.01).
Figure 6.
Knockdown of MED1 potentiates fulvestrant-repressed cell proliferation and ERα target genes’ expression in xenograft tumors.
(A) Paraffin-embedded tissue sections of above xenograft tumors were subjected to H&E staining (top panels) and immunohistochemical (IHC) staining with antibodies against MED1 (middle panels) or Ki-67 (bottom panels). Scale bar = 100µm. (B) Western blot analyses of tumors using antibodies against MED1 and β-actin. (C) Quantification of Ki-67 positive cells in each group as shown in (A). (D) and (E) Total RNA of xenograft tumors from each group were extracted and analyzed for the mRNA expression of ERα target genes TFF1 (D) and ACP6 (E) by real-time RT-PCR. (n=6. *: P< 0.05; **: P< 0.01).