ERα-positive Breast Cancer Cell Lines Research Articles

Variation in IL6ST cytokine family function and the potential of IL6 trans-signalling in ERα positive breast cancer cells

High expression of the transmembrane receptor IL6ST (gp130) has been identified as a predictive biomarker of endocrine treatment response in ERα-positive breast cancers. To investigate its function further in this disease, this study evaluated the expression, function and signalling of IL6ST in ERα-positive breast cancer cell lines and investigated crosstalk between ERα and IL6ST. IL6ST was differentially expressed in ERα-positive breast cancer cell lines (low in MCF-7, high in ZR751 and T47D), while multiple soluble isoforms of IL6ST were identified. IL6ST is the common signal transducing receptor component for the IL6ST family of cytokines and the effects of seven IL6ST cytokines on these cell lines were studied. These cytokines caused differential growth and migration effects in these cell lines e.g. MCF-7 cells were growth-stimulated, while ZR751 cells were inhibited by IL6 and OSM.. Activation of the STAT and ERK pathways is associated with these responses. Evidence to support trans-signalling involved in cell growth and migration was obtained in both MCF-7 and ZR751 models. Interaction between cytokines and estrogen on ERα-positive cell lines growth were analysed. High expression of IL6ST (in ZR751) may lead to growth inhibition by interacting cytokines while lower expression (in MCF-7) appears associated with proliferation. High IL6ST expression is consistent with a more beneficial clinical outcome if cytokine action contributes to anti-estrogen action.

Super-enhancer-controlled positive feedback loop BRD4/ERα-RET-ERα promotes ERα-positive breast cancer.

Estrogen and estrogen receptor alpha (ERα)-induced gene transcription is tightly associated with ERα-positive breast carcinogenesis. ERα-occupied enhancers, particularly super-enhancers, have been suggested to play a vital role in regulating such transcriptional events. However, the landscape of ERα-occupied super-enhancers (ERSEs) as well as key ERα-induced target genes associated with ERSEs remain to be fully characterized. Here, we defined the landscape of ERSEs in ERα-positive breast cancer cell lines, and demonstrated that bromodomain protein BRD4 is a master regulator of the transcriptional activation of ERSEs and cognate ERα target genes. RET, a member of the tyrosine protein kinase family of proteins, was identified to be a key ERα target gene of BRD4-regulated ERSEs, which, in turn, is vital for ERα-induced gene transcriptional activation and malignant phenotypes through activating the RAS/RAF/MEK2/ERK/p90RSK/ERα phosphorylation cascade. Combination therapy with BRD4 and RET inhibitors exhibited additive effects on suppressing ERα-positive breast cancer both in vitro and in vivo, comparable with that of standard endocrine therapy tamoxifen. Furthermore, combination therapy re-sensitized a tamoxifen-resistant ERα-positive breast cancer cell line to tamoxifen treatment. Taken together, our data uncovered the critical role of a super-enhancer-associated positive feedback loop constituting BRD4/ERα–RET–ERα in ERα-positive breast cancer, and suggested that targeting components in this loop would provide a new therapeutic avenue for treating ERα-positive breast cancer in the clinic.

Modification of ERα by UFM1 Increases Its Stability and Transactivity for Breast Cancer Development.

The post-translational modification (e.g., phosphorylation) of estrogen receptor α (ERα) plays a role in controlling the expression and subcellular localization of ERα as well as its sensitivity to hormone response. Here, we show that ERα is also modified by UFM1 and this modification (ufmylation) plays a crucial role in promoting the stability and transactivity of ERα, which in turn promotes breast cancer development. The elevation of ufmylation via the knockdown of UFSP2 (the UFM1-deconjugating enzyme in humans) dramatically increases ERα stability by inhibiting ubiquitination. In contrast, ERα stability is decreased by the prevention of ufmylation via the silencing of UBA5 (the UFM1-activating E1 enzyme). Lys171 and Lys180 of ERα were identified as the major UFM1 acceptor sites, and the replacement of both Lys residues by Arg (2KR mutation) markedly reduced ERα stability. Moreover, the 2KR mutation abrogated the 17β-estradiol-induced transactivity of ERα and the expression of its downstream target genes, including pS2, cyclin D1, and c-Myc; this indicates that ERα ufmylation is required for its transactivation function. In addition, the 2KR mutation prevented anchorage-independent colony formation by MCF7 cells. Most notably, the expression of UFM1 and its conjugating machinery (i.e., UBA5, UFC1, UFL1, and UFBP1) were dramatically upregulated in ERα-positive breast cancer cell lines and tissues. Collectively, these findings implicate a critical role attributed to ERα ufmylation in breast cancer development by ameliorating its stability and transactivity.

