Breast Cancer Anti-estrogen Resistance Protein Research Articles

Characterization of the sex determining region of channel catfish (Ictalurus punctatus) and development of a sex-genotyping test

Channel catfish is an important species for aquaculture that exhibits a sexually dimorphic growth in favor of males. Genetic sexing and development of sex markers are crucial for the early identification of sex and of particular genotypes (YY males) for the production of all-male population in channel catfish aquaculture. In this study, we sequenced genomic DNA from pools of males and pools of females to better characterize the sex determining region (SDR) of channel catfish and to develop sex-specific markers for genetic sexing. Performing comparative analyses on male and female pooled genomic reads, we identified a large SDR (∼8.3 Mb) in the middle of channel catfish linkage group 4 (LG04). This non-recombining SDR contains a high-density of male-specific (Y chromosome) fixed single nucleotide polymorphisms (SNPs) along with ∼ 185 kb male-specific insertions or deletions. This SDR contains 95 annotated protein-encoding genes, including the recently reported putative channel catfish master sex determining (MSD) gene, breast cancer anti-estrogen resistance protein 1 (bcar1), located at one edge of the SDR. No sex-specific SNPs and/or indels were found in the coding sequence of bcar1, but one male-specific SNP was identified in its first intron. Based on this genomic information, we developed a PCR-based sex-specific genetic test. Genotyping results confirmed strong linkage between phenotypic sexes and the identified SDR in channel catfish. Our results confirm, using a Pool-Seq approach, that channel catfish is male heterogametic (XX-XY) with a large SDR on the LG04 sex chromosome. Furthermore, our genotyping primers can be used to identify XX, XY, and YY fish that will facilitate future research on sex determination and aquaculture applications in channel catfish.

BCAR3 promotes head and neck cancer growth and is associated with poor prognosis

Breast cancer anti-estrogen resistance protein 3 (BCAR3) is involved in anti-estrogen resistance and other important aspects of breast cancer. However, the role of BCAR3 in other solid tumors remains unclear. The relationship between the clinicopathologic characteristics of head and neck squamous cell carcinoma (HNSCC) patients and BCAR3 was analyzed using the Wilcoxon’s signed-rank test and logistic regression. The association between BCAR3 expression and clinicopathologic features and survival was analyzed using Cox regression and the Kaplan–Meier method. In vivo and in vitro assays were performed to validate the effect of BCAR3 on HNSCC growth. BCAR3-related mRNAs were determined by calculating the Pearson’s correlation coefficient based on The Cancer Genome Atlas (TCGA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and gene set enrichment analysis (GSEA) were used to predict the potential functions of BCAR3. BCAR3 expression is overexpressed in HNSCC and was shown to be associated with perineural invasion (PNI) and poor survival. BCAR3 silencing significantly attenuated the proliferation of HNSCC cells, whereas BCAR3 depletion inhibited tumor growth in vitro. GO and KEGG functional enrichment analyses, and GSEA showed that BCAR3 expression in HNSCC was associated with biological processes, such as cell adhesion, actin binding, cadherin binding, and angiogenesis. BCAR3, which promotes HNSCC growth, is associated with perineural invasion and may be a potential molecular prognostic marker of poor survival in HNSCC.

Specific PIWI-Interacting RNAs and Related Small Noncoding RNAs Are Associated With Ovarian Aging in Ames Dwarf (df/df) Mice.

The Ames dwarf (df/df) mouse is a well-established model for delayed aging. MicroRNAs (miRNAs), the most studied small noncoding RNAs (sncRNAs), may regulate ovarian aging to maintain a younger ovarian phenotype in df/df mice. In this study, we profile other types of ovarian sncRNAs, PIWI-interacting RNAs (piRNAs) and piRNA-like RNAs (piLRNAs), in young and aged df/df and normal mice. Half of the piRNAs derive from transfer RNA fragments (tRF-piRNAs). Aging and dwarfism alter the ovarian expression of these novel sncRNAs. Specific tRF-piRNAs that increased with age might target and decrease the expression of the breast cancer antiestrogen resistance protein 3 (BCAR3) gene in the ovaries of old df/df mice. A set of piLRNAs that decreased with age and map to D10Wsu102e mRNA may have trans-regulatory functions. Other piLRNAs that decreased with age potentially target and may de-repress transposable elements, leading to a beneficial impact on ovarian aging in df/df mice. These results identify unique responses in ovarian tissues with regard to aging and dwarfism. Overall, our findings highlight the complexity of the aging effects on gene expression and suggest that, in addition to miRNAs, piRNAs, piLRNAs, tRF-piRNAs, and their potential targets can be central players in the maintenance of a younger ovarian phenotype in df/df mice.

