Breast Cancer Anti-estrogen Resistance 4 Expression Research Articles

BCAR4 Expression as a Predictive Biomarker for Endocrine Therapy Resistance in Breast Cancer

BackgroundBreast cancer, particularly the estrogen receptor positive (ER+) subtype, remains a leading cause of cancer-related death among women. Endocrine therapy is the most effective treatment for ER+ breast cancer; however, the development of resistance presents a significant challenge. This study explored the role of the breast cancer antiestrogen resistance 4 (BCAR4) gene as a potential driver of resistance and a pivotal biomarker in breast cancer. Patients and MethodsThe researchers undertook a comprehensive analysis of 1743 patients spanning 6 independent cohorts. They examined the association of BCAR4 expression with patient outcomes across all breast cancer types and the PAM50 molecular subtypes. The relationship between elevated BCAR4 expression and resistance to endocrine therapy including AIs, the prevailing standard-of-care for endocrine therapy, was also investigated. ResultsThis meta-analysis corroborated the link between BCAR4 expression and adverse outcomes as well as resistance to endocrine therapy in breast cancer. Notably, BCAR4 expression is clinically significant in luminal A and B subtypes. Additionally, an association between BCAR4 expression and resistance to AI treatment was discerned. ConclusionThis study expands on previous findings by demonstrating that BCAR4 expression is associated with resistance to newer therapies. The identification of patients with intrinsic resistance to hormone therapy is crucial to avoid ineffective treatment strategies. These findings contribute to our understanding of endocrine therapy resistance in breast cancer and could potentially guide the development of more effective treatment strategies.

The Functional Role of Oncogenic LncRNA BCAR4 for Cancer Outcome.

Long non-coding RNA (lncRNA) breast cancer anti-estrogen resistance 4 (BCAR4) is a characterized oncogenic lncRNA in different cancers. This review is dedicated to summarize various molecular mechanisms of BCAR4 and demonstrate that the biological functions exerted by BCAR4 are good entry points for therapy. The molecular mechanism of BCAR4 acting on tumors is summarized by reviewing PubMed. The expression of lncRNA BCAR4 is abnormally increased in all kinds of tumors, including colorectal cancer, prostate cancer, bladder cancer, gastric cancer, chondrosarcoma, glioma, breast cancer, glioma, gastric cancer, liver cancer, cervical cancer, lung cancer, etc. Besides, BCAR4 mediates multiple processes involved in carcinogenesis, including proliferation, invasion, anti-apoptosis, migration. BCAR4 may show great clinical value in this direction as a therapeutic cancer target.

The role of long non-coding RNA BCAR4 in human cancers.

Long non-coding RNA (lncRNA) is a type of non-coding RNA with length of nucleotides > 200bp. Increasing evidences show that lncRNA breast cancer antiestrogen resistance 4 (BCAR4) plays an important role in the occurrence and development of various human cancers. It is found that BCAR4 is highly expressed in diverse tumor tissues and cells, and the high expression of BCAR4 is usually associated with poor prognosis. BCAR4 is considered as an oncogene in human cancers. By competing endogenous RNA (ceRNA) mechanism and regulating different signaling pathways, BCAR4 participates in the proliferation, apoptosis, migration, invasion, epithelial-mesenchymal transition (EMT), metastasis and tumorigenesis of different cancers. In addition, overexpression of BCAR4 promotes drug resistance of tumor cells. Therefore, BCAR4 is a promising biomarker for cancer diagnosis and prognosis, and it is a potential target for cancer therapy. This paper reviews studies focusing on the relationship between BCAR4 and cancers in recent years and aims to summarize the effect and mechanism of BCAR4 in human cancers.

Variant in BCAR4 gene correlated with the breast cancer susceptibility and mRNA expression of lncRNA BCAR4 in Chinese Han population.