Heterogeneous Nuclear Ribonucleoprotein K Is Involved in the Estrogen-Signaling Pathway

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) has been found in the nucleus, cytoplasm, and mitochondria. It is implicated in chromatin remodeling, transcription, splicing, and translation processes. Although hnRNPK has reportedly been associated with poor prognosis in colon cancer patients, it is beneficial in gastric cancer as it inhibits cancer cell proliferation. Expression of hnRNPK in ER (Estrogen receptor) -positive/PR (Progesterone receptor) -positive breast cancer was higher than other subtypes; however, the biological functions of hnRNPK in the ER-mediated signaling pathway have not been identified. In this study, we investigated the functions of hnRNPK in the estrogen-signaling pathway. We initially evaluated hnRNPK expression upon treatment with estradiol (E2) and ICI 182,780 in ERα-positive breast cancer cell line MCF-7. This initial evaluation revealed that expression of hnRNPK was increased by E2 treatment but decreased by ICI 182,780 treatment. We further evaluated the effects of estrogen-signaling pathway in hnRNPK knockdown MCF-7 cells using siRNA, which revealed that hnRNPK knockdown decreased ERα expressions and ERα target gene TFF1 by E2 treatment. In addition, we examined the interaction between hnRNPK and ERα because hnRNPK has been reported to interact with several other proteins. These interactions were detected using immunoprecipitation and proximity ligation assay. We then immunolocalized hnRNPK in breast cancer and endometrial cancer. hnRNPK expression was significantly higher in ERα-positive cancer cells in both breast and endometrial cancers. In contrast, hnRNPK expression was significantly lower in Ki-67-positive breast cancer while being significantly higher in Ki-67-positive endometrial cancer. hnRNPK has been found to function differently, depending on the type of cancer (breast or endometrial) that it is expressed in. However, further studies are required to clarify the clinical significance of hnRNPK in breast and endometrial cancer patients.

Detection of Estrogen Receptor Alpha and Assessment of Fulvestrant Activity in MCF-7 Tumor Spheroids Using Microfluidics and SERS.

Breast cancer is one of the leading causes of cancer death in women. Novel in vitro tools that integrate three-dimensional (3D) tumor models with highly sensitive chemical reporters can provide useful information to aid biological characterization of cancer phenotype and understanding of drug activity. The combination of surface-enhanced Raman scattering (SERS) techniques with microfluidic technologies offers new opportunities for highly selective, specific, and multiplexed nanoparticle-based assays. Here, we explored the use of functionalized nanoparticles for the detection of estrogen receptor alpha (ERα) expression in a 3D tumor model, using the ERα-positive human breast cancer cell line MCF-7. This approach was used to compare targeted versus nontargeted nanoparticle interactions with the tumor model to better understand whether targeted nanotags are required to efficiently target ERα. Mixtures of targeted anti-ERα antibody-functionalized nanotags (ERα-AuNPs) and nontargeted (against ERα) anti-human epidermal growth factor receptor 2 (HER2) antibody-functionalized nanotags (HER2-AuNPs), with different Raman reporters with a similar SERS signal intensity, were incubated with MCF-7 spheroids in microfluidic devices and spectroscopically analyzed using SERS. MCF-7 cells express high levels of ERα and no detectable levels of HER2. 2D and 3D SERS measurements confirmed the strong targeting effect of ERα-AuNP nanotags to the MCF-7 spheroids in contrast to HER2-AuNPs (63% signal reduction). Moreover, 3D SERS measurements confirmed the differentiation between the targeted and the nontargeted nanotags. Finally, we demonstrated how nanotag uptake by MCF-7 spheroids was affected by the drug fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). These results illustrate the potential of using SERS and microfluidics as a powerful in vitro platform for the characterization of 3D tumor models and the investigation of SERD activity.

Carcinoma-Associated Fibroblasts Promote Growth of Sox2-Expressing Breast Cancer Cells.

