ERβ mRNA Expression Research Articles

Abstract 4213: Evaluation of LMTK3 expression and tumor phenotype in estrogen-dependent colorectal cancer

Abstract Background: Incompatible physiology between genders influences development and progression of colorectal cancer (CRC). It is postulated that improved survival of women with CRC may be due to exogenous estrogen exposure. A recent study used the age of 50 as a surrogate for menopause, evaluating survival in two age groups: women ≥50 and ≤50. It was observed that women ≤50 had a survival advantage that was contributed to protection by estrogen- a mechanism lost in menopause; with no significant difference found in men. Lemur tyrosine kinase 3 (LMTK3) was recently identified, that when in association with an estrogen receptor (ER) α modulator increased growth, progression, and altered resistance to endocrine therapy was observed in breast cancer. The ER family (ERα and ERβ) exerts contrasting actions of estrogen on different target tissues, where ERβ is the dominant receptor expressed in the gastrointestinal tract. The different ER distribution between breast and colon cancer led to the hypothesis that LMTK3 overexpression should stabilize ERβ and decrease tumorigenesis in CRC. Methods: CRC cell lines were characterized for LMTK3 mRNA (qRT-PCR) and protein expression (Western blot) in both transfected and non-transfected cell lines. Overexpression of LMTK3 was achieved utilizing pCMV6-XL4-LMTK3. Overexpressing LMTK3 CRC cells were evaluated for alterations to cell viability by MTT, colony formation (CFAs), invasion assays and chemosensitivity to 5-Fluororuacil, oxaliplatin, and tamoxifen. Results: CRC cell lines demonstrated a range of LMTK3, ERα and ERβ mRNA and protein expression. Caco-2, HCT116 and LoVo cells demonstrated estrogen sensitivity as evidenced by treatment with ER antagonist, tamoxifen. CFA demonstrated that the presence of estrogen plus overexpression of LMTK3 decreased cell viability by 27, 28 and 36% in Caco2, HCT116 and LoVo cells respectively, had limited impact on chemosensitivity, and decreased their ability to invade. Conclusion: This study demonstrates that estrogen mediated cell signaling and LMTK3 play an important role in decreasing CRC tumorigenesis. Further studies are underway to better understand estrogen and LMTK3's role in CRC. Citation Format: Sybil Magturo, Georgios Giamas, Snay Mallick, Austin Layton, Justin Stebbing, Heinz-Josef Lenz, Melissa J. LaBonte. Evaluation of LMTK3 expression and tumor phenotype in estrogen-dependent colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4213. doi:10.1158/1538-7445.AM2014-4213

Abstract 622: S-equol, an estrogen receptor β agonist, inhibits tumor growth and progression of breast cancer

Abstract Breast cancer is the primary cause of cancer-associated mortality in women worldwide. Estrogen and the Estrogen Receptors (ER) play a significant role in breast cancer, with over two-thirds of breast cancers expressing ERα. Current endocrine therapy, such as aromatase inhibitors, target estrogen biosynthesis and anti-estrogens target ERα. However, therapeutic resistance frequently arises. In addition to the importance of ERα, ERβ has also been shown to play a critical, but opposing role in breast cancer. ERβ has been shown to inhibit the growth of ERα-positive breast cancer cells. The ratio of ERα to ERβ, in addition to the cross talk between ER's and growth factor signaling, has been implicated in the development of therapeutic resistance. Recently, several plant-derived compounds that exhibit ERβ agonist activity have been identified. S-equol is a potent ERβ agonist and a metabolite from the soy isoflavone daidzein, and has been previously shown to alleviate menopausal symptoms in a clinical trial. Activation of ERβ, or its over expression, shifts the balance of ER's from the oncogenic action of ERα to the tumor suppressor activity of ERβ, and therefore may be a valuable therapeutic approach in the treatment of breast cancer. In this study we sought out to determine the efficacy of the ERβ agonist, S-equol, in inhibiting the growth and progression of breast tumors using a syngeneic mouse model. We used mouse mammary tumor cells expressing endogenous ERβ, and to determine the contribution of ERβ, cells with knockdown of ERβ were generated using shRNA. ERβ mRNA and protein expression was analyzed using qRT-PCR and western blot respectively. Syngeneic tumors were established and mice were treated with either a vehicle control or S-equol. Treatment with S-equol reduces tumor volume and inhibits the progression of mouse mammary tumor cells in tumors expressing ERβ. Our mechanistic studies show that S-equol reduces the expression of ERα, and increases the expression of p53 and p27 in an ERβ dependent manner. Additionally, S-equol modulates the expression of inflammatory molecules involved in aromatase expression and promotes the differentiation of cancer stem cells. In conclusion, this study suggests that targeting ERβ may be a valuable strategy in treatment of breast cancer. Citation Format: Cathy Samayoa, Naveen K. Krishnegowda, Samaya R. Krishnan, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. S-equol, an estrogen receptor β agonist, inhibits tumor growth and progression of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 622. doi:10.1158/1538-7445.AM2014-622

