A Rapid, Extensive, and Transient Transcriptional Response to Estrogen Signaling in Breast Cancer Cells

A Rapid, Extensive, and Transient Transcriptional Response to Estrogen Signaling in Breast Cancer Cells

Highlights from Recent Cancer Literature

Rottlerin induces Wnt co-receptor LRP6 degradation and suppresses both Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells

Hormone-regulated transcriptomes: Lessons learned from estrogen signaling pathways in breast cancer cells

Abstract #18 The role of endogenous estrogens in breast cancer etiology has been well established. Dysregulated estrogen signaling increases the rate of cell proliferation and thus the risk of breast cancer. However, despite widespread agreement that estrogens are involved in breast cancer etiology, the molecular mechanisms of estrogen action, especially those by which estrogen signaling stimulates mammary tumorigenesis and breast cancer progression have not been well established.
 Recently, we have identified and cloned a 36-kDa novel isoform of estrogen receptor α (ER-α36) that is generated from a promoter located in the first intron of the original 66-kDa ER-α (ER-α66) gene. The ER-α36 differs from the ER-α66 by lacking both transcriptional activation domains (AF-1 and AF-2) but retains the DNA-binding, dimerization and most of the ligand-binding domains. ER-α36 is mainly expressed on the plasma membrane and mediates membrane-initiated estrogen signaling that activates the MAPK/ERK pathway and stimulates cell proliferation. ER-α36 inhibits the genomic estrogen signaling mediated by the AF1 and AF2 domains of ER-α66 and ligand-dependent and -independent transactivation activities of ER-β. We designed and constructed shRNA expression vectors to express shRNAs specific for ER-α36 or ER-α66. We found that ER-positive breast cancer cells MCF7 and T47D expressing ER-α36 shRNA that successfully knockdown about 90% ER-α36 expression failed to respond to estrogen stimulation. However, estrogen strongly stimulated cell growth in MCF7 and T47D cells with ER-α66 knockdown. We found that some estrogen responsive genes such as c-Myc, CyclinD1 and progesterone receptor (PR) were induced through the extra-nuclear estrogen signaling pathway while pS2 was induced by the nuclear pathway. Our results for the first time demonstrated that ER-α36, not the original ER-α36 is the estrogen receptor that actually mediates estrogen-stimulated cell proliferation in breast cancer cells.
 Among 800 cases of human breast cancer specimens examined, ER-α36 is expressed in most of the breast cancer specimens, and even in 40% of ER-negative breast cancer cases that lack expression of ER-α66. In addition, ER-α36 expressing patients responded poorly to anti-estrogen Tamoxifen treatment. In vitro study revealed that breast cancer cells that highly express ER-α36 exhibited estrogen hypersensitivity and Tamoxifen resistance, consistent with the clinical evidence that breast cancers expressing ER-α36 exhibit higher degree of malignancy and poorer survival rate.
 Taken together, our results demonstrated that ER-α36 is a novel player in mitogenic estrogen signaling that plays an important role in estrogen stimulated mammary carcinogenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 18.

