Targeting the procoagulant tumour microenvironment in breast cancer
Blower, Emma Castle, J. Clarke, Robert B Kirwan, C. C.
Blower, Emma
Castle, J.
Clarke, Robert B
Kirwan, C. C.
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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.
Description
Date
2021
Publisher
Collections
Keywords
Type
Meetings and Proceedings
Citation
Blower E, Castle J, Clarke R, Kirwan C. 05. TARGETING THE PROCOAGULANT TUMOUR MICROENVIRONMENT IN BREAST CANCER. European Journal of Surgical Oncology . 2020 Jun;46(6):e2.