Simple SummaryThe tumor microenvironment has a strong impact on the behavior of tumor cells. One major cell type residing in the tumor microenvironment is the carcinoma-associated fibroblast (CAF). We were interested in the effect of CAFs on Sox2 (sex determining region Y (SRY)-box 2), which not only is an essential embryonal stem cell transcription factor, but also plays a role in cancer stem cell activity. We found that long-term exposure of ERα-positive breast cancer cells to the cocktail of CAF-secreted factors strongly increased Sox2 expression involving tumor-related proteins and signaling pathways. However, Sox2 was not only present in those tumor cells that express stem cell markers, but was equally abundant in other tumor cells. By being widely expressed, Sox2 may have functions in non-stem cells. In fact, Sox2 was found to regulate ERα expression, to act anti-apoptotically, to promote cellular growth and to protect cells against the anti-estrogen fulvestrant.CAFs (Carcinoma-associated fibroblasts) play an important role in cancer progression. For instance, they promote resistance to anti-estrogens, such as fulvestrant. Here, we show that, in ERα-positive breast cancer cell lines, the cocktail of factors secreted by CAFs (CAF-CM) induce the expression of the embryonal stem cell transcription factor Sox2 (sex determining region Y (SRY)-box 2). Long-term exposure to CAF-CM was able to give rise to very high Sox2 levels both in the absence and presence of fulvestrant. IL-6 (interleukin-6), a major component of CAF-CM, failed to raise Sox2 expression. In MCF-7 sublines established in the presence of CAF-CM, almost all cells showed Sox2 expression, whereas long-term treatment of T47D cells with CAF-CM resulted in a ~60-fold increase in the proportions of two distinct populations of Sox2 high and low expresser cells. Exposure of BT474 cells to CAF-CM raised the fraction of Sox2 high expresser cells by ~3-fold. Cell sorting based on CD44 and CD24 expression or ALDH (aldehyde dehydrogenase) activity revealed that most Sox2 high expresser cells were not CD44hi/CD24lo- or ALDH-positive cells suggesting that they were not CSCs (cancer stem cells), though CD44 played a role in Sox2 expression. Functionally, Sox2 was found to protect CAF-CM-treated cells against apoptosis and to allow higher growth activity in the presence of fulvestrant. Mechanistically, the key drivers of Sox2 expression was found to be STAT3 (Signal transducer and activator of transcription 3), Bcl-3 (B-cell lymphoma 3) and the PI3K (Phosphoinositide 3-kinase)/AKT pathway, whose activities/expression can all be upregulated by CAF-CM. These data suggest that CAF-CM induces Sox2 expression in non-CSCs by activating proteins involved in growth control and drug resistance, leading to higher protection against apoptosis.

Abstract 5843: XBP1 regulates the expression of cell cycle associated genes in ERα positive breast cancer cells

Abstract Estrogen receptor α (ERα)-positive breast cancer is a heterogeneous disease with distinct molecular features. Over the time of treatment, endocrine resistance develops in these types of cancers which is major challenge in recent times and finding a better treatment options against these resistant cancers is urgent. Several studies have suggested the association of XBP1 in therapy resistance in ERα-positive breast cancers. Indeed, XBP1 physically interacts with ER and activates ER-dependent transcription in a ligand-independent manner. Moreover, overexpression of XBP1 in ERα-positive breast cancer cells confers estrogen independent growth and reduced sensitivity to anti-estrogen. Despite growing evidence about the involvement of XBP1 in ERα-positive breast cancer, molecular targets of XBP1 is poorly understood. The aim of current study is to identify the XBP1-regulated targets in ERα-positive breast cancer cells. XBP1 deficient sub-clones in MCF7 cells were generated by using CRISPR-CAS9 mediated gene knockout strategy. Cell viability and anti-estrogen sensitivity were determined by MTS assay, cell proliferation was evaluated by colony formation assay, cell death was determined by flow cytometry. XBP1-regulated genes were identified by analysing transcriptomic data from control and XBP1-knockdown T47D cells (GSE49955). Relative expression of genes were analysed by qPCR. RRM2 overexpression clones were generated by transfecting RRM2 vector using nucleofector. Deficiency of XBP1 in MCF7 cells showed decreased growth and proliferation as compared to control MCF7 cells. Deletion of XBP1 in MCF7 cells demonstrated compromised E2-mediated cell growth and reduced activation of E2-dependent genes. Moreover, XBP1 deficiency in MCF7 cells increases sensitivity towards anti-estrogens including tamoxifen and fulvestrant. Expression of cell cycle associated genes RRM2, CDC6 and TOP2A were found significantly reduced in all ERα-positive breast cancer cell lines with compromised function of XBP1. RRM2, CDC6 and TOP2A have been previously reported as direct targets of XBP1 in other cell types (i.e., muscle, pancreatic β-cell, and plasma cell) and helper T-cells. Furthermore, overexpression of RRM2 in XBP1 deficient MCF7 sub-clones rescued the cell growth and proliferation same as control MCF7 clones. Therefore, this study has revealed XBP1 regulated new targets in ERα-positive breast cancer cells which could be instrumental in providing new insights for better therapeutic potential in this context. Citation Format: David Barua, Afrin Sultana, Md Nahidul Islam, Ananya Gupta, Sanjeev Gupta. XBP1 regulates the expression of cell cycle associated genes in ERα positive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5843.

