Targeting the procoagulant tumour microenvironment in breast cancer

Abstract

Introduction: Coagulation factors, in particular tissue factor (TF), are produced by breast tumour cells and cancer-associated fi broblasts (CAFs). These signal via protease-activated receptors 1 and 2 (PAR1/2) on the cell membrane and induce invasion, angiogenesis and metastasis. Direct oral anticoagulants (DOACs), including rivaroxaban and Dabigatran, inhibit the TF–factor VIIa–factor Xa complex and thrombin respectively, and could therefore be repurposed as anticancer drugs. Aim: We aim to demonstrate that creating a procoagulant tumour micro environment will increase breast cancer cell proliferation and migration in vitro, with this eff ect being abrogated by DOACs. The mechanism by which this occurs, in terms of downstream phosphorylated proteins will also be examined. Materials and Methods: Breast cancer cell lines (BT474, MCF-7 and MDA-MB-231) were co-cultured with recombinant clotting factors (thrombin, TF, factor Xa (FXa), factor VIIa (FVIIa)) in the presence or absence of the anti-TF antibody 10H10, the direct FXa inhibitor rivaroxaban and the direct thrombin inhibitor dabigatran. Proliferation and migration were assessed in vitro using sulforhodamine B assays and wound closure assays visualised using the Incucyte respectively. The eff ect on downstream phosphorylated proteins was assessed by Western blotting. Results: 10 and 40 units/ml of thrombin increases proliferation in BT474 cells (p<0.0001). TF, FVIIa and FXa in combination increases proliferation in MCF-7 cells (p=0.05). Conversely, FXa at 0.1 and 10 units/ml reduces proliferation in MDA-MB-231 cells (p=0.33, p=0.001). 10H10 decreases proliferation in MCF-7 cells (p=0.02), with a trend for decrease in BT474 cells. 10 units/ml of FXa and TF, FVIIa and FXa in combination increases migration of MDA-MB-231 cells (p=0.05 and p=0.007 respectively), whilst there is a trend towards increased migration with 40 units/ml of thrombin in MDA-MB-231 and MCF-7 cells. DOACs had no eff ect on proliferation or migration in vitro. In BT474 cells, thrombin and TF, FVIIa and FXa in combination appear to cause a relative increase in pERK within 1 minute and pAkt within 15 and 5 minutes respectively. Rivaroxaban and dabigatran both appear to cause a decrease in pAkt within 1 minute. Conclusions: Breast cancer cell line in vitro experiments have shown that thrombin and the TF–FVIIa–FXa complex increase proliferation, whilst 10H10 reduces proliferation. FXa independently and in combination with TF and FVIIa promotes migration. These aff ects appear to be occurring via the PAR-1/2 pathway. Targeting the procoagulant tumour microenvironment is a promising strategy in reducing breast cancer cell proliferation and migration.