Estrogen-responsive Finger Protein Research Articles

Abstract 4385: ZCCHC3 and Efp are prognostic factors for triple-negative breast cancer and potentially modulate cell division-related pathway

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and limited therapeutic strategies, lacking expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. We previously demonstrated that estrogen responsive finger protein (Efp)/TRIM25 functions as a ubiquitin ligase that degrades cell cycle checkpoint 14-3-3 sigma and promotes cell growth of ER-positive breast cancer [Urano et al., Nature 417:871-875, 2002]. In TNBC cells, we further showed that Efp regulates cell cycle-related gene expression and promotes cell proliferation [Sato et al., Biochem Biophys Res Commun 624:81-88, 2022]. Efp also exerts a distinct function to activate cellular RNA sensors such as RIG-I (retinoic acid inducible gene I), which plays a role in the innate immunity [Gack et al., Nature 446:916-920, 2007]. Another immune-related gene zinc finger CCHC domain-containing protein 3 (ZCCHC3) was recently suggested as Efp interactor, although its pathological relevance in breast cancer remains elusive. We here aimed to clarify the role of ZCCHC3 in TNBC. Immunohistochemical analysis of TNBC tissues from over 100 Japanese patients showed that a positive ZCCHC3 immunoreactivity (IR) was significantly associated with shorter disease-free survival (DFS) and positively correlated with Efp IR. Notably, patients with both ZCCHC3 and Efp IR positivity showed much shorter DFS than those with ZCCHC3 positivity alone. Functional analysis of ZCCHC3 by siRNA silencing demonstrated that ZCCHC3 promotes the proliferation, migration, and cell cycle progression of MDA-MB-231 TNBC cells. We established long-term culturable TNBC patient-derived cells (TNBC-PDC) using spheroid culture technique and revealed that the spheroid growth was suppressed by ZCCHC3-specific siRNA treatment. Moreover, ZCCHC3-specific siRNA injection suppressed in vivo tumor growth of MDA-MB-231 cells inoculated in immunodeficient mice. RNA sequencing analysis of ZCCHC3-silenced TNBC cells showed that cell division-related pathway was enriched among downregulated genes, including non-structural maintenance of chromosomes condensin I complex subunit H (NCAPH). NCAPH silencing inhibited the proliferation of TNBC-PDCs and MDA-MB-231 cells. Overall, ZCCHC3 and Efp are potential poor prognostic factors and can be applied to alternative therapeutic options for TNBC. Citation Format: Akihiro Fujimoto, Kazuhiro Ikeda, Toshihiko Takeiwa, Keiichi Kinowaki, Takuya Ogura, Hidetaka Kawabata, Akihiko Osaki, Kuniko Horie, Satoshi Inoue. ZCCHC3 and Efp are prognostic factors for triple-negative breast cancer and potentially modulate cell division-related pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4385.

Efp/TRIM25 and Its Related Protein, TRIM47, in Hormone-Dependent Cancers.

Increasing attention has been paid to the biological roles of tripartite motif-containing (TRIM) family proteins, which typically function as E3 ubiquitin ligases. Estrogen-responsive finger protein (Efp), a member of the TRIM family proteins, also known as TRIM25, was originally identified as a protein induced by estrogen and plays critical roles in promoting endocrine-related cancers, including breast cancer, endometrial cancer, and prostate cancer. The pathophysiological importance of Efp made us interested in the roles of other TRIM family proteins that share a similar structure with Efp. Based on a phylogenetic analysis of the C-terminal region of TRIM family proteins, we focused on TRIM47 as a protein belonging to the same branch as Efp. TRIM47 is a poor prognostic factor in both breast cancer and prostate cancer. Atypical lysine-27-like poly-ubiquitination was involved in the underlying mechanism causing endocrine resistance in breast cancer. We also discuss the functions of Efp and TRIM47 in other types of cancers and innate immunity by introducing substrates the are modified by poly-ubiquitination.

