Current medical treatment of estrogen receptor-positive breast cancer

Approximately 80% of breast cancers(BC) are estrogen receptor(ER)-positive and thus endocrine therapy(ET) should be considered complementary to surgery in the majority of patients. The advantages of oophorectomy, adrenalectomy and hypophysectomy in women with advanced BC have been demonstrated many years ago, and currently ET consist of(1) ovarian function suppression(OFS), usually obtained using gonadotropinreleasing hormone agonists(Gn RHa);(2) selective estrogen receptor modulators or down-regulators(SERMs or SERDs); and(3) aromatase inhibitors(AIs), or a combination of two or more drugs. For patients aged less than 50 years and ER+ BC, there is no conclusive evidence that the combination of OFS and SERMs(i.e., tamoxifen) or chemotherapy is superior to OFS alone. Tamoxifen users exhibit a reduced risk of BC, both invasive and in situ, especially during the first 5 years of therapy, and extending the treatment to 10 years further reduced the risk of recurrences. SERDs(i.e., fulvestrant) are especially useful in the neoadjuvant treatment of advanced BC, alone or in combination with either cytotoxic agents or AIs. There are two types of AIs: type Ⅰ are permanent steroidal inhibitors of aromatase, while type Ⅱ are reversible nonsteroidal inhibitors. Several studies demonstrated the superiority of the third-generation AIs(i.e., anastrozole and letrozole) compared with tamoxifen, and adjuvant therapy with AIs reduces the recurrence risk especially in patients with advanced BC. Unfortunately, some cancers are or became ET-resistant, and thus other drugs have been suggested in combination with SERMs or AIs, including cyclin-dependent kinase 4/6 inhibitors(palbociclib) and mammalian target of rapamycin(m TOR) inhibitors, such as everolimus. Further studies are required to confirm their real usefulness.

Adjuvant tamoxifen switched to exemestane treatment in postmenopausal women with estrogen receptor-positive early breast cancer:A pragmatic,multicenter,and prospective clinical trial in China

Objective:This post-approval safety study assessed the efficacy and safety of exemestane after 2-3 years of tamoxifen treatment among postmenopausal women with estrogen receptor-positive(ER+)early breast cancer in China.Methods:Enrolled patients had received 2-3 years of tamoxifen and were then switched to exemestane for completion of 5 consecutive years of adjuvant endocrine therapy.The primary endpoint was the time from enrollment to the first occurrence of locoregional/distant recurrence of the primary breast cancer,appearance of a second primary or contralateral breast cancer,or death due to any cause.Other endpoints included the proportion of patients experiencing each event,incidence rate per annum,relationships between human epidermal growth factor receptor 2 status and time to event,and relationship between disease history variables and time to event.Results:Overall,558 patients were included in the full analysis set:397(71.1%)completed the study,20experienced an event,and 141 discontinued[47 owing to an adverse event(AE);37 no longer willing to participate].Median duration of treatment was 29.5(range,0.1-57.7)months.Median time to event was not reached.Eventfree survival probability at 36 months was 91.4%(95%CI,87.7%-95.1%).The event incidence over the total exposure time of exemestane therapy was 3.5 events/100 person-years(20/565).Multivariate analysis showed an association between tumor,lymph node,and metastasis stage at initial diagnosis and time to event[hazard ratio:1.532(95%CI,1.129-2.080);P=0.006].Most AEs were grade 1 or 2 in severity,with arthralgia(7.7%)being the most common treatment-related AE.Conclusions:This study supports the efficacy and safety of exemestane in postmenopausal Chinese women with ER+breast cancer previously treated with adjuvant tamoxifen for 2-3 years.No new safety signals were identified in the Chinese population.

