1. Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy
- Author
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Po-Hung Chen, David K. Ann, Tsung-Ming Chen, Cheng-Ying Chu, Yi-Ching Lee, Ping-Sheng Lin, Tsu Yi Chao, Hsing Jien Kung, Cheng-Han Hsieh, Yuan-Li Huang, Che-Chang Chang, Chi Tai Yeh, I-Hsuan Lin, Jing Wen Shih, Yun Yen, Tsung-Han Hsieh, and Chia Hsiung Cheng
- Subjects
Male ,Hydrolases ,Nude ,Drug Resistance ,Medicine (miscellaneous) ,Polyethylene Glycols ,Small hairpin RNA ,Prostate cancer ,Gene Knockout Techniques ,Mice ,Models ,TREM1 ,CRISPR ,2.1 Biological and endogenous factors ,Molecular Targeted Therapy ,Aetiology ,Precision Medicine ,Pharmacology, Toxicology and Pharmaceutics (miscellaneous) ,Inbred BALB C ,Chemokine CCL2 ,Cancer ,Gene knockdown ,Mice, Inbred BALB C ,Tumor ,Prostate Cancer ,Up-Regulation ,arginine starvation ,Female ,CCL2 ,Biotechnology ,Signal Transduction ,Research Paper ,Urologic Diseases ,Oncology and Carcinogenesis ,Mice, Nude ,Breast Neoplasms ,Biology ,Argininosuccinate Synthase ,Arginine ,Models, Biological ,Cell Line ,Downregulation and upregulation ,DU145 ,Cell Line, Tumor ,Breast Cancer ,medicine ,Genetics ,Animals ,Humans ,ADI resistance ,CRISPR/Cas9 ,PI3K/AKT/mTOR pathway ,Inflammation ,Prevention ,Prostatic Neoplasms ,medicine.disease ,Biological ,Xenograft Model Antitumor Assays ,Triggering Receptor Expressed on Myeloid Cells-1 ,Drug Resistance, Neoplasm ,Cancer cell ,Cancer research ,Neoplasm ,CRISPR-Cas Systems - Abstract
Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.
- Published
- 2021