ER mRNA Research Articles

Gastric vagal afferent satiety signals are modulated by endogenous and exogenous oestradiol

Food intake is reduced during oestrus in rodents through a reduction in meal size. This is just after the peak in plasma oestradiol (E2) levels. Exogenous E2 reduces meal size and overall food intake in ovariectomized rats [1]. Whilst E2 can act on the arcuate nucleus to reduce food intake, the E2 receptors, ER , ER and GPR30 are also expressed in the cell bodies of vagal afferents located in the no dose ganglia [2]. Furthermore, E2 can sensitize cultured vagal neurons [3]. This suggests there may also be a peripheral site of action on food intake because activation of mechanosensitive gastric vagal afferents (GVAs) induces satiety. However, whether E2 can act on GVAs to modulate satiety signals and whether the reduction in food intake during oestrus has a vagal origin is unknown. The oestrous cycle stage of 8wk old female C57BL/6 mice was determined using vaginal cell cytometry (N = 3/cycle stage) [4]. Single fibre recordings of GVA mechanoreceptors were made [5] in the absence and presence of E2 (10—1000 pM). Recordings were also taken after pre-incubation with the ER selective antagonist, fulvestrant. No dose ganglia ER , ER and GPR30 mRNA levels were quantified by quantitative RT-PCR. Tension receptor response to stretch (3 g) was increased in mice currently in oestrus (p < 0.05 vs. dioestrus). There was no difference in the response of mucosal receptors to mucosal stroking (50mg) at any oestrous cycle stage. E2 dose dependently potentiated mucosal and tension receptor responses to mucosal stroking (10—1000mg, p < 0.001) and stretch (1—5 g, p < 0.05) respectively. The potentiation caused by E2 on tension and mucosal receptors was blocked by pre-incubation with fulvestrant. All three E2 receptors were present in the no dose ganglia, but there was 60 and 25 times more ER mRNA present than ER or GPR30 respectively (p < 0.001). Together this data suggests that the reduction in food intake observed during the oestrus stage may be due to an ER mediated E2 induced potentiation of tension receptor mechanosensitivity.

Abstract 2098: Histone deacetylation underlying hMAPK-induced ER mRNA repression

Abstract Estrogen receptor (ER)-negative breast cancer is more aggressive and associated with both shorter disease-free and overall survival than ER+ breast cancer. While anti-estrogen therapies have greatly improved treatment for ER+ breast cancer, both de-novo and acquired resistance occurs. Anti-estrogen therapies are not effective in ER- breast cancers, thus identifying mechanisms underlying lack of ER expression in ER- breast cancers is imperative. Hypermethylation of the ER promoter has been demonstrated to repress ER, and is found in ∼ 25% of ER- breast cancers. Previously we demonstrated that hyperactivation of MAPK (hMAPK) downstream of overexpressed EGFR or overexpression/amplification of Her2 represses ER protein and mRNA expression. Abrogation of hMAPK in ER- breast cancer cell lines and primary cultures causes re-expression of ER and restoration of anti-estrogen responses. hMAPK-mediated ER mRNA repression involves repression of transcription. We found that the ER mRNA synthesis rate and ER promoter activity, assessed with run-on assays and ER promoter reporter constructs, respectively, were significantly reduced in hMAPK-MCF-7 cell line models, with little to no ER expression, compared to control-transfected MCF-7 (coMCF-7) cells with high ER expression. Co-transfection of dnERK1/2 abrogated this repression restoring promoter activity to levels comparable with coMCF-7s but promoter bashing did not identify specific sequences responsible for this hMAPK repression. Thus we hypothesized that epigenetic mechanisms may be involved in hMAPK mediated repression of ER transcription; however, we could not detect ER promoter methylation in our hMAPK-MCF-7 models, nor in several breast cancer cell lines with hMAPK. In this study, we performed an epigenetic compound screen in the ER- SUM 149 breast cancer cell line, which presents hMAPK, and had been previously demonstrated to re-express ER with MAPK inhibition. Three candidate compounds, all HDAC inhibitors, were identified, validated in individual luciferase assays, and evaluated with rtPCR and Western blotting to verify their ability to modulate ER expression and estrogen signaling. They have been validated in additional ER- cell lines and clinical tumor samples lacking ER promoter methylation, as well. Finally, they have been used in our hMAPK-MCF-7 cells to demonstrate the role of hMAPK in histone deacetylation of the ER promoter. While HDAC inhibitors have been evaluated previously for their ability to sensitize anti-estrogen resistant ER+ breast cancers and re-express ER in ER- breast cancer cell lines exhibiting ER promoter methylation, histone deacetylation of ER in ER- breast cancers lacking ER promoter methylation has not been previously identified. Collectively, these data link histone deacetylation as an underlying mechanism in hMAPK-repression of ER mRNA suggesting this is may be a common epigenetic mechanism in the generation of ER-negative breast cancer. Citation Format: Amy J. Plotkin, Claude-Henry Volmar, Nagi Ayad, Dorraya El-Ashry. Histone deacetylation underlying hMAPK-induced ER mRNA repression. [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 2098. doi:10.1158/1538-7445.AM2014-2098

