Dataset Information

Regulation of p130(Cas)/BCAR1 expression in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells by EGR1 and NAB2.

ABSTRACT: Elevated levels of p130(Cas)/BCAR1 (Crk-associated substrate/breast cancer antiestrogen resistance 1) are found in aggressive breast tumors and are associated with tamoxifen resistance of mammary cancers. p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways frequently deregulated in breast cancer. To elucidate mechanisms leading to p130(Cas) up-regulation in mammary carcinomas and during acquired tamoxifen resistance, the regulation of p130(Cas)/BCAR1 was studied. Because multiple putative binding motifs for the inducible transcription factor EGR1 were identified in the 5' region of BCAR1, the p130(Cas)/BCAR1 regulation by EGR1 and its coregulator NAB2 was investigated. Overexpression or short interfering RNA (siRNA)-mediated down-regulation of EGR1 or NAB2, and chromatin immunoprecipitations indicated that EGR1 and NAB2 act in concert to positively regulate p130(Cas)/BCAR1 expression in breast cancer cells. p130(Cas) depletion using siRNA showed that, in tamoxifen-sensitive MCF-7 cells, p130(Cas) regulates EGR1 and NAB2 expression, whereas in the derivative tamoxifen-resistant TAM-R cells, only NAB2 levels were influenced. BCAR1 messenger RNA and p130(Cas) protein were upregulated by phorbol esters following the kinetics of late response genes in MCF-7 but not in TAM-R cells. Thus, in MCF-7 cells, we identified a positive feedback loop where p130(Cas) positively regulates EGR1 and NAB2, which in turn induce p130(Cas) expression. Importantly, compared with MCF-7, enhanced NAB2 expression and increased EGR1 binding to the BCAR1 5' region observed in TAM-R may lead to the constitutively increased p130(Cas)/BCAR1 levels in TAM-R cells. The uncovered differences in this EGR1/NAB2/p130(Cas) network in MCF-7 versus TAM-R cells may also contribute to p130(Cas) up-regulation during acquired tamoxifen resistance.

SUBMITTER: Kumbrink J

PROVIDER: S-EPMC3306256 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Regulation of p130(Cas)/BCAR1 expression in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells by EGR1 and NAB2.

Kumbrink Joerg J Kirsch Kathrin H KH

Neoplasia (New York, N.Y.) 20120201 2

Elevated levels of p130(Cas)/BCAR1 (Crk-associated substrate/breast cancer antiestrogen resistance 1) are found in aggressive breast tumors and are associated with tamoxifen resistance of mammary cancers. p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways frequently deregulated in breast cancer. To elucidate mechanisms leading to p130(Cas) up-regulation in mammary carcinomas and during acquired tamoxifen resistance, the regulation of p130(Cas)/BCAR1 w ...[more]