Efp promotes growth of triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is characterized by its high ability of invasiveness and metastasis, namely lacking expression of estrogen receptor (ER), progesterone receptor, and HER2. We previously demonstrated that estrogen responsive finger protein (Efp) plays a tumor-promotive role in ER-positive breast cancer, yet it remains to be addressed whether Efp contributes to TNBC pathophysiology. We here found that Efp mRNA and protein were abundantly expressed in TNBC patient-derived cells and MDA-MB-231 cells. Efp silencing significantly decreased the growth and migration of both TNBC cell models. Cell-cycle profiling showed a decrease in the S phase population upon Efp silencing. Moreover, exogenous Efp expression increased the growth, migration, and the percentages of S phase population of TNBC cells. Transcriptomic analysis in the Efp-silenced TNBC cells identified several candidate Efp targets including cell cycle-related genes CDCA7 and HELLS, whose contribution to cell growth were validated by siRNA-mediated gene silencing. These results suggest that Efp plays a tumor-promotive role in TNBC and can be a potential therapeutic target for the aggressive disease.

Leveraging transcriptome and epigenome landscapes to infer regulatory networks during the onset of sexual maturation

BackgroundDespite sexual development being ubiquitous to vertebrates, the molecular mechanisms underpinning this fundamental transition remain largely undocumented in many organisms. We designed a time course experiment that successfully sampled the period when Atlantic salmon commence their trajectory towards sexual maturation.ResultsThrough deep RNA sequencing, we discovered key genes and pathways associated with maturation in the pituitary-ovarian axis. Analyzing DNA methylomes revealed a bias towards hypermethylation in ovary that implicated maturation-related genes. Co-analysis of DNA methylome and gene expression changes revealed chromatin remodeling genes and key transcription factors were both significantly hypermethylated and upregulated in the ovary during the onset of maturation. We also observed changes in chromatin state landscapes that were strongly correlated with fundamental remodeling of gene expression in liver. Finally, a multiomic integrated analysis revealed regulatory networks and identified hub genes including TRIM25 gene (encoding the estrogen-responsive finger protein) as a putative key regulator in the pituitary that underwent a 60-fold change in connectivity during the transition to maturation.ConclusionThe study successfully documented transcriptome and epigenome changes that involved key genes and pathways acting in the pituitary – ovarian axis. Using a Systems Biology approach, we identified hub genes and their associated networks deemed crucial for onset of maturation. The results provide a comprehensive view of the spatiotemporal changes involved in a complex trait and opens the door to future efforts aiming to manipulate puberty in an economically important aquaculture species.

Transcriptomic analysis of hormone-sensitive patient-derived endometrial cancer spheroid culture defines Efp as a proliferation modulator

Estrogen-responsive endometrial cancer (EC) is prevalent in uterine cancer. Its precise molecular mechanisms remain to be elucidated partly because of limited availability of estrogen-sensitive EC models recapitulating clinical pathophysiology. We previously established EC patient-derived cancer cell (EC-PDC) spheroid culture with high expression of estrogen receptor α (ERα). Using this EC-PDC, we study the transcriptional regulation and function of estrogen-responsive finger protein (Efp), a prototypic tripartite motif (TRIM) protein that modulates protein degradation and RNA processing. Intense estrogen-dependent EFP mRNA induction and high ERα occupancy to EFP estrogen responsive element (ERE) were observed in EC-PDC. Luciferase reporter gene assay showed that the ERE facilitates EFP transcriptional activity estrogen-dependently. siRNA-mediated Efp silencing in EC-PDC resulted in suppressed spheroid proliferation and altered gene expression profile, featuring downregulation of genes related to cell cycle (e.g., CDK6) and inflammation/immune responses (e.g., IL10RA, IL26, and IL6ST) while unaffected expression of cancer stemness-related markers. Taken together, EC-PDC spheroid culture is a powerful EC tool that enables to dissect Efp-mediated ERα signaling pathways as an estrogen-sensitive EC model. This study provides an insight into alternative EC therapeutic strategies targeting ERα-Efp axis.

