Denticleless homolog-mediated ubiquitin-proteasome degradation of forkhead box O1 contributes to development of carboplatin resistance in retinoblastoma.

Abstract

This study aimed to investigate the role of forkhead box O1 (FOXO1) in carboplatin-resistant retinoblastoma (RB) cells, focusing on its subcellular distribution and regulation through ubiquitin-dependent degradation mediated by denticleless homolog (DTL). The study sought to elucidate the molecular mechanisms underlying carboplatin resistance in RB and explore potential therapeutic strategies to overcome chemoresistance. Carboplatin-resistant RB cell lines (Y79/CBP and WERI-Rb-1/CBP) were established by incremental drug exposure. Bioinformatics analysis of the GSE111168 dataset identified differentially expressed genes associated with ubiquitination pathways. DTL expression was modulated using adeno-associated virus-mediated knockdown and overexpression. FOXO1 protein levels, subcellular localization, and ubiquitination were assessed via western blotting, immunofluorescence, and coimmunoprecipitation (Co-IP). The effects of DTL knockdown and LOM612 treatment on cell proliferation, apoptosis, and tumor growth were evaluated in vitro and in vivo using xenograft models. FOXO1 expression and nuclear localization were significantly reduced in carboplatin-resistant RB cells, with elevated levels of FOXO1 ubiquitination. Proteasome inhibitors preserved FOXO1 protein levels, implicating the ubiquitin-proteasome system in its degradation. DTL was identified as a significantly overexpressed gene in both resistant cells and patient-derived samples. Silencing DTL increased FOXO1 protein expression and nuclear accumulation, while Co-IP confirmed the interaction between DTL and FOXO1, mediated by the WD40 domain of DTL. Combined DTL knockdown and LOM612 treatment synergistically inhibited cell proliferation and invasion, promoted apoptosis in vitro, and significantly reduced tumor growth and induced apoptosis in vivo. DTL-mediated ubiquitination and degradation of FOXO1 play a critical role in carboplatin resistance in RB. Dual targeting of DTL and FOXO1 nuclear translocation may represent a promising therapeutic strategy to overcome chemoresistance and improve clinical outcomes in RB.