ERβ mRNA Research Articles

MON-008 Estrogen-Responsive and -Unresponsive Gene Expressions Promoted by Enhancers Specifically Hypomethylated in Endometriotic Cells May Become a Molecular Marker in Endometriosis Lesions

BACKGROUND: Endometriosis is an estrogen-dependent, inflammatory disease, and the role of estrogen is obvious because the symptoms associated with endometriosis often disappear after menopause, and GnRH agonists or progestin relieve the pelvic lesions and endometriosis-associated pain. However, there are limitations to these treatments that target the estrogen reduction in endometriotic lesions. We sought to define an aberrant gene expression derived from an epigenetic background in endometriosis.Objective: In the hope of overcoming the limitations of endocrine treatments in endometriosis, we examined estrogen receptor (ER)-dependent and -independent gene expressions promoted by active enhancers specifically hypomethylated in endometriotic cells. Patients: Institutional Review Boards approved this project. We obtained the informed consent from all patients. The chocolate cyst lining in ovaries of patients with endometriosis was the source of endometriotic tissue. As the control, the eutopic endometrial tissues were obtained from uteri of premenopausal women who had uterine leiomyoma.Methods: Stromal cells were prepared from endometriotic and endometrial tissues. Gene expression was examined using RT-PCR. The potential function of hypomethylated gene sequence as an active enhancer was evaluated by ChIP analysis using anti-H3K4me1 and anti-H3K27ac antibodies and eRNA expression analysis. Using ChIP-seq and ChIA-PET analysis in silico, ER-specific loci within gene bodies and the up- and downstream regions were extracted. ER-dependent gene expression was examined using estradiol or SERM.Results: ER expression in endometriotic cells.1) Relative expression of ERα mRNA was estimated to be one tenth of that in endometrial cells. 2) Relative expression of ERβ1 mRNA was 40-fold higher than that in endometrial cells, which is at a comparable level of the ERα. 3) ERβ2 mRNA expression was at a comparable level of the ERβ1. From our DNA methylation and gene expression analysis, 6 genes were selected and classified into 3 categories: estrogen-responsive genes with specific methylation (ESR1 and ESR2) or without any methylation (TGFα and GREB1), and estrogen-unresponsive but upregulated genes depending on specific hypomethylation (GATA6 and CYP19). 4) ChIP-seq and ChIA-PET analysis in silico suggested the presence of ER-specific loci within gene bodies and the up- and downstream in estrogen-responsive genes. 5) ChIP and eRNA expression analysis predicted active enhancer regions both in estrogen-responsive and -unresponsive genes. 6) In response to estrogen, TGFα and GREB1 expressions were upregulated, but ESR1 and ESR2 showed marginal responses.Conclusion: We focused on estrogen-responsive and -unresponsive genes linked to the epigenetic environment of endometriotic lesions, and revealed a facet of gene expression in endometriotic cells.

Detection and Characterization of Estrogen Receptor Beta Expression in the Brain with Newly Developed Transgenic Mice

Two types of nuclear estrogen receptors, ERα and ERβ, have been shown to be differentially involved in the regulation of various types of behaviors. Due to a lack of tools for identifying ERβ expression, detailed anatomical distribution and neurochemical characteristics of ERβ expressing cells and cellular co-expression with ERα remain unclear. We have generated transgenic mice ERβ-RFPtg, in which RFP was inserted downstream of ERβ BAC promotor. We verified RFP signals as ERβ by confirming: (1) high ERβ mRNA levels in RFP-expressing cells collected by fluorescence-activated cell sorting; and (2) co-localization of ERβ mRNA and RFP proteins in the paraventricular nucleus (PVN). Strong ERβ-RFP signals were found in the PVN, medial preoptic area (MPOA), bed nucleus of the stria terminalis, medial amygdala (MeA), and dorsal raphe nucleus (DRN). In the MPOA and MeA, three types of cell populations were identified; those expressing both ERα and ERβ, and those expressing exclusively either ERα or ERβ. The majority of PVN and DRN cells expressed only ERβ-RFP. Further, ERβ-RFP positive cells co-expressed oxytocin in the PVN, and tryptophan hydroxylase 2 and progesterone receptors in the DRN. In the MeA, some ERβ-RFP positive cells co-expressed oxytocin receptors. These findings collectively suggest that ERβ-RFPtg mice can be a powerful tool for future studies on ERβ function in the estrogenic regulation of social behaviors.

