IDENTIFICATION OF ERK SUBSTRATES THAT DRIVE PROLIFERATION IN RAS TUMORS

描述(申请人提供)：ERK(细胞外信号调节激酶)是RAS-RAF-MEK-ERK通路的末端激酶，控制着许多生物学过程。ERK的不适当激活发生在许多癌症中，这些癌症包含RAS、RAF或受体酪氨酸激酶(如EGFR和FGFR)的致癌驱动突变。ERK通过底物蛋白的磷酸化来调节生物过程。ERK底物已经通过单个基因方法和最近基于发现的蛋白质组学方法进行了鉴定，尽管许多底物的功能作用尚不清楚。我们采用了三部分功能基因组学方法，鉴定了在线虫生殖系发育中在七个不同生物学过程中发挥作用的30种底物：(1)生物信息鉴定的候选底物，其中包含与人类同源基因保守的ERK对接位点；(2)RNAi使用敏化的遗传背景测试了候选底物在依赖ERK的生物过程中的功能；(3)验证了RNAi阳性HIT作为哺乳动物ERK2的强大体外底物，并针对一个亚集，证明了ERK依赖的磷酸化在体内发生。我们的工作得出三个结论：(A)多个ERK底物控制单个生物过程；(B)底物具有分子多样性，影响细胞机械的许多部分，如翻译、RNA代谢、细胞骨架；(3)底物协同作用促进特定过程。不受管制的增殖是癌症的一个关键方面。在哺乳动物细胞培养/癌症领域的单个基因研究中，流行的观点是Ras-ERK信号通过转录因子底物促进增殖。然而，蛋白质组的发现和我们的功能基因组方法表明，只有~12%的ERK底物与转录调控有关，其余的影响广泛的细胞机制。我们认为，一些介导增殖的ERK底物仍有待发现，特别是在非转录细胞机制中发挥作用的基因产物，可以在功能筛选中识别。最近，一种RAS驱动的、ERK依赖的线虫生殖系肿瘤模型被报道，这为鉴定促进增殖的ERK底物提供了机会。我们建议应用我们成熟的三部分功能方法来识别线虫蛋白，与人类同源物，这是ERK底物，在RAS驱动，ERK依赖的肿瘤模型中控制增殖。ERK底物与人类同源物的发现将进一步加深我们对Ras-ERK信号如何促进增殖的理解，提供一套在哺乳动物细胞培养模型中进行测试的基因，可能识别与癌症相关的基因组改变的基因作为ERK底物，并可能提供可用于癌症治疗方法的框架/特异性基因。

DESCRIPTION (provided by applicant): ERK (extracellular signal regulated kinase) is the terminal kinase of the RAS-RAF-MEK-ERK pathway that controls numerous biological processes. Inappropriate ERK activation occurs in many cancers that contain oncogenic driver mutations in RAS, RAF or receptor tyrosine kinases such as EGFR and FGFR. ERK regulates biological processes through phosphorylation of substrate proteins. ERK substrates have been identified through individual gene approaches and more recently through discovery based proteomic approaches, although the functional role of many substrates is not known. We have taken a three part functional genomics approach that identified 30 substrates that function in seven different biological processes in C. elegans germline development: (1) bioinformatically identified candidate substrates that contain ERK docking sites conserved in position with the human ortholog; (2) RNAi tested function of the candidate substrates in ERK dependent biological processes using sensitized genetic backgrounds; (3) validated that the RNAi positive hit as a robust in vitro substrate of mammalian ERK2 and for a subset, demonstrated that ERK dependent phosphorylation occurs in vivo. Three conclusions came from our work: (A) Multiple ERK substrates control individual biological processes; (B) Substrates are molecularly diverse, affecting many parts of the cellular machinery, such as translation, RNA metabolism, cytoskeleton; (3) Substrates function cooperatively to promote a given process. Deregulated proliferation is a key aspect of cancer. The prevailing view from individual gene studies in the mammalian cell culture/cancer field is that RAS-ERK signaling promotes proliferation through transcription factor substrates. However the proteomic discovery and our functional genomic approach indicate that only ~12% of ERK substrates are associated with transcriptional control, the remaining affecting a wide range of cellular machinery. We propose that a number of ERK substrates that mediate proliferation remain to be discovered, particularly gene products that act in non- transcriptional cellular machinery, which can be identified in functional screens. Recently, a RAS-driven, ERK- dependent germline tumor model was reported for C. elegans, which provides an opportunity to identify ERK substrates that promote proliferation. We propose to apply our proven three part functional approach to identify C. elegans proteins, with human orthologs, that are ERK substrates that control proliferation in the RAS-drive, ERK-dependent tumor model. Discovery of ERK substrates with human orthologs will further our understanding of how RAS-ERK signaling promotes proliferation, provide a set of genes to be tested in mammalian cell culture models, may identify genes with cancer associated genomic alterations as ERK substrates, and may provide a framework/specific genes that can be used in cancer therapeutic approaches.