Hormone-responsive Human Breast Cancer Cells Research Articles

Identification of Antiestrogen-Bound Estrogen Receptor α Interactomes in Hormone-Responsive Human Breast Cancer Cell Nuclei.

Estrogen receptor alpha (ERα) is a ligand-inducible transcription factor which mediates estrogen actions in hormone-responsive tumors and is targeted by effective anticancer therapies based on the ERα antagonist ligands, selective estrogen receptor modulators (such as Tamoxifen/TAM) or disruptors (such as Fulvestrant/ICI). Despite its importance for cancer therapy, including acquired resistance to endocrine therapy, the molecular basis of ERα response to different ligands is not fully known to date. Interaction proteomics shows great potential to identify and characterize molecular mechanisms of disease based on physical and functional protein-protein interaction networks. Tandem affinity purification coupled to mass spectrometry is applied here for mapping in hormone-responsive breast cancer cells nuclei, the ERα interactomes, induced by each of the two classes of antiestrogens. The results provide new insights on the molecular bases for antiestrogen-mediated control of ERα function and reveal new potential ways to overcome endocrine therapy resistance in cancer.

Natural and synthetic progestins enrich cancer stem cell-like cells in hormone-responsive human breast cancer cell populations in vitro.

Clinical trials and studies have shown that combination estrogen/progestin hormone replacement therapy, but not estrogen therapy alone or placebo, increases breast cancer risk in postmenopausal women. Using animal models, we have previously shown that both natural and synthetic progestins (including medroxyprogesterone acetate [MPA], a synthetic progestin used widely in the clinical setting) accelerate the development of breast tumors in vivo and increase their metastasis to lymph nodes. Based on these observations, we have hypothesized that progestin-induced breast cancer tumor growth and metastasis may be mediated by an enrichment of the cancer stem cell (CSC) pool. In this study, we used T47-D and BT-474 hormone-responsive human breast cancer cells to examine the effects of progestin on phenotypic and functional markers of CSCs in vitro. Both natural and synthetic progestins (10 nM) significantly increased protein expression of CD44, an important CSC marker in tumor cells. MPA increased the levels of both CD44 variants v3 and v6 associated with stem cell functions. This induction of CD44 was blocked by the antiprogestin RU-486, suggesting that this process is progesterone receptor (PR) dependent. CD44 induction was chiefly progestin dependent. Because RU-486 can bind other steroid receptors, we treated PR-negative T47-DCO-Y cells with MPA and found that MPA failed to induce CD44 protein expression, confirming that PR is essential for progestin-mediated CD44 induction in T47-D cells. Further, MPA treatment of T47-D cells significantly increased the activity of aldehyde dehydrogenase (ALDH), another CSC marker. Finally, two synthetic progestins, MPA and norethindrone, significantly increased the ability of T47-D cells to form mammospheres, suggesting that enrichment of the CD44high, ALDHbright subpopulation of cancer cells induced by MPA exposure is of functional significance. Based on our observations, we contend that exposure of breast cancer cells to synthetic progestins leads to an enrichment of the CSC pool, supporting the development of progestin-accelerated tumors in vivo.

Biomarker or Pathophysiology? The Role of DNA Methylation in Postpartum Depression

