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Abstract：
OBJECTIVE: To investigate the influence of β-arrestin2 on the docetaxel resistance in castration-resistant prostate cancer (CRPC) and elucidate the underlying molecular mechanisms.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: Our findings indicate that the β-arrestin2/hnRNP A1/PKM2 pathway could be a promising target for treating docetaxel-resistant CRPC.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: Our findings indicate that the β-arrestin2/hnRNP A1/PKM2 pathway could be a promising target for treating docetaxel-resistant CRPC.
摘要：
目的：探讨β-arrestin2对去势抵抗性前列腺癌(CRPC)多西他赛耐药的影响及其分子机制。
方法：PC3和DU145细胞具有稳定的β-arrestin2过表达，C4-2细胞具有稳定的β-arrestin2敲低，通过使用慢病毒和嘌呤霉素选择构建。采用MTT法和集落形成法研究β-arrestin2表达对CRPC细胞多西他赛耐药的影响。糖酵解分析用于评估由β-arrestin2调节的糖酵解能力。GO富集分析,利用GEO和TCGA的公开数据，进行了基因集富集分析和Spearman相关检验，以探讨潜在的生物学功能和机制。免疫印迹法检测PKM2、磷酸化PKM2、磷酸化ERK1/2和hnRNPA1的表达。进行了功能阻断实验，以证实PKM2和hnRNPA1在稳定的β-arrestin2过表达的细胞中通过沉默PKM2或hnRNPA1表达调节β-arrestin2的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实验结果。
结果：β-Arrestin2显著降低CRPC细胞对多西他赛刺激的敏感性，通过增强PKM2的磷酸化和表达。此外,β-arrestin2通过ERK1/2信号通路增加PKM2磷酸化，并通过hnRNPA1依赖性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。
结论：我们的研究结果表明，β-arrestin2/hnRNPA1/PKM2通路可能是治疗多西他赛耐药CRPC的一个有希望的靶点。
方法：PC3和DU145细胞具有稳定的β-arrestin2过表达，C4-2细胞具有稳定的β-arrestin2敲低，通过使用慢病毒和嘌呤霉素选择构建。采用MTT法和集落形成法研究β-arrestin2表达对CRPC细胞多西他赛耐药的影响。糖酵解分析用于评估由β-arrestin2调节的糖酵解能力。GO富集分析,利用GEO和TCGA的公开数据，进行了基因集富集分析和Spearman相关检验，以探讨潜在的生物学功能和机制。免疫印迹法检测PKM2、磷酸化PKM2、磷酸化ERK1/2和hnRNPA1的表达。进行了功能阻断实验，以证实PKM2和hnRNPA1在稳定的β-arrestin2过表达的细胞中通过沉默PKM2或hnRNPA1表达调节β-arrestin2的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实验结果。
结果：β-Arrestin2显著降低CRPC细胞对多西他赛刺激的敏感性，通过增强PKM2的磷酸化和表达。此外,β-arrestin2通过ERK1/2信号通路增加PKM2磷酸化，并通过hnRNPA1依赖性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。
结论：我们的研究结果表明，β-arrestin2/hnRNPA1/PKM2通路可能是治疗多西他赛耐药CRPC的一个有希望的靶点。
