Increased Estrogen Receptor Research Articles

Formononetin inhibits neuroinflammation and increases estrogen receptor beta (ERβ) protein expression in BV2 microglia

Formononetin is a bioactive non-steroidal polyphenol found in a variety of plants. In this study we evaluated the effects of formononetin on neuroinflammation in LPS-stimulated BV2 microglia. Results showed that formononetin significantly reduced the production of TNF-α, IL-6 and IL-1β, nitrite and PGE2, as well as protein levels of iNOS and COX-2. Reporter gene assays showed that formononetin produced inhibition of NF-κB luciferase activity in HEK293 cells stimulated with TNF-α. Immunoblotting experiments revealed an inhibition of IKKα phosphorylation, with the resultant attenuation of phosphorylation and degradation of IκBα following LPS stimulation. Formononetin also produced an inhibition of nuclear translocation and DNA binding by NF-κB following LPS stimulation. RNAi experiments showed that transfection of BV2 microglia with ERβ siRNA resulted in the loss of anti-inflammatory action of formononetin. MTT assay and MAP2 immunoreactivity experiments showed that formononetin produced significant neuroprotective activity by preventing BV2 microglia conditioned media-induced toxicity to HT22 neurons. Investigations on the effect of formononetin on MCF7 breast cancer cells revealed that, while the compound significantly increased ER-luciferase activity, its effects on proliferation were modest. This study has established that formononetin inhibits neuroinflammation by targeting NF-κB signalling pathway in BV2 microglia, possibly through mechanisms involving ERβ. Formononetin appears to modulate ERβ in MCF7 breast cancer cells with limited proliferative effect. Formononetin could therefore serve as a chemical scaffold for the development of novel compounds which have selective neuroprotective actions in the brain.

Previous estradiol treatment in ovariectomized mice provides lasting enhancement of memory and brain estrogen receptor activity

• Ovariectomized mice received continuous estradiol or vehicle treatment. • After 40 days all treatments were terminated. • One month after treatments ended memory and estrogen receptor activity were assayed. • Previous estradiol enhanced hippocampus-dependent memory. • Previous estradiol increased estrogen receptor activation in hippocampus and cortex.

Estradiol Activates PI3K/Akt/GSK3 Pathway Under Chronic Neurodegenerative Conditions Triggered by Perinatal Asphyxia.

Perinatal asphyxia (PA) remains as one of the most important causes of short-term mortality, psychiatric and neurological disorders in children, without an effective treatment. In previous studies we have observed that the expression of different neurodegenerative markers increases in CA1 hippocampal area of 4-months-old male rats born by cesarean section and exposed for 19 min to PA. We have also shown that a late treatment with 17β estradiol (daily dose of 250 μg/kg for 3 days) was able to revert the brain alterations observed in those animals. Based on these previous results, the main aim of the present study was to explore the mechanism by which the estrogenic treatment is involved in the reversion of the chronic neurodegenerative conditions induced by PA. We demonstrated that estradiol treatment of adult PA exposed animals induced an increase in estrogen receptor (ER) α and insulin-like growth factor receptor (IGF-1R) protein levels, an activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 beta/β-catenin signaling pathway and an increase in Bcl-2/Bax ratio in the hippocampus in comparison to PA exposed animals treated with vehicle. Taking together, our data suggest that the interaction between ERα and IGF-IR, with the subsequent downstream activation, underlies the beneficial effects of estradiol observed in late treatment of PA.

Uterine progesterone signaling is a target for metformin therapy in PCOS-like rats.

Impaired progesterone (P4) signaling is linked to endometrial dysfunction and infertility in women with polycystic ovary syndrome (PCOS). Here, we report for the first time that elevated expression of progesterone receptor (PGR) isoforms A and B parallels increased estrogen receptor (ER) expression in PCOS-like rat uteri. The aberrant PGR-targeted gene expression in PCOS-like rats before and after implantation overlaps with dysregulated expression of Fkbp52 and Ncoa2, two genes that contribute to the development of uterine P4 resistance. In vivo and in vitro studies of the effects of metformin on the regulation of the uterine P4 signaling pathway under PCOS conditions showed that metformin directly inhibits the expression of PGR and ER along with the regulation of several genes that are targeted dependently or independently of PGR-mediated uterine implantation. Functionally, metformin treatment corrected the abnormal expression of cell-specific PGR and ER and some PGR-target genes in PCOS-like rats with implantation. Additionally, we documented how metformin contributes to the regulation of the PGR-associated MAPK/ERK/p38 signaling pathway in the PCOS-like rat uterus. Our data provide novel insights into how metformin therapy regulates uterine P4 signaling molecules under PCOS conditions.