Combined Use of Immunoreactivities of RIG-I with Efp/TRIM25 for Predicting Prognosis of Patients With Estrogen Receptor-positive Breast Cancer

BackgroundWe previously identified estrogen-responsive finger protein (Efp) as an estrogen-induced gene, and showed that the positive immunoreactivity of Efp is a poor prognostic factor for patients with breast cancer. We also demonstrated that Efp has distinctive roles in innate immunity by activating pattern recognition receptor retinoic acid-inducible gene I (RIG-I). The clinical value of RIG-I protein expression in breast cancer had not been evaluated in relationship with patients’ prognosis. Patients and MethodsTissue samples of estrogen receptor-positive invasive breast cancer were obtained from 145 female patients with breast cancer who underwent surgical treatment. Immunoreactivities of RIG-I and Efp were analyzed with the antibodies generated for the present study. ResultsPositive immunoreactivity of RIG-I was correlated with lower disease-free survival (P = .032) and was an independent poor prognostic factor (P = .043). RIG-I immunoreactivity was positively correlated with that of Efp (P = .0004). Patients with positive immunoreactivities of both RIG-I and Efp proteins were associated with a lower disease-free survival rate (P = .005). ConclusionsPositive immunoreactivity of RIG-I has clinical significance as a poor prognostic factor in patients with estrogen receptor-positive breast cancer. A positive correlation of RIG-I and Efp immunoreactivities was observed, and the combination of their immunoreactivities can be used to predict patients’ prognosis.

TRIM25 enhances cell growth and cell survival by modulating p53 signals via interaction with G3BP2 in prostate cancer.

Prostate cancer growth is promoted by the gene regulatory action of androgen receptor (AR) and its downstream signals. The aberrant dysfunction of tumor suppressor p53 has an important role in the prognosis of cancer. We previously found that androgen treatments translocate p53 to the cytoplasm. The mechanism of this translocation depends on sumoylation of p53 by complex of SUMO E3 ligase RanBP2 with androgen-induced GTPase-activating protein-binding protein 2 (G3BP2). Here, we identified tripartite motif-containing protein 25 (TRIM25)/estrogen-responsive finger protein (Efp) as a novel interacting partner of G3BP2 protein complex. Then, we demonstrated that TRIM25 knockdown resulted in p53 downstream activation for cell cycle inhibition and apoptosis induction in LNCaP and 22Rv1 cells. In contrast, overexpression of TRIM25 promoted prostate cancer cell proliferation and inhibited apoptosis by docetaxel treatment in LNCaP cells. We observed that p53 activity was reduced by mechanism of G3BP2-mediated nuclear export in TRIM25-overexpressing prostate cancer cells. We also found TRIM25 is important for G3BP2/RanBP2-mediated p53 modification. Clinically, we newly demonstrated that TRIM25 is a prognostic factor for prostate cancer patients. Expression of TRIM25 is significantly associated with cytoplasmic p53 expression and G3BP2. Moreover, TRIM25 knockdown results in reduced tumor growth and increased p53 activity in the mouse xenograft model of prostate cancer. Thus, our findings show that overexpression of TRIM25 promoted prostate cancer cell proliferation and cell survival by modulating p53 nuclear export mechanism with G3BP2 interaction.

