Functional and Regulatory Effects of Factor V Leiden and Factor V rs6028 in Breast Cancer

Abstract

Background/Objectives: Cancer progression and the hemostatic system are closely linked. Coagulation factor V (FV) has a key function in coagulation, with both pro- and anticoagulant functions. FV gene (F5) expression and F5 variants have been linked to breast cancer progression. The direct impact of F5 variants on FV expression and functional effects in breast cancer are unknown. We aimed to investigate whether the F5 variants FV Leiden (F5 rs6025) and F5 rs6028 influenced FV expression, coagulant activity, and apoptosis in breast cancer cells. Methods: MDA-MB-231 cells were transfected with overexpression plasmids containing F5 wild type, F5 rs6025 or F5 rs6028. We investigated the functional impact of the F5 variants on F5 mRNA, FV protein, FV coagulant activity, and apoptosis in vitro, and examined the potential of the variants as transcriptional regulators of F5 expression in silico. Results: Increased F5 mRNA, FV protein, and apoptosis were observed in cells transfected with F5 wild-type overexpression plasmid compared to empty vector. F5 mRNA, protein, coagulant activity, and apoptosis were further increased with the F5 rs6025 and F5 rs6028 variants compared to F5 wild type. Cis-expression quantitative trait loci analyses indicated a regulatory effect of F5 rs6028, and putative transcription factor binding sites for several transcription factors overlapped with the position of F5 rs6025. Conclusions: Our study demonstrated that F5 rs6025 and F5 rs6028 have a regulatory effect on FV synthesis that might affect apoptosis in breast cancer. The F5 variants might therefore enhance the tumor suppressor function of FV in breast cancer.

Keywords: Factor V; Factor V Leiden; breast cancer; gene expression regulation; single nucleotide polymorphisms.

Figure A1

Transfection efficiency. The wild-type factor V plasmid, pMT2-FV-wt, the empty vector pMT2, and the F5 variant plasmids pFV-rs6025 and pFV-rs6028 were each co-transfected into MDA-MB-231 cells with Lipofectamine3000 (Thermo Scientific, Waltham, MA, USA). (A) Co-transfection with an equal amount of a plasmid expressing tissue factor pathway inhibitor (TFPI) (pTOPO-TFPIα). After 24 h of incubation, cells were harvested, RNA was isolated from cell lysates with the RNAqueous™ Total RNA Isolation Kit (Thermo Fisher Scientific), and cDNA synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative RT-PCR (qPCR) was performed with a TFPI-specific TaqMan assay and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific). All samples were run in triplicate on the Applied Biosystems QuantStudio 12K Flex Real-Time System (Thermo Scientific). Relative TFPI mRNA expression was calculated using the comparative CT (ΔΔCT) method with hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the endogenous control. TFPI mRNA expression in wells co-transfected with the variant FV plasmids was calculated as fold change relative to TFPI mRNA expressed in the wells co-transfected with the wild-type FV plasmid. Mean results and SEM from two independent experiments (n = 6) are shown. (B) Co-transfection with an equal amount of a plasmid expressing green fluorescent protein (GFP), pmaxGFPTM Vector (Lonza, Slough, UK). Twenty-four hours post transfection, cells were examined for GFP fluorescence in a Nikon ECLIPSE Ts2-FL microscope (Nikon, Tokyo, Japan) and cells counted and transfection efficiency calculated using ImageJ 1.54g. Mean results and SEM from two independent experiments (n = 6) are shown. (C) Pictures from one representative experiment are shown.

Figure 1

Functional effects of F5 variants on F5 mRNA, FV protein, and FV coagulant activity. (A) F5 mRNA expression and (B) FV protein levels in MDA-MB-231 cells transfected with pMT2, pFV-wt, pFV-rs6025, or pFV-rs6028 for 48 h. (C) FV coagulant activity in MDA-MB-231 cells transfected with pFV-wt, pFV-rs6025, or pFV-rs6028 for 48 h. mRNA expression was measured with qPCR and normalized against endogenous control. FV protein levels in media were measured with FV ELISA and corrected for the total protein concentration in cell lysates. FV coagulant activity was measured in cell media with one-stage assay. F5 mRNA, FV protein, and FV coagulant activity were calculated relative to pFV-wt. Mean values and SEM were calculated from three independent experiments. Significant differences were calculated using the Kruskal–Wallis test followed by the Wilcoxon rank sum test and marked with * (p < 0.05), ** (p < 0.01), *** (p < 0.001).

Figure 2

Functional effects of F5 variants on apoptosis. MDA-MB-231 cells were transfected with pMT2, pFV-wt, pFV-rs6025, or pFV-rs6028. Apoptosis was measured by detection of nucleosomes 48 h post transfection. The values for the F5 variants were calculated relative to pFV-wt. Mean values and SEM were calculated from three independent experiments. Significant differences were calculated using the Kruskal–Wallis test followed by the Wilcoxon rank sum test and marked with * (p < 0.05), ** (p < 0.01).

Figure 3

Regulatory effects of F5 variants. (A) Cis-eQTL analysis of F5 rs6028 and (B) F5 rs6025 using the FIVEx tool [20]. The dashed line illustrates the significance threshold. (C) ChIP-Seq peaks in the position for F5 rs6025 (highligted) using Remap [18] in the UCSC Genome Browser [19]. Abbreviations: ESR1, Estrogen Receptor 1; CTCF, CCCTC-binding factor; YY1AP1, YY1 Associated Protein 1; ESRRA, Estrogen Related Receptor Alpha; ESRRG, Estrogen Related Receptor Gamma; ZNF750, Zinc Finger Protein 750; EZH2, Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit; CREB1, CAMP Responsive Element Binding Protein 1; CEBPA, CCAAT Enhancer Binding Protein Alpha; NCOA1, Nuclear Receptor Coactivator 1; NRIP1, Nuclear Receptor Interacting Protein 1; SMARCA4, SWI/SNF Related Matrix Associated Actin Dependent Regulator Of Chromatin Subfamily A Member 4; RAD21, RAD21 Cohesin Complex Component; NCBI, The National Center for Biotechnology Information.