Heregulin controls ERα and HER2 signaling in mammospheres of ERα-positive breast cancer cells and interferes with the efficacy of molecular targeted therapy

Estrogen receptor (ER)α and the human epidermal growth factor receptor (HER) family are inversely expressed in ERα-positive cancer in association with resistance to hormonal therapy, but the mechanism underlying their relationship remains unknown. We analyzed the effect of HER family ligands on the expression of ER and the HER family in ERα-positive MCF-7 and T47D breast cancer cell lines in 3D spheroid culture. Here, we demonstrated for the first time that heregulin-1β (HRG), a HER3 and HER4 ligand, most effectively regulated ER/HER family expression by decreasing ERα mRNA expression and increasing HER family mRNA expression. HRG treatment attenuated fulvestrant-mediated growth inhibition, and promoted the migration of MCF-7 cells. Moreover, HRG increased the CD44+/CD24− cell fraction and side population cells, both of which are recognized as prospective breast cancer stem cell markers. HRG activated both phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and mitogen-activated protein kinase (MAPK) pathways. Inhibitors of these pathways reduced the growth of MCF-7 cells, but the addition of HRG has different effects on these pathways. HRG blocked the inhibitory effect of mTOR inhibitors, such as rapamycin and everolimus, on cell growth but not that of a PI3K inhibitor. Furthermore, HRG slightly decreased the inhibitory effect of an AKT inhibitor on cell growth. In contrast, HRG enhanced the MEK inhibitor-induced inhibition of cell growth. These findings suggest that HRG-stimulated signaling pathways allow ERα-positive breast cancer cells to escape from growth inhibition caused by everolimus, via MAPK signaling and/or other signaling pathways. Everolimus improves progression-free survival in combination with exemestane as second-line therapy for metastatic hormone receptor-positive breast cancer. Our study suggests that HRG is a novel target for ERα-positive breast cancer therapy.

Steroidal N-Sulfonylimidates: Synthesis and biological evaluation in breast cancer cells

Unique derivatives of androstene and estrane series containing N-sulfonylimidate pendants were prepared from 17α-ethynyl steroids via Cu-catalyzed azide–alkyne cycloaddition to tosyl azide in the presence of alcohols. The synthesized compounds were screened for cytotoxicity against human breast cancer cell lines and ERα agonist activity. The hit compound 3,17β-dimethoxy-17α-[iso-propyl-2′-N-tosylacetimidate]estra-1,3,5(10)-triene (4n) had no ERα-mediated hormonal activity and was found to exhibit potent cytotoxic effect in an ERα-positive breast cancer cell line. N-Sulfonylimidate 4n displayed high antiproliferative potency against triple-negative MDA-MB-231 breast cancer cells, while it was non-toxic towards normal mammary epithelial cells. Compound 4n was found to alter activity of various signaling pathways (NF-κB, Slug, cyclin D1, ERK) supporting the growth and invasiveness of tumor cells.

A temperature-sensitive phase-change hydrogel of tamoxifen achieves the long-acting antitumor activation on breast cancer cells.

Background: Breast cancer is one of the foremost threats to female health nowadays. Tamoxifen, an antagonist of estrogen receptor-α (ERα), is the first choice for endocrine-dependent breast cancer (ERα-positive breast cancer) treatment. However, ERα has an important function in the normal physical regulation of estrogen, and current oral administration of tamoxifen has potential side effects on normal endocrine secretion. In the present work, we aim to develop novel approaches to increase the antitumor effect of tamoxifen on breast cancer cells and decrease the potential side effects in the human body during treatment.Methods: A temperature-sensitive phase-change hydrogel for tamoxifen (Tam-Gel) was generated. After establishing subcutaneous tumors formed by MCF-7, an ERα-positive breast cancer cell line, in nude mice, an intratumoral injection of Tam-Gel was performed to examine whether Tam-Gel facilitated the slow-release or antitumor effect of tamoxifen. A metastatic breast cancer model was established using the intrahepatic growth of MCF-7 cells in immunodeficient rats.Results: Tam-Gel can transform from liquid to hydrogel at room temperature. An intratumoral injection of Tam-Gel facilitated the slow-release or antitumor effect of tamoxifen. Once Tam-Gel, but not Tam-Sol, was administered by intratumoral injection, it significantly decreased the uptake of radionuclide probes (18F-fluoroestradiol or 18F-fluorodeoxyglucose) by cells in rats’ livers and the intrahepatic growth of MCF-7 cells in rats’ livers.Conclusion: A novel slow-release system was successfully prepared to facilitate the long-term release of tamoxifen in breast cancer tissues, and achieved an antitumor effect in the long term.