BCAR1 promotes proliferation and cell growth in lung adenocarcinoma via upregulation of POLR2A

BackgroundThis study was designed to investigate the effects of a novel carcinogenetic molecule, p130cas (breast cancer antiestrogen resistance protein 1 or BCAR1) on proliferation and cell growth in lung adenocarcinoma. The study also aimed to identify the possible underlying signal networks of BCAR1.MethodsFirst, we evaluated proliferation, cell colony formation, apoptosis, and cell cycle after BCAR1 was knocked out (KO) using CRISPR‐Cas9 technology in H1975 and H1299 human lung adenocarcinoma cells. Subsequently, BCAR1 was upregulated in 293T cells and immunoprecipitation‐mass spectrometry (IP‐MS) was used with bioinformatics analysis to screen for potential networks of BCAR1 interacting proteins. Ultimately, we validated the correlated expressions of BCAR1 and a selected hub gene, RNA polymerase II subunit A (POLR2A), in 54 lung adenocarcinoma tissues, as well as in H1975 and H1299 cells.ResultsCell proliferation of H1975 and H1299 was significantly inhibited following BCAR1‐KO. Colony formation of H1975 cells was also significantly decreased following BCAR1‐KO. IP‐MS demonstrated 419 potential proteins that may interact with BCAR1. Among them, 68 genes were significantly positively correlated to BCAR1 expression, as verified by TCGA. Six hub genes were revealed by PPI String. High expression of POLR2A, MAPK3, MOV10, and XAB2 predicted poor prognosis in lung adenocarcinoma, as verified by the K‐M plotter database. POLR2A and MAPK3 are involved in both catalytic activity and transferase activity. POLR2A and BCAR1 were significantly increased in lung cancer tissues as compared with matched normal tissues. High expression of POLR2A was significantly positively correlated to BCAR1 overexpression and predicted poor prognosis in 54 lung cancer cases. POLR2A expression was significantly decreased following BCAR1‐KO in H1975 and H1299 cells.ConclusionsBCAR1 promotes proliferation and cell growth, probably via upregulation of POLR2A and subsequent enhancement of catalytic and transferase activities. However, additional robust studies are required to elucidate the mechanisms involved.

High Expression of BCAR1 by Circulating Tumor Cells and Tumor Tissues Is Predictive of a Poor Prognosis of Early-Stage Lung Adenocarcinoma Potentially Due to Regulation of Epithelial-Mesenchymal Transition.