Tumor biology in estrogen receptor-positive,human epidermal growth factor receptor type 2-negative breast cancer:Mind the menopausal status

Breast cancer is not one disease,but can be categorized into four major molecular subtypes according to hormone receptor [estrogen receptor(ER) and progesterone receptor(Pg R)] and human epidermal growth factor receptor type 2(HER2) expression status. Ki67 labeling index and/or multigene assays are used to classify ERpositive,HER2-negative breast cancer into luminal A and luminal B(HER2-negative) subtypes. To date,most studies analyzing predictive or prognostic factors in ER-positive breast cancer have been performed in postmenopausal women,mainly using patients and samples in adjuvant aromatase inhibitor trials. In contrast,even the clinical roles of Pg R and Ki67 have been little analyzed so far in premenopausal women. Pg R is one of the estrogen-responsive genes,and it has been reported that plasma estradiol levels are related to expression levels of estrogen-responsive genes including PGR in ER-positive breast cancer. In this article,biological differences,especially differences in expression of Pg R and Ki67 in ER-positive breast cancer between pre- and postmenopausal women are discussed. Clinical roles of Pg R and Ki67 in ER-positive breast cancer differ between pre- and postmenopausal women. We suggest that the mechanisms of development and estrogen-dependent growth of ER-positive breast cancer might differ according to menopausal status.

Osteolytic effects of tumoral estrogen signaling in an estrogen receptor-positive breast cancer bone metastasis model

Aim:Estrogen receptorα-positive(ER+)subtypes of breast cancer have the greatest predilection for forming osteolytic bone metastases(BMETs).Because tumor-derived factors mediate osteolysis,a possible role for tumoral ERαsignaling in driving ER+BMET osteolysis was queried using an estrogen(E2)-dependent ER+breast cancer BMET model.Methods:Female athymic Foxn1nu mice were inoculated with human ER+MCF-7 breast cancer cells via the left cardiac ventricle post-E2 pellet placement,and age-and dose-dependent E2 effects on osteolytic ER+BMET progression,as well as direct bone effects of E2,were determined.Results:Osteolytic BMETs,which did not form in the absence of E2 supplementation,occurred with the same frequency in young(5-week-old)vs.skeletally mature(16-week-old)E2(0.72 mg)-treated mice,but were larger in young mice where anabolic bone effects of E2 were greater.However,in mice of a single age and across a range of E2 doses,anabolic E2 bone effects were constant,while osteolytic ER+BMET lesion incidence and size increased in an E2 dose-dependent fashion.Osteoclasts in ER+tumor-bearing(but not tumor-naive)mice increased in an E2-dose dependent fashion at the bone-tumor interface,while histologic tumor size and proliferation did not vary with E2 dose.E2-inducible tumoral secretion of the osteolytic factor parathyroid hormone-related protein(PTHrP)was dose-dependent and mediated by ERα,with significantly greater levels of secretion from ER+BMET-derived tumor cells.Conclusion:These results suggest that tumoral ERαsignaling may contribute to ER+BMET-associated osteolysis,potentially explaining the greater predilection for ER+tumors to form clinically-evident osteolytic BMETs.

Clinicopathological Features and Long-Term Prognostic Role of Human Epidermal Growth Factor Receptor-2 Low Expression in Chinese Patients with Early Breast Cancer:A Single-Institution Study

Objective This study aimed to comprehensively analyze and compare the clinicopathological features and prognosis of Chinese patients with human epidermal growth factor receptor 2(HER2)-low early breast cancer(BC)and HER2-IHC0 BC.Methods Patients diagnosed with HER2-negative BC(N=999)at our institution between January2011 and December 2015 formed our study population.Clinicopathological characteristics,association between estrogen receptor(ER)expression and HER2-low,and evolution of HER2 immunohistochemical(IHC)score were assessed.Kaplan-Meier method and log-rank test were used to compare the long-term survival outcomes(5-year follow-up)between the HER2-IHC0 and HER2-low groups.Results HER2-low BC group tended to demonstrate high expression of ER and more progesterone receptor(PgR)positivity than HER2-IHC0 BC group(P<0.001).The rate of HER2-low status increased with increasing ER expression levels(Mantel-Haenszelχ^(2)test,P<0.001,Pearson’s R=0.159,P<0.001).Survival analysis revealed a significantly longer overall survival(OS)in HER2-low BC group than in HER2-IHC0 group(P=0.007)in the whole cohort and the hormone receptor(HR)-negative group.There were no significant differences between the two groups in terms of disease-free survival(DFS).The discordance rate of HER2 IHC scores between primary and metastatic sites was 36.84%.Conclusion HER2-low BC may not be regarded as a unique BC group in this population-based study due to similar clinicopathological features and prognostic roles.