PMID: 22431919

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Project description:Introduction: Although changes in microRNA (miRNA) expression correlate with breast cancer diagnostic markers, comparatively little is known about miRNA regulation by selective estrogen receptor modulators (SERMs), e.g., tamoxifen (TAM), or their role in endocrine-resistance. Methods: A microarray approach was used to identify miRNAs differentially expressed and regulated by 4-hydroxyTAM (4-OHT) in MCF-7/TAM-sensitive versus LY2 TAM/endocrine-resistant human breast cancer cells after 6 h treatment. The expression of specific miRNAs was validated by quantitative, real-time PCR. Control miRNAs and time-course studies showed important gene-, time-, and cell- specific differences in miRNA expression. Results: 97 miRNAs were differentially expressed in MCF-7 versus LY2 cells. Bioinformatic analyses to impute the biological significance of these miRNAs by identifying 36 predicted gene targets of these miRNAs from amongst those reported to be regulated by MCF-7 cells was performed and agreement was found in direction of anticipated regulation for 12 of those putative targets. Among the miRNAs differentially expressed in MCF-7 versus LY2 cells, and that putatively target mRNAs regulated by 4-OHT in MCF-7 cells, are: miRs- 10a, 21, 22, 29a, 93, 125b, 181, 200a, 200b, 200c; 205, 221, and 222. Q-PCR assessment showed agreement with microarray data: miR- 10a, 22, 125b, 181, and 222 are higher in LY2 whereas miR-21 and miR-200a are lower in LY2 than MCF-7. 4-OHT increased miR-10a, miR-21, miR-22, miR-125b, miR-181a, miR-200a, and miR-222 in MCF-7 cells and reduced miR-10a in LY2 cells. E2 reduced the miR-21expression in MCF-7 cells. Treatment with ICI 182,780 revealed that ER suppresses miR-10a, miR-21, miR-22, miR-200a, miR-221, and miR-222 expression in MCF-7 cells. Time-dependent changes in select miRNA expression were observed in control, E2, and 4-OHT-treated MCF-7 cells. ESR1/ERα was significantly lower in LY2 than MCF-7, commensurate with higher let-7 family member, miR-221, and miR-222 expression in LY2. Reflecting inverse association with miR-200 family expression, the expression of ZEB1 was higher in LY2 than MCF-7 cells and concomitantly, E-cadherin expression was absent in LY2 cells indicating that LY2 have undergone epithelial to mesenchymal transition. Conclusions: Our studies identifying miRNAs with opposite expression between the two cell lines, indicate the involvement of these miRNAs in endocrine resistance. The purpose of this experiment is: 1. To identify miRNAs that are regulated (up or down) by 4-OHT in MCF-7 and LY2 cell lines (comparison of values from EtOH versus 4-OHT treated cells). 2. To identify miRNAs that are differentially regulated under basal (EtOH) conditions between MCF-7 and LY2 cell lines. 3. To identify miRNAs that are differentially regulated by 4-OHT in MCF-7 versus LY2 cells.

Project description:A functional genetic screen to identify genes causing tamoxifen resistance in an estrogen-dependent human breast cancer cell model was performed. By insertion of defective retrovirus into the genome, individual genetic changes were introduced at random in ZR-75-1 cells. Subsequently, infected cells were selected for their ability to proliferate while being exposed to tamoxifen, and from these cultures stable cell lines were established. The retrovirus insertion sites were mapped enabling the identification of several candidate breast cancer anti-estrogen resistance (BCAR) genes. By cDNA transfection of estrogen-dependent cells resulting in their transformation into a tamoxifen-resistant phenotype, proof was provided for the causative role of BCAR genes.A functional genetic screen to identify genes causing tamoxifen resistance in an estrogen-dependent human breast cancer cell model was performed. By insertion of defective retrovirus into the genome, individual genetic changes were introduced at random in ZR-75-1 cells. Subsequently, infected cells were selected for their ability to proliferate while being exposed to tamoxifen, and from these cultures stable cell lines were established. The retrovirus insertion sites were mapped enabling the identification of several candidate breast cancer anti-estrogen resistance (BCAR) genes identified. By cDNA transfection of estrogen-dependent cells resulting in their transformation into a tamoxifen-resistant phenotype, proof was provided for the causative role of BCAR genes (Van Agthoven et al, Breast Cancer Res Treat DOI:10.1007/s10549-008-9969-5, 2008). To elucidate the mechanisms how these individual BCAR genes induce cell proliferation in growth-arrested ZR-75-1 cells, we assessed the gene expression patterns between the different groups of cell lines. Keywords: Expression profiling, reference design RNA was isolated from two independent cultures of 69 tamoxifen-resistant cell lines. Detailed information regarding these cell lines with respect to the viral integration sites, was described previously (Van Agthoven et al, Breast Cancer Res Treat DOI:10.1007/s10549-008-9969-5, 2008). Each sample was hybridized in duplicate/triplicate and once in a dye-swap. The mixture of cell lines used as a reference was detailed previously (Jansen et al, J Clin Oncol 23, 732, 2005).

Dataset Information

Regulation of p130(Cas)/BCAR1 expression in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells by EGR1 and NAB2.

Publications

Regulation of p130(Cas)/BCAR1 expression in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells by EGR1 and NAB2.

Similar Datasets

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