Integrative approaches to elucidate p53 transcriptional networks during carcinogenesis

阐明致癌过程中 p53 转录网络的综合方法

基本信息

批准号：
10673070
负责人：
LAURA D ATTARDI
金额：
$ 93.78万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-14 至 2029-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10673070
关键词：
Acute Affect Alternative Splicing Alveolar Biological Assay CRISPR screen Cell Differentiation process Cell Proliferation Cells DNA Damage Disease Early Diagnosis Early treatment Event Evolution Genes Genetic Transcription Goals Human Individual KRASG12D Kinetics Lung Adenocarcinoma Malignant Neoplasms Mediating Molecular Mus Mutant Strains Mice Mutate Pathway interactions Play Primary carcinoma of the liver cells Proteome Proteomics RNA Splicing RNA-Binding Proteins Research Role TP53 gene Testing Therapeutic Transcriptional Activation Domain Tumor Suppression Tumor Suppressor Genes Tumor Suppressor Proteins Work Zinc Fingers cancer cell cancer therapy carcinogenesis improved in vivo insight lung injury lung regeneration mouse model novel posttranscriptional programs response single-cell RNA sequencing small hairpin RNA standard of care targeted treatment tumor tumor barcoding and sequencing tumor microenvironment

项目摘要

PROJECT SUMMARY/ABSTRACT The TP53 tumor suppressor gene is mutated in over half of all human cancers, but the mechanisms through which p53 suppresses cancer in vivo remain incompletely understood. Notably, there are no standard-of-care cancer therapies based on the p53 pathway. In this proposal, we strive to deconstruct the pathways through which p53 suppresses cancer to illuminate pathways dysregulated upon p53 loss that could ultimately be targeted therapeutically. Our previous work using transcriptional activation domain mutant mice suggested that the transcriptional programs underlying p53 acute DNA damage responses are not essential for tumor suppression, prompting us to search for new mechanisms of p53-mediated tumor suppression. We thus performed unbiased in vivo shRNA and CRISPR/Cas9 screens for p53 target genes important for tumor suppression. We identified several p53 target genes with tumor suppressor activity, including Zmat3 – the top hit in both screens. We found that Zmat3 expression is highly p53-dependent across contexts and that Zmat3 suppresses lung adenocarcinoma (LUAD) and hepatocellular carcinoma (HCC) in autochthonous mouse models. Zmat3 encodes a zinc finger RNA-binding protein that we found acts by modulating alternative splicing, revealing a new branch of p53-mediated tumor suppression. As recent work has revealed a critical role for alternative splicing in cancer, we hypothesize that studying p53 pathways at the post-transcriptional level, such as through splicing and proteomics analyses, will yield novel insights into p53-mediated tumor suppression. In Theme 1, we propose to identify p53-dependent splicing and proteome changes, including both Zmat3-dependent and Zmat3-independent ones, that could explain tumor suppression in mouse LUAD and HCC. We will also identify genes that cooperate with Zmat3 to suppress cancer downstream of p53. We will test the importance of genes found in these analyses for LUAD and HCC suppression using a quantitative in vivo tumor assay known as Tuba-seq. In Theme 2, we will pursue our observation that p53 repurposes a role in lung regeneration, in which it drives alveolar type 1 cell differentiation upon lung injury, to suppress LUAD. Through single cell (sc)RNA-seq and scATAC-seq analyses, we will ask how p53 status dictates the evolutionary path of KrasG12D-expressing alveolar type 2 cells and how p53 transcriptional programs change with cell state across LUAD evolution in mouse models. We will also ask how cells in the tumor microenvironment (TME) affect cancer cell trajectories in wild-type and p53-deficient tumors. To define genes functionally important for cancer cell state transitions and crosstalk between cancer cells and TME components, we will employ scPerturb-seq. Studies of LUAD evolution, in which we express KrasG12D and analyze subsequent events at both the cancer cell and TME levels through a detailed kinetic analysis, will deconstruct p53-mediated tumor suppression in vivo at an unprecedented molecular depth. Collectively, these studies will provide crucial new insight into how to modulate p53 pathways in therapeutic strategies for cancer.

项目概要/摘要 TP53 肿瘤抑制基因在超过一半的人类癌症中发生突变，但其机制通过值得注意的是，p53 在体内抑制癌症的机制尚不完全清楚，目前尚无治疗标准。基于 p53 通路的癌症治疗在本提案中，我们努力解构通过的通路。 p53 抑制癌症，阐明 p53 缺失后失调的通路，最终可能是我们之前使用转录激活域突变小鼠的研究表明， p53急性DNA损伤反应的转录程序对于肿瘤来说并不是必需的抑制，促使我们寻找 p53 介导的肿瘤抑制的新机制。对对肿瘤重要的 p53 靶基因进行无偏见的体内 shRNA 和 CRISPR/Cas9 筛选我们鉴定了几个具有肿瘤抑制活性的 p53 靶基因，包括 Zmat3（顶部）。我们发现 Zmat3 的表达在不同环境中都高度依赖于 p53，并且 Zmat3 抑制本地小鼠的肺腺癌 (LUAD) 和肝细胞癌 (HCC) Zmat3 编码一种锌指 RNA 结合蛋白，我们发现它通过调节替代蛋白发挥作用。剪接揭示了 p53 介导的肿瘤抑制的一个新分支，正如最近的工作揭示了一个关键的那样。为了了解选择性剪接在癌症中的作用，我们致力于研究转录后 p53 通路水平，例如通过剪接和蛋白质组学分析，将对 p53 介导的肿瘤产生新的见解在主题 1 中，我们建议识别 p53 依赖性剪接和蛋白质组变化，包括 Zmat3 依赖型和 Zmat3 独立型均可解释小鼠 LUAD 中的肿瘤抑制我们还将鉴定与 Zmat3 合作抑制 p53 下游癌症的基因。将使用定量方法测试这些分析中发现的基因对于 LUAD 和 HCC 抑制的重要性称为 Tuba-seq 的体内肿瘤测定在主题 2 中，我们将继续观察 p53 重新利用 a 在肺再生中的作用，在肺损伤时驱动肺泡 1 型细胞分化，以抑制 LUAD。通过单细胞 (sc)RNA-seq 和 scATAC-seq 分析，我们将询问 p53 状态如何决定表达 KrasG12D 的肺泡 2 型细胞的进化路径以及 p53 转录程序如何变化小鼠模型中 LUAD 进化的细胞状态我们还将询问肿瘤中的细胞如何。微环境 (TME) 影响野生型和 p53 缺陷肿瘤中的癌细胞轨迹。对于癌细胞状态转换以及癌细胞与 TME 之间的串扰具有重要功能我们将使用 scPerturb-seq 来研究 LUAD 进化，其中我们表达 KrasG12D 和通过详细的动力学分析，分析癌细胞和 TME 水平的后续事件，将总的来说，这些以前所未有的分子深度解构了体内 p53 介导的肿瘤抑制。研究将为如何在癌症治疗策略中调节 p53 通路提供重要的新见解。