Cellular events during ovine implantation and impact for gestation.

The establishment of pregnancy in sheep includes elongation of the blastocyst into a filamentous conceptus, pregnancy recognition, production of histotroph, attachment of the conceptus to the endometrium for implantation, and development of synepitheliochorial placentation. These processes are complex, and this review describes some of the molecular events that underlie and support successful pregnancy. The free-floating sheep blastocyst elongates into a filamentous conceptus and metabolizes, or is responsive to, molecules supplied by the endometrium as histotroph. Amongst these molecules are SPP1, glucose and fructose, and arginine that stimulate the MTOR nutrient sensing system. The placental trophectoderm of elongating conceptuses initiate pregnancy recognition and implantation. The mononucleate cells of the trophectoderm secrete IFNT, which acts on the endometrial LE to block increases in estrogen receptor α to preclude oxytocin receptor expression, thereby preventing oxytocin from inducing luteolytic pulses of PGF2α. In addition, IFNT increases expression of IFN stimulated genes in the endometrial stroma, including ISG15, a functional ubiquitin homologue. Implantation is the initial step in placentation, and includes sequential pre-contact, apposition, and adhesion phases. Implantation in sheep includes downregulation of Muc1 and interaction of GLYCAM1, galectin 15 (LGALS15) and SPP1 with lectins and integrins (αvβ3). Sheep have synepitheliochorial placentation in which mononucleate trophectoderm cells fuse to form binucleate cells (BNCs). BNCs migrate and fuse with endometrial LE cells to form trinucleate syncytial cells, and these syncytia enlarge through continued BNC fusion to form syncytial plaques that form the interface between endometrial and placental tissues within the placentome. The placentae of sheep organize into placentomal and interplacentomal regions. In placentomes there is extensive interdigitation of endometrial and placental tissues to provide hemotrophic nutrition to the fetus. In interplacentomal regions there is epitheliochorial attachment of endometrial LE to trophectoderm, mediated through focal adhesion assembly, and areolae that take up histotroph secreted by endometrial GE.

Circulating endothelial progenitor cells in pregnant women with premature rupture of membranes: potential association with placental disorders.

The frequency of preterm labour has risen over the last few years. Plasma oestrogen concentrations differ between patients who deliver before term and those who deliver at term. Oestrogen can influence the kinetics of circulating endothelial progenitor cells (cEPCs). Here, we attempted to identify the potential association of cEPCs with the incidence of complications typical of prematurity. The study groups consisted of 60 pregnant women with premature rupture of membranes (PROM; less than 37 weeks) and 50 term pregnant women (more than 38 weeks). cEPCs were isolated from term pregnant women and pregnant women with PROM and then migratory, proliferative, tubulogenic and functional properties of these cells along with serum secretion of important EPC chemotactic cytokines were analysed. In addition, the effect of 17β-oestradiol on biological features of cEPCs harvested from pregnant women was investigated. Our results showed that an increased concentration of oestrogen in women with PROM was associated with increased numbers of cEPCs, with these cells having increased oestrogen receptor α expression together with augmented proliferative, migratory and colony-formation properties. 17β-oestradiol induced proliferation, migration and angiogenic secretory activity of cEPCs from pregnant women. Overall, circulation mobilisation of EPCs in pregnant women may be associated with placental disorders.

