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 4

UPR induces procoagulant EVs from pancreatic cancer cells.

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UPR induces procoagulant EVs from pancreatic cancer cells. (A and B) EVs...
(A and B) EVs were isolated from the supernatants of HPAF-II cells exposed to vehicle (DMSO) or 2.5 mg/mL tunicamycin. Isolated EVs were incubated with nonimmune IgG, (A) anti-TF antibody (IIID8), or (B) anti-FXIIa antibody (3F7) prior to evaluation of thrombin generation. Error bars represent the mean ± SEM of 3 samples, ****P < 0.0001, **P < 0.01, *P = 0.01 (1-way ANOVA). (C and D) BxPC3 cells were incubated with either 5 μM MKC3946 (C) or 1 μM GSK2606414 (D) for 4 hours and subsequently exposed to DMSO (Control) or 2.5 mg/mL tunicamycin (TM). EVs were isolated from supernatants and incubated with either nonimmune IgG or anti-TF antibody prior to evaluation of thrombin generation. Error bars represent the mean ± SEM of 3 samples, ****P ≤ 0.0001, ***P < 0.001 (1-way ANOVA). (E) HPAF-II cells were incubated with either 1 μM GSK2606414 or 5 μM MKC3946 for 1 hour prior to incubation with 2.5 mg/mL tunicamycin. EVs were isolated, lysed, and evaluated for protein concentration. Equal concentrations of proteins within EV lysates were subsequently separated by SDS-PAGE and analyzed for TF using Western blot analysis. Loading of total protein was assessed using Instant Blue (left panel). Quantification of 3 independent experiments (right panel). **P < 0.01, *P = 0.01 (1-way ANOVA).