Genome-wide CRISPR screen identified Rad18 as a determinant of doxorubicin sensitivity in osteosarcoma.

Abstract

BACKGROUND:

Osteosarcoma (OS) is a malignant bone tumor mostly occurring in children and adolescents, while chemotherapy resistance often develops and the mechanisms involved remain challenging to be fully investigated.

METHODS:

Genome-wide CRISPR screening combined with transcriptomic sequencing were used to identify the critical genes of doxorubicin resistance. Analysis of clinical samples and datasets, and in vitro and in vivo experiments (including CCK-8, apoptosis, western blot, qRT-PCR and mouse models) were applied to confirm the function of these genes. The bioinformatics and IP-MS assays were utilized to further verify the downstream pathway. RGD peptide-directed and exosome-delivered siRNA were developed for the novel therapy strategy.

RESULTS:

We identified that E3 ubiquitin-protein ligase Rad18 (Rad18) contributed to doxorubicin-resistance in OS. Further exploration revealed that Rad18 interact with meiotic recombination 11 (MRE11) to promote the formation of the MRE11-RAD50-NBS1 (MRN) complex, facilitating the activation of the homologous recombination (HR) pathway, which ultimately mediated DNA damage tolerance and leaded to a poor prognosis and chemotherapy response in patients with OS. Rad18-knockout effectively restored the chemotherapy response in vitro and in vivo. Also, RGD-exosome loading chemically modified siRad18 combined with doxorubicin, where exosome and chemical modification guaranteed the stability of siRad18 and the RGD peptide provided prominent targetability, had significantly improved antitumor activity of doxorubicin.

CONCLUSIONS:

Collectively, our study identifies Rad18 as a driver of OS doxorubicin resistance that promotes the HR pathway and indicates that targeting Rad18 is an effective approach to overcome chemotherapy resistance in OS.

• Authors: Mingrui Du, Jintao Gu, Chenlin Liu, Nannan Liu, Zhe Yu, Chengpei Zhou, Wei Heng, Zhengcong Cao, Feilong Wei, Kailong Zhu, Yingwen Wang, Wei Zhang, Xiaochang Xue, Yong Zhang, Jixian Qian
• Journal: Journal of experimental & clinical cancer research : CR (J Exp Clin Cancer Res) Vol. 41 Issue 1 Pg. 154 (Apr 23 2022) ISSN: 1756-9966 [Electronic] England
• PMID: 35459258 (Publication Type: Journal Article)
• Copyright: © 2022. The Author(s).

Chemical References

• Antibiotics, Antineoplastic
• DNA-Binding Proteins
• RAD18 protein, human
• Rad18 protein, mouse
• Doxorubicin
• Ubiquitin-Protein Ligases

Topics

• Adolescent
• Animals
• Antibiotics, Antineoplastic (metabolism, pharmacology, therapeutic use)
• Bone Neoplasms (drug therapy, genetics, pathology)
• Clustered Regularly Interspaced Short Palindromic Repeats
• DNA-Binding Proteins (genetics, metabolism)
• Doxorubicin (metabolism, pharmacology, therapeutic use)
• Humans
• Mice
• Osteosarcoma (drug therapy, genetics, metabolism)
• Ubiquitin-Protein Ligases (genetics, metabolism, pharmacology, therapeutic use)