ERα is a negative regulator of PD-L1 gene transcription in breast cancer

The programmed death-ligand 1 (PD-L1) expression by tumors results in potent antitumor immune suppression through binding to programmed death-1 (PD-1) on T cells and subsequent inhibition of T cells activity. Although recent pathological studies have shown that PD-L1 is actively expressed in certain ERα-negative breast cancer, little is known about whether ER signaling regulates PD-L1 gene expression. Here, we investigated the relationship between ERα and PD-L1 in breast cancer. Analysis of Comprehensive Cell Line Encyclopedia (CCLE) data showed that the average mRNA level of PD-L1 in ERα-positive breast cancer cell lines was significantly lower than that in ERα-negative breast cancer cell lines. E2 treatment inhibited PD-L1 mRNA expression in hormone-depleted ERα-positive MCF7 cells. Moreover, ectopic expression of ERα in triple-negative MDA-MB-231 cells reduced PD-L1 mRNA and protein expression. Consistently, analysis of The Cancer Genome Atlas (TCGA) data revealed an inverse correlation between ERα and PD-L1 expression in ERα-positive breast cancer. Taken together, our results identify ERα as a negative regulator of PD-L1 gene transcription in breast cancer cells, suggesting that ERα loss-of-function may facilitate the immune evasion of breast cancer cells via up-regulation of PD-L1.

Abstract B36: Progesterone receptor signaling in estrogen receptor-positive breast cancer

Abstract Approximately 65% of breast cancers are estrogen receptor alpha (ERα)-positive and depend on ERα signaling for growth. Clinical data indicate that ERα-positive breast cancers have a better prognosis when another hormone receptor, the progesterone receptor (PR), is coexpressed. Recent studies show that in the presence of its ligand progesterone, PR redirects ERα chromatin binding and transcriptional activity, thereby impacting prognosis and therapeutic response. To further examine PR function, we knocked out the PGR gene in the ERα-positive breast cancer cell lines MCF-7 and T47D using CRISPR-mediated gene editing. Knockout clones from each model were analyzed for ER- and PR-mediated transcriptional activity and gene expression profile, as well as response to selective estrogen receptor degraders (SERDs). The T47D cell line expresses high levels of PR in the presence or absence of estrogen, and stimulation with progesterone induces robust PR phosphorylation and modulation of PR target gene expression. Furthermore, progesterone attenuates estradiol-induced cell proliferation in a dose-dependent manner. Knockout of PR in this model abrogates the ability of progesterone to induce gene expression changes or attenuate estradiol-induced cell proliferation. In contrast, the MCF-7 cell line expresses low basal levels of PR, and is refractory to stimulation with progesterone. Interestingly, in estrogenic growth media, knockout of PR in this model results in basal gene expression changes that resemble a gene signature associated with tamoxifen resistance. Consistent with these findings, knockout of PR reduces the response to SERDs in a 5-day cell proliferation assay. ChIP-seq and RNA-seq analyses are ongoing to examine how loss of PR impacts ERα chromatin binding and transcriptional output. Citation Format: Amy Young, Jane Guan, Anneleen Daemen, Lori Friedman, Kui Lin. Progesterone receptor signaling in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B36.

ZBTB7A governs estrogen receptor alpha expression in breast cancer.

ZBTB7A, a member of the POZ/BTB and Krüppel (POK) family of transcription factors, has been shown to have a context-dependent role in cancer development and progression. The role of ZBTB7A in estrogen receptor alpha (ERα)-positive breast cancer is largely unknown. Approximately 70% of breast cancers are classified as ERα-positive. ERα carries out the biological effects of estrogen and its expression level dictates response to endocrine therapies and prognosis for breast cancer patients. In this study, we find that ZBTB7A transcriptionally regulates ERα expression in ERα-positive breast cancer cell lines by binding to the ESR1 promoter leading to increased transcription of ERα. Inhibition of ZBTB7A in ERα-positive cells results in decreased estrogen responsiveness as demonstrated by diminished estrogen-response element-driven luciferase reporter activity, induction of estrogen target genes, and estrogen-stimulated growth. We also report that ERα potentiates ZBTB7A expression via a post-translational mechanism, suggesting the presence of a positive feedback loop between ZBTB7A and ERα, conferring sensitivity to estrogen in breast cancer. Clinically, we find that ZBTB7A and ERα are often co-expressed in breast cancers and that high ZBTB7A expression correlates with improved overall and relapse-free survival for breast cancer patients. Importantly, high ZBTB7A expression predicts a more favorable outcome for patients treated with endocrine therapies. Together, these findings demonstrate that ZBTB7A contributes to the transcriptional program maintaining ERα expression and potentially an endocrine therapy-responsive phenotype in breast cancer.

Estrogen Receptor Mediates the Radiosensitivity of Triple-Negative Breast Cancer Cells.