Abstract 4498: Afatinib, an irreversible ErbB family inhibitor, demonstrates activity against HER2 mutated cervical cancer in vitro

Abstract Uterine cervical cancer (UCC) is the second leading cause of cancer deaths among women worldwide and results in 275,000 deaths annually. Functional characterization of cancer-associated genetic alterations may lead to new therapeutic approaches using molecularly targeted therapies which have the potential to improve patient outcomes. Consistent with this view, whole exome sequencing studies in a variety of human tumors including cervical cancer have recently identified somatic mutations in genes that encode for the ErbB family receptors. Importantly, some of these mutations have been shown to be “drivers” and to correlate with tumor sensitivity to the exposure to ErbB tyrosine kinase inhibitors in vitro as well as in vivo. In this study we have evaluated whether genetic alterations in the c-erbB2 gene determine the sensitivity of UCC primary cell lines to Afatinib, an EGFR, HER2, and HER4 irreversible tyrosine kinase inhibitor. Ten primary UCC cell lines, (two harboring c-erbB2 gene mutations in the extracellular HER2/neu domain [i.e., the S280F and E375D mutations-ref genome hg18] and eight harboring wild type c-erbB2), established as long term cultures in vitro, were analyzed in our study. The effect of Afatinib on cell growth, cell-cycle distribution and signaling were assessed using flow cytometry and western blot analysis following incubation of primary UCC cell lines with scalar concentrations of Afatinib for 72 hours in vitro. We found that despite similar ErbB2 mRNA expression levels by qRT-PCR in the mutated versus the wild type c-erbB2 groups, IC50 values in response to Afatinib were significantly lower in the group of mutated cell lines than in the non-mutated control UCC (MEAN±SEM = 0.55±0.11 vs. 1.64±0.09 μM, P<0.05). Furthermore, Afatinib growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells blocked in the G1 cell cycle phase as well as a dose-dependent dephosphorylation of HER2, S6, AKT and ERK in both c-erbB2 mutated and wild type UCC cell lines. Our data suggests that Afatinib, a recently FDA approved drug, is highly effective against c-erbB2 mutated UCC in vitro and could represent a valid therapeutic option for patients harboring c-erbB2 mutated advanced/recurrent cervical cancers unresponsive to radiation and/or chemotherapy. Citation Format: Salvatore Lopez, Emiliano Cocco, Bellone Stefania, Ileana Bortolomai, Elena Bonazzoli, Roberta Nicoletti, Carlton Schwab, Diana P. English, Corrado Terranova, Roberto Angioli, Alessandro D. Santin. Afatinib, an irreversible ErbB family inhibitor, demonstrates activity against HER2 mutated cervical cancer in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4498. doi:10.1158/1538-7445.AM2014-4498

Dysregulation of estrogen receptor beta (ERβ), aromatase (CYP19A1), and ER co-activators in the middle frontal gyrus of autism spectrum disorder subjects.

BackgroundAutism spectrum disorders (ASD) are much more common in males than in females. Molecular alterations within the estrogen receptor (ER) signaling pathway may contribute to the sex difference in ASD, but the extent of such abnormalities in the brain is not known.MethodsPostmortem middle frontal gyrus tissues (13 ASD and 13 control subjects) were used. The protein levels were examined by western blotting. The gene expression was determined by qRT-PCR.ResultsGene expression analysis identified a 35% decrease in ERβ mRNA expression in the middle frontal gyrus of ASD subjects. In addition, a 38% reduction in aromatase (CYP19A1) mRNA expression was observed in ASD subjects. We also found significant decreases in ER co-activators that included a 34% decrease in SRC-1, a 77% decrease in CBP, and a 52% decrease in P/CAF mRNA levels in ASD subjects relative to controls. There were no differences in the mRNA levels of TIF-2, AIB-1 (ER co-activators), ER co-repressors (SMRT and nCoR) and ERα in the middle frontal gyrus of ASD subjects as compared to controls. We observed significant correlations between ERβ, CYP19A1, and co-activators in the study subjects. Immunoblot analysis further confirmed the changes in ERβ and aromatase at the protein level in the control and ASD subjects.ConclusionsThese results, for the first time, provide the evidence of the dysregulation of ERβ and co-factors in the brain of subjects with ASD.Electronic supplementary materialThe online version of this article (doi:10.1186/2040-2392-5-46) contains supplementary material, which is available to authorized users.