Long non-coding RNA (LncRNA) Breast cancer anti-estrogen resistance 4 (BCAR4) has been shown to participate in the biological progress of various cancers including breast cancer. Genetic Polymorphisms in BCAR4 may have an influence on the progress of breast cancer, but it is rarely studied. In our epidemiology study, three tagging SNPs (rs4561483, rs11649623 and rs13334967) in lncRNA BCAR4 were selected for genotyping among 487 breast cancer cases and 489 healthy controls. And quantitative real-time PCR (qRT-PCR) was performed to evaluate the relative mRNA expression of BCAR4 in different genotypes of the significant locus rs13334967. We found that BCAR4 rs13334967 is associated with lower breast cancer risk both in codominant model (AT vs AA, OR 0.632, 95% CI 0.429-0.931, TT vs AA, OR 0.731, 95% CI 0.511-0.990) and dominant model (AT + TT vs AA, OR 0.798, 95% CI 0.571-0.970). The further results of qRT-PCR displayed that carriers with rs13334967 AT, TT genotype have lower BCAR4 mRNA expression compared with AA genotype. The research study implied that BCAR4 rs13334967 was correlated with the susceptibility to breast cancer and may impact the mRNA expression of BCAR4. LncRNA BCAR4 may be a potential biomarker and therapeutic target for breast cancer.

Long non-coding RNA BCAR4 promotes liver cancer progression by regulating proliferation, migration and invasion.

Liver cancer (LC) is one of the primary contributors of cancer-associated death worldwide. Long non-coding RNAs (lncRNAs) have been shown to participate in almost every aspect of cell biology and serve fundamental roles in carcinogenesis and cancer progression, including in LC. However, the clinical significance and functional role of the lncRNA breast cancer anti-estrogen resistance 4 (BCAR4) in LC have not yet been identified. The present study measured the expression levels of BCAR4 in LC cells and tissues, and discovered that BCAR4 was upregulated in LC tissues compared with adjacent normal tissues. Furthermore, high BCAR4 expression was associated with the presence of multiple tumors and advanced Tumor-Node-Metastasis stages (III/IV). Survival analysis found that high BCAR4 expression indicated poor overall survival (OS) and progression-free survival (PFS). By analyzing the risk factors of poor OS and PFS using univariate analysis and multivariate analysis, high BCAR4 expression was revealed to be an independent risk factor of poor prognosis. In addition, the role of BCAR4 was further investigated in vitro, which revealed overexpression of BCAR4 to markedly promote the proliferation, migration and invasion of LC cells. Conversely, the loss of BCAR4 expression repressed the proliferation, migration and invasion of LC cells. In conclusion, BCAR4 is overexpressed in LC and is associated with LC progression. Therefore, BCAR4 may be used as a potential prognostic marker in LC.

LncRNA BCAR4 promotes liver cancer progression by upregulating ANAPC11 expression through sponging miR‑1261.

Liver cancer is a malignant tumor that occurs in the liver and can be divided into primary and secondary liver cancer. Long non-coding RNA (lncRNA) breast cancer anti-estrogen resistance 4 (BCAR4) has been demonstrated to promote the development of various types of cancer. However, the function of lncRNA BCAR4 in liver cancer remains unclear. In the present study, the expression of lncRNA BCAR4 was notably elevated in liver cancer compared with adjacent non-tumor tissues. Functional in vitro assays demonstrated that knockdown of lncRNA BCAR4 inhibited the proliferation, migration and invasion of Huh-7 cells. In addition, lncRNA BCAR4 was demonstrated to directly bind to microRNA (miR)-1261, and miR-1261 expression negatively correlated with the expression of lncRNA BCAR4. Through bioinformatics analysis, lncRNA BCAR4 was predicted to target anaphase-promoting complex subunit 11 (ANAPC11) through miR-1261. In addition, the results demonstrated that lncRNA BCAR4 increased the expression of ANAPC11 by inhibiting miR-1261 expression. Consistently, overexpression of ANAPC11 or inhibition of miR-1261 significantly rescued liver cancer cell proliferation induced by knockdown of lncRNA BCAR4. Collectively, the results of the present study demonstrated that lncRNA BCAR4 may promote liver cancer development by directly binding to miR-1261 and targeting ANAPC11.

