Project 4: The role of codon bias in RAS tumorigenesis

项目 4：密码子偏倚在 RAS 肿瘤发生中的作用

• 批准号: 9074410
• 负责人: CHRISTOPHER M COUNTER
• 金额: $ 68.71万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-22 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9074410
• 关键词: Accounting; Affect; Alleles; Animal Model; Apoptosis; Base Sequence; Cancer Etiology; Carcinogen exposure; Carcinogens; Cells; Characteristics; Codon Nucleotides; Development; Disease; Drosophila genus; Engineering; Epidemiology; Exons; Family; Fostering; Frequencies; Genes; Growth; HRAS gene; Human; KRAS2 gene; Link; Lung; Lung Adenoma; Lung Neoplasms; Malignant - descriptor; Malignant Neoplasms; Messenger RNA; Modeling; Molecular; Mus; Mutate; Mutation; Oncogenic; Phenotype; Protein Isoforms; Proteins; Public Health; RAS Family Gene; Research; Resistance; Role; Severities; Signal Transduction; Silent Mutation; Staging; Testing; Time; Tissues; Translations; Tumor Burden; Urethane; Variant; actionable mutation; cancer cell; clinically relevant; fly; malignant phenotype; neoplastic cell; novel; protein expression; response; screening; senescence; tumor; tumorigenesis; tumorigenic

ABSTRACT The RAS family of genes, comprised of KRAS, NRAS, and HRAS, are mutated in upwards of a third of all human cancers, yielding proteins that remain in a constitutively active, oncogenic state that are well established to cause this disease. The proteins encoded by these three genes are nearly identical, activated by and signal through the same proteins, and capable of causing cancer in mice. Despite this uniformity, KRAS is the most commonly mutated of the three, and the type of mutations in KRAS, as well as the other RAS genes, varies extensively in human cancers. To understand these phenomena, we compared the nucleotide sequence of the RAS genes, finding that KRAS has many rare codons that limit protein expression, HRAS has many common codons that foster protein expression, while NRAS has a mixture of rare and common codons and intermediate protein expression. Focusing on KRAS, the most commonly mutated RAS gene, we show that introducing silent mutations to convert rare codons to common in one exon of this gene reduced both the number of lung tumors and the mutations detected in Kras of mice exposed to a carcinogen. To determine the mechanism responsible for this result, in aim 1 we will activate an inducible oncogenic Kras gene with common codons in the lungs of mice to identify the stage of tumorigenesis sensitive to perturbing the inherent rare codon bias of Kras. Once identified, we will then hone in on the cellular feature changed, and in turn, the molecular response underlying this effect. Converting rare codons to common also altered the type of oncogenic mutations recovered in Kras after carcinogen exposure. To determine the underlying mechanism, in aim 2 we will similarly engineer mice with an inducible Kras gene encoded by common versus native codons with different oncogenic mutations. As above, these Kras alleles will be activated in the lungs of mice to determine how different oncogenic mutations in the backdrop of altered codon usage impacts tumorigenesis, tumor cell characteristics, and cellular signaling. Completion of these two aims will elucidate the mechanism by which codon bias influences the frequency and type of mutations arising in Kras during tumorigenesis. Despite the advantage afforded to Kras by rare codons in early tumorigenesis, we show that established cancer cells overcome the poor translation of Kras mRNA imposed by rare codons to increase Kras protein expression, which was linked to increased tumorigenic activity and resistance to chemotherapeutics. To identify how cancer cells achieve this feat, we screened for and identified codon- dependent modifiers of oncogenic Ras in the model organism Drosophila. We will capitalize on these candidate modifiers in aim 3 to elucidate how cancer cells overcome poor translation of Kras, and in turn, whether such changes promote more malignant phenotypes. In summary, this research will reveal how this novel feature of KRAS, codon bias, impacts tumorigenesis.

抽象的 RAS 基因家族由 KRAS、NRAS 和 HRAS 组成，超过三分之一的基因发生突变。 所有人类癌症，产生的蛋白质保持在组成性活性、致癌状态，并且很好 确定会导致这种疾病。这三个基因编码的蛋白质几乎相同， 由相同的蛋白质激活并通过相同的蛋白质发出信号，并且能够在小鼠中引起癌症。尽管如此 均匀性，KRAS 是三者中最常见的突变，以及 KRAS 突变的类型 与其他 RAS 基因一样，在人类癌症中差异很大。为了理解这些现象，我们 比较了RAS基因的核苷酸序列，发现KRAS有许多稀有密码子限制 蛋白质表达，HRAS 有许多促进蛋白质表达的常见密码子，而 NRAS 有一个 稀有密码子和常见密码子以及中间蛋白表达的混合物。聚焦KRAS，最 常见突变的 RAS 基因，我们证明引入沉默突变将稀有密码子转换为 该基因的一个外显子中常见，减少了肺部肿瘤的数量以及在 暴露于致癌物质的小鼠的克拉斯。为了确定导致这一结果的机制，目标 1 我们将用小鼠肺部的常见密码子激活可诱导致癌的 Kras 基因，以识别 肿瘤发生阶段对扰乱 Kras 固有的稀有密码子偏向敏感。一旦确定， 然后我们将深入研究细胞特征的变化，进而研究其背后的分子反应 影响。将稀有密码子转换为常见密码子也改变了在 接触致癌物质后的克拉斯。为了确定潜在机制，在目标 2 中，我们将类似地 用普通密码子和天然密码子编码的诱导型 Kras 基因改造小鼠 致癌突变。如上所述，这些 Kras 等位基因将在小鼠肺部被激活，以确定 密码子使用改变背景下的不同致癌突变如何影响肿瘤发生、肿瘤 细胞特征和细胞信号传导。完成这两个目标将阐明其机制 密码子偏倚影响肿瘤发生过程中 Kras 中发生突变的频率和类型。 尽管稀有密码子在早期肿瘤发生中为 Kras 提供了优势，但我们表明，已建立的 癌细胞克服了稀有密码子造成的 Kras mRNA 翻译不佳的问题，从而增加了 Kras 的数量 蛋白质表达，这与致瘤活性和抗性的增加有关 化疗。为了确定癌细胞如何实现这一壮举，我们筛选并鉴定了密码子 模式生物果蝇中致癌 Ras 的依赖修饰物。我们将利用这些 目标 3 中的候选修饰符阐明癌细胞如何克服 Kras 的不良翻译，进而， 这些变化是否会促进更多的恶性表型。总之，这项研究将揭示如何 KRAS 的这一新特征（密码子偏倚）会影响肿瘤发生。