Subtype-specific clinical and prognostic relevance of tumor-expressed F5 and regulatory F5 variants in breast cancer: the CoCaV study.

Essentials The role of coagulation factor V (encoded by F5) in cancer pathogenesis is unknown. The clinical significance of tumor-expressed F5 was evaluated in breast cancer patient cohorts. F5 was expressed in human breast tumors, and the expression was higher than in normal tissue. High F5 expression was associated with aggressive tumors, but also with survival in breast cancer. SUMMARY: Background Tumor expression of certain coagulation factors has been linked to cancer progression. Single nucleotide polymorphisms (SNPs) in F5 (encoding the FV protein) have been found to be associated with breast cancer; however, the role of coagulation factor V (FV) in cancer pathogenesis remains undiscovered. Objectives We aimed to investigate the clinical significance of FV and the regulatory role of F5 gene variants in breast cancer. Patients/Methods A Scandinavian 503-sample breast cancer cohort and three public breast cancer datasets (GOBO, TCGA and KM plotter) were used to determine associations between F5 gene expression (tumor-specific), circulating FV, F5 SNPs, clinical characteristics and breast cancer survival. Immunohistochemistry (IHC) was used to detect FV antigen in tumors. Results F5 expression was 2-fold higher in breast tumors compared with normal tissue, and the presence of FV antigen in breast tumors was confirmed by IHC staining. F5 expression was increased in patients with hormone receptor negative tumors, triple negative tumors, HER2-enriched and basal-like tumors. In patients with basal tumors, high expression of F5 was associated with improved overall survival (hazard ratio, HR = 0.52, 95% confidence interval, 0.31-0.86). SNPs in F5 were associated with tumor size and luminal A tumors. The rs6427202-rs9332542 C-G haplotype, previously associated with breast cancer, displayed a cis-regulatory effect on F5 expression in tumors and plasma FV antigen levels. In silico mining supported this regulatory function. Conclusions FV was a possible marker of aggressive breast cancer, yet also a predictor of favorable outcome. Evaluation of FV expression may be clinically useful for prognosis and treatment decisions in aggressive breast cancer.

Effect of hypoxia on tissue factor pathway inhibitor expression in breast cancer.

UNLABELLED: ESSENTIALS: A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF-7 and SK-BR-3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia-inducible factor 1α may be a target for the therapy of cancer-related coagulation and thrombosis. BACKGROUND: Activation of coagulation is a common finding in patients with cancer, and is associated with an increased risk of venous thrombosis. As a hypoxic microenvironment is a common feature of solid tumors, we investigated the role of hypoxia in the regulation of tissue factor (TF) pathway inhibitor (TFPI) expression in breast cancer. OBJECTIVES: To explore the transcriptional regulation of TFPI by hypoxia-inducible factor (HIF)-1α in breast cancer cells and their correlation in breast cancer tissues. METHODS AND RESULTS: MCF-7 and SK-BR-3 breast cancer cells were cultured in 1% oxygen or treated with cobalt chloride (CoCl2 ) to mimic hypoxia. Time-dependent and dose-dependent downregulation of TFPI mRNA (quantitative RT-PCR) and of free TFPI protein (ELISA) were observed in hypoxia. Western blotting showed parallel increases in the levels of HIF-1α protein and TF. HIF-1α inhibitor abolished or attenuated the hypoxia-induced downregulation of TFPI. Luciferase reporter assay showed that both hypoxia and HIF-1α overexpression caused strong repression of TFPI promoter activity. Subsequent chromatin immunoprecipitation and mutagenesis analysis demonstrated a functional hypoxia response element within the TFPI promoter, located at -1065 to -1060 relative to the transcriptional start point. In breast cancer tissue samples, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and that of HIF-1α. CONCLUSIONS: This study demonstrates that HIF-1α is involved in the transcriptional regulation of the TFPI gene, and suggests that a hypoxic microenvironment inside a breast tumor may induce a procoagulant state in breast cancer patients.

Identifying microRNAs regulating B7-H3 in breast cancer: the clinical impact of microRNA-29c.

BACKGROUND: B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer. METHODS: MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3'-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients). RESULTS: We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3'-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts. CONCLUSIONS: We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.