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 7

UPR-induced production of procoagulant EVs involves ER to Golgi transport.

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UPR-induced production of procoagulant EVs involves ER to Golgi transpor...
(A) HPAF-II or (B) BxPC3 cells were incubated with vehicle (DMSO) or brefeldin A for 1 hour prior to stimulation with vehicle (DMSO) or 2.5 mg/mL tunicamycin for 4 hours before analysis of TF in EVs. (C) HPAF-II cells were exposed to 3 μM brefeldin A for 1 hour followed by 2.5 mg/mL tunicamycin or DMSO for 4 hours. EVs were isolated from supernatants and evaluated using annexin V or anti-TF antibodies by flow cytometry. Error bars represent the mean ± SEM of 3 samples, *P < 0.01, ***P < 0.001 (1-way ANOVA). (D) HPAF-II cells were exposed to 3 μM brefeldin A for 1 hour followed by 2.5 mg/mL tunicamycin or DMSO for 4 hours. EVs were isolated from supernatants and incubated in the presence of nonimmune IgG or IgG directed at TF (IIID8). Samples were subsequently evaluated for thrombin generation. Error bars represent the mean ± SEM of 3 samples, ****P ≤ 0.0001 (1-way ANOVA). (E) HPAF-II cells were exposed to 1.5 μM Golgicide A for 1 hour then 2.5 mg/mL tunicamycin or vehicle (DMSO) for 4 hours. EVs were isolated from supernatants and subsequently evaluated for thrombin generation. Error bars represent the mean ± SEM of 3 samples, **P < 0.01, ***P < 0.001 (1-way ANOVA). (F) HPAF-II cells were exposed to 40 nM of either control siRNA or siRNA directed at Arf1 for 48 hours and subsequently exposed to either DMSO or 2.5 mg/mL tunicamycin for 4 hours. EVs were isolated from supernatants and evaluated for thrombin generation. Error bars represent the mean ± SEM of 3 samples, ****P ≤ 0.0001 (1-way ANOVA). (G) Schematic model of TF trafficking to the cell surface and to EVs following activation of the UPR in pancreatic adenocarcinoma cells. Increased protein translation with malignant transformation results in increased abundance of unfolded proteins, activation of ER stress receptors, and increased TF trafficking.