Exploratory clinical study of chidamide,an oral subtype-selective histone deacetylase inhibitor,in combination with exemestane in hormone receptor-positive advanced breast cancer

Objective: The recurrence or progression under endocrine therapy in hormone receptor-positive(HR+)advanced breast cancer(ABC) remained a critical clinical challenge.Chidamide is an oral subtype-selective histone deacetylase(HDAC) inhibitor with multiple functions in tumor growth inhibition and microenvironment modulation via epigenetic reprogramming.The purpose of this study was to evaluate the safety,pharmacokinetics(PK),and preliminary efficacy of chidamide in combination with exemestane in HR+ ABC patients.Methods: Eligible patients were postmenopausal women with HR+ ABC recurrent or progressed to at least one endocrine therapy.Blood samples were obtained in the run-in period and the first day of combination treatment for PK analysis.In combination treatment,patients were given exemestane 25mg daily and chidamide 30mg twice a week(BIW) until progression of disease or intolerable toxicities.A treatment cycle was defined as 4 weeks.Safety,PK parameters,and preliminary efficacy were evaluated.Results: A total of 20 patients were enrolled between July and December,2015.The median number of treatments cycle was 5.2(20.8 weeks) with 2 patients still on treatment at the data cut-off date of October,2017.The treatment-related adverse events(AE) ≥ grade 3 in more than 2 patients were neutropenia(35%),thrombocytopenia(30%),and leucopenia(20%).The plasma exposure of exemestane was consistent in the presence or absence of chidamide.A slight increase in chidamide exposure was noted in the presence of exemestane,probably due to the inter-and intra-patient variations.The best response in 16 evaluable patients was assessed by Response Evaluation Criteria in Solid Tumors(RECIST),including 4 patients with partial response,10 patients with stable disease.The median progression-free survival(PFS) was 7.6 months.Conclusions: The combination of chidamide with exemestane was generally well tolerated with promising preliminary efficacy in HR+ ABC patients.The overall results from this study encourage further pivotal trial in this patient population.

New insights into estrogenic regulation of O^6-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells： Co-degradation of ER-α and MGMT proteins by fulvestrant or O^6-benzylguanine indicates fresh avenues for therapy

Endocrine therapy using estrogen receptor-u （ER-α） antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O^6-methylguanine DNA methyltransferase （MGMT）, a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines （MCF-7, T47D） and ER- negative cell lines （MDAMB- 468, MDAMB-231）, and established inhibitors of ER-α and MGMT, namely, ICI-182,780 （Faslodex） and O^6- benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements （EREs） and two antioxidant-responsive elements （AREs）. MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 （nuclear factor-erythroid 2-related factor-2） increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this dragresistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O^6-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O^6-benzylguanine also induced a specific loss of ER-a and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-a and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-a proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated. Fulvestrant enhanced the cytotoxicity of MGMT-targeted alkylating agents, namely, temozolomide and BCNU by 3 to 4-fold in ER-α positive cells, but not in ER-negative cells. We conclude that MGMT and ER-α proteins exist as a complex and are co-targeted for ubiquitin-conjugation and subsequent proteasomal degradation. The findings offer a clear rationale for combining alkylating agents with endocrine therapy.

Polyubiquitination inhibition of estrogen receptor alpha and its implications in breast cancer

Estrogen receptor alpha(ERα) is detected in more than 70% of the cases of breast cancer. Nuclear activity of ERα, a transcriptional regulator, is linked to the development of mammary tumors, whereas the extranuclear activity of ERα is related to endocrine therapy resistance. ERα polyubiquitination is induced by the estradiol hormone, and also by selective estrogen receptor degraders, resulting in ERα degradation via the ubiquitin proteasome system. Moreover, polyubiquitination is related to the ERα transcription cycle, and some E3-ubiquitin ligases also function as coactivators for ERα. Several studies have demonstrated that ERα polyubiquitination is inhibited by multiple mechanisms that include posttranslational modifica-tions, intera-ctions with coregula-tors, a-nd forma-tion of specific protein complexes with ERα. These events are responsible for an increase in ERα protein levels and deregulation of its signaling in breast cancers. Thus, ERα polyubiquitination inhibition may be a key factor in the progression of breast cancer and resistance to endocrine therapy.