EpigenomicsVol. 5, No. 5 EditorialBiomarker or pathophysiology? The role of DNA methylation in postpartum depressionMary Kimmel, Zachary Kaminsky & Jennifer L PayneMary KimmelJohns Hopkins School of Medicine, Department of Psychiatry, 550 N Broadway, Suite 305, Baltimore, MD 21205, USASearch for more papers by this author, Zachary KaminskyJohns Hopkins School of Medicine, Department of Psychiatry, 550 N Broadway, Suite 305, Baltimore, MD 21205, USASearch for more papers by this author & Jennifer L PayneJohns Hopkins School of Medicine, Department of Psychiatry, 550 N Broadway, Suite 305, Baltimore, MD 21205, USA. Search for more papers by this authorEmail the corresponding author at Jpayne5@jhmi.eduPublished Online:24 Sep 2013https://doi.org/10.2217/epi.13.51AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: biomarkerdepressionDNA methylationestrogenpostpartumReferences1 Campbell SB, Cohn JF. Prevalence and correlates of postpartum depression in first-time mothers. J. Abnorm. Psychol.100(4),594–599 (1992).Crossref, Google Scholar2 O’Hara MW. Postpartum depression: what we know. J. Clin. Psychol.65(12),1258–1269 (2009).Crossref, Medline, Google Scholar3 Dietz PM, Williams SB, Callaghan WM, Bachman DJ, Whitlock EP, Hornbrook MC. Clinically identified maternal depression before, during, and after pregnancies ending in live births. Am. J. Psychiatry164(10),1515–1520 (2007).Crossref, Medline, Google Scholar4 Yonkers KA, Ramin SM, Rush AJ et al. Onset and persistence of postpartum depression in an inner-city maternal health clinic system. Am. J. Psychiatry158(11),1856–1863 (2001).Crossref, Medline, CAS, Google Scholar5 Payne JL, Roy PS, Murphy-Eberenz K et al. Reproductive cycle-associated mood symptoms in women with major depression and bipolar I disorder. J. Affect. Disord.99,221–229 (2007).Crossref, Medline, Google Scholar6 Viguera AC, Tondo L, Koukopoulos AE, Reginaldi D, Lepri B, Baldessarini RJ. Episodes of mood disorders in 2,252 pregnancies and postpartum periods. Am. J. Psychiatry168(11),1179–85 (2011).Crossref, Medline, Google Scholar7 Forty L, Jones L, Macgregor S et al. Familiality of postpartum depression in unipolar disorder: results of a family study. Am. J. Psychiatry163(9),1549–53 (2006).Crossref, Medline, Google Scholar8 Murphy-Eberenz K, Zandi PP, March D et al. Is perinatal depression familial? J. Affect. Disord.90(1),49–55 (2006).Crossref, Medline, Google Scholar9 Payne JL, MacKinnon DF, Mondimore FM et al. Familial aggregation of postpartum mood symptoms in families with bipolar disorder. J. Bipolar Disord.10(1),38–44 (2008).Crossref, Medline, Google Scholar10 Mahon PB, Payne JL, MacKinnon DF et al. Genome-wide linkage and follow-up association study of postpartum mood symptoms. Am. J. Psychiatry166(11),1229–1237 (2009).Crossref, Medline, Google Scholar11 Bloch M, Schmidt PJ, Danaceau M, Murphy J, Nieman L, Rubinow DR. Effects of gonadal steroids in women with a history of postpartum depression. Am. J. Psychiatry157(6),924–930 (2000).Crossref, Medline, CAS, Google Scholar12 Guintivano J, Arad M, Gould TD, Payne JL, Kaminsky ZA. Antenatal prediction of postpartum depression with blood DNA methylation biomarkers. Mol. Psychiatry doi:10.1038/mp.2013.62 (2013) (Epub ahead of print).Medline, Google Scholar13 Nassa G, Tarallo R, Ambrosino C et al. A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei. Proteomics11,159–165 (2011).Crossref, Medline, CAS, Google Scholar14 Cao S, Iyer JK, Lin V. Identification of tetratricopeptide repeat domain 9, a hormonally regulated protein. Biochem. Biophys. Res. Commun.345,310–317 (2006).Crossref, Medline, CAS, Google Scholar15 Schmidt MV, Trümbach D, Weber P et al. Individual stress vulnerability is predicted by short-term memory and AMPA receptor subunit ratio in the hippocampus. J. Neurosci.30(50),16949–16958 (2010).Crossref, Medline, CAS, Google Scholar16 Dennis CL, Dowswell T. Psychosocial and psychological interventions for preventing postpartum depression. Cochrane Database Syst. Rev.2,CD001134 (2013).Medline, Google ScholarFiguresReferencesRelatedDetailsCited ByGenetics of perinatal obsessive–compulsive disorderProtocol for Pilot Study on Self-Management of Depressive Symptoms in Pregnancy2 August 2019 | Nursing Research, Vol. 69, No. 1Neuroactive Steroids and Perinatal Depression: a Review of Recent Literature9 August 2018 | Current Psychiatry Reports, Vol. 20, No. 9DNA methylation: conducting the orchestra from exposure to phenotype?6 September 2016 | Clinical Epigenetics, Vol. 8, No. 1Bidirectional psychoneuroimmune interactions in the early postpartum period influence risk of postpartum depressionBrain, Behavior, and Immunity, Vol. 49 Vol. 5, No. 5 Follow us on social media for the latest updates Metrics Downloaded 188 times History Published online 24 September 2013 Published in print October 2013 Information© Future Medicine LtdKeywordsbiomarkerdepressionDNA methylationestrogenpostpartumFinancial & competing interests disclosureThis work was funded in part by a NARSAD 2010 Young Investigator Award to Z Kaminsky and K23 MH074799-01A2 to JL Payne. Z Kaminsky and JL Payne are coinventors listed on a patent application for DNA methylation at biomarker loci related to postpartum depression. In the past year, JL Payne has conducted consulting work for AstraZeneca and Johnson & Johnson. JL Payne also has a grant from Corcept Pharmaceuticals. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

