Dataset Information

Progesterone receptor activation downregulates GATA3 by transcriptional repression and increased protein turnover promoting breast tumor growth.

ABSTRACT:

Introduction

The transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required.

Methods

Chromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments.

Results

In this study, we demonstrate that progestin-activated progesterone receptor (PR) reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth.

Conclusions

In the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.

SUBMITTER: Izzo F

PROVIDER: S-EPMC4303201 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Publications

Progesterone receptor activation downregulates GATA3 by transcriptional repression and increased protein turnover promoting breast tumor growth.

Izzo Franco F Mercogliano Florencia F Venturutti Leandro L Tkach Mercedes M Inurrigarro Gloria G Schillaci Roxana R Cerchietti Leandro L Elizalde Patricia V PV Proietti Cecilia J CJ

Breast cancer research : BCR 20141206 6

<h4>Introduction</h4>The transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required.<h4>Methods</h4>Chromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promote ...[more]

PMID: 25479686

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Project description:BackgroundThe transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus.MethodsImmunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ERα agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq).ResultsGATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERα and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation, albeit with a similar distribution across the genome, comparing to T47D cells.ConclusionsWe propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of breast cancer cells to estrogen signaling.

Project description:BACKGROUND:The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells. METHODS:U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice. RESULTS:Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis. CONCLUSIONS:Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.

Dataset Information

Progesterone receptor activation downregulates GATA3 by transcriptional repression and increased protein turnover promoting breast tumor growth.

Introduction

Methods

Results

Conclusions

Publications

Progesterone receptor activation downregulates GATA3 by transcriptional repression and increased protein turnover promoting breast tumor growth.

Similar Datasets

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