Abstract 4018: Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients

Abstract Introduction: Circulating Tumor Cells (CTCs) represent an important biological link in the spread of breast cancer from primary to metastatic disease. CTCs have already been established as strong predictors of prognosis in patients with metastatic breast cancer. Triple-negative breast cancers have a more aggressive clinical outcome than other forms of breast cancer. The aim of our work was to develop a quantitative real-time multiplex RT-qPCR assay for studying the expression of ER, PR, HER-2 and EGFR in CTC of breast cancer patients. Materials and Methods: A quadruplex quantitative real time RT-qPCR assay for ER, PR, HER-2 and EGFR was developed based on the de novo in-silico design of eight primers and four dual hybridization probes. All primers and probes were designed to avoid cross-reactions and primer dimer formation and to match the assay conditions, such as amplicon sizes, and melting temperatures. The specificity of all primer and hybridization probe sequences was first tested by homology searches in the nucleotide database (NCBI, nucleotide BLAST). Each probe set included a 3′-fluorescein (F) donor probe and a 5′- LC acceptor probe that was different for each gene set: ER at 610nm, PR at 640nm, HER-2 at 670nm, EGFR at 705nm. A color compensation test was performed by using pure dye spectra so that spectral overlap between dyes was corrected. The LightCycler 2.0 platform, (Roche, Diagnostics) was used since it allows detection of six multiple reporter dyes in the same capillary. The assay was designed, so that only 1 μL of cDNA is used and the total reaction volume is 10 μL. Results: The assay sensitivity was evaluated by performing spiking experiments. For this reason known numbers of MCF7 and SKBR-3 cells, as well as external quantification calibrators ranging from 105 copies/μL to 10 copies/μL, were used. The specificity was evaluated by carefully designed cross reaction studies, performed for each gene target in the presence of all other targets. Our assay, under optimized conditions has shown a very high specificity for each analyte. Conclusions: We report for the first time a highly sensitive, specific and reproducible quantitative multiplex real-time RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR. The assay is currently validated in CTC isolated from a large number of breast cancer patients. Citation Format: Areti D. Strati, Evi S. Lianidou. Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients. [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 4018. doi:10.1158/1538-7445.AM2014-4018

Transcriptional repression of ER through hMAPK dependent histone deacetylation by class I HDACs.

Anti-estrogen therapies are not effective in ER- breast cancers, thus identifying mechanisms underlying lack of ER expression in ER- breast cancers is imperative. We have previously demonstrated that hyperactivation of MAPK (hMAPK) downstream of overexpressed EGFR or overexpression/amplification of Her2 represses ER protein and mRNA expression. Abrogation of hMAPK in ER- breast cancer cell lines and primary cultures causes re-expression of ER and restoration of anti-estrogen responses. This study was performed to identify mechanisms of hMAPK-induced transcriptional repression of ER. We found that ER promoter activity is significantly reduced in the presence of hMAPK signaling, yet did not identify specific promoter sequences responsible for this repression. We performed an epigenetic compound screen in an ER- breast cancer cell line that expresses hMAPK yet does not exhibit ER promoter hypermethylation. A number of HDAC inhibitors were identified and confirmed to modulate ER expression and estrogen signaling in multiple ER- cell lines and tumor samples lacking ER promoter methylation. siRNA-mediated knockdown of HDACs 1, 2, and 3 reversed the mRNA repression in multiple breast cancer cell lines and primary cultures and ER promoter-associated histone acetylation increased following MAPK inhibition. These data implicate histone deacetylation downstream of hMAPK in the observed ER mRNA repression associated with hMAPK. Importantly, histone deacetylation appears to be a common mechanism in the transcriptional repression of ER between ER- breast cancers with or without ER promoter hypermethylation.