Wnt signaling in idiopathic carpal tunnel syndrome

Carpal tunnel syndrome (CTS) is the most frequently reported entrapment neuropathy; however, the exact pathological mechanism of CTS remains unknown. In a recent paper published in the Journal of Orthopaedic Research, we investigated the associations between Wnt signaling and the etiology of idiopathic CTS (ICTS). We compared the expression levels of genes encoding Wnt1, 2, 3, 4, 5a, 5b, 6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11, and 16 in the flexor tenosynovium between ICTS patients and controls, and we also evaluated whether an association exists between Wnt signaling and cell proliferation factors, such as estrogen-responsive finger protein, epidermal growth factor receptor, heparin binding-epidermal growth factor-like growth factor, insulin-like growth factor-1, and vascular endothelial growth factor (VEGF). To compare the cell proliferation potency, expression levels of MIB-1 protein were also measured. We found that Wnt9a gene expression in the flexor tenosynovium is more prominent in ICTS patients. A positive correlation was observed in only ICTS patient group for the gene expression of Wnt9a and VEGF in the flexor tenosynovium. There were no relationships between the expression levels of Wnt9a and fibroblast proliferation in either group. These results indicate that Wnt9a may be involved in the expression of VEGF in ICTS.

Enhanced expression of Wnt9a in the flexor tenosynovium in idiopathic carpal tunnel syndrome.

This study aimed to clarify the association between abnormal Wnt signaling and the cause of idiopathic carpal tunnel syndrome (ICTS) and whether an association exists between Wnt signaling and cell proliferation in the flexor tenosynovium. The subjects included nine patients with ICTS; the controls were nine patients with distal radius fractures without any symptoms of carpal tunnel syndrome. We extracted mRNA from the flexor tenosynovium and compared the expression levels of genes encoding 17 types of Wnt in both subjects and controls via quantitative real-time polymerase chain reaction (PCR). Expression levels of factors involved in cell proliferation, such as estrogen-responsive finger protein, epidermal growth factor receptor, heparin binding-epidermal growth factor-like growth factor, insulin-like growth factor-1, and vascular endothelial growth factor (VEGF) were also measured using quantitative real-time PCR. In addition, we compared the Wnt and MIB-1 protein expression levels to clarify the effect of Wnt on cell proliferation. Quantitative real-time PCR revealed significantly greater expression of the gene encoding Wnt9a in subjects with ICTS than in controls and also revealed a positive correlation between the expression of genes encoding Wnt9a and VEGF in subjects with ICTS. Quantitative evaluation using immunohistochemical staining also indicated more marked Wnt9a expression in subjects than in controls. However, there was no relationship between the expression of Wnt9a and the cell proliferation index MIB-1. These results indicate that Wnt9a expression is enhanced in ICTS and that Wnt9a may be involved in VEGF expression in ICTS.

Molecular characterization of tripartite motif protein 25 (TRIM25) involved in ERα-mediated transcription in the Korean rose bitterling Rhodeus uyekii

Tripartite motif-containing 25 (TRIM25), also known as estrogen-responsive finger protein (EFP), plays an essential role in cell proliferation and innate immunity. In the present study, we isolated and characterized the TRIM25 cDNA of the Korean rose bitterling Rhodeus uyekii, designated RuTRIM25. It encodes an open reading frame of 669 amino acids containing an N-terminal RBCC motif composed of a RING domain, two B boxes, and a coiled-coil domain and a C-terminal B30.2 (PRY/SPRY) domain. RuTRIM25 shows strong homology (79.7%) to zebrafish TRIM25 and shared 32.4-28.8% homology with TRIM25 from other species, including mammals. RuTRIM25 mRNA was expressed ubiquitously. It was highly expressed in the ovary, spleen, and liver and moderately in the stomach and intestine of normal Korean rose bitterling. The intracellular localization of RuTRIM25 in HEK293T cells was diffusely localized in the cytoplasm and its RING domain deletion mutant (RuTRIM25ΔR) was detected diffusely with some aggregates in the cytoplasm. RuTRIM25, but not RuTRIM25ΔR, is ubiquitinated in vivo. Ectopic expression of RuTRIM25 synergistically activated the estrogen receptor (ER)-mediated luciferase reporter activity in a dose-dependent manner in HEK293T cells. Together, these results suggest that the RuTRIM25 regulates the ER-mediated transcription in fish similarly to its mammalian counterpart.

