Abstract 4491: HSP70 leverages STUB1 to modulate N-Myc protein turnover in lethal prostate cancer

Abstract Background: Imbalances in protein homeostasis (proteostasis) are associated with a wide range of diseases, including neurodegeneration and tumorigenesis. Prostate cancer has proven to be a key model for understanding how proteostasis regulates tumor progression. Neuroendocrine prostate cancer (NEPC) is the most aggressive type of prostate cancer with no effective treatment. The amplification of N-Myc (encoded by MYCN) is a key modification of NEPC. However, it has proven challenging to identify therapeutic strategies that reduce N-Myc transcriptional activity or levels owing to the presence of intrinsically disordered functional domains and lack of enzymatically active sites. In this study, we sought to take a different approach and identify the molecular chaperones involved in N-Myc proteostasis. Methods: N-Myc was knocked down by siRNA. Cell proliferation and N-Myc expression were assessed using cell viability assays and Western blotting, respectively. Rapid immunoprecipitation pull-down assays followed by label-free mass spectrometry were used to identify N-Myc-binding proteins. Co-immunoprecipitation (Co-IP) and Proximity Ligation Assay (PLA) were performed to detect the interaction between HSP70 and STUB1 with N-Myc. In vitro ubiquitination assays were conducted to detect the ubiquitination of N-Myc. Site-directed mutagenesis (SDM) was used to construct N-Myc, STUB1, and ubiquitination deletion or point mutation plasmids. Cycloheximide (CHX) chase assays were employed to examine the half-life of N-Myc. Results: Knockdown of N-Myc by siRNA significantly inhibited the growth of H660, UCDCaP-CR, and CWR22Rv1 cells. A total of 779 proteins were identified as N-Myc-binding proteins. Among them, several HSP70 family proteins, such as HSPA1B (HSP70), HSPA6, and HSPA8 (HSC70), were the most enriched. The HSP70/STUB1 complex interacted with N-Myc and regulated N-Myc protein turnover. This interaction appeared to occur at a degron ‘SELILKR’ in N-Myc. The K416 and K419 of N-Myc seem to be sites for ubiquitination by STUB1, where it added K11-linked polyubiquitin chains for further proteasome degradation. Conclusions: The HSP70 interacts with N-Myc at a conserved binding motif and regulates N-Myc protein turnover through STUB1. Source of Funding: This work was supported in part by grants from National Institutes of Health R37CA249108 (C.L.), R01CA251253 (C.L.), R21CA277171 (C.L.), Department of Defense HT9425-23-1-0144 (C.L.), HT9425-23-1-0325 (C.L.) Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Shu Ning, Allen C. Gao, Chengfei Liu. HSP70 leverages STUB1 to modulate N-Myc protein turnover in lethal prostate cancer [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 4491.