Estrogen receptor β agonist inhibits proliferation of endometrial stromal cells in an estrogen-receptor independent manner

The finding that estrogen receptor ER? agonists induce regression of ectopic endometrial implants in a rodent model has rekindled interest in the role of ER? in the growth and maintenance of human endometriosis. We hypothesize that the ER? agonist may have a direct effect on the human endometrium. We examined the mRNA and protein expression of ER? in both eutopic and ectopic endometrium and performed a luciferase reporter assay for the presence of functional ER? in human endometrial stromal cells. Diarylpropionitrile (DPN - a ER? specific agonist) significantly inhibited endometrial stromal cell proliferation at 10 M by 25% but not at 10 nM. ER? mRNA is present in both eutopic and ectopic endometrium; however, protein expression is absent in both stroma and epithelium. Our search for functional ER? similarly showed an absence in endometrial stromal cells. These results suggest that the regression of endometriosis might be ER?-independent.

Inverse Regulation of EGFR/HER1 and HER2-4 in Normal and Malignant Human Breast Tissue

Cross-talk between the estrogen and the EGFR/HER signalling pathways has been suggested as a potential cause of resistance to endocrine therapy in breast cancer. Here, we determined HER1-4 receptor and neuregulin-1 (NRG1) ligand mRNA expression levels in breast cancers and corresponding normal breast tissue from patients previously characterized for plasma and tissue estrogen levels. In tumours from postmenopausal women harbouring normal HER2 gene copy numbers, we found HER2 and HER4, but HER3 levels in particular, to be elevated (2.48, 1.30 and 22.27 –fold respectively; P<0.01 for each) compared to normal tissue. Interestingly, HER3 as well as HER4 were higher among ER+ as compared to ER- tumours (P=0.004 and P=0.024, respectively). HER2 and HER3 expression levels correlated positively with ER mRNA (ESR1) expression levels (r=0.525, P=0.044; r=0.707, P=0.003, respectively). In contrast, EGFR/HER1 was downregulated in tumour compared to normal tissue (0.13-fold, P<0.001). In addition, EGFR/HER1 correlated negatively to intra-tumour (r=-0.633, P=0.001) as well as normal tissue (r=-0.556, P=0.006) and plasma estradiol levels (r=-0.625, P=0.002), suggesting an inverse regulation between estradiol and EGFR/HER1 levels. In ER+ tumours from postmenopausal women, NRG1 levels correlated positively with EGFR/HER1 (r=0.606, P=0.002) and negatively to ESR1 (r=-0.769, P=0.003) and E2 levels (r=-0.542, P=0.020). Our results indicate influence of estradiol on the expression of multiple components of the HER system in tumours not amplified for HER2, adding further support to the hypothesis that cross-talk between these systems may be of importance to breast cancer growth in vivo.

Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG).

BackgroundDiscrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer.Patients and MethodsFormalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes.ResultsIn a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01). ESR1 clusters were observed in 9.5% (57 gain, 38 amplification) of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression) in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification) fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49–0.64, p = 0.003). Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066) and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029).ConclusionsESR1 gene deletion and amplification do not constitute per se prognostic markers, instead they can be classified to distinct prognostic groups according to their protein-mediated functional status.

Assessment of RNA in human breast tissue sampled by random periareolar fine needle aspiration and ductal lavage and processed as fixed or frozen specimens

Ductal lavage (DL) and random periareolar fine needle aspiration (RPFNA) have both been proposed as minimally invasive techniques to sample breast tissue during breast cancer prevention trials. Laser capture microdissection (LCM), linear RNA amplification and quantitative real-time polymerase chain reaction (qPCR) theoretically overcome the limitations of small specimen size obtained with DL and RPFNA. In order to test the yield, relative stability and amplifiability of RNA from fixed and archived RPFNA and DL specimens, breast tissue was sampled from individual high risk women (n=9) by both DL and RPFNA. RPFNA samples showed good RNA/cDNA yield and amplification while only 2 of 9 of the paired DL specimens had cDNA of adequate quality for subsequent PCR. One and two rounds of linear amplification provided approximately a 200- and 20,000-fold enrichment of RNA, respectively. PCR analysis consistently detected ER and COX-1 mRNA in the majority of RPFNA samples examined while pS2, PCNA, VEGF and survivin expression varied with subject. RNA yield and/or stability was greater for fixed and archived RPFNA than DL specimens of breast tissue. In a subsequent study examining an expanded biomarker gene panel in fixed vs. frozen RPFNA samples, mRNA profiles and ranked relative mRNA abundance were similar (r=0.89) for frozen and fixed RPFNA specimens. In summary, frozen RPFNA samples may be optimal for RNA endpoints in human breast cancer prevention trials but fixed RPFNA specimens allow similar analyses with greater convenience.

