RAS and PP2A activities converge on epigenetic gene regulation [RNA-Seq]

RAS-mediated human cell transformation requires inhibition of the tumor suppressor Protein Phosphatase 2A (PP2A). Both RAS and PP2A mediate their effects by phosphoregulation, but phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, based on mass spectrometry phosphoproteome data we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168 and TP53BP1. Mechanistically, we validate co-regulation of NuRD chromatin repressor complex by RAS and PP2A. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter de-repression and activation of oncogenic transcription. Notably, transcriptional de-repression by PP2A inhibition was associated with increased euchromatin and decrease in global DNA methylation. Further, targeting of RAS- and PP2A-regulated epigenetic proteins decreased viability of KRAS-mutant human lung cancer cells. Collectively the results indicate that epigenetic protein complexes involved in oncogenic gene expression constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Further, this work provides a resource for future studies focusing on phosphoregulation as a previously unappreciated layer of regulation of epigenetic gene regulation in cancer, and in other RAS/PP2A-regulated cellular processes. Overall design: Characterization of global gene expression by RNA sequencing (RNA-seq), 72h post siRNA (Ctrl, PP2A and RAS) transfections.

RAS介导的人类细胞转化，需要抑制肿瘤抑制因子蛋白磷酸酶2A（Protein Phosphatase 2A，PP2A）。RAS与PP2A均通过磷酸化调控发挥生物学功能，但二者在人类癌症中活性交汇的磷酸化蛋白靶点及细胞过程，尚未得到系统性分析。
本研究基于质谱磷酸化蛋白质组学数据，发现RAS与PP2A共同调控的磷酸化位点，显著富集于参与表观遗传基因调控的蛋白之中。例如，RAS与PP2A可共同调控HDAC1/2、KDM1A、MTA1/2、RNF168及TP53BP1上的相同磷酸化位点。从机制层面，本研究验证了RAS与PP2A对NuRD染色质阻遏复合物的共同调控作用。
与二者在癌症中已知的协同效应一致，RAS激活与PP2A抑制可导致表观遗传报告基因去阻遏，并激活致癌转录过程。值得注意的是，PP2A抑制所引发的转录去阻遏，与常染色质水平升高及全局DNA甲基化水平降低密切相关。此外，靶向调控受RAS与PP2A调控的表观遗传蛋白，可降低KRAS突变型人类肺癌细胞的存活率。
综上，本研究结果表明，参与致癌基因表达调控的表观遗传蛋白复合物，是癌症中RAS过度激活与PP2A抑制的关键交汇节点。此外，本研究为后续研究提供了宝贵资源：后续可聚焦于磷酸化调控这一此前未被充分认知的癌症表观遗传基因调控层面，以及其他受RAS/PP2A调控的细胞过程。
实验整体设计：在小干扰RNA（small interfering RNA，siRNA，分为对照组、PP2A靶向组与RAS靶向组）转染72小时后，通过RNA测序（RNA-seq）对全局基因表达进行表征分析。