The unfolded protein response links ER stress to cancer-associated thrombosis
Oluwatoyosi Muse, … , Jeffrey I. Zwicker, Robert Flaumenhaft
Oluwatoyosi Muse, … , Jeffrey I. Zwicker, Robert Flaumenhaft
Published August 31, 2023
Citation Information: JCI Insight. 2023;8(19):e170148. https://doi.org/10.1172/jci.insight.170148.
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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 3

UPR induction in pancreatic cancer cells stimulates production of TF-bearing EVs.

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UPR induction in pancreatic cancer cells stimulates production of TF-bea...
(A) HPAF-II cells were exposed to vehicle (DMSO), tunicamycin (2.5 mg/mL), thapsigargin (0.8 μM), or triptolide (0.2 μM) for 4 hours. Supernatants were collected and EVs isolated as described in the Methods. EVs were subsequently stained for TF and evaluated by flow cytometry. Error bars represent the mean ± SEM of 4 samples. *P < 0.01 (1-way ANOVA). (B) EVs isolated from HPAF-II cells following exposure to 2.5 mg/mL tunicamycin for 4 hours and evaluated using transmission electron microscopy (TEM). (C and D) EVs were generated and isolated as described in B and subsequently stained for CD9 (C) and TF (D). (E and F) HPAF-II cells were exposed to either 5 μM IRE1α inhibitor MKC3946 (E) or 1 μM of PERK inhibitor GSK2606414 (F) for 1 hour followed by 2.5 mg/mL tunicamycin for 4 hours. Supernatants were collected and EVs evaluated for binding of annexin V or anti-TF antibody using flow cytometry. Error bars represent the mean ± SEM of 4 samples. **P < 0.005, ***P < 0.001 (1-way ANOVA). Statistically significance differences were observed for EV TF expression between tunicamycin and DMSO, tunicamycin and MKC3946 or GSK2606414 alone, and tunicamycin alone and the presence of MKC3946 or GSK2606414 with tunicamycin. HPAF-II cells were exposed to 40 nM of either control siRNA or siRNA directed at (G) XBP1 or (H) PERK for 72 hours and subsequently exposed to vehicle or tunicamycin. EVs were isolated from supernatants and evaluated for thrombin generation. Error bars represent the mean ± SEM of 9 samples, *P = 0.01 (1-way ANOVA).