USP35 Acts as a Dual Stabilizer of CDCA8 and PD-L1 to Coordinate the Progression and Immune Evasion in Non-Small Cell Lung Cancer

INTRODUCTION: Deubiquitinating enzymes (DUBs) play a crucial role in cancer, but their roles in immune modulation and the progression of non-small cell lung cancer (NSCLC) remain unclear. METHODS: USP35 expression was first analyzed using data from public databases (TIMER3.0, GEPIA) and was validated in a cohort of paired NSCLC tumor and adjacent normal tissues (n = 45). To explore the functional role of USP35 in NSCLC, siRNA was used to silence USP35 expression in A549 and H23 cells. CCK-8, wound healing, and Transwell assays were performed to assess the effect of USP35 on NSCLC cell proliferation, migration, and invasion. Putative USP35-interacting proteins were identified using the BioGRID and STRING databases, and these interactions were validated by co-immunoprecipitation (Co-IP). The effects of USP35 on ubiquitination and protein stability were assessed by IP-WB and cycloheximide chase assays. To explore its role in immune modulation, NSCLC cells were co-cultured with activated peripheral blood mononuclear cells (PBMCs), and tumor cell-mediated cytotoxicity as well as PBMC cytokine profiles were measured. RESULTS: Significantly elevated USP35 expression was correlated with a poor prognosis of NSCLC patients. Knockdown of USP35 notably inhibited the malignant behaviors of NSCLC cells. USP35 promoted NSCLC by directly interacting with and deubiquitinating the cell cycle regulator CDCA8. Deubiquitination stabilized CDCA8 and enhanced intrinsic tumor aggressiveness. Mechanistically, USP35 interacted with the immune checkpoint protein PD-L1 and removed ubiquitin chains from it, thereby preventing proteasomal degradation. This deubiquitination stabilized PD-L1, establishing a USP35-mediated mechanism of immune evasion. Consequently, knockdown of USP35 enhanced T-cell-mediated cytotoxicity and pro-inflammatory cytokine production, an effect that was partially reversed by restoring PD-L1 expression. DISCUSSION: USP35 was overexpressed in NSCLC and exerted dual pro‑tumor functions. USP35 promoted cancer cell proliferation, migration, and invasion by deubiquitinating and stabilizing CDCA8 and by mediating immune evasion through maintaining PD‑L1 stability. Computational and functional analyses revealed that USP35 was inversely correlated with CD8+ T‑cell infiltration, and that USP35 depletion enhanced T‑cell‑mediated cytotoxicity by reducing PD‑L1. CONCLUSION: The findings of this study demonstrated a potential dual role of USP35 in NSCLC, contributing to the targeted therapy of the cancer.