Investigating the role of BCAR4 in ovarian physiology and female fertility by genome editing in rabbit

Breast Cancer Anti-estrogen Resistance 4 (BCAR4) was previously characterised in bovine species as a gene preferentially expressed in oocytes, whose inhibition is detrimental to in vitro embryo development. But its role in oogenesis, folliculogenesis and globally fertility in vivo remains unknown. Because the gene is not conserved in mice, rabbits were chosen for investigation of BCAR4 expression and function in vivo. BCAR4 displayed preferential expression in the ovary compared to somatic organs, and within the ovarian follicle in the oocyte compared to somatic cells. The transcript was detected in follicles as early as the preantral stage. Abundance decreased throughout embryo development until the blastocyst stage. A lineage of genome-edited rabbits was produced; BCAR4 expression was abolished in follicles from homozygous animals. Females of wild-type, heterozygous and homozygous genotypes were examined for ovarian physiology and reproductive parameters. Follicle growth and the number of ovulations in response to hormonal stimulation were not significantly different between genotypes. Following insemination, homozygous females displayed a significantly lower delivery rate than their heterozygous counterparts (22 ± 7% vs 71 ± 11% (mean ± SEM)), while prolificacy was 1.8 ± 0.7 vs 6.0 ± 1.4 kittens per insemination. In conclusion, BCAR4 is not essential for follicular growth and ovulation but it contributes to optimal fertility in rabbits.

Long noncoding RNA BCAR4 promotes glioma cell proliferation via EGFR/PI3K/AKT signaling pathway.

Long noncoding RNA Breast Cancer Antiestrogen Resistance 4 (BCAR4) has been identified to be oncogenic in several cancers. In our study, we demonstrated that BCAR4 expression was significantly upregulated in glioma tissues compared with paired nontumor tissues. In addition, higher BCAR4 level was associated with poor overall survival in patients with glioma. Besides, we also discovered that knockdown of BCAR4 inhibited cell proliferation, whereas BCAR4 overexpression promoted this process. Intriguingly, we proved a cellular transformation of normal human astrocyte cells (NHAs) in response to enforced expression of BCAR4. In addition, we revealed that BCAR4 affected cell proliferation in glioma cells by promoting cell cycle progression and inhibiting cell apoptosis. Mechanistically, we uncovered that BCAR4 activated PI3K/AKT signaling pathway in glioma through upregulating EGFR and interacting with it. Moreover, activating PI3K/AKT pathway could reverse the repressive effects caused by BCAR4 silence on the biological behaviors of glioma cells, whereas inhibition of this pathway rescued the impact of BACR4 upregulation in NHAs. These findings disclosed that BCAR4 contributes to glioma progression by enhancing cell growth via activating EGFR/PI3K/AKT pathway, providing potent evidence that BCAR4 could be an effective new target for treatment and prognosis of glioma patients.

The Value of LncRNA BCAR4 as a Prognostic Biomarker on Clinical Outcomes in Human Cancers.

Background: This updated meta-analysis aimed to analyze available data to explore the prognostic value of long noncoding RNA breast cancer anti-estrogen resistance 4 (BCAR4) in various human malignancies. Methods: Literature retrieval was performed by systematic searching several authoritative databases, including Pubmed, PMC database, Web of Science, the Cochrane Library, Embase, and CNKI database up to Feb 10, 2019. Data were extracted and subsequently crosschecked, and discrepancies were discussed to reach consensus. Quality of the eligible studies was evaluated by Newcastle-Ottawa Scale (NOS). The fixed- or random-effects model was used to calculate the pooled the hazard ratios (HRs) or odds ratios (ORs) and the 95% confidence interval (95% CI). Publication bias was detected by using Begg's funnel plot and Egger's test. Results: A total 1,128 cancer patients from thirteen studies were included and pooled in the present meta-analysis. High expression levels of BCAR4 were correlated with unfavorable overall survival (OS) (HR=2.23, 95% CI: 1.84-2.71), but not progression-free survival (PFS) (HR=1.30, 95% CI: 0.80-2.11). Subgroup stratified analysis showed that tumor type, sample size, follow-up months, and survival analysis method did not alter the predictive value of BCAR4 on OS in various cancers. Furthermore, elevated BCAR4 level was markedly correlated with advanced clinical stage (III/IV) (OR=3.28, 95% CI: 2.33-4.60), and dramatically predicted lymph node metastasis (OR=3.00, 95% CI: 1.95-4.63, P<0.00001) and distant metastasis (OR=3.36, 95% CI: 1.88-5.98, P<0.0001), but not associated with age, gender or tumor size. No obvious heterogeneity was noted for correlation between BCAR4 expression and OS across these studies. Conclusions: High expression of BCAR4 was correlated with unfavorable overall survival outcome and clinical features including metastasis and progression, implicating an independent prognostic value for BCAR4 in human cancers.

