Breast cancer oestrogen independence mediated by BCAR1 or BCAR3 genes is transmitted through mechanisms distinct from the oestrogen receptor signalling pathway or the epidermal growth factor receptor signalling pathway

INTRODUCTION: Tamoxifen is effective for endocrine treatment of oestrogen receptor-positive breast cancers but ultimately fails due to the development of resistance. A functional screen in human breast cancer cells identified two BCAR genes causing oestrogen-independent proliferation. The BCAR1 and BCAR3 genes both encode components of intracellular signal transduction, but their direct effect on breast cancer cell proliferation is not known. The aim of this study was to investigate the growth control mediated by these BCAR genes by gene expression profiling. METHODS: We have measured the expression changes induced by overexpression of the BCAR1 or BCAR3 gene in ZR-75-1 cells and have made direct comparisons with the expression changes after cell stimulation with oestrogen or epidermal growth factor (EGF). A comparison with published gene expression data of cell models and breast tumours is made. RESULTS: Relatively few changes in gene expression were detected in the BCAR-transfected cells, in comparison with the extensive and distinct differences in gene expression induced by oestrogen or EGF. Both BCAR1 and BCAR3 regulate discrete sets of genes in these ZR-75-1-derived cells, indicating that the proliferation signalling proceeds along distinct pathways. Oestrogen-regulated genes in our cell model showed general concordance with reported data of cell models and gene expression association with oestrogen receptor status of breast tumours. CONCLUSIONS: The direct comparison of the expression profiles of BCAR transfectants and oestrogen or EGF-stimulated cells strongly suggests that anti-oestrogen-resistant cell proliferation is not caused by alternative activation of the oestrogen receptor or by the epidermal growth factor receptor signalling pathway

Relevance of BCAR4 in tamoxifen resistance and tumour aggressiveness of human breast cancer

Background:Breast cancer anti-oestrogen resistance 4 (BCAR4) was identified in a search for genes involved in anti-oestrogen resistance in breast cancer. We explored whether BCAR4 is predictive for tamoxifen resistance and prognostic for tumour aggressiveness, and studied its function.Methods:BCAR4 mRNA levels were measured in primary breast tumours, and evaluated for association with progression-free survival (PFS) and clinical benefit in patients with oestrogen receptor (ERα)-positive tumours receiving tamoxifen as first-line monotherapy for advanced disease. In a separate cohort of patients with lymph node-negative, ERα-positive cancer, and not receiving systemic adjuvant therapy, BCAR4 levels were evaluated for association with distant metastasis-free survival (MFS). The function of BCAR4 was studied with immunoblotting and RNA interference in a cell model.Results:Multivariate analyses established high BCAR4 mRNA levels as an independent predictive factor for poor PFS after start of tamoxifen therapy for recurrent disease. High BCAR4 mRNA levels were associated with poor MFS and overall survival, reflecting tumour aggressiveness. In BCAR4-expressing cells, phosphorylation of v-erb-b2 erythroblastic leukaemia viral oncogene homolog (ERBB)2, ERBB3, and their downstream mediators extracellular signal-regulated kinase 1/2 and v-akt murine thymoma viral oncogene homolog (AKT) 1/2, was increased. Selective knockdown of ERBB2 or ERBB3 inhibited proliferation, confirming their role in BCAR4-induced tamoxifen resistance.Conclusion:BCAR4 may have clinical relevance for tumour aggressiveness and tamoxifen resistance. Our cell model suggests that BCAR4-positive breast tumours are driven by ERBB2/ERBB3 signalling. Patients with such tumours may benefit from ERBB-targeted therapy

CITED2 and NCOR2 in anti-oestrogen resistance and progression of breast cancer

Background:Endocrine therapies of breast cancer are effective but ultimately fail because of the development of treatment resistance. We have previously revealed several genes leading to tamoxifen resistance in vitro by retroviral insertion mutagenesis. To understand the manner in which these genes yield tamoxifen resistance, their effects on global gene expression were studied and those genes resulting in a distinct gene expression profile were further investigated for their clinical relevance.Methods:Gene expression profiles of 69 human breast cancer cell lines that were made tamoxifen resistant through retroviral insertion mutagenesis were obtained using oligonucleotide arrays and analysed with bioinformatic tools. mRNA levels of NCOR2 and CITED2 in oestrogen receptor-positive breast tumours were determined by quantitative RT-PCR. mRNA levels were evaluated for association with metastasis-free survival (MFS) in 620 patients with lymph node-negative primary breast cancer who did not receive systemic adjuvant therapy, and with clinical benefit in 296 patients receiving tamoxifen therapy for recurrent breast cancer.Results:mRNA expression profiles of most tamoxifen-resistant cell lines were strikingly similar, except for the subgroups of cell lines in which NCOR2 or CITED2 were targeted by the retrovirus. Both NCOR2 and CITED2 mRNA levels were associated with MFS, that is, tumour aggressiveness, independently of traditional prognostic factors. In addition, high CITED2 mRNA levels were predictive for a clinical benefit from first-line tamoxifen treatment in patients with advanced disease.Conclusions: Most retrovirally targeted genes yielding tamoxifen resistance in our cell lines do not impose a distinctive expressi

miR-371a-3p, miR-373-3p and miR-367-3p as Serum Biomarkers in Metastatic Testicular Germ Cell Cancers Before, During and After Chemotherapy

