24 research outputs found

    Silencing MED1 Sensitizes Breast Cancer Cells to Pure Anti-Estrogen Fulvestrant In Vitro and In Vivo

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    <div><p>Pure anti-estrogen fulvestrant has been shown to be a promising 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. Here, we’ve reported that knockdown of estrogen receptor coactivator MED1 sensitized fulvestrant resistance breast cancer cells to fulvestrant treatment. We found that MED1 knockdown further promoted cell cycle arrest induced by fulvestrant. Using an orthotopic xenograft mouse model, we found that knockdown of MED1 significantly reduced tumor growth in mice. Importantly, knockdown of MED1 further potentiated tumor growth inhibition by fulvestrant. Mechanistic studies indicated that combination of fulvestrant treatment and MED1 knockdown is able to cooperatively inhibit the expression of ER target genes. Chromatin immunoprecipitation experiments further supported a role for MED1 in regulating the recruitment of RNA polymerase II and transcriptional corepressor HDAC1 on endogenous ER target gene promoter in the presence of fulvestrant. These results demonstrate a role for MED1 in mediating resistance to the pure anti-estrogen fulvestrant both <i>in vitro</i> and <i>in vivo</i>.</p> </div

    MED1 knockdown in combination with fulvestrant treatment dramatically inhibits the growth of orthotopic tumor xenografts.

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    <p>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: (<b>A</b>) Tumor volumes were measured twice per week by caliper (n=8). Red arrows indicate the time of fulvestrant treatments. (<b>B</b>) Tumors were harvested and the representative images of tumors in each group were shown. (<b>C</b>) Tumor weights were calculated and shown as a box-plot with median and whiskers from minimum to maximum (n=8). (<b>D</b>) Representative tumor bioluminescence images of each group at indicated time point were shown (*: P< 0.05; **: P< 0.01).</p

    Knockdown of MED1 potentiates fulvestrant-repressed cell proliferation and ERα target genes’ expression in xenograft tumors.

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    <p>(<b>A</b>) 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>B</b>) Western blot analyses of tumors using antibodies against MED1 and β-actin. (<b>C</b>) Quantification of Ki-67 positive cells in each group as shown in (A). (<b>D</b>) and (<b>E</b>) Total RNA of xenograft tumors from each group were extracted and analyzed for the mRNA expression of ERα target genes <i>TFF1</i> (D) and <i>ACP6</i> (E) by real-time RT-PCR. (n=6. *: P< 0.05; **: P< 0.01).</p

    Knockdown of MED1 sensitizes breast cancer cells to fulvestrant treatment.

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    <p>(<b>A</b>) 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>B</b>) (<b>C</b>) (<b>D</b>) 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). (<b>E</b>) 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. (<b>F</b>) 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).</p

    Silencing MED1 promotes down-regulation of ERα target genes induced by fulvestrant.

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    <p>(<b>A</b>) and (<b>B</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 <i>TFF1</i> and <i>Cyclin D1</i> (A) and EGF-induced ERα target genes <i>ACP6</i> and <i>LIF</i> (B) was measured by real-time RT-PCR after normalization to that of <i>GAPDH</i>. (n=3. *: P< 0.05; **: P< 0.01).</p

    MED1 Knockdown promotes fulvestrant-induced cell cycle arrest.

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    <p>(<b>A</b>) 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>B</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).</p

    FBI-1 Enhances ETS-1 Signaling Activity and Promotes Proliferation of Human Colorectal Carcinoma Cells

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    <div><p>In this study, we investigated a potential regulatory role of FBI-1 in transcription factor activity of ETS-1. The protein interaction was identified between ETS-1 and FBI-1 in lovo cells. The accumulating data showed that FBI-1 promoted the recruitment of ETS-1 to endogenous promoter of its target genes and increase ETS-1 accumulation in the nuclear. Our work also indicated that the FBI-1 enhances ETS-1 transcription factor activity via down-regulating p53-mediated inhibition on ETS-1. Further, FBI-1 plays a role in regulation of colorectal carcinoma cells proliferation. These findings supported that FBI-1 might be a potential molecule target for treating colorectal carcinoma.</p></div

    FBI-1 would potentially interact with ETS-1 and P53 in vivo.

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    <p>(A) Interaction of FLAG-FBI-1 with exogenous ETS-1 or p53 in vivo. Lovo cells were transfected with FLAG-tagged FBI-1 or FLAG empty vector. Then, cell lysates were immunoprecipitated (IP) by anti-FLAG beads, and the precipitates were then immunoblotted (IB) with anti-FLAG antibody, anti-ETS-1 antibody, or anti-p53 antibody. (B) Lovo cells were transfected with FLAG-ETS-1 vector or FLAG empty vector. The IP analysis was performed with anti-FLAG antibody, and the IB analysis was performed with anti-FLAG antibody, anti-FBI-1 antibody, or anti-P53 antibody.</p

    The proposed models for certain roles of FBI-1 function in Lovo cells.

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    <p>ETS-1 would be activated in presence of HGF and be blocked by ARQ-197. FBI-1 may modulate ETS-1 activity through potential protein interaction, recruitment to endogenous MMP1 promoter, or cytoplasmic/nucleus translocation. The regulatory effect of FBI-1 on ETS-1 is also through regulating P53 activity.</p

    Effect of FBI-1 on ETS-1 cytoplasmic/nucleus localization.

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    <p>(A–B) The cells were fractionated into cytoplasmic and nuclear fractions. The fractions were examined with anti-ETS-1 antibody, anti-P53 antibody and anti-FBI-1 antibody. The Lamin A/C and tubulin were used as the nuclear and cytoplasmic indicator, respectively.</p
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