Long noncoding RNA Breast cancer antiestrogen resistance 4 is associated with cancer progression and its significant prognostic value.

Breast cancer antiestrogen resistance 4 (BCAR4) is a novel long noncoding RNA. It was originally identified in a screen for genes responsible for the development of resistance to antiestrogens in breast cancer cells and plays a major role in various tumors. However, the clinical diagnostic role of BCAR4 in tumors is not completely understood. This current meta-analysis aimed to comprehensively explore the potential role of BCAR4 as a prognostic biomarker in a number of cancers. Five public databases PubMed, EMBASE, Web of Science, Wiley Online Library, and Medline were used to search for articles. Nine studies comprising 1,293 patients were included in this meta-analysis. The results of analysis showed that BCAR4 expression in human cancer was significantly associated with poor overall survival (hazard ratio [HR] = 1.98, confidence interval [CI]: [1.71-2.29]), p < 0.00001, and high BCAR4 expression was associated with clinical stage (OR and its 95% CI was 3.30 [1.99-5.46], p < 0.00001), distant metastasis (OR = 3.83, 95% CI: 2.15-6.82, p < 0.00001), and lymph node metastasis (OR and its 95% CI was 2.91 [1.62-5.25], p = 0.0004) in patients with cancer. Furthermore, the results revealed the prognostic significance of BCAR4 in gastrointestinal malignancy, breast cancer, and osteosarcoma (HR and its 95% CI were 2.05 [1.56-2.68], p < 0.00001; 1.78 [1.46-2.16], p < 0.00001; and 2.47 [1.41-4.34], p < 0.00001, respectively). This meta-analysis indicated the potential value of BCAR4 as a biomarker for predicting a poor prognosis in patients with cancer.

Up-regulated BCAR4 contributes to proliferation and migration of cervical cancer cells

OBJECTIVESRecently, thousands of long non-coding RNAs (lncRNAs) involved in development and metastasis of malignant tumors have been identified. Long non-coding RNA (lncRNA) breast cancer anti-estrogen resistance 4 (BCAR4) has been proved to promote proliferation and metastasis in multiple tumors. However, the function and significance of BCAR4 in cervical cancer are still unclear. METHODSIn this study, we concentrated on the biological function and clinical significance of BCAR4 in cervical cancer. More specifically, BCAR4 expression was evaluated in cervical cancer tissues and cell lines by qRT-PCR. Moreover, the prognostic factors were assessed by Kaplan-Meier analysis and Cox proportional hazards models. Additionally, functional assays were conducted and the potential mechanism was explored. RESULTSOur study showed that BCAR4 expression was significantly up-regulated in cervical cancer tissue and cell lines. Moreover, patients with high BCAR4 expression showed worse survival outcomes and overexpression of BCAR4 might be an independent prognostic factor in cervical cancer. Furthermore, overexpression of BCAR4 remarkably promoted the proliferation, motility of cervical cancer cells and silencing BCAR4 significantly suppressed the proliferation, migration and invasion of cervical cancer cells. Additionally, overexpression of BCAR4 promoted epithelial-mesenchymal transition (EMT) process and silencing BCAR4 suppressed EMT process in cervical cancer cells. CONCLUSIONSThe results indicated that BCAR4 might play a crucial role in cervical cancer progression and act as an underlying biomarker for the diagnosis and prognosis of cervical cancer.

