A hypercoagulant tumour microenvironment promotes breast cancer progression, with effects inhibited by anticoagulants

Abstract

Background: Breast cancer patients have a four-fold increased risk of developing a venous thromboembolism (VTE). VTE is associated with increased mortality despite adjusting for cancer stage. Tissue Factor (TF) is expressed by breast cancer-associated ?broblasts as well as breast cancer epithelial cells. TF signals to promote cancer growth and metastasis. Rivaroxaban, a licensed oral anticoagulant that inhibits this TF-Factor VIIa (FVIIa)-Factor Xa (FXa) complex, could potentially be repurposed to target this procoagulant tumour microenvironment in breast cancer. Methods: Recombinant coagulation factors, lentivirally transduced TF over-expressing ?broblasts (TFF) and their control (CF) or conditioned media (TFFCM and CFCM), were cultured with oestrogen receptor positive (ER+) breast cancer cells (MCF-7) +/- Rivaroxaban or anti-TF antibody 10H10. Proliferation (sulforhodamine-B/EdU assay), migration (scratch/transwell assay) and stem cell activity (mammosphere forming e?ciency (MFE) assay) were assessed. The underlying mechanism was analysed with western blotting and quantitative PCR.Results: Recombinant TF,FVIIa and FXa versus control promoted proliferation and migration in MCF-7 cells (p<0.001), with these e?ects abrogated by Rivaroxaban (p<0.05). Recombinant TF,FVIIa and FXa increased phospho-ERK (p<0.01), CXCL8 (p<0.05) and VEGFA (p<0.0001) expression as compared to control, with CXCL8 and VEGFA inhibited by Rivaroxaban (p<0.01).TFFCM promoted proliferation, migration and stem cell activity in MCF-7 cells (p<0.05) as compared to CFCM, with these e?ects abrogated by 10H10 (proliferation, migration, MFE:p<0.05) and Rivaroxaban (migration, MFE:p<0.05). The cancer-promoting e?ects of TFFCM versus CFCM were associated with increased VEGFA (p<0.05) expression; which was reversed by 10H10 and Rivaroxaban (p<0.05). 3D co-culture of MCF-7s with TFF as compared to CF promoted cancer cell migration (p=0.04) and stem cell activity (MFE:p<0.0001), with these e?ects abrogated by 10H10 (migration:p=0.01, MFE:p=0.0028) and Rivaroxaban (migration:p= 0.0341, MFE:p=0.0003).Conclusion: A procoagulant microenvironment promotes proliferation, migration and stem cell activity in ER+ breast cancer in vitro which can be targeted by anticoagulants. Rivaroxaban could potentially be repurposed as anticancer therapy.