Estrogen receptor subtype expression and regulation is altered in papillary thyroid cancer after menopause

Estrogen receptors can regulate growth in papillary thyroid cancer and may affect prognosis after menopause. This study examines changes of estrogen receptor subtype ratio expression in papillary thyroid cancer cell lines derived from pre- and postmenopausal women. Cells were harvested from papillary thyroid cancer and non-papillary thyroid cancer thyroid tissue (control) from pre- (n = 9) and postmenopausal women (n = 11). Protein expression of estrogen receptor α, estrogen receptor β, and phosphorylated extracellular signal-regulated kinase and protein kinase B were analyzed. Matrix metalloproteinase-2 activity was determined as a measure of tumor invasiveness. Mitochondrial retrograde signaling was altered with ethidium bromide to determine its effect on estrogen receptor α protein expression. Estrogen receptor α expression was increased in postmenopausal papillary thyroid cancer cells compared with controls but was unchanged in premenopausal papillary thyroid cancer. Estrogen receptor β expression did not change in either group. Increased matrix metalloproteinase-2 activity was observed only in postmenopausal papillary thyroid cancer. Premenopausal papillary thyroid cancer cells demonstrated increased extracellular signal-regulated kinase and unchanged protein kinase B activation. Conversely, postmenopausal papillary thyroid cancer cells had decreased extracellular signal-regulated kinase and increased protein kinase B activation. Ethidium bromide treatment resulted in increased estrogen receptor α protein expression only in premenopausal papillary thyroid cancer cells. Increased estrogen receptor α expression may be involved in papillary thyroid cancer aggressiveness after menopause. This process may be regulated by differential activation of intracellular pathways and differing sensitivities to mitochondrial signaling regulation.

Breast cancer in Iraq is associated with a unimodally distributed predominance of luminal type B over luminal type A surrogates from young to old age

BackgroundBreast cancer has recently increased in post-menopausal Iraqi women. In Western countries at high-risk for breast cancer, there is a bimodal increase in estrogen receptor (ER) positive tumors with a peak of low proliferation rate luminal A over higher proliferation rate luminal B tumors after 60 years of age. The aim of this study was to analyze in Iraqi women whether shifts are occurring in immunohistochemical (IHC) surrogates of molecular breast cancer subtypes toward a high-risk profile.MethodsAge specific and age standardized womens breast cancer incidence was estimated for the years 2006 through 2012. IHC results of ER, PR, HER2, and Ki67 testing were analyzed on the breast cancers of 125 Arabic and 725 Kurdish women by frequency of distribution and by age.ResultsBetween 2006 and 2012, age standardized incidence of breast cancer in Iraq increased from 30 to 40/100,000 women with the increase specifically occurring in women ≥ 60 years old (P < 0.001). Breast cancers in Kurdish women ≥ 60 years old may also have increased (P = 0.047) with urban exceeding rural rates by 2:1. For both Kurdish and Arabic women, there was a marked predominance of luminal B tumors at all ages in which luminal B and luminal A tumors were asymmetric skewed toward older age but with no late luminal A age peak.ConclusionsOlder Iraqi women do not show the bimodal shift toward higher rates of luminal A breast cancers seen in the West. The modest increase in age standardized incidence of breast cancer in Iraqi is being seen specifically in older women and may be better attributed to a trend for care in urban cancer centers rather than changing tumor characteristics.

Clinicopathologic study of invasive micropapillary carcinoma of the breast.

Invasive micropapillary carcinoma (IMPC) is a rare subtype of breast carcinoma. It is presumed to be more aggressive than invasive ductal carcinoma (IDC), though it is uncertain whether the prognoses of IMPC and IDC differ. In this retrospective study, we compared the clinicopathologic characteristics and survival between 170 female patients with IMPC (pure or mixed with IDC) and 728 with pure IDC. The IMPC patients had higher clinical stages and histologic grades, higher incidences of lymphovascular invasion and axillary lymph node extracapsular extension, and a higher degree of lymph node involvement than IDC patients. Moreover, IMPC was associated with increases in estrogen receptor (ER) and progesterone receptor (PR) positivity and HER-2 overexpression. Although locoregional recurrence-free survival (LRRFS) and recurrence-free survival (RFS) were poorer in IMPC patients than IDC patients, overall survival and distant metastasis survival did not differ between the two groups. Multivariate analysis revealed that IMPC was an independent prognostic factor for LRRFS in breast cancer, and IMPC patients had poorer clinicopathologic characteristics and poorer RFS and LRRFS than IDC patients. We therefore suggest that to improve treatment decisions, patients with breast carcinoma be tested for the presence of this specific subtype.

Can MTHFR C677T and A1298C Polymorphisms Alter the Risk and Severity of Sporadic Breast Cancer in Brazilian Women?

IntroductionPolymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR) modify the risk and severity of sporadic breast cancer (BC). In this context, the MTHFR C677T and A1298C polymorphisms have been associated with risk and severity of sporadic BC. Patients and MethodsIn total, 253 women with BC and 257 controls were enrolled in this study. Polymorphisms were analyzed using restriction fragment length polymorphism - polymerase chain reaction. Epidemiology, tumor characteristics, and reproductive factors were considered in the analysis. Statistical tests included the χ2 test, the Fisher exact test, and the Mann-Whitney and Kruskal-Wallis tests, or parametric equivalents. ResultsMTHFR polymorphisms were not a risk factor for BC. The 677CC genotype was associated with distant metastasis (odds ratio [OR] = 5.311; 95% confidence interval [CI] = 1.124-25.09) and lower estrogen receptor expression, whereas the 1298AA genotype was associated with stage 0 (OR = 0.244; 95% CI = 0.077-0.771) and increased estrogen receptor expression. In haplotype analysis, 677CC/1298AA was associated with hypertension (OR = 1.979; 95% CI = 1.036-3.782), and 677CT/1298AC was associated with invasive carcinoma of no special type (OR = 0.472; 95% CI = 0.243-0.918) and stage 0 (OR = 3.476; 95% CI = 1.341-10.47). ConclusionThe MTHFR C677T and A1298C polymorphisms do not alter the risk of BC, but are associated with the clinical severity of BC.

Bisphenol S (BPS) Alters Maternal Behavior and Brain in Mice Exposed During Pregnancy/Lactation and Their Daughters.

Estrogenic endocrine disrupting chemicals have been shown to disrupt maternal behavior in rodents. We investigated the effects of an emerging xenoestrogen, bisphenol S (BPS), on maternal behavior and brain in CD-1 mice exposed during pregnancy and lactation (F0 generation) and in female offspring exposed during gestation and perinatal development (F1 generation). We observed different effects in F0 and F1 dams for a number of components of maternal behavior, including time on the nest, time spent on nest building, latency to retrieve pups, and latency to retrieve the entire litter. We also characterized expression of estrogen receptor α in the medial preoptic area (MPOA) and quantified tyrosine hydroxylase immunoreactive cells in the ventral tegmental area, 2 brain regions critical for maternal care. BPS-treated females in the F0 generation had a statistically significant increase in estrogen receptor α expression in the caudal subregion of the central MPOA in a dose-dependent manner. In contrast, there were no statistically significant effects of BPS on the MPOA in F1 dams or the ventral tegmental area in either generation. This work demonstrates that BPS affects maternal behavior and brain with outcomes depending on generation, dose, and postpartum period. Many studies examining effects of endocrine disrupting chemicals view the mother as a means by which offspring can be exposed during critical periods of development. Here, we demonstrate that pregnancy and lactation are vulnerable periods for the mother. We also show that developmental BPS exposure alters maternal behavior later in adulthood. Both findings have potential public health implications.

Differences in the mechanisms of action of BDE-47 and its metabolites on OVCAR-3 and MCF-7 cell apoptosis.

Data concerning possible carcinogenic action of polybrominated diphenyl ethers (PBDEs) in hormone-dependent tissues are limited. Our earlier studies showed that 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) stimulated OVCAR-3 and MCF-7 cell proliferation, while its hydroxylated metabolites (5-OH-BDE-47 and 6-OH-BDE-47) increased estrogen receptors protein expression and extracellular signal-regulated kinase 1/2 and protein kinase Cα phosphorylation in these cell lines. In addition to cell proliferative disorder, a failure in the regulation of apoptosis can also lead to the formation and development of tumors. Therefore, in the present study, we investigated the effect of BDE-47 and its metabolites (2.5-50 ng ml-1 ) on the expression of apoptosis regulatory genes and proteins, caspase-8 and -9 activity and DNA fragmentation induced by extracellular signal-regulated kinase inhibitor (PD098059) and protein kinase Cα inhibitor (Gӧ 6976) in ovarian (OVCAR-3) and breast (MCF-7) cancer cells. In OVCAR-3 cells, BDE-47 upregulated expression of most of the investigated genes and increased protein expression of tumor necrosis factor (TNF)-α, TNF receptor 1, caspase-6, Bcl-xl and caspase-8 activity. Whereas in MCF-7 cells, BDE-47 resulted in the downregulation of most of the investigated genes, and decreased caspase-8 and -9 activity. In both OVCAR-3 and MCF-7 cells, the expression of most of the investigated genes were downregulated by metabolites. Exposure of OVCAR-3 cells to 5-OH-BDE-47 corresponded with a decrease in the protein expression of caspase-6, caspase-9 and Bcl-xl and treatment with 6-OH-BDE-47 decreased Bcl-xl and TNF receptor 1 expression in OVCAR-3 cells and caspase-9 expression in MCF-7 cells. Hydroxylated metabolites of BDE-47 have strong inhibitory effects on apoptosis in ovarian and breast tumor cells and thus should be considered potential carcinogens in hormone-dependent cancers. Copyright © 2016 John Wiley & Sons, Ltd.

