Estradiol-regulated MicroRNAs Control Estradiol Response in Breast Cancer Cells

Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). E2-inducible proteins c-Myc and E2Fs are required for optimal ERα activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERα-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERα-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome

Estradiol-regulated microRNAs control estradiol response in breast cancer cells.

Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). E2-inducible proteins c-Myc and E2Fs are required for optimal ERalpha activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERalpha-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERalpha-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

BACKGROUND: Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. METHODS: MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. RESULTS: We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. CONCLUSION: E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Subcellular Localization of Activated AKT in Estrogen Receptor- and Progesterone Receptor-Expressing Breast Cancers : Potential Clinical Implications

Activated v-AKT murine thymoma viral oncogene homolog 1 (AKT)/protein kinase B (PKB) kinase (pAKT) is localized to the plasma membrane, cytoplasm, and/or nucleus in 50% of cancers. The clinical importance of pAKT localization and the mechanism(s) controlling this compartmentalization are unknown. In this study, we examined nuclear and cytoplasmic phospho-AKT (pAKT) expression by immunohistochemistry in a breast cancer tissue microarray (n = 377) with ≈15 years follow-up and integrated these data with the expression of estrogen receptor (ER)α, progesterone receptor (PR), and FOXA1. Nuclear localization of pAKT (nuclear-pAKT) was associated with long-term survival (P = 0.004). Within the ERα+/PR+ subgroup, patients with nuclear-pAKT positivity had better survival than nuclear-pAKT–negative patients (P ≤ 0.05). The association of nuclear-pAKT with the ERα+/PR+ subgroup was validated in an independent cohort (n = 145). TCL1 family proteins regulate nuclear transport and/or activation of AKT. TCL1B is overexpressed in ERα-positive compared with ERα-negative breast cancers and in lung metastasis–free breast cancers. Therefore, we examined the possible control of TCL1 family member(s) expression by the estrogen:ERα pathway. Estradiol increased TCL1B expression and increased nuclear-pAKT levels in breast cancer cells; short- interfering RNA against TCL1B reduced nuclear-pAKT. Overexpression of nuclear-targeted AKT1 in MCF-7 cells increased cell proliferation without compromising sensitivity to the anti-estrogen, tamoxifen. These results suggest that subcellular localization of activated AKT plays a significant role in determining its function in breast cancer, which in part is dependent on TCL1B expression