Methylseleninic acid (MSA) inhibits 17^|^beta;-estradiol-induced cell growth in breast cancer T47D cells via enhancement of the antioxidative thioredoxin/ thioredoxin reductase system

The purpose of this study was to clarify the cell growth inhibitory mechanism of human breast cancer cells caused by selenium (Se) compounds. In the presence of 17β-estradiol (E(2)) at physiological concentrations, growth of estrogen receptor α (ERα)-positive T47D cells was markedly inhibited by 1 × 10(-6) mol/L methylseleninic acid (MSA) with no Se related toxicity.Under conditions where cell growth was inhibited, MSA decreased ERα mRNA levels and subsequent protein levels; further decreasing expression of estrogen-responsive finger protein (Efp) which is a target gene product of ERα and promotes G2/M progression of the cell cycle. Therefore, the decline in Efp expression is presumed to be involved in G2 arrest. Coincidentally, the antioxidative thioredoxin/ thioredoxin reductase (Trx/TrxR) system in cells was enhanced by the synergistic action of E(2) and MSA. It has been reported that ROS-induced oxidative stress enhanced ERα expression. E(2) increased production of intracellular ROS in T47D cells. Meanwhile, MSA significantly decreased E(2)-induced ROS accumulation. From these results, activation of the Trx/TrxR system induced by the coexistence of MSA and E(2) suppresses oxidative stress and decreases expression of ERα, and finally induces the growth arrest of T47D cells through disruption of ERα signaling.

Abstract A68: The role of estrogen-responsive finger protein (Efp) and 14-3-3 sigma (σ) in breast carcinogenesis and their prognostic implication

Abstract Background: 14-3-3 sigma (σ) is a negative regulator of the cell cycle and contributes to G2/M arrest. In many malignant tumors, the 14-3-3 σ is considered to be an important tumor suppressor of which the decreased expression has been reported. The level of the 14-3-3 σ was reported to be decreased either by hypermethylation at its promoter site or ubiquitin-mediated proteolysis by the estrogen-responsive ring finger protein (Efp). In this study, we tried to investigate which mechanism plays more important role in the 14-3-3 σ regulation, how the change of the 14-3-3 σ expression affect the biology of human breast cancer, and hence to extend our understanding of the role of the 14-3-3 σ in human breast cancer. Materials and Methods: In order to examine the role of ubiquitin-associated proteolysis by the Efp, we examined the change of the level of 14-3-3 σ protein after Efp silencing using siRNA in MCF-7 breast cancer cell line. We also examined the Efp and 14-3-3 σ expression in 220 human breast carcinoma tissues by immunohistochemistry. To investigate the role of methylation in regulation of 14-3-3 σ, we examined the methylation status of 14-3-3 σ promoter in human breast carcinoma tissue by methylation-specific polymerase chain reaction. Other clinicopathological variables were also evaluated together with the follow-up data to assess the prognostic significance of those two molecules. Results: Silencing of the Efp with siRNA in a MCF-7 breast cancer cell line resulted in increased expression of the 14-3-3 σ. Expression of the Efp and 14-3-3 σ in human breast cancer tissue was not very common with 19.8% and 20.7% of the total cases being positive, respectively. The Efp-positive cases were more frequently 14-3-3 sigma-negative. Hypermethylation of the 14-3-3 σ was common in breast cancer (64.9%) and had a tendency of negative association with the 14-3-3 σ positivity. The Efp expression was negatively associated with axillary lymph node metastasis (p=0.021) or positive p53 status (p=0.009). The 14-3-3 σ expression was positively associated with increasing histologic and nuclear grade (p=0.012 and p=0.033), and positive p53 (p=0.001). The 14-3-3 σ was negatively correlated with ER or PR expression (p=0.020 and p=0.032) and hypermethylation (p=0.072). Positive 14-3-3 σ expression was significantly correlated with poor prognosis. A multivariate analyses of disease-free survival (p=0.008) and disease-specific survival (p=0.009) showed that the 14-3-3 σ was a independent prognostic marker. Conclusion: Our data clearly demonstrated that 14-3-3 σ expression was negatively correlated with Efp expression in vitro and excised breast cancer tissues demonstrated similar tendency. 14-3-3 σ was also negatively correlated with hypermethylation in human breast cancer tissue. These results suggest that 14-3-3 σ may be regulated by both mechanisms i.e. ubiquitin-mediated proteolysis by Efp and down-regulation by hypermethylation in human breast cancers. Interestingly, 14-3-3 σ turned out to be a significant poor prognostic indicator in breast cancer, suggesting a hither to unrecognized role of 14-3-3 σ in breast cancer in addition to a well established role as tumor suppressor. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A68.