Effects of UHRF1 on Estrogen Receptor and Proliferation, Invasion and Migration of BCPAP Cells in Thyroid Papillary Carcinoma

To investigate the effects of ubiquitin-like PDH and ring finger domain 1 (UHRF1) on the expression ratio of estrogen receptor (ER) α/ERβ, and to explore the experimental mechanism of UHRF1 affecting the proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma. The protein and mRNA expressions of UHRF1, ERα and ERβ in normal thyroid Nthy-ori3-1 cells and thyroid papillary carcinoma BCPAP cells were detected by Western blot and qRT-PCR. BCPAP cells were treated with Scrambled siRNA and UHRF1 siRNA, respectively. The expressions of ER α and ER β mRNAs were detected by qRT-PCR. MTT and Transwell were used to determine the proliferation, invasion and migration in each group of BCPAP cells. Compared with Nthy-ori3-1 cells, the expressions of UHRF1 and ERα proteins and mRNAs in BCPAP cells were significantly up-regulated ( P<0.05), while the expressions of ERβ protein and mRNA were significantly down-regulated ( P<0.05). Compared with the control group and Scrambled siRNA group, the expression of ER α mRNA in BCPAP cells transfected with UHRF1 siRNA was significantly decreased ( P<0.05), while the expression of ER β mRNA was significantly increased ( P<0.05). The proliferation, invasion and migration of BCPAP cells transfected with UHRF1 siRNA were significantly decreased ( P<0.05). UHRF1 upregulates ERα/ERβ expression ratio and promotes proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma.

23P Clinical, pathological and gene expression features of HER2-low breast cancer

Novel antibody-drug conjugates against HER2 are showing high activity in clinically HER2-negative (cHER2-) breast cancer (BC) with low HER2 expression. However, the clinical and molecular features of cHER2-/HER2-low BC are yet to be elucidated. We collected retrospective data from 8 multicenter cHER2- BC datasets, including 4 clinical trials. HER2 status in each study was determined using standard FDA-approved antibodies and ISH-techniques and classified according to the ASCO/CAP guidelines. For this study, tumors were regrouped in HER2 0 (0 score) and HER2-low (1+ or 2+ with ISH-negativity). The following variables were compared between the 2 groups in all patients and according to hormone receptor (HR) status: age, grade, ki67, histotype, tumor size, HR, HER2 and nodal status. nCounter-based PAM50 subtypes distribution and the expression of the 50 PAM50 genes, including ERBB2, were also compared. A total of 3,136 patients with cHER2- disease (57.4% HER2-low and 42.6% HER2 0) were evaluated. Overall, 888 (28.3%) tumor samples came from metastatic sites. No statistically significant differences were found regarding clinicopathological variables between HER2-low and HER2 0. Within HR-positive (+) disease (n=2,497), 63.2% and 36.8% of tumors were HER2-low and HER2 0, respectively. Subtype distribution was similar across HR+/HER2-low and HR+/HER2 0. A total of 45/50 PAM50 genes were found differentially expressed between HR+/HER2-low and HR+/HER2 0 (False Discovery Rate [FDR]<5%). High expression of luminal (e.g. ESR1 and FOXA1) and ERBB2, and low expression of proliferation-related genes (e.g. MKI67) was found in HER2-low compared to HER2 0. Within triple negative BC (TNBC) (n=622), 34.2% were HER2-low and 65.8% were HER2 0. Subtype distribution was similar across TNBC/HER2-low and TNBC/HER2 0. No PAM50 gene was found differentially expressed between TNBC/HER2-low and TNBC/HER2 0 (FDR≥5%). Finally, ERBB2 mRNA levels were higher in HER2-low/HR+ tumors than HER2-low/TNBC (p<0.001). HER2-low disease within clinically HER2- BC is frequent. However, significant differences exist according to HR status. Compared to HER2-low/TNBC, HER2-low/HR+ disease is a more distinct biological entity and has higher ERBB2 expression.

2O ERBB3 mRNA expression in breast cancer (BC): A SOLTI biomarker discovery analysis

Recently, HER3 has received attention as new anti-HER3 antibody-drug conjugates (e.g. U3-1402) are showing activity in BC. The most common method to determine HER3 expression is immunohistochemistry (IHC)-based assays. However, technical limitations exist when using IHC, such as different sensitivities of the antibodies and its subjectivity in scoring and cut-off determination. To overcome those limitations, we developed an mRNA-based ERBB3 expression assay using FFPE BC tissues and the Nanostring nCounter platform.