BackgroundThis study aimed to evaluate differences in the radiosensitivities of triple-negative breast cancer (TNBC) and luminal-type breast cancer cells and to investigate the effects of estrogen receptor (ER) expression on the biological behaviors of the cells.Material/MethodsColony-forming assays were performed to detect differences in radiosensitivities in breast cancer cell lines. Gene transfection technology was used to introduce the expression of ERα in TNBC cells to compare the difference in radiosensitivity between the TNBC cells and ERα transfected cells. CCK-8 assays were used to observe changes in the proliferation of TNBC cells after ERα transfection. Immunofluorescence was used to detect the number of γH2AX foci in nuclei. Flow cytometry was used to detect changes in cell cycle distribution and apoptosis. Western blotting was used to detect changes in autophagy-associated proteins.ResultsThe radioresistance of the TNBC cell line MDA-MB-231 (231 cells) was greater than that of ERα-positive luminal-type breast cancer cell line MCF-7. Moreover, 231 cell proliferation and radioresistance decreased after ERα transfection. Interestingly, ERα-transfected 231 cells showed increased double-stranded breaks and delayed repair compared with 231 cells, and ERα-transfected 231 cells showed increased G2/M phase arrest and apoptosis after irradiation compared with those in 231 cells. ERα transfection in 231 cells reduced autophagy-related protein expression, suggesting that autophagy activity decreased in 231 ER-positive cells after irradiation.ConclusionsTNBC cells were more resistant to radiation than luminal-type breast cancer cells. ERα expression may have major roles in modulating breast cancer cell radiosensitivity.

Cytotoxic Activity on MCF-7 Cells and in Silico Study of Sapogenin Steroids From Trigonella foenum-graecum L.

Trigonella foenum-graecum (TFG) is one of medicinal plants containing several steroidal sapogenins, such as diosgenin, yamogenin, gitogenin, tigogenin and trigoneoside, and also alkaloid trigonellin and some flavonoids such as vitexin, isovitexin, orientin, isoorientin, which has many activities, such as antidiabetic, estrogenic, and also anticancer. As phytoestrogen, TFG was predicted to have potency as Selective Estrogen Receptor Modulators (SERMs) which is used for hormonal-dependent breast cancer treatment. This experiment was carried out to investigate interaction of some sapogenin steroids and flavonoids in TFG to estrogen receptor alpha (ERα) and its activity to breast cancer cell line as confirmation. In silico prediction was carried out to investigate their estrogenic activity by analyzing their binding affinity to ERα using AutoDock Vina program. In vitro activity confirmation of TFG extract and its fractions were carried out using MTT assay on Erα-positive human breast cancer cell line, MCF-7. Results showed that free binding energies of diosgenin and yamogenin were -6.4 kcal/mol, estradiol was -6.0 kcal/mol, and tamoxifen was -5.1 kcal/mol. While cytotoxicity assay showed that ethyl acetate fraction gave the lowest IC50 of 41.81 ppm, with total steroid content of 20.03 ppm. From these results, we can conclude that diosgenin and yamogenin have greater binding affinity to ERα comparing to estradiol and tamoxifen. In vitro assay confirmation showed that ethyl acetate fraction has cytotoxic effect on MCF-7 cells.Keywords: Trigonella foenum-graecum, sapogenin steroids, MCF-7, estrogen receptor alpha, binding affinity

Genetic Polymorphisms in the Long Noncoding RNA MIR2052HG Offer a Pharmacogenomic Basis for the Response of Breast Cancer Patients to Aromatase Inhibitor Therapy.

Genetic risks in breast cancer remain only partly understood. Here, we report the results of a genome-wide association study of germline DNA from 4,658 women, including 252 women experiencing a breast cancer recurrence, who were entered on the MA.27 adjuvant trial comparing the aromatase inhibitors (AI) anastrozole and exemestane. Single-nucleotide polymorphisms (SNP) of top significance were identified in the gene encoding MIR2052HG, a long noncoding RNA of unknown function. Heterozygous or homozygous individuals for variant alleles exhibited a ∼40% or ∼63% decrease, respectively, in the hazard of breast cancer recurrence relative to homozygous wild-type individuals. Functional genomic studies in lymphoblastoid cell lines and ERα-positive breast cancer cell lines showed that expression from MIR2052HG and the ESR1 gene encoding estrogen receptor-α (ERα) was induced by estrogen and AI in a SNP-dependent manner. Variant SNP genotypes exhibited increased ERα binding to estrogen response elements, relative to wild-type genotypes, a pattern that was reversed by AI treatment. Further, variant SNPs were associated with lower expression of MIR2052HG and ERα. RNAi-mediated silencing of MIR2052HG in breast cancer cell lines decreased ERα expression, cell proliferation, and anchorage-independent colony formation. Mechanistic investigations revealed that MIR2052HG sustained ERα levels both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated, proteasome-dependent degradation of ERα. Taken together, our results define MIR2052HS as a functionally polymorphic gene that affects risks of breast cancer recurrence in women treated with AI. More broadly, our results offer a pharmacogenomic basis to understand differences in the response of breast cancer patients to AI therapy. Cancer Res; 76(23); 7012-23. ©2016 AACR.