Ligand-dependent transcription of estrogen receptor α is mediated by the ubiquitin ligase EFP

Estrogen-mediated ubiquitylation and subsequent degradation of the estrogen receptor α (ERα) appears to be involved in the transcriptional activity of ERα. We show that the estrogen-responsive finger protein (EFP) interacts with and ubiquitylates ERα. EFP promoted the ubiquitylation of ERαin vitro and in vivo and consequently promoted the degradation of ERα. The interaction between EFP and ERα was greatly enhanced in the presence of estrogen. The action of EFP on ERα in the presence of estrogen resulted in a robust interaction between ERα and Tip60, one of the transcriptional coactivators, leading to activation of ERα transcriptional activity. However, a dominant negative mutant of EFP lacking the RING domain prolonged the half-life of ERα and inhibited the transcription by ERα. Our results indicate that EFP functions as a cofactor for ERα-mediated transcription, thus suggesting that ERα-mediated transcription is closely linked to the ubiquitylation of ERα.

The Interferon-inducible Ubiquitin-protein Isopeptide Ligase (E3) EFP Also Functions as an ISG15 E3 Ligase

The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3sigma protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3sigma in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3sigma in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations.

Increasing 14‐3‐3 sigma expression with declining estrogen receptor α and estrogen‐responsive finger protein expression defines malignant progression of endometrial carcinoma

14-3-3 sigma (sigma) is a negative regulator of the cell cycle and contributes to G2 arrest. Lack of its expression due to hypermethylation of CpG islands has been reported in some carcinomas. A recent study showed that 14-3-3 sigma was down-regulated through proteolysis by estrogen-responsive finger protein (Efp). Here, we investigated the expression of 14-3-3 sigma, hormone receptors, Efp and p53 in 86 cases of endometrial adenocarcinoma and 46 cases of normal or non-neoplastic endometria by means of immunohistochemistry and methylation-specific polymerase chain reaction. In normal endometrium, 14-3-3 sigma was overexpressed in the mid- to late-secretory phase due to hypomethylation. In endometrial adenocarcinoma, 14-3-3 sigma expression was low in low grade endometrioid adenocarcinoma due to hypermethylation, and increased significantly with increasing histological grade due to hypomethylation. 14-3-3 sigma expression inversely correlated with estrogen receptor alpha, progesterone receptor and Efp, and positively correlated with myometrial invasion and lymph node metastasis. These results suggest that 14-3-3 sigma was one of the menstrual cycle-related proteins regulated by epigenetic methylation, and its expression was influenced by epigenetic methylation or hormone receptors in progression of endometrial adenocarcinoma, and therefore was more than just a cell-cycle regulator.