The pioneer transcription factor FOXA1 is a critical determinant for estrogen receptor (ER) function in hormone-dependent breast cancers. Upon estrogen stimulation, liganded ER binds to poised enhancer regions across the genome that are demarcated by FOXA1 and histone modifications such as H3K4me1 and H3K27ac. In a recent publication, we show that proinflammatory signaling, caused by the cytokine TNFa, drives FOXA1 to latent enhancer binding sites to promote chromatin accessibility for subsequent ER binding upon estrogen ligation. These latent enhancers, activated by the combined treatment of estrogen and TNFa, induced the expression of a unique transcriptome with clinical significance. The effects of TNFa treatment on FOXA1 chromatin redistribution and subsequent gene expression occur within 40 minutes, which points to a rapid signaling cascade that culminates in either changes in FOXA1's posttranslational modifications (PTMs) or its binding partners. To understand how proinflammatory TNFa signaling can redirect FOXA1 to new sites across the genome, we started by characterizing the posttranslational modifications (PTMs) of FOXA1. We immunoprecipitated FOXA1 from MCF-7 breast cancer cells that were treated by E2, TNFa or E2+ TNFa, and then examined their posttranslational status using semi-quantitative and quantitative mass spectrometry approaches. Several phosphorylation sites and acetylation sites have been identified near the DNA binding domain of FOXA1, and acetylation of lysine 295 (K295) was found specifically enriched in TNFa treatment. To test if acetylation of FOXA1 at K295 changes its binding preference and genomic distribution, we used the programmable properties of CRISPR/Cas9 to create specific knockin mutations to mimic or prevent acetylation of K295 in MCF-7 cells. More specifically, we mutated K295 to glutamine (K295Q) to mimic acetylation and essentially “lock” FOXA1 into a permanently acetylated state and, for comparison, we created another cell line where K295 was mutated to arginine (K295R) to prevent acetylation of FOXA1. Our preliminary data shows changes in the genomic redistribution of FOXA1 in the knock-in cell lines resulting in altered gene expression programs. These data suggest that inflammation-based acetylation of FOXA1 can affect estrogen signaling pathways in breast cancer cells by altering the enhancer landscape of FOXA1 and consequently the estrogen receptor. Supported by a grant from the NIH/NCI (R00 CA204628) to H.L.F Citation Format: Shen Li, Raul Mendez-Giraldez, Joseph P. Garay, Kamila Wisniewska, Colby A. Tubbs, Charles M. Perou, Hector L. Franco. Cytokine-induced post-translational modifications of FOXA1 affect enhancer selection and estrogen signaling in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4496.

<div>Abstract<p>Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and <i>in vivo</i> in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells.</p>Significance:<p>Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells.</p></div>

<div>Abstract<p>Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and <i>in vivo</i> in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells.</p>Significance:<p>Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells.</p></div>

While exposure to estrogens is a major risk factor of breast and endometrial cancer, it well established that estrogens are beneficial for bone health. We have previously shown that carotenoids inhibit estrogen signaling in breast and endometrial cancer cells. The aim of this study was to compare the effects of various phytonutrients, (carotenoid derivatives, polyphenols, isothiocyanates) on estrogenic activity in breast cancer cells and osteoblast-like cells. All the tested phytonutrients inhibited estrogen response element (ERE) transactivation in breast cancer cells. In contrast, these compounds either did not affect or enhanced ERE activity and the expression of several bone-forming genes. These results were obtained using two osteoblast-like cell lines, MG-63 human osteosarcoma cells stably transfected with estrogen receptor-α (ERα) and MC3T3-E1 mouse calvaria-derived cells expressing endogenous ER. Phytonutrients-induced ERE inhibition in breast cancer cells, and its potentiation in osteoblast-like cells were associated both with a decrease and a rise in total and nuclear ERα levels, respectively. Phytonutrients activated the electrophile/antioxidant response element (EpRE/ARE) transcription system to various extents in both cancer and bone cell lines. Overexpression of Nrf2, the major EpRE/ARE activating transcription factor, mimicked the effects of phytonutrients, causing inhibition and enhancement of ERE transactivation in breast cancer cells and in osteoblast-like cells, respectively. Moreover, reduction in Nrf2 levels by RNAi led to a decrease in the phytonutrient potentiation of ERE activity transactivation in osteoblast-like cells. These findings suggest that the enhancement and inhibition of estrogen signaling by phytonutrients in bone-derived cells and breast cancer cells, respectively, is partially mediated by the activation of the Nrf2/ARE system.

Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

: The aim of this project is preclinical testing of a novel Ras antagonist, farnesylthiosalicylate (FTS in complex with a cyclodextrin (CD) carrier. Ras is involved in estrogen and growth factor signaling in breast cancer cells. inhibition of Ras should prevent growth of breast cancer cells. We have tested FTS-CD against several breast cancer cell lines in vitro and found that: * FTS-CD is effective against estrogen receptor positive breast cancer in vitro. * FTS-CD is effective against Tamoxifen resistant cells in vitro. * FTS-CD interferes with estrogen signaling. * FTS-CD induces profound changes in cell signaling, manifested as increase in apoptosis and decrease in proliferation.

Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells. SIGNIFICANCE: Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1234/F1.large.jpg.

Williams syndrome transcription factor (WSTF) acts as an activator of estrogen receptor signaling in breast cancer cells and the effect can be abrogated by 1α,25-dihydroxyvitamin D3

We previously reported that quinacrine (QC) has anticancer activity against breast cancer cells. Here, we report the mechanism of action of QC and its ability to inhibit Wnt-TCF signaling in two independent breast cancer cell lines. QC altered Wnt-TCF signaling components by increasing the levels of adenomatous polyposis coli (APC), DAB2, GSK-3β and axin and decreasing the levels of β-catenin, p-GSK3β (ser 9) and CK1. QC also reduced the activity of the Wnt transcription factor TCF/LEF and its downstream targets cyclin D1 and c-MYC. Using a luciferase-based Wnt-TCF transcription factor assay, it was shown that APC levels were inversely associated with TCF/LEF activity. Induction of apoptosis and DNA damage was observed after treatment with QC, which was associated with increased expression of APC. The effects induced by QC depend on APC because the inhibition of Wnt-TCF signalling by QC is lost in APC-knockdown cells, and consequently, the extent of apoptosis and DNA damage caused by QC was reduced compared with parental cells. Further, previously we have shown that QC inhibits topoisomerase, therefore we tested the effect of another topoisomerase inhibitor, etoposide, on Wnt signaling. Interestingly, etoposide treatment also reduced TCF/LEF activity, β-catenin and cyclin D1 levels commensurate with induction of DNA damage and apoptosis. Lycopene, a plant-derived antioxidant, synergistically increased QC activity and inhibited Wnt-TCF signaling in cancer cells without affecting the MCF-10A normal breast cell line. Collectively, the data suggests that QC-mediated Wnt-TCF signal inhibition depends on APC and that the addition of lycopene synergistically increases QC anticancer activity. Note: This abstract was not presented at the meeting. Citation Format: Ranjan Preet, Rajasubramaniam Shanmugam, Purusottam Mohapatra, Dipon Das, Shakti R. Satapathy, Michael D. Wyatt, Chanakya N. Kundu. Lycopene synergistically enhances quinacrine action to inhibit Wnt-TCF signaling in breast cancer cells through APC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2777. doi:10.1158/1538-7445.AM2014-2777

Mechanisms for cancer-related inflammation remain to be fully elucidated. Non-apoptotic functions of Fas signaling have been proposed to play an important role in promoting tumor progression. It has yet to be determined if targeting Fas signaling can control tumor progression through suppression of cancer-related inflammation. In the current study we found that breast cancer cells with constitutive Fas expression were resistant to apoptosis induction by agonistic anti-Fas antibody (Jo2) ligation or Fas ligand cross-linking. Higher expression of Fas in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. To determine whether blockade of Fas signaling in breast cancer could suppress tumor progression, we prepared an orthotopic xenograft mouse model with mammary cancer cells 4T1 and found that blockade of Fas signaling in 4T1 cancer cells markedly reduced tumor growth, inhibited tumor metastasis in vivo, and prolonged survival of tumor-bearing mice. Mechanistically, blockade of Fas signaling in cancer cells significantly decreased systemic or local recruitment of myeloid derived suppressor cells (MDSCs) in vivo. Furthermore, blockade of Fas signaling markedly reduced IL-6, prostaglandin E2 production from breast cancer cells by impairing p-p38, and activity of the NFκB pathway. In addition, administration of a COX-2 inhibitor and anti-IL-6 antibody significantly reduced MDSC accumulation in vivo. Therefore, blockade of Fas signaling can suppress breast cancer progression by inhibiting proinflammatory cytokine production and MDSC accumulation, indicating that Fas signaling-initiated cancer-related inflammation in breast cancer cells may be a potential target for treatment of breast cancer.