Ubiquitin proteasome system in immune and Diffuse Large B-Cell Lymphoma (DLBCL) cancer cells

Abstract

ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous type of non-Hodgkin lymphoma, and its small tumor size complicates sampling and diagnosis. To evaluate the potential of peripheral immune cells’ proteome as diagnostic biomarkers, we used mass spectrometry (MS)-based proteomics to analyze immune cells isolated from DLBCL patients. The findings revealed significant suppression of the cytoskeleton and activation of the immune response in the immune cells of DLBCL patients. Additionally, the significant dysregulation of the proteins belonging to the ubiquitin-proteasome system (UPS) suggests that DLBCL cells may affect UPS activity in immune cells. Our meta-analysis study further demonstrated the importance of the UPS in DLBCL. To investigate UPS and proteasome inhibitor (PI) effect on immune cells and DLBCL cancer cells, we treated peripheral blood mononuclear cells (PBMCs) and DLBCL cell line (DB cell) with Bortezomib (BTZ), a proteasome inhibitor. Results showed that healthy donors PBMCs are less sensitive to BTZ and exhibit lower levels of polyubiquitinated proteins compared to DLBCL cells. Proteomic and immunoblotting analysis demonstrated down regulation of proteasome-related proteins in treated PBMCs, while BTZ treated DB cells exhibited increased proteasome subunit levels. To investigate DLBCL cell adaptation to BTZ, we generated BTZ-adapted DB cells and analyzed their proteome. Results indicated decreased levels of polyubiquitinated proteins in BTZ-adapted DB cells, suggesting adaptation to BTZ. Immunoblotting showed elevated levels of proteasome subunits and autophagy-related proteins in BTZ-adapted cells compared to parental BTZ sensitive cells. Upon BTZ withdrawal from the culture medium of cells, polyubiquitinated protein levels increased in cells. We also examined extracellular vesicles (EVs) derived from BTZ-adapted cells by MS-based proteomics to understand their role in transmitting resistance to sensitive cells. Results showed that EVs contain proteins associated with endoplasmic III reticulum and DNA repair, indicating that adapted cells might help sensitive cells to feel the presence of BTZ and repair drug-induced DNA damage. To assess the impact of DB-derived EVs on immune cell polyubiquitination, we cultured healthy donor PBMCs with conditioned medium, small EVs, and large EVs from DB cells. Our results showed that these DB-derived EVs increased polyubiquitination in immune cells. Proteomic analysis revealed that surface proteins in the EVs may induce polyubiquitination in immune cells. In conclusion, our study highlights the complex interactions between DLBCL cells and peripheral immune cells, particularly concerning the UPS regulation and resistance to BTZ. The dysregulation of UPS proteins in the immune cells of patients, along with increased polyubiquitination in PBMCs treated with DLBCL-derived secretome, suggests that DLBCL cells may alter the proteome and UPS regulation in immune cells through their secretome. Furthermore, the role of EVs in transmitting proteins involved in drug resistance offers valuable insights into the interactions between tumor cells, immune cells, and other tumor cells. This research paves the way for future studies investigating the UPS as a diagnostic biomarker in the immune cells of cancer patients and exploring EVs as therapeutic targets, ultimately contributing to more effective treatment strategies for DLBCL.