Different epigenetic mechanisms of ERα implicated in the fate of fulvestrant-resistant breast cancer

Approximately 70% of breast cancers express estrogen receptor α (ERα), which plays critical roles in breast cancer development. Fulvestrant has been effectively used to treat ERα-positive breast cancer, although resistance remains a critical problem. To elucidate the mechanism of resistance to fulvestrant, we established fulvestrant-resistant cell-lines named MFR (MCF-7 derived fulvestrant resistance) and TFR (T-47D derived fulvestrant resistance) from the ERα-positive luminal breast cancer cell lines MCF-7 and T-47D, respectively. Both fulvestrant-resistant cell lines lost sensitivity to estrogen and anti-estrogens. We observed diminished ERα expression at both the protein and mRNA levels. To address the mechanism of gene expression regulation, we examined epigenetic alteration, especially the DNA methylation level of ERα gene promoters. MFR cells displayed high methylation levels upstream of the ERα gene, whereas no change in DNA methylation was observed in TFR cells. Hence, we examined the gene expression plasticity of ERα, as there are differences in its reversibility following fulvestrant withdrawal. ERα gene expression was not restored in MFR cells, and alternative intracellular phosphorylation signals were activated. By contrast, TFR cells exhibited plasticity of ERα gene expression and ERα-dependent growth; moreover, these cells were resensitized to estrogen and anti-estrogens. The difference in epigenetic regulation among individual cells might explain the difference in the plasticity of ERα expression. We also identified an MFR cell-activating HER/Src-Akt/MAPK pathway; thus, the specific inhibitors effectively blocked MFR cell growth. This finding implies the presence of multiple fulvestrant resistance mechanisms and suggests that the optimal therapies differ among individual tumors as a result of differing epigenetic mechanisms regulating ERα gene expression.

Abstract 3737: Inhibition of O-GlcNAc transferase in tamoxifen resistant breast cancer cells

Abstract O-linked N-acetyl-glucosamine transferase (OGT) is an enzyme that catalyzes addition of the O-GlcNAc modification to a wide range of intracellular proteins. The O-GlcNAc modification is a product of the hexosamine biosynthetic pathway, which requires glucose and glutamine as substrates. Uptake of both of these nutrients is often up-regulated in cancer, which in turn leads to an increase in the total protein O-GlcNAcylation. Increased OGT expression has also been reported in most cancer types, including the most frequently diagnosed cancer in women, breast cancer. Many of the breast cancers rely on estrogen receptor alpha (ERα) for proliferation and have shown a strong response to the ERα inhibition, most commonly achieved by treatment with tamoxifen. However, while efficient, prolonged exposure to tamoxifen commonly causes resistance and relapse of the disease. It is therefore vital to uncover mechanisms which contribute to the resistance in order to develop adequate treatment strategy for these patients. Here, we have investigated the effect of targeting OGT in an isogenic pair of ERα-positive tamoxifen-sensitive MCF7 and tamoxifen-resistant TAMR breast cancer cell lines. OGT inhibition decreased viability and triggered cell death in both cell lines. These responses were associated with over 50% reduction in ERα expression in both MCF7 and TAMR cells. Reduced O-GlcNAcylation has previously been reported to induce endoplasmic reticulum stress and activation of transcription factor C/EBP homologous protein (CHOP), which promotes cell death. Targeting OGT resulted in a strong increase of CHOP expression, which appeared more prominent in the TAMR cells. Finally, targeting OGT induced a very pronounced cell cycle arrest in the G2/M phase in the TAMR cells, while the MCF7 cell lined showed a very modest response. Taken together, these results indicate that targeting OGT leads to a differential response in the tamoxifen-sensitive and resistant breast cancer cells. Currently, we are using an expanded panel of tamoxifen-resistant cell lines to perform expression microarrays, metabolic flux assays and DNA damage response analysis in order to uncover the cause of the differential response to OGT targeting. This may help us identify potential therapeutic combinations that might be suitable for treatment of tamoxifen-resistant cancers. Citation Format: Anna Barkovskaya, Lina Prasmickaite, Ian G. Mills, Gunhild M. Mælandsmo, Siver A. Moestue, Harri M. Itkonen. Inhibition of O-GlcNAc transferase in tamoxifen resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3737.

Interleukin-6 is a potential therapeutic target in interleukin-6 dependent, estrogen receptor-α-positive breast cancer.

