The unfolded protein response links ER stress to cancer-associated thrombosis
Oluwatoyosi Muse, Rushad Patell, Christian G. Peters, Moua Yang, Emale El-Darzi, Sol Schulman, Anna Falanga, Marina Marchetti, Laura Russo, Jeffrey I. Zwicker, Robert Flaumenhaft
Oluwatoyosi Muse, Rushad Patell, Christian G. Peters, Moua Yang, Emale El-Darzi, Sol Schulman, Anna Falanga, Marina Marchetti, Laura Russo, Jeffrey I. Zwicker, Robert Flaumenhaft
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Research Article Hematology

The unfolded protein response links ER stress to cancer-associated thrombosis

Abstract

Thrombosis is a common complication of advanced cancer, yet the cellular mechanisms linking malignancy to thrombosis are poorly understood. The unfolded protein response (UPR) is an ER stress response associated with advanced cancers. A proteomic evaluation of plasma from patients with gastric and non–small cell lung cancer who were monitored prospectively for venous thromboembolism demonstrated increased levels of UPR-related markers in plasma of patients who developed clots compared with those who did not. Release of procoagulant activity into supernatants of gastric, lung, and pancreatic cancer cells was enhanced by UPR induction and blocked by antagonists of the UPR receptors inositol-requiring enzyme 1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK). Release of extracellular vesicles bearing tissue factor (EVTFs) from pancreatic cancer cells was inhibited by siRNA-mediated knockdown of IRE1α/XBP1 or PERK pathways. Induction of UPR did not increase tissue factor (TF) synthesis, but rather stimulated localization of TF to the cell surface. UPR-induced TF delivery to EVTFs was inhibited by ADP-ribosylation factor 1 knockdown or GBF1 antagonism, verifying the role of vesicular trafficking. Our findings show that UPR activation resulted in increased vesicular trafficking leading to release of prothrombotic EVTFs, thus providing a mechanistic link between ER stress and cancer-associated thrombosis.

Authors

Oluwatoyosi Muse, Rushad Patell, Christian G. Peters, Moua Yang, Emale El-Darzi, Sol Schulman, Anna Falanga, Marina Marchetti, Laura Russo, Jeffrey I. Zwicker, Robert Flaumenhaft

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Figure 6

UPR does not mediate increased TF synthesis but promotes thrombin generation at the surface of pancreatic cancer cells.

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UPR does not mediate increased TF synthesis but promotes thrombin genera...
(A) HPAF-II cells were incubated with either 5 μM MKC3946 or 1 μM GSK2606414 for 1 hour prior to exposure to 2.5 mg/mL tunicamycin or vehicle (DMSO) for 4 hours. After a 1-hour incubation, cells were lysed and TF transcript levels quantified using quantitative PCR. (B and C) HPAF-II cells were incubated in the presence of vehicle. Not significant (1-way ANOVA). (B) MKC3946 or (C) GSK2606414 for 1 hour prior to stimulation with either vehicle (DMSO) or 2.5 mg/mL tunicamycin for 4 hours. TF in cells was then analyzed by Western blot analysis. (D) HPAF-II cells were exposed to 2.5 mg/mL tunicamycin for 4 hours. The supernatant was removed, cells were washed, and cells’ surface factor Xa (FXa) activity was evaluated using a FXa assay as described in Supplemental Methods. Error bars represent the mean ± SEM of 3 samples, **P < 0.005, ***P < 0.001, ****P < 0.0001 (1-way ANOVA). (E and F) HPAF-II cells were exposed to either 5 μM MKC3946 (E) or 1 μM GSK2606414 for 1 hour (F) followed by 2.5 mg/mL tunicamycin for 4 hours. The supernatant was removed, cells were washed, and thrombin generation on cells’ surfaces was evaluated. Error bars represent the mean ± SEM. ****P ≤ 0.0001, ***P < 0.0005, *P = 0.01 (1-way ANOVA).