Abstract 4316: Investigation the role of DDI2 in autophagy

Abstract

Abstract Background: The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are critical intracellular protein degradation pathways. In response to proteasome dysfunction, cells initiate compensatory autophagy to manage proteotoxic stress, which impacts cancer therapy efficacy. Despite current insights, the exact molecular mechanisms underlying the induction of autophagy due to impaired proteasome function remain unclear. The transcription factor nuclear factor erythroid-2-related factor 1 (NRF1) is the master regulator of proteasome subunit genes, which is activated by the protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome inhibition. DDI2 also acts as an ubiquitin shuttling factor, and its depletion causes the accumulation of highly ubiquitinated proteins, resulting in increased sensitivity to proteasome inhibition in cancer cells. However, cancer cells might trigger compensatory autophagy in response to DDI2 depletion to degrade ubiquitinated protein aggregates. Here, we demonstrated that DDI2 depletion plays a role in autophagy induction. Methods: A panel of cell lines of different origins (EW16 and ES1 human Ewing sarcoma, MIA PaCa-2 human pancreatic cancer, and NIH-3T3 murine fibroblasts) either control or DDI2-deficient were examined for autophagy activity following treatment with the autophagy inhibitor chloroquine (CQ). Through total proteome analysis, we identified an increase in cellular communication network factor 1 (CCN1) protein levels in DDI2-depleted cells, which plays a role in autophagy induction. To determine whether CCN1 is required and sufficient to induce autophagy, we applied small interfering RNA (siRNA), and viral constructs to knockdown and overexpress CCN1. Results: The autophagic flux measurements in four different DDI2-deficient cell lines revealed elevated LC3B-II protein levels, a known autophagy marker, following CQ treatment compared to their control. Western blotting analysis confirmed increased CCN1 protein levels in the same cell lines. The autophagy induction observed in DDI2-deficient cells was diminished by CCN1-siRNA, demonstrating that CCN1 may be required for autophagy induction. Furthermore, overexpressing CCN1 in wild-type cells resulted in increased LC3B-II protein levels, indicating that CCN1 alone is sufficient to induce autophagy. Conclusion: Targeting autophagy is a promising therapeutic strategy to improve the effects of anti-cancer therapies. We demonstrated that upon depletion of ubiquitin shuttle factor DDI2, secretory protein CCN1 accumulates leading to the subsequent induction of autophagy. These findings improve our understanding of the molecular basis governing DDI2’s role in crosstalk between protein degradation pathways. Targeting DDI2 in combination with autophagy inhibition could further potentiate these drugs as anti-cancer therapies. Citation Format: Nayyerehalsadat Hosseini, Holly A. Byers, Senthil K. Radhakrishnan. Investigation the role of DDI2 in autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4316.