Estrogen receptor alpha gene amplification in breast cancer:25 years of debate

Twenty-five years ago,Nembrot and colleagues reported amplification of the estrogen receptor alpha gene(ESR1) in breast cancer,initiating a broad and still ongoing scientific debate on the prevalence and clinical significance of this genetic aberration,which affects one of the most important genes in breast cancer.Since then,a multitude of studies on this topic has been published,covering a wide range of divergent results and arguments.The reported prevalence of this alteration in breast cancer ranges from 0% to 75%,suggesting that ESR1 copy number analysis is hampered by technical and interpreter issues.To date,two major issues related to ESR1 amplification remain to be conclusively addressed:(1) The extent to which abundant amounts of messenger RNA can mimic amplification in standard fluorescence in situ hybridization assays in the analysis of strongly expressed genes like ESR1,and(2) the clinical relevance of ESR1 amplification:Such relevance is strongly disputed,with data showing predictive value for response as well as for resistance of the cancer to anti-estrogen therapies,or for subsequent development of cancers in the case of precursor lesions that display amplification of ESR1.This review provides a comprehensive summary of the various views on ESR1 amplification,and highlights explanations for the contradictions and conflicting data that could inform future ESR1 research.

Soy isoflavone extracts stimulate the growth of nude mouse xenografts bearing estrogen-dependent human breast cancer cells(MCF-7)

We explored the effects of different lifetime exposures to soy isoflavone extracts on the growth of estrogen- dependent human breast cancer cells （MCF-7） implanted into athymic mice of different ovarian statuses. The athymic mice, ovariectomized or not, were implanted with MCF-7 cells. Mice were fed with low, moderate and high doses of soy isoflavone extract, at dietary concentrations of 6.25, 12.5 and 25 g/kg, in different reproductive models, respectively. The expression of ki-67 was detected by immunohistochemistry, pS2 expression in tumors was analyzed by real-time PCR. Estrogen level in the serum was measured by chemiluminescence enzyme im- munoassay. Total genistein and daidzein levels in serum and urine were determined by liquid chromatography- electrospray tandem mass spectrometry （LC-ES/MS/MS）. In Group A, on week 4, nude mice were exposed to different doses of soy iosflavone extracts. In Group B, the experimental diets were given to the nude mice follow- ing ovariectomy and tumor implantation. In both groups, 6.25 and 12.5 g/kg soy isoflavone extracts stimulated the growth of MCF-7 xenografts, increased pS2 expression, proliferation and estrogen level in serum. In both Group B （postmenopausal mouse model） and Group C （premenopausal mouse model）, soy isoflavone extracts at doses of 6.25 and 12.5 g/kg showed stimulatory effects on the growth of MCF-7 tumors. In conclusion, administration of soy isoflavone extracts at doses of 6.25 and 12.5 g/kg during adolescence or later in life stimulated tumor growth in both menopausal and postmenopausal mouse models.

Level of Adherence to Breast Cancer Molecular Subtyping among Women with Breast Cancer Attending Tertiary Health Facilities

Background: Breast cancer is a genetically and clinically heterogeneous disease with multiple subtypes. The classification of these subtypes has evolved over the years. The most common and widely accepted classification of breast cancer is from an immunohistochemical perspective, based on the expression of the following hormone receptors: Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor (HER2). Accordingly, the following four subtypes of breast cancer are widely recognized—Luminal A, Luminal B, HER2 Enriched and Triple Negative. Breast cancer management approaches include surgery, chemotherapy, radiotherapy and targeted hormone therapy necessitated by molecular subtyping. Aims: This study aimed to determine the level of adherence to breast cancer molecular subtyping among women with breast cancer attending tertiary health facilities in Imo State. Methodology: Immunohistochemistry reports of women with breast cancer attending tertiary health facilities in Imo State were retrieved from patient’s case files. Tissue blocks were also retrieved from tissue block archives of both hospitals for women who did not take up immunohistochemistry services after their initial diagnosis and also those whose immunohistochemistry reports were not found in their case files. Results: Among the 121 women that participated in the study, there were in all 74 (61.2%) had molecular subtyping of their tumour blocks. Up to 45 (37.2%) did not go for molecular subtyping of their tumour blocks while 2 (1.7%) were not sure whether they had or not. Conclusion: It, therefore, depicts that the rate of uptake was found as 61.2% among the participants and there is a need to create more awareness of the importance of molecular subtyping, which necessitates the use of targeted hormone therapy.