IntroductionInterleukin-6 (IL-6) is an important growth factor for estrogen receptor-α (ERα)-positive breast cancer, and elevated serum IL-6 is associated with poor prognosis.MethodsThe role of the phosphorylated signal transducer and activator of transcription 3 pathway was investigated in ERα-positive breast cancer. A panel of cell lines was treated with exogenous IL-6. An IL-6 specific gene signature was generated by profiling ten ERα-positive breast cancer cell lines alone or following treatment with 10 ng/mL recombinant IL-6 or human marrow stromal cell-conditioned media, with or without siltuximab (a neutralizing anti-IL-6 antibody) and grown in three-dimensional tumor microenvironment-aligned cultures for 4 days, 5 days, or 6 days. The established IL-6 signature was validated against 36 human ERα-positive breast tumor samples with matched serum. A comparative MCF-7 xenograft murine model was utilized to determine the role of IL-6 in estrogen-supplemented ERα-positive breast cancer to assess the efficacy of anti-IL-6 therapy in vivo.ResultsIn eight of nine ERα-positive breast cancer cell lines, recombinant IL-6 increased phosphorylation of tyrosine 705 of STAT3. Differential gene expression analysis identified 17 genes that could be used to determine IL-6 pathway activation by combining their expression intensity into a pathway activation score. The gene signature included a variety of genes involved in immune cell function and migration, cell growth and apoptosis, and the tumor microenvironment. Validation of the IL-6 gene signature in 36 matched human serum and ERα-positive breast tumor samples showed that patients with a high IL-6 pathway activation score were also enriched for elevated serum IL-6 (≥10 pg/mL). When human IL-6 was provided in vivo, MCF-7 cells engrafted without the need for estrogen supplementation, and addition of estrogen to IL-6 did not further enhance engraftment. Subsequently, we prophylactically treated mice at MCF-7 engraftment with siltuximab, fulvestrant, or combination therapy. Siltuximab alone was able to blunt MCF-7 engraftment. Similarly, siltuximab alone induced regressions in 90% (9/10) of tumors, which were established in the presence which were established in the presence of hMSC expressing human IL-6 and estrogen.ConclusionGiven the established role for IL-6 in ERα-positive breast cancer, these data demonstrate the potential for anti-IL-6 therapeutics in breast cancer.

Abstract LB-152: A new role for ERα: Gene silencing via DNA methylation

Abstract As a master transcription factor, estrogen receptor-α (ERα) regulates expression of a multitude of genes. We hypothesized that ERα may also regulate gene expression via DNA methylation since methylation of specific CpG sites associates with ERα-positive status in human breast cancer. We tested this hypothesis by identifying genes normally silenced in ERα-positive T47D and MCF-7 luminal breast cancer cell lines but which became expressed (or derepressed) upon exposure to the demethylating agent decitabine and antagonizing ERα activity. These genes were identified using a combination of Agilent gene expression microarrays and assessed according to the Significance Analysis of Microarrays (SAM) method at a 2-fold change and 5% FDR. Selected genes were validated by pyrosequencing to quantitate CpG methylation, lentiviral re-introduction of ERα, real-time PCR and immunoblotting. In a short-term study, T47D cells were treated with decitabine and the ER antagonist fulvestrant (FUL) for 96 h. Array analysis identified 87 genes that were derepressed by decitabine and FUL, of which 31 (36%) were markers of the basal subtype of breast cancer. In long-term studies (8 - 36 weeks), MCF-7 and T47D cells were FUL-treated or estrogen-deprived (ED) to derive multiple antihormone-resistant cell lines (MCF-7/FUL, T47D/FUL, and 2 independent T47D/ED), all of which lost ≥ 95% of ERα. Comparing all of the resistant cell lines and decitabine-treated T47D cells by array analysis revealed that 58 genes were up-regulated by both loss of ERα and decitabine treatment, 35 (60%) of which were basal markers. Two basal breast cancer subtype markers, LCN2 and IFI27 which are involved in epithelial-mesenchymal transition and interferon signaling, respectively, were selected for verification. First, LCN2 and IFI27 expression increased ∼132- and 343-fold, respectively, while their CpG methylation levels significantly decreased upon ERα loss in all T47D antihormone-resistant cell lines. Second, LCN2 and IFI27 expression decreased while their methylation levels increased upon estrogen re-exposure or lentiviral ERα re-introduction in the T47D/ED cell lines. Moreover, high CpG methylation levels of LCN2 and IFI27 directly associated with ERα-positive status but inversely correlated with their expression levels across a panel of 12 breast cancer cell lines. Therefore, ERα targets specific genes, such as basal markers, for DNA methylation-mediated silencing. Mechanistically, ERα-dependent DNA methylation may occur as a consequence of long-term transcriptional repression. Although not well appreciated, estrogen-bound ERα represses transcription of more genes than it activates. Thus, ERα may direct DNA methylation by interacting with multicomponent corepressor complexes, which could then recruit DNA methyltransferases to target promoters. Moreover, ERα-dependent silencing of basal markers may promote the prognostically more favorable luminal breast cancer subtype. Citation Format: Eric A. Ariazi, John C. Taylor, Emmanuelle Nicolas, Michael J. Slifker, Jeff Boyd. A new role for ERα: Gene silencing via DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-152. doi:10.1158/1538-7445.AM2015-LB-152