Objective:To clarify the clinical significance of breast cancer anti-estrogen resistance protein 1 (BCAR1) expression in circulating tumor cells (CTCs) in the peripheral blood and tumor tissues in patients with early stage lung adenocarcinoma (ES-LUAD).Methods:The study cohort included 60 patients with stage I LUAD (50 IA and 10 IB) who underwent surgery from November 2015 to November 2018 and 31 healthy controls. The expression levels of BCAR1 and markers of epithelial-mesenchymal transition (EMT) in peripheral blood CTCs were detected using CanPatrolTM technology before surgery, and immunohistochemical analysis was used to detect BCAR1 expression in tumor tissues collected from 40 patients. The predictive power of BCAR1 expression in CTCs and tumor tissues on disease-free survival (DFS) was analyzed. The Cancer Genome Atlas (TCGA) database was used to study BCAR1 expression and overall survival as validation. The Gene Expression Profiling Interactive Analysis online tool was used to analyze the correlations between the expression levels of BCAR1 and EMT molecular markers.Results:Both the number and detection rates of BCAR1-negative CTCs and BCAR1-positive CTCs in peripheral blood of lung cancer patients were significantly higher as compared with healthy controls (p < 0.05). BCAR1-positive CTCs more commonly co-expressed both epithelial and mesenchymal markers. Kaplan–Meier analysis demonstrated that patients with BCAR1(++) CTCs in peripheral blood before surgery were more prone to recurrence or metastasis after 2 years. COX analysis showed that patients with higher abundance of BCAR1(++) CTCs had a poorer prognosis (hazard ratio [HR] = 1.712, 95% confidence interval [CI] = 1.077–2.272, p = 0.023). Furthermore, high BCAR1 expression in tumor tissues was predictive of a poor prognosis (HR = 2.654, 95% CI = 1.239–5.686, p = 0.012), as validated by TCGA database (HR = 2.217, 95% CI = 1.069–4.595, p = 0.032). In addition, BCAR1 expression in LUAD tissues from TCGA was significantly positively correlated with the expression of both epithelial markers (e.g., ck8/18/19) and mesenchymal markers (e.g., vimentin and twist).Conclusion:BCAR1 may have a “dual impact” on EMT markers in tumor tissues and CTCs due to micro-environmental disparities, resulting in important clinical significance, which can potentially guide accurate treatment of LUAD.

Molecular and disease association of gestational diabetes mellitus affected mother and placental datasets reveal a strong link between insulin growth factor (IGF) genes in amino acid transport pathway: A network biology approach.

Discerning the relationship between molecules involved in diseases based on their underlying biological mechanisms is one of the greatest challenges in therapeutic development today. Gestational diabetes mellitus (GDM) is one of the most common complications during pregnancy, which adversely affects both mothers and offspring during and after pregnancy. We have constructed two datasets of (GDM associated genes from affected mother and placenta to systematically analyze and evaluate their interactions like gene-gene, gene-protein, gene-microRNA (miRNA), gene-transcription factors, and gene-associated diseases to enhance our current knowledge, which may lead to further advancements in disease diagnosis, prognosis, and treatment. The results identify the key genes with respect to maternal dataset as insulin receptor, insulin (INS), leptin (LEP), glucokinase, and hepatocyte nuclear factor 1 alpha, whereas from placenta include insulin-like growth factor 1, growth hormone receptor, and breast cancer anti-estrogen resistance protein 1, which are found to be highly enriched in pancreas, ovary, adipocyte, heart, and placental tissues. The key transcription factors include Sp1 transcription factor, pancreatic and duodenal homeobox 1, and hepatocyte nuclear factor 4 alpha, whereas miRNA includes has-miR-5699-5p and has-miR-3158-3p. The study also reveals that GDM has associations with diseases like type I and II diabetes mellitus, obesity, and preeclampsia. More significantly, we could trace out a significant connection between the key molecules like LEP and placental growth hormone from mother and placental dataset, which plays a critical role in INS secretion, INS signaling, and β-cell dysfunction pathways.

RNA-Seq-based transcriptome analysis of Korean rose bitterling (Rhodeus uyekii) exposed to synthetic estrogen 17-α-ethinylestradiol (EE2)

The potential impact of natural and synthetic estrogens on aquatic ecosystems has become a subject of great interest in recent years. One synthetic estrogen, 17-alpha-ethinylestradiol (EE2), is present in municipal sewage discharges and causes gonad alterations in various fish species. To understand the possible damage caused by EE2, male Rhodeus uyekii were exposed to 100ng/L EE2 for 7days. RNA-Seq was performed to assess the effects of EE2 on gene expression in hepatic and skin tissues. The analysis revealed that EE2 induced the expression of various genes, including sex hormone genes, anti-Mullerian hormone, vitellogenin, and estrogen receptor alpha; cancer genes, breast cancer anti-estrogen resistance protein 3, caveolin 2, and Smad2; and apoptotic genes, p53, Bcl-2, TNF-α, and WDR36. These results suggest that the synthetic estrogen EE2 disturbs the endocrine system and regulates both carcinogenic and apoptotic gene expressions in R. uyekii.