Effects of illegal cyanide fishing on vitellogenin in the freshwater African catfish, Clarias gariepinus (Burchell, 1822)

The effects of cyanide, used in illegal fishing, on one of the most economically important Nile fishes, the African catfish (Clarias gariepinus), were studied. Cyanide impacts were evaluated in terms of biochemical, molecular and histopathological characteristics. After exposure to sublethal concentration (0.05mg/l) of potassium cyanide (KCN) for two and four weeks, GOT (glutamate oxaloacetate transaminase) was significantly increased in both male and female, while GPT (glutamate pyruvate transaminase), total plasma protein, phosphoprotein phosphorus (Vgt) in serum, vitellogenin gene expression (Vtg mRNA) and estrogen receptors (ER mRNA) were significantly decreased in female. On the other hand, male C. gariepinus showed a significant increase in Vtg and Vtg mRNA. Liver, testis and ovaries showed distinct histopathological changes. It was concluded that, cyanide caused damaging effects to fish and can cause serious disturbance in the natural reproduction and a drastic decline in fish population. Therefore, it is recommended that, the use of cyanide compounds must be prohibited to conserve the fisheries resources.

Abstract P6-10-03: The PKC inhibitor PKC412 antagonizes breast cancer cell growth and enhances tamoxifen sensitivity

Abstract Background: AIB1 (SRC-3, NCoA3), a member of the p160/steroid receptor coactivators family, plays a critical role in cell growth and proliferation. In estrogen receptor-alpha positive (ER+) breast cancer (BC) cells, it coactivates estrogen- and additional transcription factors-dependent gene transcription, reducing the antagonistic activity of tamoxifen and resulting in tamoxifen resistance (TR). We have previously shown that BC patients whose tumors expressed high levels of both AIB1 and HER-2 had worse outcomes with tamoxifen therapy, suggesting that AIB1 may be an important diagnostic and therapeutic target. Our findings that knocking down AIB1 attenuates ER signaling and inhibits breast cancer cell growth further indicate that the manipulation of AIB1 level could be an approach to treating BC and overcoming TR. Recently, it has been shown that protein kinase C (PKC) isoforms phosphorylate AIB1 and prevent its proteasome-mediated degradation. The present study was carried out to test if the multi-targeted kinase and PKC inhibitor PKC412 (midostaurin) is capable of promoting degradation of AIB1, inhibiting BC cell growth, and promoting tamoxifen antagonistic activity. Methods: The ER+ MCF7, T47D, and ZR75-B BC cells and their tamoxifen-resistant derivatives (TR) were used. The Methylene Blue assay was employed to measure cell viability of BC cell lines after treatment with PKC412 or the combination of PKC412 with tamoxifen. To determine the impact of PKC412 on AIB1 and ER proteins and mRNA levels, we used immunoblotting and RT-qPCR, respectively. Results: PKC412 successfully inhibited the growth of MCF7, T47D, and ZR75-B cells, and their tamoxifen-resistant derivatives. Treatment with PKC412 depleted AIB1 protein and reduced the level of ER protein without significant alteration in AIB1 mRNA level. Consequently, ER signaling was disrupted, as reflected by decreased expression of ER target genes such as GREB1. PKC412 also enhanced tamoxifen's antagonistic activity in the parental cell lines and sensitized tamoxifen-resistant MCF7 and ZR75-B cells to tamoxifen. Conclusions: The results of this study suggest that the multi-targeted kinase and PKC inhibitor PKC412 can post-translationally destabilize AIB1 protein and ER, inhibiting BC cell growth and viability. PKC412 enhances tamoxifen's antagonistic activity on BC growth; furthermore, it sensitizes tamoxifen-resistant cells to tamoxifen. Thus, PKC412 is a promising agent to treat BC and, in combination with tamoxifen, to delay or overcome TR. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-03.