Background: LDH (lactate dehydrogenase), AFP (alpha-fetoprotein) and \xce\xb2-HCG (human \nchorionic gonadotropin) are used in diagnosis and follow-up of testicular germ cell cancer (TGCC) \npatients. Our aim was to investigate the association between levels of miR-371a-3p, miR-373-3p \nand miR-367-3p and clinical features in metastatic TGCC. Methods: relative levels of miR-371a-3p, \nmiR-373-3p and miR-367-3p were evaluated in serum of metastatic TGCC patients. A prospectively \nincluded and a retrospectively selected cohort were studied (total patient number = 109). Blood \nsamples were drawn at start of chemotherapy and during follow-up. Serum microRNA (miR) \nlevels were determined using the ampTSmiR test. Results: at start of chemotherapy, miR-371a-3p, \nmiR-373-3p and miR-367-3p levels were positively correlated to LDH. The median level of these miRs \nwas higher in patients who developed a relapse after complete biochemical remission (n = 34) than \nin those who had complete durable remission (n = 60). Higher levels of miR-367-3p were found in \npatients with refractory disease (n = 15) compared to those who had complete response. miR levels \ndecreased during the first week of chemotherapy in patients with complete response and stayed \nbelow threshold after one year of treatment. Conclusion: high miR levels at start of chemotherapy \nare associated with worse clinical outcome and can assist in early diagnosing of relapses

Cellular origin of microRNA-371a-3p in healthy males based on systematic urogenital tract tissue evaluation

Background: The microRNA-371a-3p (miR-371a-3p) has been reported to be an informative liquid biopsy (serum and plasma) molecular biomarker for both diagnosis and follow-up of patients with a malignant (testicular) germ cell tumor ((T)GCT). It is expressed in all histological cancer elements, with the exception of mature teratoma. However, normal testis, semen, and serum of males with a disrupted testicular integrity without a TGCT may contain miR-371a-3p levels above threshold, of which the cellular origin is unknown. Objectives: Therefore, a series of relevant tissues (frozen and formalin-fixed paraffin-embedded (FFPE), when available) from the complete male urogenital tract (i.e., kidney to urethra and testis to urethra) and semen was investigated for miR-371a-3p levels using targeted quantitative RT-PCR (qRT-PCR). Materials and methods: In total, semen of males with normospermia (n = 11) and oligospermia (n = 3) was investigated, as well as 88 samples derived from 32 different patients. The samples represented one set of tissues related to the entire male urogenital tract (11 anatomical locations), three sets for 10 locations, and four sets for six locations. Results: All testis parenchyma (n = 17) cases showed low miR-371a-3p levels. Eight out of 14 (57%) semen samples showed detectable miR-371a-3p levels, irrespective of the amount of motile spermatozoa, but related to sperm concentration and matched Johnsen score (Spearman’s rho correlation coefficient 0.849 and 0.871, p = 0.000, respectively). In all other tissues investigated, miR-371a-3p could not be detected. Discussion: This study demonstrates that the miR-371a-3p in healthy adult males is solely derived from the germ cell compartment. Conclusions: The observation is important in the context of applying miR-371a-3p as molecular liquid biopsy biomarker for diagnosis and follow-up of patients with malignant (T)GCT. Moreover, miR-371a-3p might be an informative seminal biomarker for testicular germ cell composition

High TWIST1 mRNA expression is associated with poor prognosis in lymph node-negative and estrogen receptor-positive human breast cancer and is co-expressed with stromal as well as ECM related genes

Introduction: The TWIST homolog 1 (TWIST1) is a transcription factor that induces epithelial to mesenchymal transition (EMT), a key process in metastasis. The purpose of this study was to investigate whether TWIST1 expression predicts disease progression in a large breast cancer cohort with long-term clinical follow-up, and to reveal the biology related to TWIST1 mediated disease progression.Methods: TWIST1 mRNA expression level was analyzed by quantitative real-time reverse polymerase chain reaction (RT-PCR) in 1,427 primary breast cancers. In uni- and multivariate analysis using Cox regression, TWIST1 mRNA expression level was associated with metastasis-free survival (MFS), disease-free survival (DFS) and overall survival (OS). Separate analyses in lymph node-negative patients (LNN, n = 778) who did not receive adjuvant systemic therapy, before and after stratification into estrogen receptor (ER)-positive (n = 552) and ER-negative (n = 226) disease, were also performed. The association of TWIST1 mRNA with survival endpoints was assessed using Kaplan-Meier analysis. Using gene expression arrays, genes showing a significant Spearman rank correlation with TWIST1 were used to identify overrepresented Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-annotated biological pathways.Results: Increased mRNA expression level of TWIST1 analyzed as a continuous variable in both uni- and multivariate analysis was associated with shorter MFS in all patients (hazard ratio (HR): 1.17, 95% confidence interval, (95% CI):1.09 to 1.26; and HR: 1.17, 95% CI: 1.08 to 1.26; respectively), in LNN patients (HR: 1.22, 95% CI: 1.09 to 1.36; and HR: 1.21, 95% CI: 1.07 to 1.36; respectively) and in the ER-positive subgroup of LNN patients (HR: 1.34, 95% CI: 1.17 to 1.53; and HR: 1.32, 95% CI: 1.14 to 1.53; respectively). Similarly, high TWIST1 expression was associated with shorter DFS and OS in all patients and in the LNN/ER-positive subgroup. In contrast, no association of TWIST1 mRNA expression with MFS, DFS or OS was observed in ER-negative patients. Genes h

Clinical significance of the nuclear receptor co-regulator DC-SCRIPT in breast cancer: an independent retrospective validation study

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