Expression of estrogen-responsive finger protein (Efp) is associated with advanced disease in human epithelial ovarian cancer

Objective. The estrogen-responsive ring finger protein (Efp) gene, one of estrogen receptor (ER) target genes, is considered to be essential for estrogen-dependent cell proliferation. To understand the estrogenic action on ovarian cancer, we studied the relationships between Efp and ERs expressions and the correlations of Efp expression with clinicopathological parameters in epithelial ovarian cancer. Methods. The protein expressions for Efp, ERα and ERβ were examined by immunoblotting in 12 ovarian cancer cell lines. Efp mRNA expressions were evaluated by quantitative RT-PCR in 12 ovarian cancer cell lines. A total of 100 surgical specimens diagnosed as epithelial ovarian cancer were examined immunohistochemically using antibodies for Efp, ERα and ERβ. Results. Efp protein was detected in 8 out of 12 cell lines. In Efp protein-positive cell lines, Efp mRNA was expressed higher than that in negative ( P = 0.021). All of the Efp protein-positive cell lines simultaneously expressed either ERα or ERβ protein. By immunohistochemical staining, Efp immunoreactivity was detected in 63 out of 100 ovarian cancer specimens and positive signals were in the cytoplasm of carcinoma cells. There were significant correlations between Efp and ERα, ERβ immunoreactivity (Efp and ERα, P = 0.022; Efp and ERβ, P = 0.032). Efp expression was significantly higher in a subgroup with serous adenocarcinoma ( P = 0.010) and with advanced disease ( P = 0.026). No significant relationship was detected between Efp immunoreactivity and overall survival. Conclusion. The expression of Efp was detected in human epithelial ovarian cancer and high expression of Efp was correlated with advanced disease and serous adenocarcinoma, and ERs status.

Estrogen-Responsive Finger Protein as a New Potential Biomarker for Breast Cancer

Estrogen-responsive finger protein (Efp) is a member of RING finger-B box-Coiled Coil family and is also a downstream target of estrogen receptor alpha. Previously, Efp was shown to mediate estrogen-induced cell growth, which suggests possible involvement in the development of human breast carcinomas. In this study, we examined expression of Efp in breast carcinoma tissues and correlated these findings with various clinicopathologic variables. Thirty frozen specimens of breast carcinomas were used for immunohistochemistry and laser capture microdissection/real-time PCR of Efp. Immunohistochemistry for Efp was also done in 151 breast carcinoma specimens fixed with formalin and embedded in paraffin wax. Efp immunoreactivity was detected in breast carcinoma cells and was significantly associated with the mRNA level (n = 30). Efp immunoreactivity was positively associated with lymph node status or estrogen receptor alpha status and negatively correlated with histologic grade or 14-3-3sigma immunoreactivity (n = 151). Moreover, Efp immunoreactivity was significantly correlated with poor prognosis of breast cancer patients, and multivariate analyses of disease-free survival and overall survival for 151 breast cancer patients showed that Efp immunoreactivity was the independent marker. Our data suggest that Efp immunoreactivity is a significant prognostic factor in breast cancer patients. These findings may account for an oncogenic role of Efp in the tumor progression of breast carcinoma.

Expression of estrogen-responsive finger protein (Efp) is associated with advanced disease in human epithelial ovarian cancer

3143 Background: The estrogen-responsive ring finger protein (Efp) gene is one of the estrogen receptor (ER) target genes identified in the MCF-7 human breast cancer cell line using genomic binding-site cloning. Efp is considered to be essential for estrogen-dependent cell proliferation. In the present study, we studied correlations of Efp expression with clinicopathological parameters, Efp’s significance as a prognostic indicator, and relationships between Efp and ERs expressions in human epithelial ovarian cancer. Methods: A total of 100 epithelial ovarian cancer cases were examined immunohistochemically using antibodies for Efp, ERα, and ERβ. The correlation between Efp immunoreactivity and clinicopathological parameters was examined. The expression of Efp, ERα and ERβ and mRNA expressions of Efp, ERα, and ERβ were evaluated by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively, in 12 ovarian cancer cell lines. Results: Efp immunoreactivity was detected in the cytoplasm of carcinoma cells in 63 out of 100 cases (63%). Efp expression was significantly higher in carcinomas with serous differentiation (P=0.0102) and advanced carcinomas (P=0.0259). No significant relationship was detected between Efp immunoreactivity and overall survival. There were high correlations between Efp and ERα and β immunoreactivity (Efp and ERα, P=0.022; Efp and ERβ, P=0.0323). In immunoblotting, Efp protein was detected in 8 out of 12 cell lines; furthermore, ERα and/or ERβ protein were detected in all 8 cell lines. Significantly higher mRNA levels were detected in the 8 cancer cell lines with Efp-immunoreactive bands than in the other 4 lines without Efp-immunoreactive bands (P=0.021). Conclusion: The expression of Efp is associated with advanced disease and serous histology, and is also correlated with ER status. Our results suggest that Efp could be a prognostic indicator in human epithelial ovarian cancer. No significant financial relationships to disclose.