Single-Cell Gene Profiling Reveals Social Status-Dependent Modulation of Nuclear Hormone Receptors in GnRH Neurons in a Male Cichlid Fish.

Gonadotropin-releasing hormone (GnRH) is essential for the initiation and maintenance of reproductive functions in vertebrates. To date, three distinct paralogue lineages, GnRH1, GnRH2, and GnRH3, have been identified with different functions and regulatory mechanisms. Among them, hypothalamic GnRH1 neurons are classically known as the hypophysiotropic form that is regulated by estrogen feedback. However, the mechanism of action underlying the estrogen-dependent regulation of GnRH1 has been debated, mainly due to the coexpression of low levels of estrogen receptor (ER) genes. In addition, the role of sex steroids in the modulation of GnRH2 and GnRH3 neurons has not been fully elucidated. Using single-cell real-time PCR, we revealed the expression of genes for estrogen, androgen, glucocorticoid, thyroid, and xenobiotic receptors in GnRH1, GnRH2, and GnRH3 neurons in the male Nile tilapia Oreochromis niloticus. We further quantified expression levels of estrogen receptor genes (ERα, ERβ, and ERγ) in three GnRH neuron types in male tilapia of two different social statuses (dominant and subordinate) at the single cell level. In dominant males, GnRH1 mRNA levels were positively proportional to ERγ mRNA levels, while in subordinate males, GnRH2 mRNA levels were positively proportional to ERβ mRNA levels. These results indicate that variations in the expression of nuclear receptors (and possibly steroid sensitivities) among individual GnRH cells may facilitate different physiological processes, such as the promotion of reproductive activities through GnRH1 neurons, and the inhibition of feeding and sexual behaviors through GnRH2 neurons.

Identification of Novel C-Terminally Truncated Estrogen Receptor β Variant Transcripts and Their Distribution in Humans.

The nuclear receptor genes, including estrogen receptor β (ERβ), contain non-conventional internal and terminal exons, and alternative choice of the exons yields multiple mRNA and protein variants with unique structures and functions. However, the genomic structure of the intronic and 3'-downstream regions of the human ERβ gene and the presence of novel ERβ variants with non-conventional sequences have not been re-examined in about 20 years. Therefore, we attempted to re-characterize the structure of the human ERβ gene and identify novel non-conventional exons and distinct splice variants. Rapid amplification of cDNA 3'-end and RT-PCR cloning were used to isolate human ERβ mRNA variants from the testis. The identified cDNA sequences were mapped on the human genome assembly. Expression profiles of the variants were assessed by RT-PCR analysis. We cloned multiple ERβ mRNA variants with novel nucleotide sequences from the testis and identified several alternative splice sites, 3'-elongation of conventional coding exons, and novel terminal exons in the human ERβ gene. The variants encode C-terminally truncated ERβ proteins termed ERβ6, ERβ7, ERβEx. 4L, and ERβEx. 6L. Furthermore, we identified exon 7-defective forms of ERβ2/βcx, ERβ4, ERβ6, and ERβ7. Subsequently, we noted distinct expression patterns of the variants in human peripheral organs and brain subregions. This study clarified complicated genomic organization and splicing patterns of the human ERβ gene that contribute to the distinct heterogeneity of human ERβ mRNAs and proteins.

Spatial and temporal changes in the expression of steroid hormone receptors in mouse model of endometriosis.

Endometriosis is recognized as a steroid hormone-dependent disorder. However, controversies exist regarding the status of the steroid hormone receptor expression in endometriotic tissues. The purpose of this study was to determine the ontogeny of cellular changes in the expression of estrogen receptors (ERα, ERβ), G protein-coupled estrogen receptor 1 (GPER1), and progesterone receptors (PRs) in endometriosis using a mouse model. We used the autologous uterine tissue transfer mouse model and studied the mRNA and protein expression of ERα, ERβ, GPER1, and PR in ectopic lesions at 2, 4, and 8weeks of induction of endometriosis. As compared to endometrium of controls, in the ectopic endometrium, ERα is reduced while ERβ was elevated in stromal cells; however, Gper1 and PR levels are reduced in both stromal and epithelial cells in a time-specific manner. There is a high inter-animal variation in thelevels of these receptors in ectopic endometrium as compared to controls; the levels also varied by almost 100-fold within the same lesion resulting in "micro-heterogeneity." The expression of all these receptors also deferred between two lesions from the same animal. In the endometriotic tissue, there is extensive inter-animal and intra-lesion heterogeneity in the expression of ERα, ERβ, GPER1, and PR. These changes are not due to the influence of the peritoneal environment but appear to be tissue intrinsic. We propose that the variable outcomes in hormonal therapy for endometriosis could be possibly due to heterogeneity in the expression of steroid hormone receptors in the ectopic endometrium.