Increased TNF α, IL-6 and ErbB2 mRNA expression in peripheral blood leukocytes from breast cancer patients.

Obesity has been associated with increased incidence and mortality of breast cancer. The precise relation between obesity and breast cancer is yet to be determined, with few studies linking them with altered serum levels adipokines and inflammatory cytokines. The relevance of the expression of genes encoding for adipokines and inflammatory cytokines in the peripheral blood and their contribution to obesity and breast cancer has not been fully investigated. We aim to identify potential transcriptional biomarkers in blood samples that may assist to underpin the link between obesity and breast cancer. Therefore, have investigated whether or not the expression levels, of selected genes [tumor necrosis factor-α (TNFα), interleukin 6 (IL-6), adiponectin, leptin, C-reactive protein (CRP), parathyroid hormone (PTH), tumor protein 53 (TP53) and erythroblastic leukemia viral oncogene 2 (ErbB2)] were altered in blood samples of lean, overweight/obese and breast cancer subjects. Blood samples were obtained from 37 lean, 19 overweight/obese and 12 breast cancer patients. Real-time polymerase chain reaction assays were performed to detect TNFα, IL-6, adiponectin, leptin, CRP, PTH, TP53 and ErbB2 gene transcripts. Transcript levels of TNFα were significantly higher by 1.4-fold and 2.1-fold in blood cells of overweight/obese and breast cancer patients, respectively, compared with lean control subjects. Transcript levels of IL-6 were significantly higher by 2.3-fold in blood cells from breast cancer patients compared with lean control subjects with normal body mass index, and no significant difference was found in the expression level of IL-6 transcripts between overweight/obese and lean control subjects. The ErbB2 transcript levels were significantly higher by 4.72-fold compared to lean control subjects and were also significantly higher compared to overweight/obese subjects. Breast cancer and obesity are associated with altered mRNA levels of cytokines and tumor marker in peripheral blood.

Different action of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolites on ERα and ERβ gene and protein expression

In our previously published data we showed that PBDEs act as endocrine disruptors in ovarian follicles by altering steroid secretion.In this study we try to answer a question if BDE-47 and its hydroxylated metabolites (5-OH-BDE-47 and 6-OH-BDE-47) can act as endocrine disruptors in the ovary by changing the expression of the steroid nuclear receptors, estrogen receptor alpha (ERα) and beta (ERβ), androgen receptor (AR), and receptors associated with the metabolism of xenobiotics and steroid hormones, constitutive androstane receptor (CAR) and pregnane X-receptor (PXR), in porcine ovarian follicles. Expression of mRNA was evaluated by real-time PCR, whereas protein level by western blotting.CAR and PXR mRNAs were not expressed in porcine ovarian follicular cells. BDE-47 and its hydroxylated metabolites had no effect on the expression of AR mRNA and protein. Decreased expression of ERβ mRNA and protein under BDE-47 influence and increase both ERα and ERβ gene and protein expression in cells exposed to hydroxylated metabolites was noted. These findings indicate that BDE-47, by altering the ratio of ERα to ERβ toward ERα, and the hydroxylated metabolites of BDE-47, by increase estrogen receptors expression, may result in excessive ovarian exposure to estrogens.

An emerging water contaminant, semicarbazide, exerts an anti-estrogenic effect in zebrafish (Danio rerio).

To determine the endocrine disrupting effect of semicarbazide, an emerging water contaminant, the changes in transcript levels of hepatic estrogen-response genes including vitellogenin-1 (vtg-1), estrogen receptor α (ERα), and estrogen receptor β (ERβ) were measured in male and female zebrafish exposed to semicarbazide with or without exogenous 17β-estradiol (E2). Exposure of male zebrafish to semicarbazide for 96 h or 28 days resulted in no significant induction in hepatic vtg-1, ERα, or ERβ mRNA expression, indicating that semicarbazide has no estrogenic effect. However, a remarkable anti-estrogenic effect of semicarbazide was demonstrated: semicarbazide treatment of female zebrafish for 96 h and 28 days resulted in significant decreases in transcript levels of vtg-1, ERα, and ERβ, as well as decreases in the gonadosomatic index level after 28 days. Moreover, semicarbazide exposure significantly inhibited the induction of vtg-1, ERα and ERβ mRNA by E2 when male zebrafish were co-exposed for 28 days.

