Mechanisms of action of the Smyd3 methyltransferase in cancer cells

描述(申请人提供)：我们的首要目标是研究蛋白质翻译后修饰(PTM)网络在癌症中的重要性。由PTM赖氨酸甲基化定义的信号系统最近已成为癌症通路的潜在关键调节因子。然而，与蛋白质激酶等在肿瘤发生中具有公认作用的酶相比，赖氨酸甲基转移酶(KMT)酶促进癌症的机制尚不清楚。我们相信，表征参与癌症相关信号网络中细胞蛋白翻译后控制的新型KMT可以为细胞转化的基本机制提供新的见解，并为癌症治疗寻找新的治疗靶点。SMYD3是一种KMT，它在多种人类癌症中表达上调。我们和其他人已经证明，SMYD3的瞬时过表达可以促进几种癌细胞系的致瘤潜力。然而，对SMYD3的内源性底物以及这种酶在细胞和体内的整体作用模式的了解仍然不清楚。我们假设SMYD3通过甲基化和调控包括非组蛋白蛋白在内的关键细胞效应因子来促进癌细胞的增殖和存活。我们将首先通过探索SMYD3在体内癌症中的作用模式来测试这一想法，这是以前从未做过的。在目标1中，我们将使用一个新的条件突变Smyd3等位基因来检验SMYD3缺失抑制肿瘤发生的假设。由于SMYD3水平在具有致癌RAS的肿瘤中更具特异性，我们将重点关注肺腺癌和胰腺导管癌的小鼠模型，这两种致命的人类癌症中RAS途径经常被激活。在目标2中，我们将分析SMYD3在癌细胞中作用的分子机制。我们最近发现MEKK2(MAP3K2)是SMYD3底物。MEKK2与RAF蛋白属于同一家族，介导细胞对包括EGF在内的各种生长因子的反应，调节JNK和NF-kB通路等信号网络。我们将检验这样一种假设，即SMYD3介导的MEKK2甲基化构成了癌细胞对压力和生长因子做出反应的新机制。目标3的目标是使用我们为在蛋白质组范围内发现功能相关的SMYD3底物而开发的一种新的化学、生物-蛋白质组学策略来鉴定SMYD3的新底物。最有希望的靶点在调控癌细胞途径中的作用将使用分子方法和小鼠遗传学相结合的方法进行研究。基于越来越多的证据表明SMYD3在癌症中的作用，一些制药公司和学术实验室正在开发SMYD3抑制剂。我们的研究有能力确定SMYD3是影响广泛患者的许多癌症类型的有吸引力的治疗靶点。此外，关键的SMYD3分子靶点的识别可能会识别新的生物标记物和有希望的新的候选癌症治疗靶点。

DESCRIPTION (provided by applicant): Our overarching goal is to investigate the importance of protein post-translational modification (PTM) networks in cancer. The signaling system defined by the PTM lysine methylation has recently emerged as a potential key regulator of cancer pathways. However, in comparison to enzymes like protein kinases that have well-established roles in tumorigenesis, the mechanisms by which lysine methyltransferase (KMT) enzymes contribute to cancer are poorly understood. We believe that characterizing novel KMTs involved in the post-translational control of cellular proteins in cancer-relevant signaling networks can provide novel insights into the basic mechanisms of cellular transformation and identify new therapeutic targets for cancer treatment. SMYD3 is a KMT whose expression is up-regulated in a wide range of human cancers. We and others have shown that transient overexpression of SMYD3 can promote the tumorigenic potential of several cancer cell lines. However, knowledge of the endogenous substrates of SMYD3 and the overall mode of action of this enzyme in cells and in vivo is still obscure. We hypothesize that SMYD3 promotes the proliferation and the survival of cancer cells by methylating and regulating key cellular effectors including non-histone proteins. We will first test this idea by exploring the mode of action of SMYD3 in cancer in vivo, which has not previously been done. In Aim 1, we will use a novel conditional mutant Smyd3 allele to test the hypothesis that loss of SMYD3 inhibits tumorigenesis. Because SMYD3 levels are more specifically elevated in tumors with oncogenic RAS, we will focus on mouse models of lung adenocarcinoma and pancreas ductal carcinoma, two fatal human cancers in which the RAS pathway is often activated. In Aim 2 we will analyze the molecular mechanisms of SMYD3 action in cancer cells. We recently identified the MEKK2 (MAP3K2) kinase as a SMYD3 substrate. MEKK2 belongs to the same family as RAF proteins and mediates cellular responses to various growth factors, including EGF, to regulate signaling networks such as the JNK and NF-kB pathways. We will test the hypothesis that SMYD3-mediated methylation of MEKK2 constitutes a new mechanism by which cancer cells respond to stress and growth factors. The goal of Aim 3 is to identify new substrates of SMYD3 using a novel chemical biological- proteomic strategy we have developed for proteome-wide discovery of functionally-relevant SMYD3 substrates. The role of the most promising targets in regulation of cancer cell pathways will be investigated using a combination of molecular approaches and mouse genetics. Based on accumulating evidence implicating a role for SMYD3 in cancer, a number of pharmaceutical companies and academic laboratories are developing SMYD3 inhibitors. Our studies have the capacity to identify SMYD3 as an attractive therapeutic target in many cancer types affecting a wide range of patients. Furthermore, the identification of key SMYD3 molecular targets may identify novel biomarkers and promising new candidate therapeutic targets in cancer.