LncRNA BCAR4 promotes colon cancer progression via activating Wnt/β-catenin signaling

BCAR4 (Breast Cancer Anti-Estrogen Resistance 4) is a long noncoding RNA that was identified as an oncogene in breast cancer. In our research, we found that the expression level of BCAR4 was upregulated in colon cancer tissues compared to paired normal tissues. What's more, higher BCAR4 expression was correlated with lower survival rate in patients with colon cancer. Mechanistically, we showed that BCAR4 activated Wnt/β-catenin signaling in colon cancer by protecting β-catenin from degradation. We also showed that BCAR4 overexpression promoted cell proliferation and migration in colon cancer. However, silencing BCAR4 inhibited cell growth and promoted apoptosis. Besides, BCAR4 knockdown decreased tumor growth in vivo. These findings indicate that BCAR4 facilitated colon cancer progression by enhancing cell proliferation and inhibiting apoptosis via BCAR4/β-catenin axis. BCAR4 may be a useful new target for treatment of patients with colon cancer.

LncRNA wires up Hippo and Hedgehog signaling to reprogramme glucose metabolism.

The Hippo pathway plays essential roles in organ size control and cancer prevention via restricting its downstream effector, Yes-associated protein (YAP). Previous studies have revealed an oncogenic function of YAP in reprogramming glucose metabolism, while the underlying mechanism remains to be fully clarified. Accumulating evidence suggests long noncoding RNAs (lncRNAs) as attractive therapeutic targets, given their roles in modulating various cancer-related signaling pathways. In this study, we report that lncRNA breast cancer anti-estrogen resistance 4 (BCAR4) is required for YAP-dependent glycolysis. Mechanistically, YAP promotes the expression of BCAR4, which subsequently coordinates the Hedgehog signaling to enhance the transcription of glycolysis activators HK2 and PFKFB3. Therapeutic delivery of locked nucleic acids (LNAs) targeting BCAR4 attenuated YAP-dependent glycolysis and tumor growth. The expression levels of BCAR4 and YAP are positively correlated in tissue samples from breast cancer patients, where high expression of both BCAR4 and YAP is associated with poor patient survival outcome. Taken together, our study not only reveals the mechanism by which YAP reprograms glucose metabolism, but also highlights the therapeutic potential of targeting YAP-BCAR4-glycolysis axis for breast cancer treatment.

Abstract 3137: Breast cancer anti-estrogen resistance 4 (BCAR4) is a novel oncogene in lung cancer

Abstract BCAR4 was identified as tamoxifen resistance related gene in breast cancer due to its function to activate estrogen-independent cell growth. Instead of estrogen receptor, ERBB2 and ERBB3 genes were activated by BCAR4 in tamoxifen resistant cells. Recently, the function of BCAR4 as a long non-coding RNA (lncRNA) was reported to regulate a non-canonical Hedgehog/GLI2 pathway in breast cancer. However, the association of BCAR4 and lung cancer has not been identified. Here, we describe the oncogenic effect of BCAR4 in human lung cancer. Expression pattern of BCAR4 was examined in lung cancer cell lines, primary tumor tissues, and adjacent normal tissues. BCAR4 was highly expressed in 71% of lung cancer cell lines and especially in 43% of cancer tissues of lung adenocarcinoma patients who did not harbor the activational mutations of EGFR nor KRAS. Exogenous expression of BCAR4 protein promoted cell growth of non-tumorigenic bronchial epithelial cell line, BEAS-2B, and lung cancer cell line, NCI-H1299. We also confirmed the oncogenic feature of BCAR4 by colony forming assay using stably expressing cells. These effects were notably reduced after knockdown of BCAR4 by small interfering RNA. BCAR4 encoded a functional protein and its overexpression resulted in the enhanced migration than controls cells. This result proposed a similar role of BCAR4 in lung cancer considering its previously reported role in breast cancer metastasis. We also detected a fusion gene of BCAR4 in a lung cancer tissue by analyzing RNA-sequencing data. BCAR4 protein as well as its fusion form showed the improved tumorigenic feature in the mouse xenograft model with lung cancer cells. We compared the gene expression pattern in BCAR4 overexpressed cells to identify the possible mechanism of the oncogenic effect of BCAR4. Cyclin D1, Cyclin E, and MMP1 mRNA and protein levels were significantly increased after BCAR4 overexpression. However, downstream genes of GLI2 signaling were not altered by protein expression of BCAR4 arguing an alternative role for lncRNA BCAR4. Our results suggest a novel oncogenic role of BCAR4 protein in lung cancer. Citation Format: Kieun Bae, Minkyong Lee, Daseul Yoon, Yun-Hee Kim, Kyong-Ah Yoon. Breast cancer anti-estrogen resistance 4 (BCAR4) is a novel oncogene in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2017-3137