Endocrine disruption in thicklip grey mullet (Chelon labrosus) from the Urdaibai Biosphere Reserve (Bay of Biscay, Southwestern Europe)

Endocrine disruptors (EDs) interfere with the development and functioning of the endocrine system, causing reproductive disturbance in aquatic wildlife. The aim of the present work was to determine the presence of EDs in sediments and to investigate possible exposure and effects of EDs in the estuary of the Urdaibai Biosphere Reserve (Gernika) in comparison with the Arriluze marina. For this, gonad histology, plasma vitellogenin (VTG) protein levels and mRNA levels of vitellogenin (vtg), cyp19 aromatases, estrogen receptor (er) and retinoid X receptor (rxr) were studied in Chelon labrosus. The presence of alkylphenols (APs) in fish bile was also assessed. In sediments, estrogenic hormones were below the detection limit and levels of bisphenol A were very low. In Gernika organotin compounds were low but in Arriluze levels of up to 12μg/g were found. Moderate levels of APs and phthalate levels of up to 8μg/g were found in sediments. In fish, a high prevalence up to 33% of intersex gonads was found in Gernika, whereas only one intersex was found in Arriluze. Accordingly, mullets from Gernika showed higher concentrations of APs in bile. VTG protein levels were detected not only in females but also in some undifferentiated, male and intersex fish. mRNA of vtg was detected in one male from Gernika. mRNA of er and rxr showed significant differences between seasons. In conclusion, the present study demonstrated that C. labrosus from the Urdaibai estuary were exposed to EDs and showed clear signs of endocrine disruption.

Localization of Estrogen Receptor in the Central Lymphoid Organs of Chickens during the Late Stage of Embryogenesis

Immunological function in chicks is greatly affected by estrogen treatment during embryogenesis, but the mechanism of the estrogen effect is not fully understood. To elucidate the effect of estrogen on immune function, we observed estrogen receptor expression in the thymus and bursa of chick embryos by immunohistochemistry. We compared the distribution of estrogen receptor-positive cells with that of keratin-positive epithelial cells. Intense expression of estrogen receptors was detected in thymic and bursal lymphocytes. In peripheral lymphocytes, ER mRNA was detected by RT-PCR analysis. The results of fluorescence-activated cell sorting analysis indicated that the estrogen receptor was expressed in the cytoplasm of the lymphocytes. Furthermore, intense expression of the estrogen receptor was also confirmed in thymic Hassall's corpuscles, bursal follicle-associated epithelial cells, and the bursal interfollicular epithelium. Our results indicate that estrogen affects the differentiation of thymic and bursal lymphocytes, suggesting that the underlying role for estrogen in immune function.

Estrogen-Induced Retinal Endothelial Cell Proliferation: Possible Involvement of Pigment Epithelium-Derived Factor and Phosphoinositide 3-Kinase/Mitogen-Activated Protein Kinase Pathways

Diabetic retinopathy is a leading cause of blindness due to a progressive damage of the retina by neovascularization and other related ocular complications. However, the molecular mechanism underlying the development of diabetic retinopathy is not well understood. An increase in estrogen levels during puberty is associated with an accelerated development of diabetic retinopathy. Previously, we have introduced 17β-estradiol (E2) to rhesus retinal capillary endothelial cells (RhRECs) in culture and observed a dose- and time-dependent increase in the number of viable cells. The purpose of this present study was to investigate the molecular signaling pathway associated with this estrogen-induced proliferation of RhRECs. Estrogen receptor (ER) ER(α) and ER(β) mRNA expression, and protein synthesis were measured at 0, 3, 6, and 12 h using nested polymerase chain reaction and Western blots. Phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors were introduced into culture media to study their effects on E2-induced cell proliferation and pigment epithelium-derived factor (PEDF) synthesis. The levels of PEDF in the conditioned media were measured by enzyme-linked immunosorbent assay. Exogenous E2 induced a significant increase in the expression of ER(β) along with an increase in the number of viable RhRECs. Cotreatment of E2 with PI3K and MAPK inhibitors significantly reduced the E2-induced effect on cell proliferation and PEDF production in a dose-dependent manner. Results from the present study suggest that an E2-induced increase in the proliferation of RhRECs may be mediated by the action of ER(β.) Both PI3K and MAPK signaling pathways are involved in this E2-induced cell proliferation, which may follow changes in PEDF levels controlled by these pathways. Further studies will provide additional details on the interaction between these pathways to control changes in PEDF levels and cell proliferation.