Association of the Breast Cancer Antiestrogen Resistance Protein 1 (BCAR1) and BCAR3 Scaffolding Proteins in Cell Signaling and Antiestrogen Resistance

Most breast cancers are estrogen receptor-positive and treated with antiestrogens, but aberrant signaling networks can induce drug resistance. One of these networks involves the scaffolding protein BCAR1/p130CAS, which regulates cell growth and migration/invasion. A less investigated scaffolding protein that also confers antiestrogen resistance is the SH2 domain-containing protein BCAR3. BCAR1 and BCAR3 bind tightly to each other through their C-terminal domains, thus potentially connecting their associated signaling networks. However, recent studies using BCAR1 and BCAR3 interaction mutants concluded that association between the two proteins is not critical for many of their interrelated activities regulating breast cancer malignancy. We report that these previously used BCAR mutations fail to cause adequate loss-of-function of the complex. By using structure-based BCAR1 and BCAR3 mutants that lack the ability to interact, we show that BCAR3-induced antiestrogen resistance in MCF7 breast cancer cells critically depends on its ability to bind BCAR1. Interaction with BCAR3 increases the levels of phosphorylated BCAR1, ultimately potentiating BCAR1-dependent antiestrogen resistance. Furthermore, antiestrogen resistance in cells overexpressing BCAR1/BCAR3 correlates with increased ERK1/2 activity. Inhibiting ERK1/2 through overexpression of the regulatory protein PEA15 negates the resistance, revealing a key role for ERK1/2 in BCAR1/BCAR3-induced antiestrogen resistance. Reverse-phase protein array data show that PEA15 levels in invasive breast cancers correlate with patient survival, suggesting that PEA15 can override ERK1/2 activation by BCAR1/BCAR3 and other upstream regulators. We further uncovered that the BCAR3-related NSP3 can also promote antiestrogen resistance. Thus, strategies to disrupt BCAR1-BCAR3/NSP3 complexes and associated signaling networks could ultimately lead to new breast cancer therapies.

Abstract 1190: Gene expression profiling identified FYN as an important gene in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy .

Abstract To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer we performed microarray profiling and identified 366 genes with altered expression in 4 tamoxifen-resistant (TamRs) vs. the parental tamoxifen-sensitive MCF-7/S0.5 cell lines. Most of these genes were functionally linked to cell growth, proliferation, death and control of gene expression and included FYN, PRKCα, ITPR1, DPYD, DACH1, GBP1 and PRLR. Increased expression of FYN was observed in TamR cells and treatment with FYN-specific interfering RNA or a specific Src family kinase (SFK) inhibitor restored the anti-proliferative effect of tamoxifen in these cells. TamR cells also expressed increased levels of phosphorylated breast cancer antiestrogen resistance protein 1 (pBCAR1), an important downstream SFK substrate in the MAPK pathway. Evaluation of the association of subcellular localization of FYN in cancer tissue and clinical endpoints was made in 2 cohorts of ER+ breast cancer patients, one with advanced disease (N=47) and the other with early breast cancer (N=76). The result showed that plasma membrane-associated FYN expression in the cancer cells strongly correlated with longer progression-free survival compared to FYN expression not associated with the plasma membrane (p&amp;lt;0.0002) in the advanced disease cohort. Similarly, in the early breast cancer patients, plasma membrane associated FYN expression was associated with longer metastasis-free survival (p&amp;lt;0.003) and overall survival (p&amp;lt;0.0005) independent of tumor size, grade and node status. Our results indicate that FYN plays an important role in tamoxifen resistance, and its subcellular location in breast tumor cells may be an important novel biomarker of response to endocrine therapy in breast cancer. Citation Format: Daniel Elias Roro, Henriette Vever, Henrik Ditzel. Gene expression profiling identified FYN as an important gene in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1190. doi:10.1158/1538-7445.AM2013-1190

Breast Cancer Anti-Estrogen Resistance Protein 1 (BCAR1/p130cas) in Pulmonary Disease Tissue and Serum