Down-regulation of the Tumor Suppressor Protein 14-3-3σ Is a Sporadic Event in Cancer of the Breast

14-3-3 proteins comprise a family of highly conserved and broadly expressed multifunctional regulatory proteins that are involved in various cellular processes such as cell cycle progression, cell growth, differentiation, and apoptosis. Transcriptional expression of the sigma isoform of 14-3-3 is frequently impaired in human cancers, including carcinomas of the breast, which has led to the suggestion that this protein might be involved in the neoplastic transformation of breast epithelial cells. Here we report on the analysis of 14-3-3sigma expression in primary breast tumors using a proteomic approach complemented by immunohistochemical analysis by means of specific antibodies against this isoform. We show that the levels of expression of 14-3-3sigma were similar in non-malignant breast epithelial tissue and matched malignant tissue with only sporadic loss of expression observed in 3 of the 68 tumors examined. Moreover we show that 14-3-3sigma immunoreactivity was restricted to epithelial cells and significantly stronger in the myoepithelial cells that line the mammary ducts and lobules. The lack of expression of 14-3-3sigma in the three breast carcinomas was not associated with high levels of expression of the dominant-negative transcriptional regulator DeltaNp63 or with increased expression of estrogen-responsive finger protein, a ubiquitin-protein ligase (E3) that targets 14-3-3sigma for proteolysis. Validation of the results was performed retrospectively on an independent clinical tumor sample set using a tissue microarray containing 65 primary tumors. Our data suggest that, contrary to what was previously thought, loss of expression of 14-3-3sigma protein is not a frequent event in breast tumorigenesis.

Estrogen receptor activation at serine 305 is sufficient to upregulate cyclin D1 in breast cancer cells

Recent studies have shown that p21-activated kinase 1 (Pak1) phosphorylates estrogen receptor-α (ERα) at Ser 305 and also promotes its transactivation function. Here, we sought to investigate whether substitution of serine 305 in ER with glutamic acid (ERα-S305E), which mimics the phosphorylation state, would influence the status of ER-target genes. To explore this possibility, we generated clones overexpressing ERα-S305E in ER-negative MDA-MB-231 cells and analyzed the status of ER-regulated genes using a gene array. Results indicated that the expression of ERα-S305E is sufficient to upregulate the expression of a few but not all ER-regulated genes, i.e., cyclin D1 and zinc finger protein 147 (estrogen-responsive finger protein), while there was no significant change in the expression of remaining genes on the array. In addition, we found an increased expression as well as nuclear accumulation of cyclin D1 protein in MDA-MB-231 cells expressing ERα-S305E as compared to the level of cyclin D1 in MDA-MB-231 cells expressing WT-ERα or pcDNA. Furthermore, ERα-S305E, but not mutation of ERα-S305 to alanine, enhanced the cyclin D1 promoter activity. These findings suggest that ERα activation at S305 is sufficient to upregulate the expression of cyclin D1, an ER-regulated gene that is implicated in the progression of breast cancer. Phosphorylation of ERα by Pak1 or its upstream regulators could upregulate the expression of a subset of ER-target genes in a ligand-independent manner and hence, might contribute toward the development of hormone independence in breast cancer cells.