Increased Expression of Long Non-Coding RNA BCAR4 Is Predictive of Poor Prognosis in Patients with Non-Small Cell Lung Cancer.

Lung cancer is the most common human cancer, and the majority of lung cancer cases are categorized as non-small cell lung cancer (NSCLC). Long non-coding RNAs (lncRNAs) play key roles in the development and progression of human cancers. LncRNA breast cancer anti-estrogen resistance 4 (BCAR4) has been identified as an oncogenic lncRNA involved in the progression of breast cancer and osteosarcoma. However, the clinical significance of the lncRNA BCAR4 in NSCLC remains largely unclear. In the present study, real-time quantitative reverse transcriptase-polymerase chain reaction was used to examine the relative level of lncRNA BCAR4 in 68 cases of NSCLC tissues and their adjacent non-tumor tissues. Our data showed that the expression level of lncRNA BCAR4 was significantly higher in NSCLC tissues compared to their matched non-tumor tissues. Moreover, BCAR4 expression was significantly upregulated in NSCLC cell lines, when compared to the normal human bronchial epithelial cell line BEAS-2B. In addition, the BCAR4 expression was associated with the lymph node metastasis, distant metastasis and clinical stage, but not with the age, sex, tumor size, histological grade, and histological type. The increased expression of BCAR4 was significantly associated with poorer 5-year overall survival rate of NSCLC patients. Multivariate survival analysis indicated that BCAR4 was an independent prognostic factor for NSCLC patients. Taken together, our study suggests that the upregulation of lncRNA BCAR4 expression plays a promoting role in the malignant progression of NSCLC. Thus, BCAR4 is a potential biomarker for NSCLC progress and a therapeutic target for NSCLC.

Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) Drives Proliferation of IPH-926 lobular Carcinoma Cells.

BackgroundMost breast cancers depend on estrogenic growth stimulation. Functional genetic screenings in in vitro cell models have identified genes, which override growth suppression induced by anti-estrogenic drugs like tamoxifen. Using that approach, we have previously identified Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) as a mediator of cell proliferation and tamoxifen-resistance. Here, we show high level of expression and function of BCAR4 in human breast cancer.Methods BCAR4 mRNA expression was evaluated by (q)RT-PCR in a panel of human normal tissues, primary breast cancers and cell lines. A new antibody raised against C78-I97 of the putative BCAR4 protein and used for western blot and immunoprecipitation assays. Furthermore, siRNA-mediated gene silencing was implemented to study the function of BCAR4 and its downstream targets ERBB2/3.ResultsExcept for placenta, all human normal tissues tested were BCAR4-negative. In primary breast cancers, BCAR4 expression was comparatively rare (10%), but associated with enhanced proliferation. Relative high BCAR4 mRNA expression was identified in IPH-926, a cell line derived from an endocrine-resistant lobular breast cancer. Moderate BCAR4 expression was evident in MDA-MB-134 and MDA-MB-453 breast cancer cells. BCAR4 protein was detected in breast cancer cells with ectopic (ZR-75-1-BCAR4) and endogenous (IPH-926, MDA-MB-453) BCAR4 mRNA expression. Knockdown of BCAR4 inhibited cell proliferation. A similar effect was observed upon knockdown of ERBB2/3 and exposure to lapatinib, implying that BCAR4 acts in an ERBB2/3-dependent manner.Conclusion BCAR4 encodes a functional protein, which drives proliferation of endocrine-resistant breast cancer cells. Lapatinib, a clinically approved EGFR/ERBB2 inhibitor, counteracts BCAR4-driven tumor cell growth, a clinical relevant observation.