Effects of Paeonia lactiflora Extract on Estrogen Receptor β, TPH2, and SERT in Rats with PMS Anxiety.

This study is to investigate the effect of Paeonia lactiflora extract on PMS anxiety and on expression of estrogen receptor β (ERβ), tryptophan hydroxylase-2 (TPH2), and serotonin transporter (SERT) in the premenstrual syndrome (PMS) anxiety model rats. The vaginal smear and open field test were used to screen rats in nonreception phase of estrus cycle with similar macroscopic behaviors and regular estrus cycle. PMS anxiety model rats were prepared by electrical stimulation. RT-PCR and immunofluorescence were used to measure the expression of ERβ, TPH2, and SERT. Compared with normal rats, the total distance in the open field test of the model rats was significantly increased (P < 0.05). The model rats showed nervous alertness, irritability, and sensitivity to external stimuli. After treatment with the Paeonia lactiflora extract, the total distance of rats was significantly reduced (P < 0.05). In reception stage, there was no significant difference in the mRNA and protein expression of ERβ, TPH2, and SERT. In nonreception stage, the expression of ERβ and TPH2 in the model group was significantly decreased (P < 0.05) as compared with the control group, but not SERT. Abnormal changes of the above indicators were reversed after the administration of the Paeonia lactiflora extract. In conclusion, Paeonia lactiflora extract can increase the expression of ERβ and TPH2 and decrease SERT in PMS model rats, which may be one of the mechanisms underlying the effect of Paeonia lactiflora extract on PMS.

Ventral prostate and mammary gland phenotype in mice with complete deletion of the ERβ gene

Disagreements about the phenotype of estrogen receptor β (ERβ) knockout mouse, created by removing the DNA-binding domain of the ERβ gene or interruption of the gene with a neocassette (Oliver Smithies ERβ knockout mice [ERβOS-/-]), prompted us to create an ERβ knockout mouse by deleting the ERβ gene with the use of CRISPR/Cas9 technology. We confirmed that the ERβ gene was eliminated from the mouse genome and that no ERβ mRNA or protein was detectable in tissues of this mouse. Overall the phenotype of the ventral prostate (VP) and mammary gland (MG) in ERβcrispr-/- mice was similar to, but more severe than, that in the ERβOS-/-mice. In the VP of 6-mo-old ERβcrispr-/- mice there was epithelial hyperplasia, fibroplasia, inflammation, stromal overgrowth, and intraductal cancer-like lesions. This was accompanied by an increase in Ki67 and P63 and loss in DACH1 and PURα, two androgen receptor (AR) repressors. In the MG there was overexpression of estrogen receptor α and progesterone receptor, loss of collagen, increase in proliferation and expression of metalloproteases, and invasive epithelium. Surprisingly, by 18 mo of age, the number of hyperplastic foci was reduced, the ducts of the VP and MG became atrophic, and, in the VP, there was massive immune infiltration and massive desquamation of the luminal epithelial cells. These changes were coincident with reduced levels of androgens in males and estrogens in females. We conclude that ERβ is a tumor suppressor gene in the VP and MG where its loss increases the activity AR and ERα, respectively.

English

Recognized for its traditional roles, estrogen is ever-present in all vertebrates, regulates reproduction by binding and activating estrogen receptors (ERs), and also controls several functions of vertebrates, including reproductive immune, and central nervous systems. In order to access any other possible functions of the estrogen receptors in the development of the juvenile Hybrid grouper (Epinephelus fuscoguttatus â™€× Epinephelu polyphekadion ♂), full-length of ERs cDNA sequences were isolated and analyses were found to be 2391 bp for hgERα, 2626 bp for gERb1 and 2339 bp for hgERb2, respectively. The results of amino acid and phylogenetic analysis revealed that each hgER was grouped in consistent taxonomic groups of perciformes and demonstrated great evolutional conservation in functional domains. Real-time PCR examination discovered that the receptors expressed in all tissues examined, though, at a different level, the ERα mRNA level expressed higher than ERβ1, and ERβ2 in tissues examined. The ERα mRNA level of expression was found to be highest in the tissue of the heart, followed by muscle, and liver. The ERβ1 mRNA level was greatest in heart tissue, trailed by liver and muscle and ERβ2 was highest in the heart trailed by stomach and liver. The minimal expression was recorded in the kidney, the gill, and the brain for ERα, ERβ1, and ERβ2 respectively. These results put forward that steroid hormone estrogen receptors might be playing a significant part in the controlling of social behavior complexity, plasticity behavior, and the assessment of a gratifying inducement in Hybrid grouper. Key words: Estrogen receptors, Real-time POR, tissue expression, hybrid grouper.