The purpose of the study was to evaluate clinical presentation of breast cancer anti-estrogen resistance protein 1 (BCAR1, also known as p130cas) expression in pulmonary diseases, and to assess its potential as a molecular marker for diagnosis and prognosis. Between March 2008 and August 2010, we enrolled a total of 80 patients (group A) with non-small-cell lung cancer (NSCLC), 48 patients (group B) with pulmonary tuberculosis (including 27 cases of tuberculoma and 21 cases of cavitary pulmonary tuberculosis), and 32 patients (group C) with other benign pulmonary mass (hamartoma in 15 cases, inflammatory pseudotumor in 10 cases, fibroid tumor in 7 cases). Additionally, 160 healthy age- and sex-matched volunteers were recruited as healthy controls. Tissue BCAR1 expression was investigated by using tissue microarray and immunohistochemistry. BCAR1 and tumor markers (carcinoma embryonic antigen [CEA] and the cancer antigens CA19-9 and CA125) in serum were assayed by using ELISA and immunoradiometrics, respectively. BCAR1 expression was detected (either in the nucleus, the cytoplasm, or both) in tumor cells in 79 of the 80 NSCLC cases in group A, and in fibroblasts in 41 of the 48 pulmonary tuberculosis cases in group B. However, it was not detected in the normal adjacent tissue in 70 of the 80 cases in group A and in 47 of the 48 cases in group B. In group C, BCAR1 expression was negative in all 32 cases. Additionally, we investigated adjacent tissue with acute or chronic inflammation in 20 cases from group C, and found no expression of BCAR1. Serum BCAR1 levels were significantly higher in patients with NSCLC than in the control group, increased gradually with the progression of tumor staging, and decreased after removal of the tumors. The levels were significantly lower in bronchioloalveolar carcinoma than in other subtypes of carcinoma (Mann-Whitney U test, Z = -5.089; p < 0.001). Serum BCAR1 levels were significantly higher in patients with pulmonary tuberculosis than in the control group, were positively and significantly correlated with the diameter of the tuberculosis lesion (Spearman's rho, correlation coefficient 0.753; p < 0.001), and decreased after removal of the tuberculosis lesions. The levels were significantly higher in patients with cavitary pulmonary tuberculosis than in those with tuberculoma (517.6 ± 326.5 vs 282.2 ± 137.6; Student's t-test, t = -3.387; p = 0.001). In group C, there was no appreciable difference in serum BCAR1 levels compared with the matched controls (222.8 ± 111.0 vs 201.6 ± 35.7; Dunnett's T3 test, p = 0.993). The discrimination power of combining BCAR1 and tumor markers in NSCLC versus benign lung diseases was higher than that of sole use of BCAR1 as a marker (maximal sum of sensitivity and specificity: 1.538 vs 1.237). We conclude that a combined assay of serum BCAR1 and traditional tumor markers is potentially applicable for distinguishing NSCLC from benign lung diseases. However, the clinical utility of serum BCAR1 as a molecular marker for prognosis in NSCLC or pulmonary tuberculosis requires further clarification and verification.

Osteoclastic Differentiation of Monocytes Is Inhibited by Non-Transcriptional Effects of Estrogen Mediated by BCAR1.

