Abstract 2821: Regulation of autophagy by the DDI2-CCN1 axis

Abstract Background: The two primary mechanisms of protein degradation in cells are the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). In cases of UPS dysfunction, compensatory autophagy is activated to alleviate proteotoxic stress. This phenomenon can influence the efficacy of cancer treatments. Despite significant progress in understanding these systems, the specific molecular pathways linking proteasome dysfunction to the activation of compensatory autophagy remain poorly understood. DDI2 (DNA Damage Inducible 1 Homolog 2) functions as both a protease and a ubiquitin shuttle factor. Its loss sensitizes cancer cells to proteasome inhibition through the accumulation of polyubiquitinated proteins. However, these cells may activate autophagy to clear ubiquitinated protein aggregates. This study investigate how DDI2 depletion promotes autophagy in cancer cells to improve the efficacy of cancer treatments. Methods: Autophagy activity was assessed in various cell lines, including non-cancerous (NIH-3T3 murine fibroblasts, MRC5 human fetal lung fibroblasts) and cancerous (EW16 and ES1 human Ewing sarcoma, MIA PaCa-2 human pancreatic cancer) cell lines, either with or without DDI2 depletion. Proteomic analysis revealed an upregulation of cellular communication network factor 1 (CCN1) in DDI2-deficient cells, suggesting its potential role in autophagy activation. Then, siRNA and viral constructs were used to examine CCN1’s role in autophagy induction. Moreover, Co-immunoprecipitation (Co-IP) and confocal microscopy were applied to investigate the possible interactions between DDI2 and CCN1. Results: Autophagy flux analysis showed increased LC3B-II levels, a key marker of autophagy, in DDI2-deficient cells compared to controls. Furthermore, western blotting confirmed elevated levels of CCN1 protein in DDI2-deficient cells. Downregulation of CCN1 by siRNA resulted in reduced autophagy activity in DDI2-deficient cells, demonstrating that CCN1 is essential for the autophagic induction. In contrast, overexpression of CCN1 in wild-type cells led to an increase in LC3B-II levels, confirming that CCN1 alone can trigger autophagy activity. Co-IP analysis revealed a direct interaction between DDI2 and CCN1, and confocal microscopy further confirmed their colocalization. Conclusion: Autophagy plays a critical role in effectiveness of cancer treatments. Our findings show that the loss of DDI2 leads to the accumulation of CCN1, which consequently activates autophagy. This research highlights the interaction between protein degradation pathways, UPS and autophagy, suggests that targeting both DDI2 and autophagy could provide a promising strategy to improve cancer therapy outcomes. Citation Format: Nayyerehalsadat Hosseini, Ahmed M. Elshazly, Holly A. Byers, Janakiram R. Vangala, Senthil K. Radhakrishnan. Regulation of autophagy by the DDI2-CCN1 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2821.