Estrogenic compound attenuates angiotensin II-induced vascular smooth muscle cell proliferation through interaction between LKB1 and estrogen receptor α

The prevalence rate of cardiovascular disease is higher for males than females, and estradiol (E2) induces AMP-activated protein kinase (AMPK) activation, which is known to regulate proliferation of VSMC. We identified the estrogenic properties of nordihydroguaiaretic acid (NDGA, a lignan phytoestrogen) that inhibit VSMC proliferation and explored the underlying mechanisms. Both the phosphorylation and expression of LKB1 were increased by NDGA. In addition, NDGA significantly attenuated angiotensin II (Ang II)-induced VSMC proliferation. To elucidate the estrogenic effects, we confirmed that NDGA increased estrogen receptor α (ERα) expression, similar to treatment with E2 and estriol (E3). Furthermore, tamoxifen and ERα siRNA obstructed the effects of NDGA including ERα expression, AMPK phosphorylation and both LKB1 phosphorylation and expression. VSMC proliferation was restored by tamoxifen and ERα siRNA. LKB1 siRNA also reversed the NDGA-mediated inhibition of VSMC proliferation. The estrogenic activity of NDGA induced LKB1 translocation from nucleus to cytosol, and tamoxifen obstructed LKB1 translocation. The absence of LKB1 completely abolished the increase of ERα expression induced by NDGA. Taken together, the beneficial effects of estrogenic compound (E2 and NDGA) on inhibition of VSMC proliferation are mediated by interaction between LKB1 and ERα, suggesting a potential mechanism for females having less cardiovascular disease.

Modifications of Western-type diet regarding protein, fat and sucrose levels as modulators of steroid metabolism and activity in liver

The aim of the study was to evaluate whether the modification of the Western-type diet (high-fat, high-sucrose diet rich in saturated fatty acids) considering macronutrients content would influence hepatic metabolism and activity of steroids. For 3 weeks Wistar rat were fed the Western-type diet (21% fat, 35% sucrose, 19% protein, lard) and its modifications regarding dietary protein (10 and 19%), fat (5 and 21%) and sucrose (0 and 35%) levels. The steroid 5α-reductase type 1 (Srd5a1) and androgen receptor (Ar) gene expression as well as testosterone (T) conversion towards 5α-reduced derivatives in liver were positively correlated with body weight gain. The Western-type diets with decreased protein content regardless of the sucrose level exerted the most negative effect on the antioxidant system decreasing catalase (Cat), sodium dismutase (Sod1) and glutathione peroxidase (Gpx1) gene expression as well as Cat and Gpx activity and total antioxidant status, simultaneously intensifying lipid peroxidation. The impaired antioxidant system was accompanied by decreased level of hepatic T metabolism towards estrogens: 17β-estradiol (E2) and estriol, and increased estrogen receptor type 1 (Esr1) gene expression. Liver Esr1 mRNA level was differently correlated with T (positively) and E2 (negatively) plasma levels. Whereas the fat reduction in Western-type diet restored the plasma proportion between T and E2. In conclusion it could be stated that Western-type diet modification relating to protein, sucrose and fat content can influence hepatic steroid metabolism and activity; however the estrogens and androgens metabolism in liver would be connected with impairment of liver function or catabolic activity, respectively.

