Biomolecular Mass Spectrometry and Proteomics Group, Utrecht Institute for Pharmaceutical Science, Utrecht University, Utrecht 3584 CH, The Netherlands.
Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
J Proteome Res. 2021 Sep 3;20(9):4381-4392. doi: 10.1021/acs.jproteome.1c00331. Epub 2021 Aug 3.
Acquired resistance to MAPK inhibitors limits the clinical efficacy in melanoma treatment. We and others have recently shown that BRAF inhibitor (BRAFi)-resistant melanoma cells can develop a dependency on the therapeutic drugs to which they have acquired resistance, creating a vulnerability for these cells that can potentially be exploited in cancer treatment. In drug-addicted melanoma cells, it was shown that this induction of cell death was preceded by a specific ERK2-dependent phenotype switch; however, the underlying molecular mechanisms are largely lacking. To increase the molecular understanding of this drug dependency, we applied a mass spectrometry-based proteomic approach on BRAFi-resistant BRAF 451Lu cells, in which ERK1, ERK2, and JUNB were silenced separately using CRISPR-Cas9. Inactivation of ERK2 and, to a lesser extent, JUNB prevents drug addiction in these melanoma cells, while, conversely, knockout of ERK1 fails to reverse this phenotype, showing a response similar to that of control cells. Our analysis reveals that ERK2 and JUNB share comparable proteome responses dominated by reactivation of cell division. Importantly, we find that EMT activation in drug-addicted melanoma cells upon drug withdrawal is affected by silencing ERK2 but not ERK1. Moreover, transcription factor (regulator) enrichment shows that PIR acts as an effector of ERK2 and phosphoproteome analysis reveals that silencing of ERK2 but not ERK1 leads to amplification of GSK3 kinase activity. Our results depict possible mechanisms of drug addiction in melanoma, which may provide a guide for therapeutic strategies in drug-resistant melanoma.
获得性 MAPK 抑制剂耐药限制了黑色素瘤治疗的临床疗效。我们和其他人最近表明，BRAF 抑制剂（BRAFi）耐药的黑色素瘤细胞可以对它们获得耐药的治疗药物产生依赖性，为这些细胞创造了一个潜在的弱点，可以在癌症治疗中加以利用。在药物成瘾的黑色素瘤细胞中，已经表明这种细胞死亡的诱导之前存在一个特定的 ERK2 依赖性表型转换；然而，其潜在的分子机制在很大程度上是缺乏的。为了增加对这种药物依赖性的分子理解，我们应用了基于质谱的蛋白质组学方法对 BRAFi 耐药的 BRAF 451Lu 细胞进行研究，其中 ERK1、ERK2 和 JUNB 分别使用 CRISPR-Cas9 进行沉默。ERK2 的失活，在较小程度上，JUNB 阻止了这些黑色素瘤细胞的药物成瘾，而相反，ERK1 的敲除则未能逆转这种表型，表现出与对照细胞相似的反应。我们的分析表明，ERK2 和 JUNB 具有相似的蛋白质组反应，主要由细胞分裂的再激活主导。重要的是，我们发现药物成瘾的黑色素瘤细胞在停药后 EMT 激活受到 ERK2 沉默的影响，但不受 ERK1 沉默的影响。此外，转录因子（调节因子）富集表明 PIR 作为 ERK2 的效应物起作用，磷酸化蛋白质组分析表明 ERK2 沉默而不是 ERK1 沉默导致 GSK3 激酶活性的放大。我们的研究结果描绘了黑色素瘤中药物成瘾的可能机制，这可能为耐药性黑色素瘤的治疗策略提供指导。