Neuroprotective action of raloxifene against hypoxia-induced damage in mouse hippocampal cells depends on ERα but not ERβ or GPR30 signalling

Raloxifene is the selective estrogen receptor modulator (SERM) currently used in clinical practice to activate estrogen receptors (ERs) in bone tissue and to antagonise ERs in breast and uterine cancers. Little is known, however, about mechanisms of action of raloxifene on hypoxia-induced neuronal cell damage. The aim of the present study was to investigate the neuroprotective potential of raloxifene against hypoxia-induced damage of mouse hippocampal cells in primary cultures, with a particular focus on raloxifene interactions with the classical nuclear ERs (ERα, ERβ) and the recently identified membrane ER G-protein-coupled receptor 30 (GPR30). In this study, 18h of hypoxia increased hypoxia inducible factor 1 alpha (Hif1α) mRNA expression and induced apoptotic processes, such as loss of the mitochondrial membrane potential, activation of caspase-3 and fragmentation of cell nuclei based on Hoechst 33342 staining. These effects were accompanied by reduced ATPase and intracellular esterase activities as well as substantial lactate dehydrogenase (LDH) release from cells exposed to hypoxia. Our study demonstrated strong neuroprotective and anti-apoptotic caspase-3-independent actions of raloxifene in hippocampal cells exposed to hypoxia. Raloxifene also inhibited the hypoxia-induced decrease in Erα mRNA expression and attenuated the hypoxia-induced rise in Erβ and Gpr30 mRNA expression levels. Impact of raloxifene on hypoxia-affected Erα mRNA was mirrored by fluctuations in the protein level of the receptor as demonstrated by Western blot and immunofluorescent labelling. Raloxifene-induced changes in Erβ mRNA expression level were in parallel with ERβ immunofluorescent labeling. However, changes in Gpr30 mRNA level were not reflected by changes in the protein levels measured either by ELISA, Western blot or immunofluorescent staining at 24h post-treatment. Using specific siRNAs, we provided evidence for a key involvement of ERα, but not ERβ or GPR30 in neuroprotective action of raloxifene against hypoxia-induced cell damage. This study may have implications for the treatment or prevention of hypoxic brain injury and the administration of current or new generations of SERMs specific to ERα.This article is part of a Special Issue entitled “Sex steroids and brain disorders”.

Effects of unilaterally microinjecting ethanol in the preoptic-anterior hypothalamic areas of rats on ovulation.

Intragastric or intraperitoneal ethanol (EtOH) treatment inhibits reproductive functions in females and male rats. The area of the hypothalamus where these effects take place is unknown. As the participations of the preoptic-anterior hypothalamic area (POA-AHA) in regulating ovulation is asymmetric, this study aims to analyze the effects on 17β-estradiol(E2 ), progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) serum levels, the messenger ribonucleic acid (mRNA) expression of estrogen receptor alpha (ERα) and beta (ERβ), and ovulation resulting from unilaterally microinjecting water or an EtOH solution into either side of the POA-AHA. The treatment consisted of microinjecting a 8.6 μM EtOH solution into either side of the POA-AHA. The study was performed on groups of adult cyclic rats at 09.00 hours on diestrus-1, and sacrificed on diestrus-2 at 13.00, on proestrus at 09.00 or 17.00 or on estrus at 09.00 hours. Ovulation rates were assessed in rats sacrificed on estrus. Hormonal serum levels were measured using radioimmunoassay, and as a function of ERα and ERβ mRNA expression in each side of the POA-AHA by reverse transcriptase polymerase chain reaction. EtOH treatment blocked ovulation and the preovulatory release of LH, and lowered E2 levels. Irrespective of the treated POA-AHA side, ERα mRNA expression was consistently lower in the left POA-AHA and higher on the right. EtOH treatment in the left POA-AHA decreased FSH serum levels and lowered ERβ mRNA expression. In turn, EtOH treatment on the right POA-AHA resulted in higher FSH levels and ERβ mRNA expression. The present results show that EtOH blocks the preovulatory surge of LH on the POA-AHA. The effects of EtOH treatment of preovulatory FSH surge on the POA-AHA are asymmetric (stimulative on the right and inhibiting in the left). The effects of EtOH treatment on preovulatory LH and FSH surge could be explained by the inhibition of ERα and ERβ mRNA expression, respectively.

Inverse prognostic impact of ErbB2 mRNA and protein expression level in tumors of soft tissue sarcoma patients

