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.

BCAR4 induces antioestrogen resistance but sensitises breast cancer to lapatinib.

BACKGROUND: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression. METHODS: Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease. RESULTS: BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group. CONCLUSION: This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy.

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 expression profile, suggesting that their causative role in cell growth may be accomplished by post-transcriptional processes. The associations of NCOR2 and CITED2 with outcome in oestrogen receptor-positive breast cancer patients underscore the clinical relevance of functional genetic screens to better understand disease progression, which may ultimately lead to the development of improved treatment options.

Co-occurrence based meta-analysis of scientific texts: retrieving biological relationships between genes.

MOTIVATION: The advent of high-throughput experiments in molecular biology creates a need for methods to efficiently extract and use information for large numbers of genes. Recently, the associative concept space (ACS) has been developed for the representation of information extracted from biomedical literature. The ACS is a Euclidean space in which thesaurus concepts are positioned and the distances between concepts indicates their relatedness. The ACS uses co-occurrence of concepts as a source of information. In this paper we evaluate how well the system can retrieve functionally related genes and we compare its performance with a simple gene co-occurrence method. RESULTS: To assess the performance of the ACS we composed a test set of five groups of functionally related genes. With the ACS good scores were obtained for four of the five groups. When compared to the gene co-occurrence method, the ACS is capable of revealing more functional biological relations and can achieve results with less literature available per gene. Hierarchical clustering was performed on the ACS output, as a potential aid to users, and was found to provide useful clusters. Our results suggest that the algorithm can be of value for researchers studying large numbers of genes. AVAILABILITY: The ACS program is available upon request from the authors.