LB-OR-6 Erα Regulates the Expression of H19, a Long Non-Coding RNA, in Normal Human Luminal and Malignant Breast Epithelial Cells

Program: Late-Breaking Abstracts
Session: LB-OR-Late-Breaking Oral Session
Basic/Clinical
Saturday, June 15, 2013: 11:30 AM-1:00 PM
Presentation Start Time: 12:45 PM
Room 122 (Moscone Center)
Pratima Basak1, Sumanta Chatterjee1, Steve Weger1, Ketan Badiani2, James R Davie1, Leigh C Murphy3 and Afshin Raouf*1
1University of Manitoba, Winnipeg, MB, Canada, 2CancerCare Manitoba, Winnipeg, MB, Canada, 3University of Manitoba
The developmentally regulated and imprinted gene, H19,  is a long non-coding RNA that is expressed exclusively from the maternal allele. High expression of H19 gene in many malignant tumors including breast tumors has been reported. While previous observations have suggested that estrogen signaling enhances the expression of H19 gene in breast cancer, we and others have found that in normal human and mouse mammary glands H19 is expressed predominantly in the Estrogen Receptor alpha (ERα) negative, basal epithelial cells. In this study, we examined if estrogen regulation of H19 gene is specfic to ERα  positive breast cancer cells. To this end, we isolated purified ERα positive luminal progenitors and ERα negative, undifferentiation bipotential progenitors from normal human breast reduction samples and placed them in matrigel cultures under estrogen-depleted growth conditions. Using quantitative PCR, we observed that the progeny of the bipotential progenitors exhibited the highest expression of the H19 gene compared to the progeny of the luminal progenitors. However, addition of 17-beta estradiol to the progeny of the uminal progenitors, increased the expression of H19 gene by 4.5 fold (p = 0.04). This observation suggests that estrogen regulation of H19 gene expression is not unique to ERα  positive breast cancer cells and that H19 may have key roles in regulating the normal proliferation and differentiation of ERα positive luminal progenitors. Using ERa  positive breast cancer cells we have further determined that estrogen treatment at 10nM requires at least 10 hr to significantly increase H19 expression (2 fold, p=0.002) which reaches its apex after 24hr (4.5 fold p=0.02). Interestingly, H19 expression subsides after 48hr of exposure to estrogen. Using gain of function studies and loss of function studies, as well as specific ERα or ERβ agonists, we demonstrate that ERα alone regulates the expression of H19. Moreover, using the transcriptional inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide we found that estrogen-induced up regulation of H19 requires activation of transcription but not de novo protein synthesis, suggesting that ERα directly activates the transcription of H19 gene. Using chromatin immunoprecipitation technique we have identified 5 ER response-element half-sites within the H19 promoter to which ERα is bound after estrogen treatment. Our data therefore, suggest that estrogen signaling through the ERα, up regulates H19 gene expression in both normal and malignant human breast epithelial cells and that H19 may play a role in regulating the normal proliferation and differentiation of luminal progenitors. Understanding the molecular mechanisms that underlie estrogen’s regulation of proliferation in both normal and malignant ERα positive breast cells may lead to the identification of early diagnostic markers to detect breast cancers at an early stage.

Nothing to Disclose: PB, SC, SW, KB, JRD, LCM, AR

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: This work was supported by a generous grant support from the CancerCare Manitoba, the Manitoba Health Research Council, and the University of Manitoba tom AR.
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