Human epidermal growth factor receptor 2 (ErbB2) is overexpressed in a variety of human malignancies. Moreover, ErbB2 has been reported to influence cancer patient survival and progression of different tumor entities. However, information regarding the prognostic impact of ErbB2 in soft tissue sarcoma (STS) patients is limited and conflicting. ErbB2 mRNA and protein levels were defined by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), and the prognostic impact of ErbB2 mRNA and protein levels in tumor tissue of 124 soft tissue sarcoma patients were investigated. The median ErbB2 mRNA expression level in tumor tissue was decreased 3.9-fold compared to non-neoplastic surrounding tissue (p = 0.001). Furthermore, an increased ErbB2 mRNA expression level was associated with an improved tumor-specific survival (p = 0.01, log rank test). Multivariate Cox's proportional hazard regression analyses revealed an increased ErbB2 mRNA expression level as an independent favorable prognostic factor for tumor-specific survival of STS patients (n = 124; RR = 3.0; 95 % CI = 1.6-5.7; p < 0.001). In addition, multivariate Cox's proportional hazard regression analyses showed that an increased ErbB2 protein expression level correlated with poorer recurrence-free survival of STS patients (n = 47; RR = 9.9; 95 % CI = 1.7-59.7; p = 0.012), in particular for STS patients who received postoperative radiotherapy (n = 27; RR = 17.9; 95 % CI = 1.3-247.7; p = 0.031). This study suggests an inverse prognostic value of ErbB2 mRNA and protein expression level.

Poster #S263 DOES ROUTINE OUTCOME MONITORING IN SCHIZOPHRENIA IMPROVE THE IMPLEMENTATION OF EVIDENCE BASED CARE?

Overexpression of the epidermal growth factor receptor (EGFR) family is common in oral squamous cell carcinoma (OSCC). Therefore, we analyzed the expression profiles of the four EGFR family members (ErbB1, ErbB2, ErbB3, and ErbB4) in OSCC of Japanese patients.Sixty-eight primary tumors and 18 normal oral mucosal tissue specimens were evaluated in this study. We analyzed EGFR family members using quantitative polymerase chain reaction and immunohistochemistry, as well as their relationships with clinical factors.The expression level of ErbB1 messenger RNA (mRNA) was markedly increased in OSCC. By comparing the gene expression levels of EGFR family members in OSCC tissues that had lymph node metastasis with those in the absence of lymph node metastasis, we found that ErbB4 mRNA expression was increased significantly. There was also a significant correlation between the mRNA expression level of ErbB4 and those of ErbB2 and ErbB3 in cases with lymph node metastasis. Moreover, we confirmed protein expression of ErbB4 in the cytoplasm and membrane of tumor cells, which was stronger in cases with lymph node metastasis.ErbB4 is an independent marker for lymph node metastasis in OSCC.

Angiotensin II modulates sex steroid receptors and their metabolizing enzymes in rat cardiac fibroblast (701.10)

Sex steroids influence angiotensin II (AngII)‐induced cardiac remodeling; however, the importance of local sex steroid metabolism in this process is not known. We previously demonstrated that AngII increased aromatase levels, the enzyme that converts testosterone (T) to 17β‐estradiol, in cultured coronary vascular smooth muscle cells. In the present study we investigated the impact of AngII on sex steroid receptor and enzyme expression in primary rat cardiac fibroblasts. Fibroblasts were isolated from adult male rats and treated at P1 in 2% charcoal‐stripped FBS with AngII or vehicle (18h), followed by T (6h) in the presence or absence of anastrozole (aromatase inhibitor; 6.5h). Cardiac fibroblasts expressed receptors ERα, ERβ, and AR, as well as the enzymes aromatase and 5α‐reductase. AngII significantly reduced mRNA expression of ERβ. 5α‐reductase, AR, and ERβ levels increased when anastrazole was administered with T. This is the first demonstration that cardiac fibroblasts express the enzymes and receptors necessary for local sex steroid metabolism and action. Enzymes and receptor levels are differentially impacted by AngII. Additionally, aromatase inhibition in the presence of T alters steroid receptor and 5α‐reductase (testosterone metabolizing enzyme) levels. Given the major role of the fibroblast in pathogenic cardiac remodeling, the relative balance of T to estrogen in these cells may play an important role in the development of AngII‐mediated fibrosis.Grant Funding Source: Supported by American Heart Association

Effects of ovarian hormone treatment on the gene expression of muscarinic acetylcholine receptors in the ovariectomized rat myometrium