Identification of a Hormone-regulated Dynamic Nuclear Actin Network Associated with Estrogen Receptor α in Human Breast Cancer Cell Nuclei

Estrogen receptor alpha (ERalpha) is a modular protein of the steroid/nuclear receptor family of transcriptional regulators that upon binding to the hormone undergoes structural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ERalpha assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, leading to dynamic and coordinated regulation of hormone-responsive genes. Identification of the molecular partners of ERalpha and understanding their combinatory interactions within functional complexes is a prerequisite to define the molecular basis of estrogen control of cell functions. To this end, affinity purification was applied to map and characterize the ERalpha interactome in hormone-responsive human breast cancer cell nuclei. MCF-7 cell clones expressing human ERalpha fused to a tandem affinity purification tag were generated and used to purify native nuclear ER-containing complexes by IgG-Sepharose affinity chromatography and glycerol gradient centrifugation. Purified complexes were analyzed by two-dimensional DIGE and mass spectrometry, leading to the identification of a ligand-dependent multiprotein complex comprising beta-actin, myosins, and several proteins involved in actin filament organization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chromatin dynamics, and ribosome biogenesis. Time course analyses indicated that complexes containing ERalpha and actin are assembled in the nucleus early after receptor activation by ligands, and gene knockdown experiments showed that gelsolin and the nuclear isoform of myosin 1c are key determinants for assembly and/or stability of these complexes. Based on these results, we propose that the actin network plays a role in nuclear ERalpha actions in breast cancer cells, including coordinated regulation of target gene activity, spatial and functional reorganization of chromatin, and ribosome biogenesis.

Time-course analysis of genome-wide gene expression data from hormone-responsive human breast cancer cells.

BackgroundMicroarray experiments enable simultaneous measurement of the expression levels of virtually all transcripts present in cells, thereby providing a ‘molecular picture’ of the cell state. On the other hand, the genomic responses to a pharmacological or hormonal stimulus are dynamic molecular processes, where time influences gene activity and expression. The potential use of the statistical analysis of microarray data in time series has not been fully exploited so far, due to the fact that only few methods are available which take into proper account temporal relationships between samples.ResultsWe compared here four different methods to analyze data derived from a time course mRNA expression profiling experiment which consisted in the study of the effects of estrogen on hormone-responsive human breast cancer cells. Gene expression was monitored with the innovative Illumina BeadArray platform, which includes an average of 30-40 replicates for each probe sequence randomly distributed on the chip surface. We present and discuss the results obtained by applying to these datasets different statistical methods for serial gene expression analysis. The influence of the normalization algorithm applied on data and of different parameter or threshold choices for the selection of differentially expressed transcripts has also been evaluated. In most cases, the selection was found fairly robust with respect to changes in parameters and type of normalization. We then identified which genes showed an expression profile significantly affected by the hormonal treatment over time. The final list of differentially expressed genes underwent cluster analysis of functional type, to identify groups of genes with similar regulation dynamics.ConclusionsSeveral methods for processing time series gene expression data are presented, including evaluation of benefits and drawbacks of the different methods applied. The resulting protocol for data analysis was applied to characterization of the gene expression changes induced by estrogen in human breast cancer ZR-75.1 cells over an entire cell cycle.

Glycolysis and glucose transporter 1 as markers of response to hormonal therapy in breast cancer.

Estrogen plays a key role in the development and progression of breast cancer; hence, antiestrogens, such as tamoxifen, have a marked impact on the treatment and outcome of breast cancer patients. Estrogen-induced growth requires continuous replenishment of energy, predominantly generated by glycolysis. Previous work from this laboratory demonstrated estrogen induction and tamoxifen inhibition of glycolysis in MCF7 human breast cancer cells in vitro (Furman et al., J Steroid Biochem Mol Biol 1992;43:189-95). We present here studies of estrogen vs. tamoxifen regulation of glycolysis in orthotopic MCF7 human breast cancer xenografts in vivo. In addition we investigated mediation of this metabolic regulation through glucose transporter 1, in the same cells, in vitro, as well as in 2 other hormone-responsive human breast cancer cells. Tumor response and glycolysis were monitored noninvasively by means of magnetic resonance imaging and 13C spectroscopy, respectively. During estrogen-stimulated tumor growth (from approximately 0.5 to approximately 1.3 cm3 in 10 days), the rate of glucose metabolism through glycolysis in vivo was high at 40 +/- 4 micromole/g/min. However, treatment for 10 days with tamoxifen induced growth arrest and a concomitant decrease of 2-fold in the rate of glycolysis. In congruence, glucose transporter 1 expression was stimulated by estrogen, reaching after 72 hr a 2- to 3-fold higher level of expression relative to that in tamoxifen-treated cells. Thus, estrogen-induced changes in glycolysis appeared to be mediated via its regulation of glucose transporter 1 expression. The in vivo monitoring of glycolysis may serve as a tool to expose hormonal regulation of glucose transporter 1 expression in breast cancer tumors, as well as to assess response to hormonal therapy.