Effects of menopausal hormone therapy-based on the role of estrogens,progestogens,and their metabolites in proliferation of breast cancer cells

Menopausal hormone therapy(MHT)has been widely used for the clinical treatment of symptoms associated with menopause in women.However,the exact nature of the relationship between MHT and the increased risk of breast cancer has not been fully elucidated.The results of the Women’s Health Initiative’s randomized controlled clinical studies showed that estrogen monotherapy was associated with a lower incidence of breast cancer as compared to estrogen-progesterone combined therapy,with an elevated risk of breast cancer.The evidence currently available from randomized trials and observational studies is based on data from different populations,drug formulations,and routes of administration.Even though the risks of MHT and breast cancer have received a great deal of attention,information regarding the unpredictable toxicological risks of estrogen and progestogen metabolism needs to be further analyzed.Furthermore,the diversity and complexity of the metabolic pathways of estrogen and different progestogens as well as the association of the different estrogen and progestogen metabolites with the increased risk of breast cancer need to be adequately studied.Therefore,this review aimed to describe the biological effects of estrogen,progesterone,and their metabolites on the proliferation of breast cancer cells,based on relevant basic research and clinical trials,to improve our understanding of the biological functions of estrogen and progestogen as well as the safety of MHT.

Increased Cellular Invasion and Proliferation via Estrogen Receptor after 17-<i>β</i>-Estradiol Treatment in Breast Cancer Cells Using Stable Isotopic Labeling with Amino Acids in Cell Culture (SILAC)

17-β-estradiol (estrogen) is a steroid hormone important to human development;however, high levels of this molecule are associated with increased risk of breast cancer primarily due to estrogen’s ability to bind and activate the estrogen receptor (ER) and initiate gene transcription. Currently, estrogen mechanisms of action are classified as genomic and non-genomic and occur in an ER-dependent and ER-independent manner. In this study, we examine estrogen signaling pathways, by measuring changes in protein expression as a function of time of exposure to estrogen in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. Using a robust experimental design utilizing isotopic labeling, two-dimensional LC-MS, and bioinformatics analysis, we report genomic and non-genomic ER regulated estrogen responsive proteins. We find a little over 200 proteins differentially expressed after estrogen treatment. Cell proliferation, transcription, actin filament capping and cell to cell signaling are significantly enriched in the MCF-7 cell line alone. Translational elongation and proteolysis are enriched in both cell lines. Subsets of the proteins presented in this study are for the first time directly associated with estrogen signaling in mammary carcinoma cells. We find that estrogen affected the expression of proteins involved in numerous processes that are related to tumorigenesis such as increased cellular division and invasion in an ER-dependent manner. Moreover, we identified negative regulation of apoptosis as a non-genomic process of estrogen. This study complements gene expression studies and highlights the need for both genomic and proteomic analyses in unraveling the complex mechanisms by which estrogen affects progression of breast cancer.

LACKING EXON5 OF VARIANT ESTROGEN RECEPTOR IN HUMAN BREAST CANCER

Methods: The target sequence of ER RNA covering exon4(6(1082(1520bp) was amplified in 7 clinical human breast cancer tissues by reverse transcription and polymerase chain reaction (RTPCR) techniques. Results: PCR products were transferred to nitrocellulose membranes and hybridized using a [r32P]ATP labeled ER 29 oligonulceotide probe representing the antisense strand of the ER Cdna sequence 1271(1299. Specific bands were found at 438 and 300 base pairs in two tumors. The 300 base pair of PCR product was recovered from ER+/PR+ and ER+/PR tumor, respectively. Conclusion: Dideoxy sequence analysis revealed that they contained the variant ER completely missing exon 5.