We investigate the effects of ovarian hormone on the gene expression of muscarinic acetylcholine receptors (M1–M5) in the myometrium using real-time PCR and evaluate the relationships between their expression and that of ovarian hormone receptors (ERα, ERβ, and PgR). Wistar rats were sham operated (SO) or ovariectomized (OVX) and treated with vehicle, estradiol (E2), progesterone (P4), or both E2 and P4 for 2 days beginning on postoperative day 33.M1 and M4 mRNA expressions were not detected in the myometrium. M2 mRNA expression did not change significantly in the OVX and OVX+P4 groups compared to the SO group, but increased significantly in the OVX+E2 group and was normalized in the OVX+E2P4 group. M3 mRNA expression increased significantly in the OVX and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2 and OVX+E2P4 groups. M5 mRNA expression did not change significantly in all experimental groups. ERα mRNA expression increased significantly in the OVX, OVX+E2, and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2P4 group. The changes in ERβ mRNA expression were similar to those of M3 mRNA expression in all experimental groups. In contrast, the changes in PgR mRNA expression did not correspond with that of M2, M3, or M5 mRNA expression in any of the experimental groups.Additionally, we evaluated the relationship between the expression of muscarinic acetylcholine receptors and ovarian hormone receptors in estrus cycle. M2 mRNA expression increased significantly in diestus and metaestrus compared in proestrus and estrus. M3 mRNA expression increased significantly in only diestrus compared in the other stages. In contrast, M5 mRNA expression did not change in estrus cycle. The changes in ERα mRNA expression appeared to be similar to those of M2 in estrus cycle, but no significant difference was found. The changes in ERβ mRNA expression were similar to those of M3 mRNA expression. The change in PgR mRNA expression increased significantly in diestrus compared in metaestrus, but did not correspond with that of M2, M3, or M5 mRNA expression in estrus cycle. When acetylcholine sensitivity in the myometrium was compared between diestrus and estrus, the sensitivity is significantly lower in estrus than in diestrus.These results suggest that ovarian hormones influence the expression of M2 and M3 in the myometrium by regulating the expression of hormone receptors. E2 may upregulate M2 via ERα, but P4 may downregulate M2 by inhibiting ERα via PgR. E2 may downregulate M3 by inhibiting ERβ, but P4 may not regulate the expression of M3 and ERβ. M5 may be a constitutive muscarinic receptor in the myometrium because neither E2 nor P4 influence the expression of M5. The combination of E2 and P4 may contribute the reproduction by quieting down the acetylcholine-induced myometrial contraction.

Impact of Lycopene on Epididymal Androgen and Estrogen Receptors’ Expression in Polychlorinated Biphenyls–Exposed Rat

The aim of the study was to evaluate the androgen (AR) and estrogen receptors' (ER) expression in epididymis of polychlorinated biphenyls (PCBs)-exposed rats. The rats were assigned to groups. Group I controls were treated with corn oil 80 µL/d intraperitoneally (ip), group II were treated with 2 mg/kg/d of A1254 ip; and group III were treated with 2 mg/kg/d of A1254 ip along with simultaneous oral supplementation of 4 mg/kg/d lycopene . The treatment was given daily for 30 days. After 24 hours of treatment, the rats were killed, and the epididymal regions (caput, corpus, and cauda) were dissected out, weighed, and prepared to estimate the levels of sialic acid, glyceryl phosphoryl choline (GPC), hydrogen peroxide (H2O2), and lipid peroxidation (LPO). The messenger RNA (mRNA) expressions of AR, ERα, and ERβ were analyzed by reverse transcriptase-polymerase chain reaction, and ERα and ERβ protein expressions were analyzed by immunoblotting. The toxicity of PCBs was also confirmed by histology. There was a marked decrease in epididymal weight, sialic acid, and GPC levels, while oxidative stress markers H2O2 and LPO were increased in PCBs-treated rats. The mRNA and protein expression of AR, ERα, and ERβ were decreased in PCBs-treated groups, and the histology confirms the cytoplasmic damage in the regions of caput, corpus, and cauda in PCBs-treated rats. Simultaneous supplementation of lycopene to PCBs-exposed rats resulted in significant decrease in the oxidative stress markers as that of control, while the AR, ERα, and ERβ gene expressions were near to control. The results suggest that lycopene has ameliorative effect against PCBs-induced toxicity in epididymis.

Human uterine and placental arteries exhibit tissue-specific acute responses to 17 -estradiol and estrogen-receptor-specific agonists

The discrete regulation of vascular tone in the human uterine and placental circulations is a key determinant of appropriate uteroplacental blood perfusion and pregnancy success. Humoral factors such as estrogen, which increases in the placenta and maternal circulation throughout human pregnancy, may regulate these vascular beds as studies of animal arteries have shown that 17β-estradiol, or agonists of estrogen receptors (ER), can exert acute vasodilatory actions. The aim of this study was to compare how acute exposure to ER-specific agonists, and 17β-estradiol, altered human placental and uterine arterial tone in vitro. Uterine and placental arteries were isolated from biopsies obtained from women with uncomplicated pregnancy delivering a singleton infant at term. Vessels were mounted on a wire myograph, exposed to the thromboxane receptor agonist U46619 (10−6 M), and then incubated with incremental doses (5 min, 0.03–30 µM) of either 17β-estradiol or agonists specific for the ERs ERα (PPT), ERβ (DPN) or the G-protein-coupled estrogen receptor GPER-1 (G1). ERα and ERβ mRNA expression was assessed. 17β-estradiol, PPT and DPN each relaxed myometrial arteries (P < 0.05) in a manner that was partly endothelium-dependent. In contrast, 17β-estradiol or DPN relaxed placental arteries (maximum relaxation to 42 ± 1.1 or 47.6 ± 6.53% of preconstriction, respectively) to a lesser extent than myometrial arteries (to 0.03 ± 0.03 or 8.0 ± 1.0%) and in an endothelial-independent manner whereas PPT was without effect. G1 exposure did not inhibit the constriction of myometrial nor placenta arteries. mRNA expression of ERα and ERβ was greater in myometrial arteries than placental arteries. ER-specific agonists, and 17β-estradiol, differentially modulate the tone of uterine versus placental arteries highlighting that estrogen may regulate human uteroplacental blood flow in a tissue-specific manner.