Biomarker Analyses of Response to Cyclin-Dependent Kinase 4/6 Inhibition and Endocrine Therapy in Women with Treatment-Naïve Metastatic Breast Cancer.

Preclinical data identified the cyclin-dependent kinase 4/6 (CDK4/6) inhibitor palbociclib as synergistic with antiestrogens in inhibiting growth of hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) human breast cancer models. This observation was validated clinically in the randomized, placebo-controlled, phase III PALOMA-2 study. To determine markers of sensitivity and resistance to palbociclib plus letrozole, we performed comprehensive biomarker analyses, investigating the correlation with progression-free survival (PFS), on baseline tumor tissues from PALOMA-2. Despite a broad biomarker search, palbociclib plus letrozole demonstrated consistent PFS gains versus placebo plus letrozole, with no single biomarker or cassette of markers associated with lack of benefit from combination treatment. Palbociclib plus letrozole confers efficacy on both luminal A and B patients. Higher CDK4 levels were associated with endocrine resistance which was mitigated by the addition of palbociclib, whereas lower PD-1 levels were associated with greater palbociclib plus letrozole benefit. Tumors with more active growth factor signaling, as exemplified by increased expression of FGFR2 and ERBB3 mRNA, appeared to be associated with greater PFS gain from the addition of palbociclib. These data underscore the importance of CDK4/6 signaling in HR+/HER2- breast cancer and suggest that the interplay between steroid hormone and peptide growth factor signaling could drive dependence on CDK4/6 signaling.See related commentary by Anurag et al., p. 3.

Cytotoxic Effects of the Dual ErbB Tyrosine Kinase Inhibitor, Lapatinib, on Walker 256 Rat Breast Tumour and IEC-6 Rat Normal Small Intestinal Cell Lines.

Lapatinib is an orally administered, dual ErbB1/ErbB2 tyrosine kinase inhibitor (TKI). It is effective in ErbB2 + ve breast cancer treatment. However, lapatinib is associated with diarrhoea with an incidence of 47–75%. The mechanism of ErbB1 TKI-induced diarrhoea remains unclear. ErbB1 or epidermal growth factor receptor (EGFR) is expressed in gastrointestinal mucosa whereby the primary site for drug absorption is intestine. Thus, administration of ErbB1 oral TKI may disrupt gut homeostasis, leading to diarrhoea. Nevertheless, further investigations are required. We observed that lapatinib inhibited 50% Walker 256 breast tumour cells and IEC-6 small intestinal cell growth. Higher percentage of necrosis was observed in lapatinib-treated Walker 256. Lapatinib-treated IEC-6 showed higher percentage of late apoptosis. Only ErbB2 mRNA was detected in Walker 256 but both ErbB1 and ErbB2 mRNAs were detected in IEC-6, yet both protein staining were detected in both cells. Lapatinib exhibited cytotoxic properties on ErbB1/ErbB2 expressing cell lines, with intestinal cells being more sensitive to lapatinib compared to tumour cells. Lapatinib induced necrosis in tumour cells, while inducing late apoptosis in intestinal cells may explain lapatinib-induced diarrhoea in patients administered with the drug which could be due to apoptosis of intestinal epithelial cells leading to barrier disruption and consequently diarrhoea.

Sex-dependent regulation of estrogen receptor beta in human colorectal cancer tissue and its relationship with clock genes and VEGF-A expression.

The incidence of colorectal cancer (CRC) shows a sex-dependent difference in humans. The aim of this study was to analyze estrogen receptor beta mRNA (ERbeta) expression in patients with CRC with respect to their gender and clinicopathological features. Since cancer progression is accompanied by tumor vascularization, VEGF-A (vascular endothelial growth factor A) transcription was analyzed along with ERbeta mRNA. ERbeta mRNA was also correlated with the expression of clock genes, which are known to influence the cell cycle. ERbeta mRNA expression in females with CRC showed an inverse association with increasing tumor staging that was not observed in males. Lower levels of ERbeta mRNA were observed in females with a higher clinical stage compared with those with earlier-stage tumors. ERbeta mRNA expression showed a significant positive correlation with mRNA of clock genes period 2 and cryptochrome 2 in healthy but not in cancerous tissue in males. Expression of VEGF-A mRNA showed a negative correlation with ERbeta mRNA after splitting of the cohort according to gender and nodus involvement. We propose that gender differences in ERbeta mRNA expression in tumors during the early stages of CRC can partially explain the lower occurrence of CRC in females compared with males.