Expression of ΔDNMT3B Variants and Its Association with Estrogen/Progestogen Receptor Status in Breast Cancer

Objective： Our previous study has showed that △DNMT3B is the predominant form of DNMT3B in non-small cell lung cancer （NSCLC）. In this study, we aimed to explore the expression patterns of the △DNMT3B variants in breast cancer and to identify whether the pattern was similar to that in NSCLC or not and its clinical significance. Methods： Expression of seven △DNMT3B variants in 59 breast cancer and the corresponding normal tissue was measured using RT-PCR. The correlations between the expressions of △DNMT3B variants and the clinical parameters including ER/PR status, clincopathologic feature and survivals were analyzed. Results： There were significant differences in the expression ratios of △DNMT3B1-7 variants between breast cancer tissues and normal tissues （P〈0.001）. The positive ratio of △DNMT3B1-7 variants were 66%, 71%, 17%, 51%, 76.2%, 50% and 61% in tumor tissue, respectively; while 16%, 8.4%, 3.38%, 3.38%, 11.8%, 13.5% and 5.08% in the corresponding normal tissue, which was different from the pattern of △DNMT3B1-7 expression in NSCLC （62%, 76%, 2.5%, 46%, 18%, 27% and 16% in tumor tissue, respectively; while 18%, 11%, 0%, 3.3%, 0%, 0% and 0% in normal lung tissue, respectively; P〈0.0001）. Expressions of △DNMT3B2, 3B4 and 3B7 were higher in the patients with negative estrogen receptor （ER） than those with positive estrogen receptor （P=0.035, P=0.0141 and P=0.0219, respectively）. △DNMT3B7 expression was higher for the patients with negative progestogen receptor （PR） compared to those with positive progestogen receptor （P=0.0379）. Expression ratio of △DNMT3B5 in stage Ⅲ tumors is lower than that in stage Ⅰ/Ⅱ ones （P= 0.041）. But we did not find any relation between the △DNMT3B variants and the patients＇ survival. Conclusion： The pattern of △DNMT3B variants in breast cancer is different from that in NSCLC. Expressions of △DNMT3B2, 3B4 and 3B7 are associated with estrogen receptors status. While △DNMT3B7 is associated with progestogen receptor. No relation between the △DNMT3B variants and the patients＇ survival were found.

Effect of Estrogen on Telomerase Activity in Human Breast Cancer Cells

To investigate the effects of estrogen(E 2) on telomerase activity and its mechanism in human breast cancer cells, estrogen receptor positive MCF-7 cells were treated with different concentrations of E 2 Telomerase activity was measured by using TRAP-ELISA method, the cell cycle phases analyzed by using flow cytometry, and the expression of Cyclin D 1 detected by using immunohistochemistry method The results showed that telomerase activity levels were increased in MCF-7 cells treated with 10 -8 mol/L E 2 during the observed period ( P <0 05), and E 2 increased telomerase activity levels in a dose-dependent manner(10 -10 -10 -8 mol/L); Simultaneously, the cell cycle phases of MCF-7 cells treated with 10 -8 mol/L E 2 were changed significantly: G 0/G 1 phase decreased from 60 52% to 50 93%, S phase increased from 29 03% to 30 83%; However, the expression of Cyclin D 1 was decreased It was concluded that estrogen can upregulate telomerase activity of MCF-7 cells, and the effect can be blocked by antiestrogen tamoxifen Its mechanism may be closely associated with modulation of cell cycle phases

Leptin influences estrogen metabolism and increases DNA adduct formation in breast cancer cells

Objective: The elevated incidence of obesity has been paralleled with higher risks of breast cancer. High adiposity increases leptin secretion from adipose tissue, which in turn increases cancer cell proliferation. The interplay between leptin and estrogen is one of the mechanisms through which leptin influences breast carcinogenesis. An unbalanced estrogen metabolism increases the formations of catechol estrogen quinones, DNA adducts, and cancer mutations. This study aims to investigate the effect of leptin on some estrogen metabolic enzymes and DNA adduction in breast cancer cells.Methods: High performance liquid chromatography(HPLC) was performed to analyze the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine. Reporter gene assay, real time reverse transcription polymerase chain reaction(real time RT-PCR), and Western blot were used to assess the expression of estrogen metabolizing genes and enzymes: Cytochrome P-4501B1(CYP1B1), Nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase1(NQO1), and Catechol-O-methyl transferase(COMT).Results: Leptin significantly increased the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine.Furthermore, leptin significantly upregulated CYP1B1 promoter activity and protein expression. The luciferase promoter activities of NQO1 and m RNA levels were significantly reduced. Moreover, leptin greatly reduced the reporter activities of the COMT-P1 and COMT-P2 promoters and diminished the protein expression of COMT.Conclusions: Leptin increases DNA adduct levels in breast cancer cells partly by affecting key genes and enzymes involved in estrogen metabolism. Thus, increased focus should be directed toward leptin and its effects on the estrogen metabolic pathway as an effective approach against breast cancer.