Abstract P6-06-44: Role of uPA and PAI-1 mRNA expression as prognostic factors in molecular subtypes of breast cancer

Abstract Background: Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) determined by ELISA from fresh-frozen tumor tissue have been evaluated as prognostic factors in prospectively randomized trials in node-negative breast cancer. However, uPA and PAI-1 mRNA expression might reveal new clinically relevant information using paraffin-embedded tumor tissue. Methods: We evaluated uPA and PAI-1 mRNA expression with the Affymetrix HG-U 133A array also within molecular subgroups of breast cancer in a finding cohort of breast cancer patients (cohort A; n = 548). We validated mRNA expression in a cohort of HER2 positive breast cancer patients (cohort B, n = 347). For uPA and PAI-1, mRNA values below and above the median were compared in two different probesets. “Luminal”, “triple-negative” and “HER2 positive” subcohorts were defined by ESR1 and ERBB2 mRNA expression using pre-defined cut-offs. Results: In cohort A, elevated PAI-1 mRNA expression was associated with shorter disease-free survival (DFS) and overall survival (OS), elevated uPA mRNA expression was associated with shorter OS. Regarding different molecular subgroups, 71% (n = 388) of tumors had a luminal, 13% (n = 69) a HER2 positive and 17% (n = 91) a triple-negative subtype. Only in the HER2 positive subgroup, elevated PAI-1 mRNA expression was associated with shorter disease-free survival and overall survival (p = 0.003 for DFS and p = 0.012 for OS). Same results were found for uPA in HER2 positive patients (DFS, p = 0.027 and OS, p = 0.011). In contrast, no association between both markers and DFS and OS could be seen in the luminal or triple-negative subgroups. In the HER2 positive validation cohort B elevated uPA and PAI-1 mRNA expression showed a strong association with shorter DFS (p = 0.013 for PAI-1, p = 0.001 for uPA). Conclusion: Results of this study demonstrate that the prognostic impact of uPA and PAI-1 mRNA expression was observed mainly in patients with HER2 positive tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-44.

Predicting Degree of Benefit From Adjuvant Trastuzumab in NSABP Trial B-31

National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31 suggested the efficacy of adjuvant trastuzumab, even in HER2-negative breast cancer. This finding prompted us to develop a predictive model for degree of benefit from trastuzumab using archived tumor blocks from B-31. Case subjects with tumor blocks were randomly divided into discovery (n = 588) and confirmation cohorts (n = 991). A predictive model was built from the discovery cohort through gene expression profiling of 462 genes with nCounter assay. A predefined cut point for the predictive model was tested in the confirmation cohort. Gene-by-treatment interaction was tested with Cox models, and correlations between variables were assessed with Spearman correlation. Principal component analysis was performed on the final set of selected genes. All statistical tests were two-sided. Eight predictive genes associated with HER2 (ERBB2, c17orf37, GRB7) or ER (ESR1, NAT1, GATA3, CA12, IGF1R) were selected for model building. Three-dimensional subset treatment effect pattern plot using two principal components of these genes was used to identify a subset with no benefit from trastuzumab, characterized by intermediate-level ERBB2 and high-level ESR1 mRNA expression. In the confirmation set, the predefined cut points for this model classified patients into three subsets with differential benefit from trastuzumab with hazard ratios of 1.58 (95% confidence interval [CI] = 0.67 to 3.69; P = .29; n = 100), 0.60 (95% CI = 0.41 to 0.89; P = .01; n = 449), and 0.28 (95% CI = 0.20 to 0.41; P < .001; n = 442; P(interaction) between the model and trastuzumab < .001). We developed a gene expression-based predictive model for degree of benefit from trastuzumab and demonstrated that HER2-negative tumors belong to the moderate benefit group, thus providing justification for testing trastuzumab in HER2-negative patients (NSABP B-47).

