Introduction: Non-muscle invasive bladder cancer is characterized by a high recurrence rate and multifocal tumors that may have different driver mutations. This feature contributes greatly to the challenges in clinical management and financial burden. Increasing evidence has indicated that cancer extracellular vesicles (EV) carry oncogenic cargos and play roles in tumorigenesis. Our recent study reported that normal urothelial cells are malignantly transformed by long-term exposure to cancer EVs through inducing endoplasmic reticulum (ER) stress. This study aims to identify responsible EV-borne molecules that drive tumorigenesis.
Methods: Proteomic analysis of EVs was performed by TMT mass spectrometry and Online database (PANTHER and Human Protein Atlas) analysis. Cell lines, including grade 4 bladder cancer TCCSUP cells, and non-malignant urothelial SV-HUC cells were used. We applied a loss-of-function strategy to study the functional roles of the candidate protein in both cell lines. The impacts on SV-HUC cells after receiving EVs were assessed by Western blot, DCFDA assay, and ?H2AX staining to respectively detect ER stress signaling, reactive oxygen species (ROS) level, and DNA damage. TCCSUP was analyzed by Western blot to determine apoptosis and PDI signals. Redox state of the candidate protein was determined by immunoprecipitation followed by reduced thiol quantification.
Results: Protein disulfide isomerase (PDI) was found enriched and exclusively present in cancer EVs and was selected for further investigation. Its expression level is inversely correlated with bladder cancer patient survival. Under ER stress, TCCSUP bladder cancer cells maintain a PDI homeostasis through releasing PDI by EVs while retaining non-active reduced-PDI within the cells. Knocking down PDI in cancer cells increases their resistance to ER stress-induced apoptosis. Surprisingly, TCCSUP cells export EVs contain mostly oxidized-PDI, which can induce ER stress and oxidative stress. Correspondingly, treating normal SV-HUC cells with TCCSUP EVs results in increased cellular ROS, PERK/ER stress signaling, and DNA damage levels that could eventually lead to malignant transformation.
Conclusions: Bladder cancer cells export oxidized PDI via EVs into extracellular space to reduce cellular stress. Exposing to PDI-containing EVs in the surrounding tumor microenvironment induces oncogenic insults in normal cells. This study provides a novel mechanism for the field cancerization effect and its contribution to bladder cancer recurrence. Source of