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 5

Induction of the UPR enhances cell surface TF expression.

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Induction of the UPR enhances cell surface TF expression. (A) HPAF-II ce...
(A) HPAF-II cells were incubated in the presence of vehicle (DMSO), 2.5 mg/mL tunicamycin, or 0.2 μM triptolide for 4 hours. TF in cells was then analyzed by Western blot analysis (left panel) and quantified using densitometry (right panel). GAPDH was used as a loading control. Glycosylated (upper bands; black arrow) and deglycosylated (lower bands; gray arrow) TF were analyzed separately. *P = 0.01 (1-way ANOVA). (B) HPAF-II cells were exposed to DMSO (Control), 2.5 mg/mL tunicamycin, or 0.2 μM triptolide for 4 hours. Cells were then washed, fixed, permeabilized, and stained with antibody directed at TF (green), PE-phalloidin (red), and DAPI (blue). Cells were subsequently evaluated using 3-color immunofluorescence confocal microscopy. Arrows in magnified insets show TF-rich, actin-poor blebs. The graphs to the right represent the quantification of TF intensity and the percentage of cellular blebs as indicated. **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA). (C) HPAF-II cells were grown on grids and subsequently exposed to vehicle (DMSO) or 2.5 tunicamycin for 4 hours. Cells were washed and fixed. Fixed cells were stained with anti-TF antibody (IIID8) followed by immunogold-labeled secondary IgG and evaluated by TEM as described in the Methods. The graph to the right shows quantification of TF on the cell membrane (gold particle number per micron of membrane). **P < 0.01 (2-tailed t test).