Abstract 2030: The role of genetic variation in calcium-activated potassium channels in breast cancer patients treated with tamoxifen

Abstract Potassium channels control membrane voltage and are essential for cell proliferation. Abnormal regulation and/or expression of these channels have been reported in cancer and result in dysregulated cell cycle progression, cell proliferation and migration. In breast cancer, clinical correlations of potassium channels have been associated with brain metastases, high stage, high grade, high proliferation, nodal status and poor prognosis. Interestingly, they have been associated with increased estrogen receptor (ER) expression. The selective ER modulator, tamoxifen, blocks estrogen from binding to the ER and is commonly used to treat patients with ER-positive breast cancer. However, up to 33% of patients treated with tamoxifen relapse or dies at 15 years of follow up. Both estrogen and tamoxifen have been demonstrated to activate potassium channels which then cause an increase in cell proliferation. In preliminary animal in vivo studies, we observed a better breast cancer prognosis in those with a knockout in potassium channel genes. Therefore, any alterations in the potassium channels may help to explain why patients treated with tamoxifen fail therapy. We hypothesized that genetic variation in potassium channels may affect breast cancer risk as well as tamoxifen treatment outcome. Genotyping was conducted on 45,290 breast cancer patients and 41,880 controls of European ancestry from 41 studies with the custom Illumina Infinium array (iCOGS). More than 200,000 SNPs were genotyped with over 11 million SNPs estimated using genotype imputation with SHAPEIT and IMPUTEv2 and the 1000 Genomes Project March 2012 release as the reference panel (Michailidou et al. Nature Genetics 2015). Variants from 13 potassium channels including KCNMA1, KCNMB1-4, KCNN1-4, LRRC26, LRRC38, LRRC52 and LRRC55 were analyzed. Of these, 3 imputed SNPs from KCNN4 have shown decreased breast cancer risk with GWAS significance (rs12463319, rs12609846, rs1685191; OR 0.94, P &amp;lt; 1.0E-08). In particular, these SNPs were significant in ER-positive breast cancer (OR 0.93, P &amp;lt; 1.0E-09), but was not observed in ER-negative patients (P &amp;gt; 0.05). These findings encouraged us to now further investigate the outcomes of patients with ER-positive breast cancer treated with tamoxifen. Survival data for 49,751 patients with a median follow-up of 8 years has been completed and released in November 2015. Of these, 32,571 were ER-positive and 10,383 have been treated with tamoxifen. Current efforts are underway in order to determine the effects of genetic variations in potassium channels on the survival of patients treated with tamoxifen. Our work will provide a better understanding of the potential clinical relevance of potassium channels in tamoxifen treatment in breast cancer. Citation Format: Wing-Yee Lo, Corinna Mohr, Friederike Steudel, Marjanka Schmidt, Douglas Easton, Reiner Hoppe, Werner Schroth, Peter Ruth, Robert Lukowski, Hiltrud Brauch, in collaboration with the Breast Cancer Association Consortium. The role of genetic variation in calcium-activated potassium channels in breast cancer patients treated with tamoxifen. [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 2030.

Abstract 4588: Estrogen receptor alpha mediates HPV E6/E7 oncoproteins: Induced breast cells proliferation