Changes in Estrogen Receptor ERα and ERβ Expression in Chicken (Gallus domesticus) Adrenal Gland during Short-fasting and Refeeding

Estrogen receptors have been found in the adrenal gland of rodents, monkeys, mares and sheep, indicating a connection between sex steroids and the activity of the adrenal gland. In the present study, the expression of estrogen receptors alpha (ERalpha) and beta (ERbeta) in the chicken adrenal gland during stress induced by 24 h fasting and after refeeding was determined using reverse transcription and the polymerase chain reaction (RT-PCR). The presence of both ER mRNAs in the adrenal gland of all examined groups was found. The relative expression of ERalpha mRNA was higher than ERbeta mRNA. There were no significant differences in ERalpha mRNA expression among the examined groups. On the contrary, we observed changes in ERbeta expression during stress conditions. These findings indicate different pathways of estrogen action in the avian adrenal gland. Furthermore, changes in ERbeta level suggest that this form of estrogen receptor plays a predominant role for estrogen action in the chicken adrenal gland during stress.

Preclinical evaluation of selective inhibitors of nuclear export (SINE) in basal-like breast cancer (BLBC).

1055 Background: Basal-like breast cancers (BLBC) compose up to 15% of breast cancer (BC) and are usually triple negative characterized by lack of ER, PR, and HER-2 amplification. In addition, most BRCA1-associated BCs are BLBC and TNBC, expressing basal cytokeratins and EGFR. BLBC is characterized by an aggressive phenotype, high histological grade, and poor clinical outcomes: high recurrence and metastasis rates. CRM1 (XPO1) is the exclusive nuclear exporter of multiple Tumor Suppressor Proteins (TSP) including p53, p21, BRCA1&amp;2, pRB, FOXO. CRM1 inhibition forces nuclear accumulation of TSPs, inducing apoptosis in cancer cells. KPT-SINE are novel, small molecule, irreversible inhibitors of CRM1 with potent anti cancer activity. Methods: The Cancer Genome Atlas (TCGA) and BC cell line databases were used for mRNA analyses. MTT assay was used to determine the cytotoxic effect of KPT-SINE (KPT-185 and KPT-330) on 44 breast cell lines including luminal A, luminal B, HER2 positive, BLBC and TNBC cells. The effect of KPT-330 treatment on tumor growth was tested in vivo in the TNBC model MDA-MB-468 xenograft. Results: Analyses of nuclear pore complex (NPC)-related mRNA levels (including CRM1) showed clear separation of BCs into high and low NPC expression. BLBC subtype was enriched with high NPC transcripts while luminal BC was enriched in low NPC levels (p&lt;1.53e-20). High NPC levels had higher mutation levels in BRCA2 (cor=0.33, p=1.83e-8) and ABL1. NPC expression was inversely correlated with ER mRNA expression (cor=-0.58, p=1.37e-7). KPT-SINE showed potent cytotoxicity on &gt;75% of the cell lines (IC50 values &lt;1 μM). Only three of 24 TNBC cell lines displayed IC50 values &gt;1.5 μM upon KPT-SINE treatment. Genomic analyses on all BC lines indicated that p53, PI3K/AKT and BRCA1 or 2 status did not affect cytotoxicity. In MDA-MB-468 xenograft, KPT-330 displayed efficacy in a dose-dependent manner inhibiting nearly 100% of tumor growth compared with vehicle treated animals, and was well tolerated. Conclusions: These data show that NPC/CRM1 mRNAs are overexpressed in BLBC/TNBC and that CRM-1 mediated nuclear export inhibition by SINE represents a potentially novel and well tolerated therapy for BLBC / TNBC.