Estrogen is a major positive regulator of bone mass with important inhibitory effects on bone resorption. However, knowledge of the mechanisms underlying estrogen effects remains incomplete. We investigated whether estrogen altered osteoclastic differentiation of human monocytes in response to Receptor Activator of NF-kB (RANK) Ligand (RANKL), a critical stimulus for osteoclast formation. For these studies, human monocytic cells were isolated from peripheral blood by positive selection for CD14 expression (“CD14 cells”) and maintained in charcoal stripped medium with monocyte colony-stimulating factor. Compared to RANKL treatment alone, co-treatment with 17-β -estradiol reduced osteoclast formation, as shown by decreased development of tartrate resistant acid phosphatase (TRAP) activity, multinucleation, and resorption pit formation on bone. A possible mechanism for the estrogen effects was interference with RANKL signal transduction. We examined estrogen effects on a key event in the RANKL signaling: nuclear translocation of NF-kB following phosphorylation and degradation of IkB. Estrogen cotreatment of RANKL-stimulated CD14 cells inhibited the nuclear translocation of NF-kB observed with RANKL alone. This effect was evident after only 30 minutes of treatment. Cotreatment with estrogen also inhibited RANKL-stimulated IkB phosphorylation; this was evident within 5 minutes after treatment. The time course of the estrogen effects argued strongly for a non-transcriptional mechanism. We therefore investigated the involvement of proteins previously implicated in rapid estrogen effects, including the breast cancer anti-estrogen resistance protein 1(BCAR1, also called p130Cas), a scaffolding/adaptor protein recently reported to interact with estrogen receptors in breast cancer cells. Co-immunoprecipitation from ligand treated CD14 cells revealed an estrogen-dependent association of estrogen receptor-α (ER) with BCAR1 that was enhanced by cotreatment with RANKL. The coordinate regulation of this complex by both RANKL and estrogen suggested that BCAR1 might represent a link between the two signaling systems. In addition, in cells cotreated with estrogen and RANKL, co-immunoprecipitation revealed a further association of ER with Traf6, a critical proximal signaling intermediate for RANKL. These protein interactions were also rapid, evident 5 minutes after ligand addition. To test the functional significance of BCAR1 in estrogen effects, its expression was inhibited by transduction with BCAR1 siRNA. In cells deficient in BCAR1, estrogen no longer inhibited RANKL-stimulated NF-kB nuclear translocation. Unexpectedly, suppression of BCAR1 itself partially blocked RANKL stimulated NF-kB translocation, suggesting a role for BCAR1 even in the absence of estrogen. Furthermore, when BCAR1 deficient CD14 cells were treated with RANKL, osteoclastic differentiation, as assessed by formation of TRAP-positive multinucleated cells, was reduced compared to RANKL-treated cells transduced with control siRNA. The results suggest that BCAR1 is involved in the RANK-NF-kB pathway that mediates osteoclast differentiation signals in monocytes. In addition, BCAR1 appears necessary for estrogen inhibition of RANKL signaling. The estrogen dependent association of ER with BCAR1 may, like BCAR1 deficiency, block BCAR1 actions in the RANKL-NF-kB pathway, and thus inhibit the osteoclastic differentiation of monocytes.

Application of a newly developed ELISA for BCAR1 protein for prediction of clinical benefit of tamoxifen therapy in patients with advanced breast cancer.

Advanced breast cancer is frequently treated with the antiestrogen tamoxifen. Response is observed in approximately one half of estrogen receptor (ER)-positive patients but ultimately fails because the cancer becomes resistant. The mechanisms for resistance of breast cancer to tamoxifen treatment are not yet understood (1). The breast cancer anti-estrogen resistance protein 1 ( BCAR1 ) gene was identified in a search for genes that cause proliferation of estrogen-dependent breast cancer cells in the presence of an antiestrogen drug (2). To assess the role of the BCAR1 protein in breast cancer progression, we developed a specific and sensitive ELISA. Here we report the use of this BCAR1 ELISA to measure BCAR1 protein concentrations in human breast cancer cytosols to predict success of tamoxifen treatment for advanced disease. Primary invasive breast tumors were collected between 1978 and June 1995. Our study design was approved by the medical ethics committee of the Erasmus MC Rotterdam. Selection of samples for analysis of response to tamoxifen in recurrent breast cancer was based on the availability of stored cytosol extracts (in liquid nitrogen), which remained after routine ER and progesterone receptor analyses (3). Tissue specimens that were sampled after neoadjuvant treatment or obtained from a biopsy were not included. In addition, the samples selected were only from ER-positive (>10 fmol/mg of protein) tumors of patients who developed recurrent disease and were treated with tamoxifen as first-line therapy. Of these 592 patients, 133 had received adjuvant chemotherapy (89 receiving cyclophosphamide–methotrexate–5-fluorouracil and 44 receiving 5-fluorouracil–epirubicin–cyclophosphamide), and none had received adjuvant hormonal therapy. The median age at the start of tamoxifen treatment for recurrent disease was 61 years (range, 28–91 years); 144 patients were premenopausal and 448 were postmenopausal. During the follow-up period, 527 of 592 patients (89%) showed tumor progression, and 433 patients (73%) died. …