Characterization of BCAR4, a novel oncogene causing endocrine resistance in human breast cancer cells

Resistance to the antiestrogen tamoxifen remains a major problem in the management of estrogen receptor-positive breast cancer. Knowledge on the resistance mechanisms is needed to develop more effective therapies. Breast cancer antiestrogen resistance 4 (BCAR4) was identified in a functional screen for genes involved in tamoxifen resistance. BCAR4 is expressed in 27% of primary breast tumors. In patients treated with tamoxifen for metastized disease high BCAR4 mRNA levels are associated with reduced clinical benefit and progression-free survival. Regarding tumor aggressiveness high BCAR4 mRNA levels are associated with a shorter metastasis free survival and overall survival. In the present study, we investigated the role of BCAR4 in endocrine resistance. Forced expression of BCAR4 in human ZR-75-1 and MCF7 breast cancer cells resulted in cell proliferation in the absence of estrogen and in the presence of various antiestrogens. Inhibition of estrogen receptor 1 (ESR1) expression with small interfering RNA (siRNA), implied that the BCAR4-induced mechanism of resistance is independent of ESR1. Highly conserved BCAR4 homologues of rhesus monkey, green monkey, and the less conserved common marmoset gene induced tamoxifen-resistant cell proliferation, in contrast to the distant BCAR4 homologues of bovine and rabbit. Injection of BCAR4-expressing ZR-75-1 cells into nude mice resulted in rapidly growing tumors. In silico analysis showed that BCAR4 mRNA is highly expressed in human placenta and oocyte, and absent in other normal tissues. In conclusion, BCAR4 is a strong transforming gene causing estrogen-independent growth and antiestrogen resistance, and induces tumor formation in vivo. Due to its restricted expression, BCAR4 may be a good target for treating antiestrogen-resistant breast cancer.

Abstract 358: Identification of signaling pathways activated by BCAR4 expression in human breast cancer

Abstract Background: The development of resistance to the antiestrogen tamoxifen remains a major challenge in the management of estrogen receptor alpha (ER)-positive breast cancer. Understanding the mechanisms leading to resistance is essential to develop more efficient therapies. In a functional screening for genes causing tamoxifen resistance, we have identified the breast cancer antiestrogen resistance 4 (BCAR4) gene. BCAR4 mRNA is expressed in 27% of primary breast tumors. High BCAR4 mRNA levels predict resistance to tamoxifen treatment and poor outcome, and are associated with a shorter metastasis-free survival and overall survival. Forced expression of BCAR4 in the estrogen-dependent breast cancer cell line ZR-75-1 induced antiestrogen resistance and phosphorylation of ERBB2, ERBB3, and their down-stream mediators ERK1/2 and AKT. The knockdown of ERBB2 or ERBB3 with specific siRNA inhibited cell proliferation, confirming the role of these receptors in BCAR4-induced tamoxifen resistance in vitro. These findings suggest that tumors expressing BCAR4 may rely on ERBB2/ERBB3 signaling and patients with such tumors may be eligible to receive ERBB-targeted therapy. Although ER is functional in ZR/BCAR4 cells, the knockdown of the receptor had no effect on the proliferation rate of BCAR4-expressing cells, showing that this mechanism of resistance is independent of ER signaling. Objective: To identify signaling pathways activated by BCAR4 expression and investigate the consequences of perturbations of the pathways. Experimental design: Reverse-phase protein microarray (RPPA) using phospho-specific antibodies was done to examine the activation status of several key signaling molecules in a cellular model for endocrine resistance of breast cancer. A panel of ZR-75-1-derived cell lines, each containing a different transgene conferring anti-estrogen resistance, was analyzed by RPPA. Results: By measuring the activity of a large number of phospho-protein isoforms in cell lysates from vector-containing control and BCAR4-expressing cells (ZR/BCAR4), it was confirmed that ERBB2 and ERBB3 were strongly phosphorylated in ZR/BCAR4 cells. This was observed in estradiol-containing medium and in medium supplemented with tamoxifen. Furthermore, many established down-stream signaling molecules of ERBB2/3 were found to be activated. Conclusions: ERBB2/ERBB3signaling is strongly activated in cells with forced expression of BCAR4 and completely independent of estrogen signaling. Both ERBB2 and ERBB3 are required for BCAR4-mediated tamoxifen resistance. Our results suggest that patients with ER-positive breast tumors expressing BCAR4 may benefit from ERBB-targeted therapies combined with tamoxifen treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 358. doi:10.1158/1538-7445.AM2011-358