English

The objective of the study to evaluate the effect of Qianliening capsule (QLNC) on the expression levels of serum hormones, prostatic estrogen receptor and androgen receptor in benign prostatic hyperplasia (BPH) rats, and investigate the possible molecular mechanisms mediating its anti-BPH activity. Male Sprage-Dawley (SD) rat BPH model was generated. BPH rats were orally treated with different concentrations of QLNC. Blood and the prostatic tissues of animals were obtained. The prostatic weight (PW) and prostatic index (PI) were evaluated; the histopathological changes of prostatic tissue, the levels of serum testosterone and estradiol, the mRNA and protein expression of ER and AR in prostatic tissue were examined by microscopy with hematoxylin and eosin staining HE staining, ELISA, RT-PCR, immunohistochemistry, respectively. Compared to the model group, the PW and PI in all QLNC-treated groups have significantly lower (p&lt;0.05) serum than T/E2&nbsp;in QLNC-treated groups which was elevated significantly (p&lt;0.05 or&nbsp;p&lt;&nbsp;0.01). Pathomorphism of prostatic tissue in QLNC-treated groups improved. The mRNA and protein expression of ER and AR in QLNC-treated groups decreased significantly. QLNC has significant therapeutic effect on BPH rats. Improvement of sex hormones disorder and regulation of ER and AR are one of the mechanisms by which QLNC treats BPH. &nbsp; Key words:&nbsp;Benign prostatic hyperplasia; Qianliening capsule; sex hormones; estrogen receptor; androgen receptor.

Oncostatin M suppresses oestrogen receptor-α expression and is associated with poor outcome in human breast cancer

The most important clinical biomarker for breast cancer management is oestrogen receptor alpha (ERα). Tumours that express ER are candidates for endocrine therapy and are biologically less aggressive, while ER-negative tumours are largely treated with conventional chemotherapy and have a poor prognosis. Despite its significance, the mechanisms regulating ER expression are poorly understood. We hypothesised that the inflammatory cytokine oncostatin M (OSM) can downregulate ER expression in breast cancer. Recombinant OSM potently suppressed ER protein and mRNA expression in vitro in a dose- and time-dependent manner in two human ER+ breast cancer cell lines, MCF7 and T47D. This was dependent on the expression of OSM receptor beta (OSMRβ) and could be blocked by inhibition of the MEKK1/2 mitogen-activated protein kinases. ER loss was also necessary for maximal OSM-induced signal transduction and migratory activity. In vivo, high expression of OSM and OSMR mRNA (determined by RT-PCR) was associated with reduced ER (P<0.01) and progesterone receptor (P<0.05) protein levels in a cohort of 70 invasive breast cancers. High OSM and OSMR mRNA expression was also associated with low expression of ESR1 (ER, P<0.0001) and ER-regulated genes in a previously published breast cancer gene expression dataset (n=321 cases). In the latter cohort, high OSMR expression was associated with shorter recurrence-free and overall survival in univariate (P<0.0001) and multivariate (P=0.022) analyses. OSM signalling may be a novel factor causing suppression of ER and disease progression in breast cancer.

Differences in Gene Expression Profiles between Human Breast Tissue and Peripheral Blood Samples for Breast Cancer Detection

The purpose of this study is to check the similarities of differential gene expression of 11 genes in breast cancer tissue and blood samples from the same individual for early detection of breast cancer. We had investigated differential gene expression by qRT-PCR in 20 breast cancer patients’ tumoral tissues and corresponding blood sample. In our analysis BRCA2, HER-2, ER, PR, MET and BRAF mRNA levels were significantly over expressed in tumoral tissues. ER and PR mRNA levels were not detected in any of the peripheral blood samples, whereas KRAS and PTEN mRNA levels were not detected in any of the tumoral tissues. HER-2 (45%), EGFR (40%) and PI3KCA (30%). KRAS and PTEN mRNA levels were significantly over expressed in peripheral blood. In the correlation analysis expression of most of the genes were significantly altered in grade II and III in the tissues, where as in premenopausal women mRNA expression was significantly high in Grade II and III and ER/PR negative tumors. Our results suggests that BRCA2, ER, PR, PI3KCA, MET and BRAF differential gene expression at mRNA levels showed no diagnostic value as a marker of circulating tumour cells in breast cancer. qRT-PCR may be suitable alternative method for the determination of HER-2, EGFR, PI3KCA KRAS and PTEN mRNA status in the blood of breast cancer patients. Premenopausal women with high grade (Grade II and III) and ER/PR negative cases may be associated with proliferation/metastasis, high recurrence rate, and poor prognosis.

P1-07-06: High Concordance of Protein (by IHC), Gene (by FISH; HER-2 Only) and Microarray Readout (by TargetPrint) of ER/PR/HER2: Results from the MINDACT Trial.