Abstract The association of human papillomavirus (HPV) with cervical cancer, head and neck cancer is well established, but the involvement of HPV E6/E7 oncoproteins in breast cancer is more contentious. Previous studies suggest that high risk HPV E6/E7 oncoproteins inactivate two tumor suppressor proteins p53 and pRb signaling and increase cell proliferation. There is cumulative evidence that high risk HPV 16 E6/E7 oncoproteins may have causal roles in some human breast cancers. Moreover, some studies have identified HPV DNA in breast tissue and breast cancer specimens. Nevertheless, the route of transmission of HPV in breast cells has not been determined, and the mechanisms by which HPV involvement in breast cancer are not known. We used a non-tumorigenic (MCF12A) epithelial breast cell line transfected with HPV 16 to elucidate the role of oncoproteins E6/E7 in pro-proliferative and anti-apoptotic signaling pathways. We also used Tamoxifen treatment, which is an antagonist of the estrogen receptor in breast tissues through its active metabolite, 4hydroxytamoxifen. We demonstrate that HPV E6/E7 transfection activates the extracellular signal-regulated kinase (ERK) MAPK pathway and Akt/PKB kinase signaling pathways through activating estrogen receptor alpha in MCF12A cells. E6/E7 oncoproteins transfection alone dramatically increases estrogen receptor alpha protein expression. Treatment with tamoxifen, an antagonist of the ER, significantly abolishes E6/E7-increase in ERK and PKB phosphorylation without altering their protein levels, suggesting that the estrogen receptor is a key mediator of HPV E6/E7 mediated ERK and Akt/PKB activation. Our current work show an insightful understanding of the molecular mechanisms of HPV-elicited Akt/PKB activation and its roles in cell proliferation and survival in normal breast epithelial cells. Therefore, these findings indicate that HPV E6/E7 oncoproteins may promote the transforming activities of high-risk HPV E6/E7 proteins and cell proliferation in breast cells. Therefore, suppression of estrogen receptor signaling networks may be used as a therapeutic strategy for HPV associated lesions and cancers such as breast metastasis. Citation Format: Eva McGhee, Lucy Tran, Mengtao Li, Yong Wu, Seyung Chung, Cheryl Araniego, Karen Tate, Melanie Baker, Kamilah Evans, Mechelle Rouse, Jay Vadgama. Estrogen receptor alpha mediates HPV E6/E7 oncoproteins: Induced breast cells proliferation. [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 4588.

Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells.

Standard treatment for primary prostate cancer includes systemic exposure to chemotherapeutic drugs that target androgen receptor or antihormone therapy (chemical castration); however, drug-resistant cancer cells generally emerge during treatment, limiting the continued use of systemic chemotherapy. Patients are then treated with more toxic standard therapies. Therefore, there is an urgent need for novel and more effective treatments for prostate cancer. The cholesterol biosynthetic pathway is an attractive therapeutic target for treating endocrine-dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as the metabolic precursor of endogenous steroid hormones. In this study, we have examined the effects of RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate; Roche Pharmaceuticals internal reference: RO0488071) (RO), which is an inhibitor of 2, 3-oxidosqualene cyclase (a key enzyme in the cholesterol biosynthetic pathway), on prostate cancer cells. Exposure of both hormone-dependent and castration-resistant human prostate cancer cells to RO reduced prostate cancer cell viability and induced apoptosis in vitro. RO treatment reduced androgen receptor protein expression in hormone-dependent prostate cancer cells and increased estrogen receptor β (ERβ) protein expression in both hormone-dependent and castration-resistant prostate cancer cell lines. Combining RO with an ERβ agonist increased its ability to reduce castration-resistant prostate cancer cell viability. In addition, RO effectively suppressed the growth of aggressive castration-resistant human prostate cancer cell xenografts in vivo without any signs of toxicity to experimental animals. Importantly, RO did not reduce the viability of normal prostate cells in vitro. Our study is the first to demonstrate that the cholesterol biosynthesis inhibitor RO effectively suppresses growth of human prostate cancer cells. Our findings suggest that cholesterol biosynthesis inhibitors such as RO, when used in combination with commonly used chemotherapeutic drugs or ERβ specific ligands, could represent a novel therapeutic approach to prevent the growth of prostate cancer tumors.

Oncogenic AKT1(E17K) mutation induces mammary hyperplasia but prevents HER2-driven tumorigenesis.

One of the most frequently deregulated signaling pathways in breast cancer is the PI 3-K/Akt cascade. Genetic lesions are commonly found in PIK3CA, PTEN, and AKT, which lead to excessive and constitutive activation of Akt and downstream signaling that results in uncontrolled proliferation and increased cellular survival. One such genetic lesion is the somatic AKT1(E17K) mutation, which has been identified in 4-8% of breast cancer patients. To determine how this mutation contributes to mammary tumorigenesis, we constructed a genetically engineered mouse model that conditionally expresses human AKT1(E17K) in the mammary epithelium. Although AKT1(E17K) is only weakly constitutively active and does not promote proliferation in vitro, it is capable of escaping negative feedback inhibition to exhibit sustained signaling dynamics in vitro. Consistently, both virgin and multiparous AKT1(E17K) mice develop mammary gland hyperplasia that do not progress to carcinoma. This hyperplasia is accompanied by increased estrogen receptor expression, although exposure of the mice to estrogen does not promote tumor development. Moreover, AKT1(E17K) prevents HER2-driven mammary tumor formation, in part through negative feedback inhibition of RTK signaling. Analysis of TCGA breast cancer data revealed that the mRNA expression, total protein levels, and phosphorylation of various RTKs are decreased in human tumors harboring AKT1(E17K).