Abstract 3668: Relevance of BCAR4 in tamoxifen resistance and tumor aggressiveness of breast cancer

Abstract Background: Tamoxifen is effective in the treatment of estrogen receptor alpha (ER)-positive breast cancer. However, almost all responding patients develop resistance. Understanding the mechanisms leading to resistance is crucial for the development of more efficient therapies. In a functional screen for genes causing tamoxifen resistance, we have identified the breast cancer antiestrogen resistance 4 (BCAR4) gene. Forced expression of BCAR4 in estrogen-dependent breast cancer cell lines induced antiestrogen resistance. Objective: We explored whether BCAR4 mRNA levels in primary tumors predict tamoxifen resistance and/or tumor aggressiveness, and searched for signaling pathways involved in BCAR4-induced resistance to tamoxifen. Experimental design: BCAR4 mRNA levels were measured by quantitative RT-PCR. In 280 ER-positive primary tumors from patients receiving tamoxifen as first-line monotherapy for advanced disease, the associations between BCAR4 mRNA levels and progression-free survival (PFS) and clinical benefit were evaluated. BCAR4 mRNA levels in primary tumors from 506 patients with lymph node-negative, ER-positive cancer were evaluated for their association with distant metastasis-free survival (MFS). None of them received systemic adjuvant therapy, thereby preventing confounding effects of systemic treatment. In order to study BCAR4 function, we forced the expression of BCAR4 in estrogen-dependent ZR-75-1 breast cancer cells, and analyzed changes in phospho-protein expression. Results: In the study for the association of BCAR4 with tamoxifen resistance, high BCAR4 mRNA levels were significantly associated with a shorter PFS and poor clinical benefit (measurable tumor response or no change &amp;gt; 6 months). Multivariate analysis including the traditional predictive factors indicated that a high BCAR4 mRNA level is an independent predictive factor for PFS. In the assessment of tumor aggressiveness, high BCAR4 mRNA levels were associated with a shorter MFS and overall survival (OS). Multivariate analyses showed that a high BCAR4 mRNA level was an independent prognostic factor for MFS and OS. Forced expression of BCAR4 induced phosphorylation of ERBB2, ERBB3, and their down-stream mediators ERK1/2 and AKT in ZR-75-1 cells. Proliferation of these cells was inhibited by the knockdown of ERBB2 or ERBB3 with specific siRNAs, confirming the role of these receptors in BCAR4-induced tamoxifen resistance. The knockdown of ER had no effect on the proliferation of BCAR4-expressing cells, indicating that this mechanism of resistance is independent of ER signaling. Conclusions: Our data indicate that high BCAR4 mRNA levels predict resistance to tamoxifen treatment and poor outcome. The results of our experimental cell model also suggest that tumors expressing BCAR4 may rely on ERBB2/ERBB3 signaling. Therefore, patients with tumors expressing BCAR4 may be eligible to receive ERBB-targeted therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3668.