New vitamin D less-calcemic analog affect human bone cell line and cultured vascular smooth muscle cells similar to other less-calcemic analogs

Primary cultures of human bone and vascular cells respond to vitamin D treatment by modulation of cell proliferation measured by DNA synthesis (DNA) and energy metabolism measured by creatine kinase specific activity (CK) via binding to vitamin D receptors (VDR) which are expressed in these cells. Vitamin D compounds also modulate the response to estradiol-17β (E2) and the expression mRNAs of estrogen receptors (ERα and ERβ), VDR, 25-hydroxy vitamin D3 1-α hydroxylase (1OHase) and lipoxygenases (12LO and 15LO). We now compared our newly synthesized analog: 1α,25-dihydroxy-9-methylene-19-norvitamin D3 JK152 (JK), on bone and vascular cells compared to other analogs. Human bone cell line SaOS2 respond to JK by increased DNA and stimulated CK dose-dependently, similar to the less-calcemic analogs CB 1093 (CB) and EB 1089 (EB). JK also up-regulated the response to E2 in terms of DNA and CK. JK inhibited DNA synthesis and increased CK in primary human vascular smooth muscle cells (VSMC) dose-dependently similar to EB and CB. JK up regulated the response to E2 in terms of CK with no effect on DNA. JK similar to CB and EB stimulated mRNA expression of VDR and ERα, 12LO and 15LO, with no effect on ERβ and 1OHase mRNA expression in SaOS2 measured by real time PCR. Similar treatments of VSMC with JK, CB and EB stimulated 12LO and 15LO, VDR and ERα mRNA expression with no effect on ERβ and 1OHase mRNA expression. The results presented here demonstrate that the new vitamin D less-calcemic analog JK is similar to other analogs in its effects on human cultured cells and therefore may be used in combined hormone replacement treatment (HRT) both in vitro and in vivo.

Estrogen-dependent changes in estrogen receptor-β mRNA expression in middle-aged female rat brain

During aging, estrogen production and circulating levels of estrogen are markedly decreased in females. Although several differences exist in the process of reproductive aging between women and female rats, the results of many studies suggest that the female rat, especially the middle-aged or aged ovariectomized female, is an important animal model of hormone loss in women. In target tissues including the brain, the actions of estrogen are mediated mainly via the alpha and beta subtypes of the estrogen receptor (ER-α and ER-β). Estrogen treatment is known to change the expression of ER-α mRNA and protein in specific regions of the brain in middle-aged female rodents. In contrast, we do not know if estrogen regulates the expression of ER-β in the brain at this stage of life. In the present study, we performed in situ hybridization on brain sections of ovariectomized and estrogen-treated middle-aged female rats to reveal the effects of estrogen on the expression of ER-β throughout the brain. Our results showed that estrogen treatment decreased the number of ER-β mRNA-positive cells in the mitral cell and external plexiform layers of the olfactory bulb, central amygdaloid nucleus, medial geniculate nucleus, posterior hypothalamic nucleus, suprachiasmatic nucleus, and reticular part of the substantia nigra. As compared to the results of previous studies of young females, our data revealed that the regions in which expression of ER-β mRNA expression is affected by estrogen differ in middle age. These results suggest that the effects of estrogen on ER-β expression change with age.

Inhibition of ErbB-2 induces TFF3 downregulation in breast cancer cell lines

ErbB-2 gene plays an important role in carcinoma formation whose overexpression was observed in many types of tumors, including breast cancer. Dysregulation of Trefoil factor 3 (TFF3), which is thought to function in the development and progression of breast cancer, was found to be upregulated in ErbB2-overexpressing breast cancers and cells. However, a putative interaction between ErbB-2 and TFF3 in breast cancer remains unknown. To determine whether TFF3 has an important role in breast tumor, its levels were measured by immunohistochemistry in 130 cases of breast infiltrating duct carcinoma and 30 cases of normal breast tissue with a specific monoclonal antibody raised against human TFF3. Patients who were positive for ErbB-2 also had high expression levels of TFF3 (p < 0.05). Also, after infecting the SK-BR-3 cells with lentivirus-mediated ErbB2-specific shRNA (Lenti-ShERBB2), we detected the expressions of ErbB-2 and TFF3 by real-time polymerase chain reaction and Western blotting, respectively. Compared with the control groups, ErbB-2 mRNA expression was decreased in the Lenti-ShERBB2 infection group, and Western blotting indicated a concordant ErbB-2 protein reduction. On the other hand, TFF3 expression at both mRNA and protein levels was significantly downregulated by ErbB-2 silencing in SK-BR-3. These findings are a proof of the foundation for a certain relationships of ErbB-2 and TFF3, which may serve as novel therapeutic markers of ErbB2-overexpressing breast cancers in the future.