Effects of Puerarin on the Ovariectomy-Induced Depressive-Like Behavior in ICR Mice and Its Possible Mechanism of Action.

Daily treatment of ovariectomized (OVX) ICR mice with puerarin, a glycosyl isoflavone isolated from the root bark of Pueraria candollei var. mirifica, and 17β-estradiol attenuated ovariectomy-induced depression-like behavior, as indicated by a decrease in immobility times in the tail suspension test (TST) and the forced swimming test (FST), an increase in the uterine weight and volume, a decrease in serum corticosterone levels, and dose-dependently normalized the downregulated transcription of the brain-derived neurotrophic factor (BDNF) and estrogen receptor (Erβ and Erα) mRNAs. Like 17β-estradiol, puerarin also inhibited ovariectomy-induced suppression of neurogenesis in the dentate gyrus of the hippocampus (increased the number of doublecortin (DCX)-immunosuppressive cells). These results suggest that puerarin exerts antidepressant-like effects in OVX animals, possibly by attenuating the OVX-induced hyperactivation of the HPA axis and/or normalizing the downregulated transcription of BDNF and ER mRNA in the brain.

Blocking of Estradiol Receptors ERα, ERβ and GPER During Development, Differentially Alters Energy Metabolism in Male and Female Rats

Estradiol not only participates in the regulation of energy metabolism in adulthood, but also during the first stages of life as it modulates the alterations induced by under- and over-nutrition. The objectives of the present study were to determine: 1) If estradiol is involved in the normal programming of energy metabolism in rats; 2) If there is a specific window of time for this programming and 3) If males and females are differentially vulnerable to the action of this hormone. Estrogen receptors (ER) α, ERβ and GPER were blocked by their specific antagonists MPP, PHTPP and G15, respectively, from postnatal day (P) 1 (the day of birth) to P5 or from P5 to P13. Physiological parameters such as body weight, fat depots and caloric intake were then analysed at P90. Hypothalamic AgRP, POMC, MC4R, ERα, ERβ and GPER mRNA levels and plasma levels of estradiol, were also studied. We found that blocking ER receptors from P5 to P13 significantly decreases long-term body weight in males and hypothalamic POMC mRNA levels in females. The blocking of ERs from P1 to P5 only affected plasma estradiol levels in females. The present results indicate programming actions of estradiol from P5 to P13 on body weight in male and POMC expression in female rats and emphasize the importance of including both sexes in metabolic studies. It is necessary to unravel the mechanisms that underlie the actions of estradiol on food intake, both during development and in adulthood, and to determine how this programming differentially takes place in males and females.

The MS-lincRNA landscape reveals a novel lincRNA BCLIN25 that contributes to tumorigenesis by upregulating ERBB2 expression via epigenetic modification and RNA\u2013RNA interactions in breast cancer

The landscape of molecular subtype-specific long intergenic noncoding RNAs (MS-lincRNAs) in breast cancer has not been elucidated. No study has investigated the biological function of BCLIN25, serving as a novel HER2 subtype-specific lincRNA, in human disease, especially in malignancy. Moreover, the mechanism of BCLIN25 in the regulation of ERBB2 expression remains unknown. Our present study aimed to investigate the role and underlying mechanism of BCLIN25 in the regulation of ERBB2 expression. The transcriptional landscape across five subtypes of breast cancer was investigated using RNA sequencing. Integrative transcriptomic analysis was performed to identify the landscape of novel lincRNAs. Next, WEKA was used to identify lincRNA-based subtype classification and MS-lincRNAs for breast cancer. The MS-lincRNAs were validated in 250 breast cancer samples in our cohort and datasets from The Cancer Genome Atlas and Gene Expression Omnibus. Furthermore, BCLIN25 was selected, and its role in tumorigenesis was examined in vitro and in vivo. Finally, the mechanism by which BCLIN25 regulates ERBB2 expression was investigated in detail. A total of 715 novel lincRNAs were differentially expressed across five breast cancer subtypes. Next, lincRNA-based subtype classifications and MS-lincRNAs were identified and validated using our breast cancer samples and public datasets. BCLIN25 was found to contribute to tumorigenesis in vitro and in vivo. Mechanistically, BCLIN25 was shown to increase the expression of ERBB2 by enhancing promoter CpG methylation of miR-125b, leading to miR-125b downregulation. In turn, ERBB2 mRNA degradation was found to be abolished due to decreased binding of miR-125b to the 3’-untranslated region (UTR) of ERBB2. These findings reveal the role of novel lincRNAs in breast cancer and provide a comprehensive landscape of breast cancer MS-lincRNAs, which may complement the current molecular classification system in breast cancer.