Application of molecular endpoints in early life stage salmonid environmental biomonitoring

Molecular endpoints can enhance existing whole animal bioassays by more fully characterizing the biological impacts of aquatic pollutants. Laboratory and field studies were used to examine the utility of adopting molecular endpoints for a well-developed in situ early life stage (eyed embryo to onset of swim-up fry) salmonid bioassay to improve diagnostic assessments of water quality in the field. Coastal cutthroat trout (Oncorhynchus clarki clarki) were exposed in the laboratory to the model metal (zinc, 40μg/L) and the polycyclic aromatic hydrocarbon (pyrene, 100μg/L) in water to examine the resulting early life stage salmonid responses. In situ field exposures and bioassays were conducted in parallel to evaluate the water quality of three urban streams in British Columbia (two sites with anthropogenic inputs and one reference site). The endpoints measured in swim-up fry included survival, deformities, growth (weight and length), vitellogenin (vtg) and metallothionein (Mt) protein levels, and hepatic gene expression (e.g., metallothioneins [mta and mtb], endocrine biomarkers [vtg and estrogen receptors, esr] and xenobiotic-metabolizing enzymes [cytochrome P450 1A3, cyp1a3 and glutathione transferases, gstk]). No effects were observed in the zinc treatment, however exposure of swim-up fry to pyrene resulted in decreased survival, deformities and increased estrogen receptor alpha (er1) mRNA levels. In the field exposures, xenobiotic-metabolizing enzymes (cyp1a3, gstk) and zinc transporter (zntBigM103) mRNA were significantly increased in swim-up fry deployed at the sites with more anthropogenic inputs compared to the reference site. Cluster analysis revealed that gene expression profiles in individuals from the streams receiving anthropogenic inputs were more similar to each other than to the reference site. Collectively, the results obtained in this study suggest that molecular endpoints may be useful, and potentially more sensitive, indicators of site-specific contamination in real-world, complex exposure scenarios in addition to whole body morphometric and physiological measures.

Molecular analysis of a male breast cancer patient with prolonged stable disease under mTOR/PI3K inhibitors BEZ235/everolimus.

The mTORC1 inhibitor everolimus (Afinitor/RAD001) has been approved for multiple cancer indications, including ER+/HER2− metastatic breast cancer. However, the combination of everolimus with the dual PI3K/mTOR inhibitor BEZ235 was shown to be more efficacious than either everolimus or BEZ235 alone in preclinical models. Herein, we describe a male breast cancer (MBC) patient who was diagnosed with hormone receptor-positive (HR+)/HER2− stage IIIA invasive ductal carcinoma and sequentially treated with chemoradiotherapy and hormonal therapy. Upon the development of metastases, the patient began a 200 mg twice-daily BEZ235 and 2.5 mg weekly everolimus combination regimen. The patient sustained a prolonged stable disease of 18 mo while undergoing the therapy, before his tumor progressed again. Therefore, we sought to both better understand MBC and investigate the underlying molecular mechanisms of the patient's sensitivity and subsequent resistance to the BEZ235/everolimus combination therapy. Genomic and immunohistochemical analyses were performed on samples collected from the initial invasive ductal carcinoma pretreatment and a metastasis postprogression on the BEZ235/everolimus combination treatment. Both tumors were relatively quiet genomically with no overlap to recurrent MBC alterations in the literature. Markers of PI3K/mTOR pathway hyperactivation were not identified in the pretreatment sample, which complements previous reports of HR+ female breast cancers being responsive to mTOR inhibition without this activation. The postprogression sample, however, demonstrated greater than fivefold increased estrogen receptor and pathogenesis-related protein expression, which could have constrained the PI3K/mTOR pathway inhibition by BEZ235/everolimus. Overall, these analyses have augmented the limited episteme on MBC genetics and treatment.