IDENTIFICATION OF ERK SUBSTRATES THAT DRIVE PROLIFERATION IN RAS TUMORS

描述（由申请人提供）：ERK（细胞外信号调节激酶）是RAS-RAF-MEK-ERK通路的末端激酶，控制许多生物学过程。不适当的ERK激活发生在许多在RAS、RAF或受体酪氨酸激酶（如EGFR和FGFR）中含有致癌驱动突变的癌症中。ERK通过底物蛋白的磷酸化调节生物过程。ERK底物已通过单个基因方法和最近通过基于发现的蛋白质组学方法鉴定，尽管许多底物的功能作用尚不清楚。我们已经采取了三部分功能基因组学的方法，确定了30个底物，在七个不同的生物过程中的C。线虫生殖系发育：（1）生物信息学鉴定的候选底物，其含有与人直系同源物在位置上保守的ERK对接位点;（2）RNAi使用致敏遗传背景测试候选底物在ERK依赖性生物过程中的功能;（3）验证RNAi阳性命中作为哺乳动物ERK 2的稳健体外底物，并且对于子集，证明ERK依赖性磷酸化在体内发生。我们的工作得到了三个结论：（A）多种ERK底物控制着个体的生物学过程;（B）底物具有分子多样性，影响着细胞机器的许多部分，如翻译、RNA代谢、细胞骨架;（3）底物协同作用以促进给定的过程。增殖失控是癌症的一个关键方面。在哺乳动物细胞培养/癌症领域中，来自个体基因研究的流行观点是RAS-ERK信号传导通过转录因子底物促进增殖。然而，蛋白质组学的发现和我们的功能基因组学方法表明，只有~12%的ERK底物与转录控制相关，其余的影响广泛的细胞机制。我们提出，一些ERK底物，介导的增殖仍然有待发现，特别是基因产物，在非转录细胞机制，这可以确定在功能筛选。最近，RAS驱动的ERK依赖性生殖系肿瘤模型被报道用于C。elegans，这为鉴定促进增殖的ERK底物提供了机会。我们建议应用我们已经证明的三部分功能方法来识别C。elegans蛋白与人直系同源物，其是在RAS驱动的ERK依赖性肿瘤模型中控制增殖的ERK底物。发现ERK底物与人类直系同源物将进一步我们理解RAS-ERK信号传导如何促进增殖，提供一组待在哺乳动物细胞培养模型中测试的基因，可以将具有癌症相关基因组改变的基因鉴定为ERK底物，并且可以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.