LPS impairs steroidogenesis and ROS metabolism and induces PPAR transcriptional activity to disturb estrogen/androgen receptor expression in testicular cells

Inflammation can deregulate the testicular functions of steroidogenesis and spermatogenesis, consequently contributing to male infertility. Animals and cells treated with lipopolysaccharide (LPS) exhibit infection- and inflammation-induced testicular dysfunction. However, the precise mechanisms affecting steroidogenesis and spermatogenesis in response to LPS-treatment remain poorly understood. We isolated distinct testicular cells including spermatocytes, round spermatids and late spermatids to analyze distribution of peroxisome proliferator-activated receptor (PPAR) family, plays central roles in the regulation of metabolism. Our results suggested Pparα/Pparγ mRNA was highly expressed in late spermatids, while Pparβ mRNA was highly expressed in round spermatids. To analyze the effect of LPS on testicular cells, we established an LPS infection model using primary Sertoli cells and testicular cell lines (TM4, GC2 and MLTC1). We observed that PPARγ and SIRT1 were concentrated in the nuclear region and that the mRNA expression levels of antioxidative enzymes (Cat and Homx1) and PPARγ were upregulated in primary Sertoli cells after LPS-treatment. Moreover, luciferase reporter gene assays of the testicular cell lines revealed that the activity of the PPAR response element (PPRE) was significantly increased. Importantly, the transcriptional activity of the androgen response element was significantly reduced, whereas activity of estrogen response element was strongly induced in LPS-treated TM4 cells, consistent with the RT-PCR results. Meanwhile, the qRT-PCR results revealed that the LPS-induced upregulation of Ar mRNA in MLTC1 cells and Erβ mRNA in TM4 cells were significantly recovered after treatment with the specific PPARγ-antagonist GW9662. In addition, we also found that LPS induced alterations in enzymes involved in steroidogenesis in testicular cell lines. Taken together, our results revealed that LPS may induce PPAR transcriptional activity to disturb estrogen/androgen receptor expression and impair steroidogenesis and ROS metabolism in testicular cells.

Early-life exposure to 17β-estradiol and 4-nonylphenol impacts the growth hormone/insulin-like growth-factor system and estrogen receptors in Mozambique tilapia, Oreochromis mossambicus

It is widely recognized that endocrine disrupting chemicals (EDCs) released into the environment through anthropogenic activities can have short-term impacts on physiological and behavioral processes and/or sustained or delayed long-term developmental effects on aquatic organisms. While numerous studies have characterized the effects of EDCs on temperate fishes, less is known on the effects of EDCs on the growth and reproductive physiology of tropical species. To determine the long-term effects of early-life exposure to common estrogenic chemicals, we exposed Mozambique tilapia (Oreochromis mossambicus) yolk-sac fry to 17β-estradiol (E2) and nonylphenol (NP) and subsequently characterized the expression of genes involved in growth and reproduction in adults. Fry were exposed to waterborne E2 (0.1 and 1 μg/L) and NP (10 and 100 μg/L) for 21 days. After the exposure period, juveniles were reared for an additional 112 days until males were sampled. Gonadosomatic index was elevated in fish exposed to E2 (0.1 μg/L) while hepatosomatic index was decreased by exposure to NP (100 μg/L). Exposure to E2 (0.1 μg/L) induced hepatic growth hormone receptor (ghr) mRNA expression. The high concentration of E2 (1 μg/L), and both concentrations of NP, increased hepatic insulin-like growth-factor 1 (igf1) expression; E2 and NP did not affect hepatic igf2 and pituitary growth hormone (gh) levels. Both E2 (1 μg/L) and NP (10 μg/L) induced hepatic igf binding protein 1b (igfbp1b) levels while only NP (100 μg/L) induced hepatic igfbp2b levels. By contrast, hepatic igfbp6b was reduced in fish exposed to E2 (1 μg/L). There were no effects of E2 or NP on hepatic igfbp4 and igfbp5a expression. Although the expression of three vitellogenin transcripts was not affected, E2 and NP stimulated hepatic estrogen receptor (erα and erβ) mRNA expression. We conclude that tilapia exposed to E2 and NP as yolk-sac fry exhibit subsequent changes in the endocrine systems that control growth and reproduction during later life stages.

