PO.TB03.01 · 肿瘤生物学
RIPK2和CDK2在前列腺癌细胞中形成一个可靶向的正反馈环
RIPK2 and CDK2 form a targetable positive feedback loop in prostate cancer cells
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：前列腺癌（PC）转移是PC相关死亡的主要原因，然而靶向这一致命过程的有效疗法仍然缺乏。我们此前将RIPK2确定为PC转移中一个有前景的药物靶点，其部分功能通过非经典的RIPK2/MKK7/c-Myc磷酸化通路发挥作用（Nat. Commun.，2022）。然而，RIPK2的其他下游效应因子和上游调控因子仍缺乏充分表征。本研究旨在填补这一知识空白，该空白是开发高效低毒联合疗法的主要障碍。
方法：使用CRISPR/Cas9敲除、siRNA敲低以及野生型或突变型基因的过表达进行基因操作。通过细胞增殖、Matrigel侵袭、克隆形成和软琼脂实验评估PC细胞的转移潜能。通过蛋白质印迹和体外激酶实验评估RIPK2对CDK2的激活。使用共免疫沉淀、GST下拉、邻近连接实验、荧光共定位和荧光共振能量转移检测RIPK2-CDK2相互作用。通过RT-qPCR测量基因转录和表达。使用放线菌酮追踪和泛素化实验确定蛋白质稳定性。使用6x6矩阵评估RIPK2和CDK2抑制剂的协同效应。每项细胞实验至少使用两个独立的细胞系。
结果：RIPK2主要通过其激酶活性、胞质定位和诱导c-Myc表达增强了PC 22Rv1和PC3细胞的转移潜能。RIPK2直接结合CDK2并将其激活，导致c-Myc在S62位点的磷酸化。值得注意的是，活化的CDK2增加了RIPK2的转录和蛋白质稳定性，建立了一个正反馈环。共同抑制RIPK2和CDK2协同降低了c-Myc蛋白水平，并抑制了PC细胞侵袭和克隆形成。
结论：RIPK2和CDK2形成一个多层次的正反馈环，维持c-Myc表达并驱动PC转移进展。使用经临床评估的抑制剂低剂量共同抑制这两种激酶，代表了一种以高疗效和最小毒性克服c-Myc依赖性PC转移的有前景的治疗策略。
查看英文原文 English abstract
Background : Prostate cancer (PC) metastasis is the leading cause of PC-related mortality, yet effective therapies targeting this lethal process remain lacking. We previously identified RIPK2 as a promising drug target in PC metastasis, functioning in part through a noncanonical RIPK2/MKK7/c-Myc phosphorylation pathway ( Nat. Commun. , 2022). However, additional downstream effectors and upstream regulators of RIPK2 remain poorly characterized. This study aims to address this knowledge gap, which represents a major barrier to developing highly effective and low-toxicity combination therapies.
Methods: Genetic manipulation was performed using CRISPR/Cas9 knockout, siRNA knockdown, and overexpression of wild-type or mutant genes. The metastatic potential of PC cells was assessed by cell proliferation, Matrigel invasion, clonogenicity, and soft-agar assays. RIPK2 activation of CDK2 was evaluated by western blotting and in vitro kinase assays. RIPK2-CDK2 interaction was examined using co-immunoprecipitation, GST pulldown, proximity-ligation assay, fluorescence colocalization, and fluorescence resonance energy transfer. Gene transcription and expression were measured by RT-qPCR. Protein stability was determined using cycloheximide chase and ubiquitination assays. Synergistic effects of RIPK2 and CDK2 inhibitors are assessed with a 6x6 matrix. At least two independent cell lines were used for each cell assay.
Results: RIPK2 enhanced the metastatic potential of PC 22Rv1 and PC3 cells primarily through its kinase activity, cytoplasmic localization, and induction of c-Myc expression. RIPK2 bound directly to CDK2 and activated it, leading to phosphorylation of c-Myc at S62. Notably, active CDK2 increased both transcription and protein stability of RIPK2, establishing a positive feedback loop. Co-inhibition of RIPK2 and CDK2 synergistically reduced c-Myc protein levels and suppressed PC cell invasion and colony formation.
Conclusion: RIPK2 and CDK2 form a multi-layered positive feedback loop that sustains c-Myc expression and drives PC metastatic progression. Low-dose co-inhibition of these two kinases using clinically evaluated inhibitors represents a promising therapeutic strategy to overcome c-Myc-dependent PC metastasis with high efficacy and minimal toxicity.
利益披露 Disclosure
A. Elgehama, None.