profiling-of-estrogen-regulated-micrornas-in-breast-cancer-cells

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

University of Houston

Breast cancers are sensitive to hormones such as estrogen, which binds to and activates estrogen receptors (ER) leading to significant changes in gene expression. microRNAs (miRNA) have emerged as a major player in gene regulation, thus identification of miRNAs associated with normal or disrupted estrogen signaling is critical to enhancing our understanding of the diagnosis and prognosis of breast cancer. We have previously shown that 17β-estradiol (E2) induced activation of ERα in T47D cells results in significant changes in the expression of protein-coding genes involved in cell cycle, proliferation, and apoptosis. To identify miRNAs regulated by E2-activated ERα, we analysed their expression in T47D cells following E2-activation using both dual-color microarrays and TaqMan Low Density Arrays, and validations were carried out by real-time PCR. Although estrogen treatment results in altered expression of up to 900 protein-coding transcripts, no significant changes in mature miRNA expression levels could be confirmed. Whereas previous studies aiming to elucidate the role of miRNA in ER-positive breast cancers cell lines have yielded conflicting results, the work presented here represents a thorough investigation of and significant step forward in our understanding of ERα mediated miRNA regulation.

Estradiol-activated estrogen receptor α does not regulate mature microRNAs in T47D breast cancer cells.

MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.

MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer.

Epithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. 60% of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERbeta1). However, it is still unclear whether the ERbeta expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERbeta1 through regulating EMT can influence invasion and metastasis in basal-like cancers.

ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

Multiple sclerosis (MS), a demyelinating immune-mediated central nervous system disease characterized by increasing female penetrance, is the leading cause of disability in young adults in the developed world. Epidemiological data strongly implicate an environmental factor, acting at the population level during gestation, in the increasing incidence of female MS observed over the last 50 years, yet the identity of this factor remains unknown. Gestational exposure to bisphenol A (BPA), an endocrine disruptor used in the manufacture of polycarbonate plastics since the 1950s, has been reported to alter a variety of physiological processes in adulthood. BPA has estrogenic activity, and we hypothesized that increased gestational exposure to environmental BPA may therefore contribute to the increasing female MS risk. To test this hypothesis, we utilized two different mouse models of MS, experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice (chronic progressive) and in SJL/J mice (relapsing-remitting). Dams were exposed to physiologically relevant levels of BPA in drinking water starting 2 weeks prior to mating and continuing until weaning of offspring. EAE was induced in adult offspring. No significant changes in EAE incidence, progression, or severity were observed with BPA exposure, despite changes in cytokine production by autoreactive T cells. However, endocrine disruption was evidenced by changes in testes development, and transcriptomic profiling revealed that BPA exposure altered the expression of several genes important for testes development, including Pdgfa, which was downregulated. Overall, our results do not support gestational BPA exposure as a significant contributor to the increasing female MS risk.

Studies in experimental autoimmune encephalomyelitis do not support developmental bisphenol a exposure as an environmental factor in increasing multiple sclerosis risk.

The expression of estrogen receptor α (ERα) in breast cancer identifies patients most likely to respond to endocrine treatment. The second ER, ERβ, is also expressed in breast tumors, but its function and therapeutic potential need further study. Although in vitro studies have established that ERβ opposes transcriptional and proliferative functions of ERα, several clinical studies report its correlation with proliferative markers and poorer prognosis. The data demonstrate that ERβ opposes ERα are primarily based on transient expression of ERβ. Here, we explored the functions of constitutively expressed ERβ in ERα-positive breast cancer lines MCF7 and T47D. We found that ERβ, under these conditions heterodimerized with ERα in the presence and absence of 17β-estradiol, and induced genome-wide transcriptional changes. Widespread anti-ERα signaling was, however, not observed and ERβ was not antiproliferative. Tamoxifen antagonized proliferation and ER-mediated gene regulation both in the presence and absence of ERβ. In conclusion, ERβ's role in cells adapted to its expression appears to differ from its role in cells with transient expression. Our study is important because it provides a deeper understanding of ERβ's role in breast tumors that coexpress both receptors and supports an emerging bi-faceted role of ERβ.

Support of a bi-faceted role of estrogen receptor β (ERβ) in ERα-positive breast cancer cells.

Endocrine-disrupting chemicals (EDC) are abundant in our environment. A number of EDCs, including bisphenol A (BPA) can bind to the estrogen receptors, ERα and ERβ, and may contribute to estrogen-linked diseases such as breast cancer. Early-exposure is of particular concern; many EDCs cross the placenta and infants have measurable levels of e.g. BPA. In addition, infants are frequently fed soy-based formula that contains phytoestrogens. Effects of combined exposure to xeno- and phytoestrogens are poorly studied. Here, we extensively compared to what extent BPA, genistein and an extract of infant soy-based formula (SF) mimic estrogen-induced gene transcription and cell proliferation. We investigated ligand-specific effects on ER activation in HeLa-ERα and ERβ reporter cells; on proliferation, genome-wide gene regulation and non-ER mediated effects in MCF7 breast cancer cells; and how co-exposure influenced these effects. The biological relevance was explored using enrichment analyses of differentially regulated genes and clustering with clinical breast cancer profiles. We demonstrate that co-exposure to BPA and genistein, or SF, results in increased functional and transcriptional estrogenic effects. Using statistical modeling we determine that BPA and phytoestrogens act in an additive manner. The proliferative and transcriptional effects of the tested compounds mimic those of 17β-estradiol, and are abolished by co-treatment with an ER antagonist. Gene expression profiles induced by each compound clustered with poor-prognosis breast cancer, indicating that exposure may adversely affect breast cancer prognosis. This study accentuates that co-exposure to BPA and soy-based phytoestrogens results in additive estrogenic effects, and may contribute to estrogen-linked diseases, including breast cancer.

Article	Year
Estradiol-activated estrogen receptor α does not regulate mature microRNAs in T47D breast cancer cells. The Journal of steroid biochemistry and molecular biology\| PubMed ID: 22079223	2012
MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer. Carcinogenesis\| PubMed ID: 22562546	2012
ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR. Breast cancer research : BCR\| PubMed ID: 23158001	2012
Studies in experimental autoimmune encephalomyelitis do not support developmental bisphenol a exposure as an environmental factor in increasing multiple sclerosis risk. Toxicological sciences : an official journal of the Society of Toxicology\| PubMed ID: 23798566	2013
Support of a bi-faceted role of estrogen receptor β (ERβ) in ERα-positive breast cancer cells. Endocrine-related cancer\| PubMed ID: 24192230	2014
Co-exposure to Phytoestrogens and Bisphenol A mimic estrogenic effects in an additive manner. Toxicological sciences : an official journal of the Society of Toxicology\| PubMed ID: 24284790	2013

Anne Katchy

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