119P - ERBB2 mRNA as a predictor in HER2-positive (HER2+)/hormone receptor-positive (HR+) metastatic breast cancer (BC) treated with HER2 blockade in combination with endocrine therapy (ET): A retrospective analysis of the ALTERNATIVE and SOLTI-PAMELA trials

BackgroundThe ALTERNATIVE trial randomized 355 patients (pts) with HER2+/HR+ metastatic BC to receive as 1st-line therapy lapatinib (L) + trastuzumab (T) + aromatase inhibitors (AI) or T + AI or L + AI. The neoadjuvant PAMELA trial tested a chemo-free regimen of L + T on HER2+ pts, combined with letrozole or tamoxifen in HR+ tumors. We explored the hypothesis that gene expression may help predicting benefit from anti-HER2 therapy in combination with ET. MethodsThe expression of 55 BC-related genes was evaluated from FFPE tumors using the nCounter. The PAM50 subtype distribution and the association of the expression of each gene (continuous variable) with progression-free survival (PFS) was evaluated using univariate Cox-models. Median PFS was calculated using the Kaplan-Meier method. Clinical benefit (CB) was defined as complete or partial response or stable disease at 6 months. The Cutoff Finder tool was used to find an optimal gene expression cut-off with CB as the endpoint. Logistic regression was used to evaluate the association of gene expression with pathologic complete resonse (pCR) and CB. ResultsIn ALTERNATIVE, 60 tumors (16.9%) were analyzed: 57% HER2-enriched, 20% Luminal B, 12% Luminal A, 8% Normal-like and 3% Basal-like. Median PFS in ERBB2-high group (above the median) was higher compared to ERBB2-low group (11.0 vs 5.6 months; Hazard Ratio [HazR]=0.49; p=0.006). ERBB2 was found more expressed in pts with CB. CB rate was higher in the ERBB2-high group compared to ERBB2-low group (54% vs 22%; p=0.013). An optimal ERBB2 mRNA cutoff (2.923) for predicting CB (AUC=0.68; odds ratio [OR]=1.49, p=0.014; PFS HazR=0.46, p=0.022) was then identified. The same ERBB2 cutoff in PAMELA baseline tumor samples (n=77) was found significantly associated with pCR (43.8% in ERBB2-high vs. 11.5% in ERBB2-low; adjusted OR=4.45; p=0.041). ConclusionsERBB2 mRNA expression is a robust predictor of response and survival outcome in HER2+/HR+ BC following HER2-blockade and ET. Our study identifies a common biomarker between pCR improvement (OR ∼4.5) in early disease and CB in the advanced setting (PFS HazR of ∼0.50). Legal entity responsible for the studyInstitut d’investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). FundingHas not received any funding. DisclosureN. Chic: Travel / Accommodation / Expenses: Eisai. F. Schettini: Travel / Accommodation / Expenses: Celgene; Travel / Accommodation / Expenses: Pfizer. M. Vidal: Speaker Bureau / Expert testimony: Novartis; Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert testimony: Eisai; Speaker Bureau / Expert testimony: Daiichi Sankyo. M. Muñoz: Travel / Accommodation / Expenses: Roche. J. Cortés: Honoraria (self): Novartis; Honoraria (self): Eisai; Honoraria (self): Roche; Advisory / Consultancy: Roche/Genentech; Advisory / Consultancy: Celgene; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Biothera Pharmaceutical; Advisory / Consultancy: Merus; Advisory / Consultancy: Seattle Genetics. A. Llombart-Cussac: Advisory / Consultancy: Novartis; Advisory / Consultancy: Roche/Genentech. M. Rimawi: Advisory / Consultancy: GlaxoSmithKline; Advisory / Consultancy: Roche/Genentech; Advisory / Consultancy: MacroGenics; Advisory / Consultancy: Novartis; Advisory / Consultancy: Daiichi Sankyo. A. Prat: Advisory / Consultancy: Nanostring Techonologies. All other authors have declared no conflicts of interest.