Chu Cheng-Ying, Lee Yi-Ching, Hsieh Cheng-Han, Yeh Chi-Tai, Chao Tsu-Yi, Chen Po-Hung, Lin I-Hsuan, Hsieh Tsung-Han, Shih Jing-Wen, Cheng Chia-Hsiung, Chang Che-Chang, Lin Ping-Sheng, Huang Yuan-Li, Chen Tsung-Ming, Yen Yun, Ann David K, Kung Hsing-Jien
TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei 110, Taiwan.
CRISPR Gene Targeting Core Lab, Taipei Medical University, Taipei 110, Taiwan.
Theranostics. 2021 Jan 25;11(8):3624-3641. doi: 10.7150/thno.51795. eCollection 2021.
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. 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 and in xenograft models. The signal pathways were verified by western blotting and cytokine release. 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 and , 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. 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.
由于精氨琥珀酸合成酶1（ASS1）表达受抑制导致的精氨酸合成缺陷是肿瘤细胞中最常见的代谢缺陷之一。近年来，精氨酸剥夺疗法越来越受到关注。ADI-PEG20（聚乙二醇化精氨酸脱亚氨酶）治疗面临的一个挑战是由ASS1表达恢复和其他因素引起的耐药性发展。这项工作的目标是确定赋予治疗抗性的新因素。我们培养了多个独立衍生的对ADI耐药的克隆，包括乳腺癌（MDA-MB-231和BT-549）和前列腺癌（PC3、CWR22Rv1和DU145）细胞的衍生物。利用RNA测序（RNA-seq）和逆转录聚合酶链反应（RT-PCR）来鉴定耐药克隆中上调的基因。通过无偏倚的全基因组CRISPR/Cas9基因敲除筛选来鉴定缺失后会使这些细胞对ADI敏感的基因。利用短发夹RNA（shRNA）、CRISPR/Cas9基因敲除以及过表达方法在体外和异种移植模型中验证耐药基因的功能。通过蛋白质免疫印迹法和细胞因子释放来验证信号通路。基于对独立衍生的对ADI耐药（ADIR）克隆的无偏倚CRISPR/Cas9基因敲除筛选和RNA-seq分析，除了ASS1表达外，TREM1/CCL2轴的异常激活也一直被确定为耐药因素。与ADIR不同，过表达ASS1的MDA-MB-231细胞在体外和体内仅获得中等程度的ADI耐药性，单独过表达ASS1不会激活TREM1/CCL2轴。这些数据表明，TREM1的上调是产生强耐药性的一个独立因素，它伴随着AKT/mTOR/STAT3/CCL2信号通路的激活，并有助于细胞存活以及克服ASS1过表达的肿瘤抑制作用。重要的是，敲低TREM1或CCL2可显著使ADIR对ADI敏感化。在BT-549乳腺癌细胞系以及去势抵抗性前列腺癌细胞中也获得了类似结果。本研究揭示了精氨酸剥夺疗法耐药性的详细机制，并发现了克服耐药性的新靶点。我们发现，除了ASS1表达恢复外，TREM1/CCL2激活是乳腺癌和前列腺癌模型中完全ADI耐药所涉及的关键途径。
