Abstract
Background Growth Regulation by Estrogen in Breast cancer (GREB1) was an estrogen receptor (ER) target gene, and GREB1 expression inversely correlated with HER2 status, possibly as a surrogate marker for ER status and a predictor for tamoxifen resistance in breast cancer patients. In the present study, we examine the function and regulation of GREB1 in breast cancer, with the goal to develop GREB1 as a biomarker in breast cancer with de novo and acquired tamoxifen resistance.MethodsWe overexpressed GREB1 using adenovirus containing the full length GREB1 cDNA (Ad-GREB1) in breast cancer cell lines. The soft agar assay was used as a measure of anchorage independent growth. The effects of GREB1 on cell proliferation in MCF-7 cells transduced with Ad-GREB1 were also measured by the me olic activity using AlamarBlue assay. We tested whether there was interaction between STAT3 and ER, which could repress GREB1 expression by immunoprecipitation assay. The effects of IL-6/JAK/STAT3 cascade activation on estrogen-induced GREB1 promoter activity were determined by luciferase assay and those on gene expression were measured by real time reverse transcription polymerase chain reaction (qRT-PCR).ResultsWe found that the ability of breast cancer cells to grow in soft agar is enhanced following GREB1 transfection. In MCF-7 cells transduced with Ad-GREB1 or transfected with siRNA GREB1, the metabolic activity was increased or completely abolished, suggesting that GREB1 may function as a growth promoter in breast cancer. E2 treatment increased GREB1 promoter luciferase activity. IL-6 inhibited E2-induced GREB1 transcription activity and GREB1 mRNA expression. Constitutively expressing active STAT3 construct (STAT3-C) dramatically decreased GREB1 transcription.ConclusionsThese data indicate that overexpression of GREB1 promotes cell proliferation and increases the clonogenic ability in breast cancer cells. Moreover, Il6/STAT3 modulates estrogen-induced GREB1 transcriptional activity in breast cancer cells.
Highlights
Current endocrine therapies for breast cancer patients target the estrogen receptor (ER) by reducing its ligand-induced activation, blocking its function and inducing ER degradation
GREB1 was detected in MCF-7 cells treated with E2 for 24 and 48 hrs, while no protein isdetected in the ER-negative BT-549 cells or in MCF-7 cells grown in estrogen-free conditions in Western blotting assay (Figure 1A)
progesterone receptor (PgR) expression alone has historical precedence as a clinical prognosticator of breast cancer patient in response to hormone therapy [8,51,52], the analysis showed that GREB1 mRNA predicts disease-free survival (DFS) in Tamoxifen-treated patients better than PgR or ER (Table 1)
Summary
Current endocrine therapies for breast cancer patients target the estrogen receptor (ER) by reducing its ligand-induced activation, blocking its function and inducing ER degradation. These therapies are effective in many patients with ERpositive tumors, long-term follow up and clinical trials have demonstrated that up to 62% of breast cancers that are initially responsive to endocrine therapy eventually relapse, with the patients requiring salvage surgery [1,2,3]. Growth Regulation by Estrogen in Breast cancer (GREB1) was an estrogen receptor (ER) target gene, and GREB1 expression inversely correlated with HER2 status, possibly as a surrogate marker for ER status and a predictor for tamoxifen resistance in breast cancer patients. We examine the function and regulation of GREB1 in breast cancer, with the goal to develop GREB1 as a biomarker in breast cancer with de novo and acquired tamoxifen resistance
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