Effect of Estradiol on Histone Acetylation Dynamics in Human Breast Cancer Cells

Histone acetylation plays an important role in remodeling chromatin structure, facilitating nuclear processes such as transcription. We investigated the effect of estradiol on global histone acetylation in hormone-responsive human breast cancer cells. Pulse-chase experiments and immunoblot analyses of dynamically acetylated histones show that estradiol rapidly increases histone acetylation in estrogen receptor (ER)-positive, hormone-dependent T5, but not in ER-negative, hormone-independent MDA MB 231 breast cancer cells. The effect of estradiol on the rates of histone acetylation and deacetylation in T5 cells was determined. We found that estradiol increased the level of acetylated histones by reducing the rate of histone deacetylation, whereas the rate of histone acetylation was not altered. Enzymatic assays and immunoblot analyses of cell fractions showed that estradiol did not affect the level, subnuclear distribution, or activity of class I and II histone deacetylases. However, estradiol did alter the intranuclear distribution of ER and histone acetyltransferases, with both becoming tightly bound in the nucleus and associated with the nuclear matrix. We propose that, following the association of ER with nuclear matrix sites, ER alters the balance of histone acetyltransferases and histone deacetylases at these sites and the dynamics of acetylation of histones associated with transcriptionally active and competent chromatin.

Gene expression monitoring in hormone-responsive human breast cancer cells during estrogen-induced cell cycle progression

Gene expression monitoring in hormone-responsive human breast cancer cells during estrogen-induced cell cycle progression

Estrogen induction of TGF-alpha is mediated by an estrogen response element composed of two imperfect palindromes.

To investigate the molecular mechanisms underlying the two- to three-fold induction of human transforming growth factor-alpha (hTGF-alpha) mRNA and two- to five-fold induction of hTGF-alpha protein observed following estrogen treatment of hormone-responsive human breast cancer cell lines, the hTGF-alpha promoter was assayed for ERE-like sequences able to mediate estrogen induction of a heterologous gene. Transient co-transfection of a chloramphenicol acetyl transferase (CAT) construct consisting of either 1100 bp or 330 bp of hTGF-alpha promoter sequence and an estrogen receptor expression vector into either COS-7 cells or hormonally responsive MCF-7 human breast cancer cells resulted in a two- to five-fold induction of CAT activity by estrogen. Although no consensus estrogen response element (ERE) exists in the hTGF-alpha promoter, a sequence consisting of two imperfect ERE palindromes separated by 20 bp is located at -200 to -252. This sequence was inserted into a mouse mammary tumor virus (MMTV) based CAT construct and assayed for its ability to confer estrogen regulation of CAT expression to a heterologous promoter. Transient co-transfection of this construct with an estrogen receptor expression vector into either COS-7 cells or MCF-7 cells resulted in an average 30-fold estrogen induction of CAT activity. Gel shift assays with human recombinant estrogen receptor (ER) and 32P-labelled fragments revealed that the ER could specifically bind to this sequence. These results indicate that this 53 bp sequence can function as an ERE, and is likely to be responsible for the observed induction of TGF-alpha message and protein in response to estrogen. These data also indicate that the level of estrogen inducibility mediated by this element may be positively or negatively modulated by interaction or competition with other transcription factors.

Transcriptional regulation of c-myc oncogene expression by estrogen in hormone-responsive human breast cancer cells.

Enhanced c-myc oncogene expression associated with peptide mitogen-stimulated cell growth is primarily a result of a post-transcriptional event involving increased mRNA stability. We have recently shown that estradiol stimulates c-myc expression in estrogen receptor-positive human breast cancer cells. In this report, we show that in estrogen-responsive MCF-7 cells, estradiol stimulated the c-myc gene exclusively at the transcriptional level, increasing c-myc mRNA transcription more than 10-fold within 20 min, while having no effect on the c-myc mRNA half-life of 18 min. In addition, pretreatment of the cells with cycloheximide did not prevent induction of the c-myc oncogene, indicating a primary effect of estrogen. Furthermore, the elevated level of c-myc mRNA in estrogen-independent MDA-MB-231 cells was due primarily to a more stable c-myc mRNA with a half-life of 49 min, in the absence of enhanced transcription. These results indicate that different mechanisms of regulation of c-myc oncogene expression exist in hormone-dependent and -independent human breast cancer cells.