Promestriene Affects <i>GREB</i>1 Expression in Estrogen Sensitive Breast Cancer Cells

Promestriene (3-propyl ethyl, 17B-methyl estradiol) is a synthetic estrogen analogue with reported minimal systemic absorption which has been suggested for topical treatment of vaginal atrophy. Promestriene’s ability to stimulate proliferation and estrogen responsive gene expression was analyzed in estrogen receptor (ER+) positive breast cancer cell lines MCF-7, T-47D, and BT-474 using CFSE flow cytometric analysis, and quantitative RT-PCR analysis of GREB1 RNA expression, an estrogen responsive gene involved in estrogen receptor alpha expression. In estrogen replete conditions, Promestriene did not stimulate proliferation even at high concentrations (100,000 pg/ml). However, anti-estradiol depletion allowed low dose Promestriene (2 - 10 pg/ml) to stimulate GREB1 expression in all three cell lines at levels equal to that induced by estradiol (BT-474) or significantly higher than estradiol (MCF7 and T-47D). These findings suggest that Promestriene has the potential to support estrogen like cell signaling, a possible contraindication for use in treatment of vaginal atrophy associated with breast cancer aromatase inhibitor therapy.

Detection of Estrogen Responsive Breast Cancer Circulating Tumor Cells: Assay Development for Anti-Hormone Therapy Resistance

Recent clinical trials with histone deacetylase inhibitors (HDACi) have shown increased progression free survival by re-sensitizing resistant estrogen receptor positive (ER+) breast cancer cells to hormone suppressive therapies (HT). However, these trials lacked a sensitive, specific assay to identify and monitor HDACi/HT sensitive or resistant tumors. We tested detection of ER expression and histone acetylation of chromatin at the growth regulation by estrogen in breast cancer 1 (GREB1) gene, an estrogen-responsive gene involved in ER expression, in circulating tumor cell (CTC) as potential candidate assays for HDACi/HT sensitivity. ER+ and ER- CTC were detected and isolated from breast cancer patient peripheral blood by high speed fluorescence activated cell sorting (FACS) for use in mRNA analysis and anti-acetylated histone-mediated Chromatin Immunoprecipitation (ChIP). cDNA from mRNA and DNA extracted from the ChIP isolates were quantified by real-time PCR for GREB1. CTC isolates from patients who had an ER+ breast cancer primary contained both ER+ and ER- cells. More ER+ than ER- CTC was found in HT sensitive patients compared to HT resistant patients (p = 0.0559). GREB1 was found in acetylated histone chromatin from both ER+ and ER- CTC. The number of ER+ and ER- CTC found in peripheral blood appears to parallel patient outcomes as to their sensitivity to HT. Acetylated histone analysis can detect chromatin containing GREB1 in CTC, suggesting it may be useful as a more specific measure of HDACi effects on breast tumor cells. A larger, longitudinal data set following patients through HT/HDACi trials is needed to confirm these observations and their development for clinical use.

A Review Study on the Effects of Estrogen Level on Vaginal Candida spp.of Women with Estrogen-Related Receptor Breast Cancer

The aim of the present study have been reported to review the estrogen level in the patients with the breast cancer and healthy individuals.Breast cancer is one of the most common diseases in women worldwide that is characterized by uncontrolled growth of malignant cells in the mammary epithelial tissue.The estrogen was found at normal level in most patients with ER-positive breast cancer and in healthy individuals,while its high level was higher among patients with ER-negative breast cancer.Many studies show evidences about the role of estrogen at a high level on the development of breast cancer.The association between the estrogen levels and the presence of Candida spp.in vagina of patients with breast cancer was reviewed.