P122: Loss of DDI2 triggers autophagy through CCN1 in cancer cells

Introduction:The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are two major protein degradation pathways in cells.Dysfunction in the proteasome leads to the activation of compensatory autophagy, which mitigates proteotoxic stress and can significantly impact cancer therapy effectiveness.Although substantial progress has been made in understanding these processes, the molecular mechanisms by which impaired proteasome function triggers autophagy remain unclear.DDI2 (DNA Damage Inducible 1 Homolog 2) functions both as a protease and an ubiquitin shuttle factor.Its depletion sensitizes cancer cells to proteasome inhibition by causing the accumulation of highly ubiquitinated proteins.However, cancer cells may activate compensatory autophagy to degrade these ubiquitinated protein aggregates.This study investigates the role of DDI2 depletion in inducing autophagy in cancer cells to enhance cancer therapy effectiveness.Methods: We evaluated autophagy activity in a panel of cell lines of different origins, 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) cells, either control or DDI2-deficient, following treatment with the autophagy inhibitor chloroquine (CQ).Total proteomic analysis revealed elevated levels of cellular communication network factor 1 (CCN1) in DDI2-depleted cells compared to controls, which is a protein known to induce autophagy.To investigate whether CCN1 is required and sufficient for autophagy induction, we used small interfering RNA (siRNA) and viral constructs for knockdown and overexpression studies.We then performed coimmunoprecipitation (Co-IP) and confocal microscopy to assess potential interactions between DDI2 and CCN1.Results: Measurements of autophagic flux revealed elevated LC3B-II protein levels, a known autophagy marker, in different DDI2-deficient cell lines compared to controls.Western blot analysis further confirmed increased CCN1 protein levels in these cell lines.Notably, autophagy induction in DDI2deficient cells was significantly reduced by CCN1-siRNA, highlighting the necessity of CCN1 for autophagy activation.In contrast, CCN1 overexpression in wild-type cells led to increased LC3B-II levels, demonstrating that CCN1 alone is sufficient to induce autophagy.Additionally, co-immunoprecipitation (Co-IP) confirmed an interaction between DDI2 and CCN1, which was further validated by confocal microscopy to visualize their colocalization.Conclusion: Autophagy is a key target for improving the effectiveness of cancer therapies.Our study demonstrates that depletion of the ubiquitin shuttle factor DDI2 leads to CCN1 accumulation, which in turn induces autophagy.These findings highlight the molecular interplay between protein degradation pathways and suggest that targeting of DDI2 in combination with autophagy could enhance cancer treatment efficacy.