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.

摘要 RAS基因家族，包括KRAS、NRAS和HRAS，在超过三分之一的人中发生突变。 所有人类癌症，产生的蛋白质保持在组成性活性，致癌状态， 导致了这种疾病。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，能够在小鼠中引起癌症。尽管如此 KRAS是三者中最常见的突变，KRAS中的突变类型也是如此。 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有许多罕见的密码子， 蛋白质表达，HRAS有许多共同的密码子，促进蛋白质表达，而NRAS有一个共同的密码子。 罕见和常见密码子的混合物和中间蛋白质表达。专注于KRAS， 通常突变的RAS基因，我们表明，引入沉默突变转换罕见的密码子， 在该基因的一个外显子中存在一个共同的突变，这既减少了肺肿瘤的数量，也减少了在肺癌中检测到的突变。 暴露于致癌物的小鼠的Kras。为了确定造成这一结果的机制，在目标1中 我们将激活小鼠肺中具有共同密码子的可诱导致癌Kras基因， 肿瘤发生的阶段对扰乱Kras的固有稀有密码子偏好敏感。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。 效果将罕见密码子转换为常见密码子也改变了在基因组中恢复的致癌突变的类型。 致癌物暴露后的Kras。为了确定潜在的机制，在目标2中，我们将类似地 用可诱导的Kras基因改造小鼠，所述基因由具有不同的共同密码子与天然密码子编码 致癌突变如上所述，这些Kras等位基因将在小鼠的肺中被激活，以确定 在密码子使用改变的背景下，不同的致癌突变如何影响肿瘤发生， 细胞特性和细胞信号传导。这两个目标的完成将阐明该机制， 该密码子偏好性影响肿瘤发生过程中Kras突变的频率和类型。 尽管在早期肿瘤发生中稀有密码子为Kras提供了优势，但我们发现， 癌细胞克服了由稀有密码子施加的Kras mRNA的差翻译，以增加Kras 蛋白质表达，这与增加的致瘤活性和耐药性有关。 化疗药物为了确定癌细胞是如何实现这一壮举的，我们筛选并鉴定了密码子- 模式生物果蝇中致癌Ras的依赖性修饰物。我们将利用这些 目的3中的候选修饰剂，以阐明癌细胞如何克服Kras的不良翻译，并且反过来， 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的这种新特征，密码子偏好性，影响肿瘤发生。