Zhou Yuhao, Li Fei, Zou Bangyu, Zhou Xiaofeng, Luo Lianmin, Dong Sicheng, He Zhiqing, Zhang Zhixiong, Liao Liqiong, Liu Hongxing, Cai Chao, Gu Di, Duan Xiaolu
Department of Urology, Minimally Invasive Surgery Center, The First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology, Guangzhou Institute of Urology, Kangda Road 1, Haizhu District, Guangzhou, 510230, Guangdong, China.
Department of Pharmacy, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China.
Discov Oncol. 2023 Nov 29;14(1):215. doi: 10.1007/s12672-023-00740-0.
To investigate the influence of β-arrestin2 on the docetaxel resistance in castration-resistant prostate cancer (CRPC) and elucidate the underlying molecular mechanisms.
PC3 and DU145 cells with stable β-arrestin2 overexpression and C4-2 cells with stable β-arrestin2 knockdown, were constructed via using lentivirus and puromycin selection. MTT and colony formation assays were carried out to investigate the effect of β-arrestin2 expression on the docetaxel resistance of CRPC cells. Glycolysis analysis was used to assess the glycolytic capacity modulated by β-arrestin2. GO enrichment analysis, gene set enrichment analysis and Spearman correlation test were carried out to explore the potential biological function and mechanism via using public data from GEO and TCGA. The expressions of PKM2, Phospho-PKM2, Phospho-ERK1/2 and hnRNP A1 were detected by western blot. Functional blocking experiments were carried out to confirm the roles of PKM2 and hnRNP A1 in the regulation of β-arrestin2's biological functions via silencing PKM2 or hnRNP A1 expression in cells with stable β-arrestin2 overexpression. Finally, nude mice xenograft models were established to confirm the experimental results of cell experiments.
β-Arrestin2 significantly decreased the sensitivity of CRPC cells to docetaxel stimulation, through enhancing the phosphorylation and expression of PKM2. Additionally, β-arrestin2 increased PKM2 phosphorylation via the ERK1/2 signaling pathway and induced PKM2 expression in a post-transcriptional manner through an hnRNP A1-dependent PKM alternative splicing mechanism, rather than by inhibiting its ubiquitination degradation.
Our findings indicate that the β-arrestin2/hnRNP A1/PKM2 pathway could be a promising target for treating docetaxel-resistant CRPC.
研究β-抑制蛋白2对去势抵抗性前列腺癌（CRPC）多西他赛耐药性的影响，并阐明其潜在的分子机制。
通过慢病毒和嘌呤霉素筛选构建稳定过表达β-抑制蛋白2的PC3和DU145细胞，以及稳定敲低β-抑制蛋白2的C4-2细胞。采用MTT和集落形成试验研究β-抑制蛋白2表达对CRPC细胞多西他赛耐药性的影响。利用糖酵解分析评估β-抑制蛋白2调节的糖酵解能力。通过使用来自GEO和TCGA的公共数据进行基因本体（GO）富集分析、基因集富集分析和Spearman相关性检验，以探索潜在的生物学功能和机制。通过蛋白质免疫印迹法检测丙酮酸激酶M2（PKM2）、磷酸化PKM2、磷酸化细胞外信号调节激酶1/2（ERK1/2）和异质性核糖核蛋白A1（hnRNP A1）的表达。通过在稳定过表达β-抑制蛋白2的细胞中沉默PKM2或hnRNP A1的表达，进行功能阻断实验，以确认PKM2和hnRNP A1在调节β-抑制蛋白2生物学功能中的作用。最后，建立裸鼠异种移植模型以证实细胞实验的结果。
β-抑制蛋白2通过增强PKM2的磷酸化和表达，显著降低CRPC细胞对多西他赛刺激的敏感性。此外，β-抑制蛋白2通过ERK1/2信号通路增加PKM2磷酸化，并通过hnRNP A1依赖性的PKM可变剪接机制以转录后方式诱导PKM2表达，而不是通过抑制其泛素化降解。
我们的研究结果表明，β-抑制蛋白2/hnRNP A1/PKM2通路可能是治疗多西他赛耐药性CRPC的一个有前景的靶点。