Abstract Background Previously, the micro-array readout of ER, PR and HER2 by TargetPrint was shown to be strongly correlated with high quality immunohistochemistry (IHC)/FISH assessment, especially for ER and HER2. Concordance rates were 93% (k=0.79) for ER; 83% (k=0.65) for PR and 96% for HER2 (k=0.88) in 636 patients (Roepman et al., Clin Cancer Res, 2009). This study analysis was undertaken to further determine the correlation of microarray readout with IHC/FISH assessment both locally and centrally determined in the 1st 800 pts enrolled in the MINDACT trial. This work is essential to determine the quality of biological data in the two risk assessment methods used in MINDACT based upon which adjuvant chemotherapy decision is made, in order to exclude bias. Methods: ER/PR/HER2 IHC assessment was performed on the 1st 800 primary breast cancers (BC) of pts enrolled in the MINDACT study. The assessment was performed locally at each center (n=800) and by central review at the laboratory of the European Institute of Oncology (n=626). A tumor was classified positive for ER and PR when 1% of tumor cells showed positive staining. HER2 IHC status was scored as 0, 1+, 2+ or 3+; a score of 3+ was considered positive. In 2+ cases FISH was performed to assess final HER2 status. Gene expression data for ER, PR and HER2 were obtained by TargetPrint stratified as receptor positive or negative using previously determined and validated thresholds for ER, PR and HER2 mRNA levels (n=800). Results: Comparison of local assessment (IHC &amp; FISH for HER2) with central review indicated highly similar results for receptor readout with a concordance of 98% (k=0.90) for ER; and 96% for HER2 (k=0.80) and slightly lower for PR (90% (k=0.72)). Comparison of central assessment (IHC &amp; FISH for HER2) with micro array readout by TargetPrint indicated highly similar results for receptor readout with a concordance of 97% (k=0.88) for ER and 95% for HER2 (k=0.76). For PR the concordance was lower but still quite acceptable (85% (k=0.62)). Conclusion: Local and centrally assessed ER, PR and HER2 status in the first 800 MINDACT patient samples indicate a high level of quality for pathology in the local participating hospitals. These results exclude any bias induced by a lower quality of “traditional” pathology results as compared to the centrally assessed MammaPrint, both used for risk assessment and adjuvant chemotherapy decision in the MINDACT trial. The microarray-based assessment of ER, PR and HER2 gives results comparable to IHC &amp; FISH and provides an objective and quantitative assessment of tumor receptor status. These results indicate that TargetPrint can serve as a second pathology assessment for locally assessed parameters, especially since TargetPrint is part of a multi-profile platform for breast cancer treatment management. This work was funded by the Breast Cancer Research Foundation and the EU Framework Program VI. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-06.

Testicular toxicity of methyl thiophanate in the Italian wall lizard (Podarcis sicula): morphological and molecular evaluation

The effects of the fungicide methyl thiophanate (MT) on testis were determined in the Italian wall lizard (Podarcis sicula) using morphological and molecular analyzes. Three experimental trials were performed: an acute test using six doses, a two-week chronic test, and "ecotoxicological" exposure (3 weeks). The minimal lethal dose (LD(50)) of pure MT, reached by the acute test, was 100 mg/kg body weight. Testicular histopathology of surviving animals showed a reduced lumen and several multinucleated giant cells 24 h after injection followed by large decreases in spermatogonia (72%) and secondary spermatocytes (58%) and a loss of spermatids and sperms 7 days after. In the chronic test, a dose equivalent to 1/100 of LD(50) was injected on alternate days. Complete shutting of the lumen and a great decrease in spermatogonia (82%) were observed. In "ecotoxicological" exposure, achieved with a commercial MT compound, testis showed a decrease in primary spermatocytes (20%) and several vacuoles. An increase in germ cell apoptosis was observed in all experimental groups using TUNEL assay. A decrease in expression of androgen and estrogen receptor (AR and ER) mRNAs was seen in all experimental groups. The reduction in AR and ER mRNAs was correlated to exposure time. Indeed, in the "ecotoxicological" treatment (30 days), the decrease reached 82 and 90% for AR and ER mRNAs, respectively. These data strongly indicate that treatment with MT, damaging the seminiferous epithelium and decreasing steroid receptor expression, might render exposed lizards infertile.