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Systematic analyses of CTNNB1-, BRAF-, KRAS- and PIK3CA-induced oncogenic activities in the intestinal epithelium

CTNNB1、BRAF、KRAS 和 PIK3CA 诱导的肠上皮致癌活性的系统分析

基本信息

批准号：
269381282
负责人：
Dr. Markus Morkel
金额：
--
依托单位：
Institut für Pathologie (CCM)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/269381282?language=en
关键词：
Systematic analyses CTNNB1 BRAF KRAS

项目摘要

Colon cancer cells contain sets of recurring mutations that alter the activities of major signalling pathways, among these the Wnt/beta-Catenin, MAPK, and the PI3K pathways that regulate stem cell traits, proliferation, differentiation and apoptosis in the intestinal epithelium. The contribution of a specific oncoprotein to the cancer phenotype is difficult to dissect, since many mutated proteins contribute simultaneously to aberrant signalling. We have therefore in the last years established a series of transgenic mice that allow induction of oncogenic forms of beta-Catenin (gene symbol CTNNB1), KRAS, BRAF or PIK3CA alone or in combinations, using tetracycline-controlled transgene expression. By this approach, oncogenes can be induced in a generalized manner in the intestine, allowing to study early oncoprotein-driven signalling events and effects on cell fate within a few days. Preliminary analyses of all models, and in-depth analyses of the CTNNB1 and BRAF models, showed that following oncogene induction the intestines of mice rapidly develop characteristics of different subtypes of colon tumors. We propose here to systematically analyze cellular signalling and cell composition of the intestines of the transgenic mice. First, we propose to evaluate changes in the intestinal cell hierarchy instigated by each oncogene in a time- and spatially-resolved manner (i.e. numbers and localizations of stem cells, proliferative cells, differentiated cells, apoptotic and senescent cells), using a combination of immunohistochemical (IHC), gene expression and fluorescent-activated cell sorting (FACS) analyses. In parallel, we will assess the localisation, amount and phosphorylation status of the central signal transducers CTNNB1, MEK, ERK and AKT, using IHC and Phospho-Western analyses. The functional roles of important signalling pathways will subsequently be interrogated using interference with small molecule inhibitors in oncogene-inducible organotypic primary cell cultures derived from the mice.Second, we propose to determine comprehensive sets of genes whose expression is de-regulated following oncogene activation in intestinal stem cells and in differentiated cells, using RNA-seq of FACS-sorted cell populations. Importantly, no such analyses have been performed in intestinal stem cells before. These experiments are thus suited to shed light on novel and relevant oncoprotein-induced changes of the cancer cell transcriptome. In summary, this proposal aims to disentangle and isolate the effects of the recurring CTNNB1, KRAS, BRAF and PIK3CA oncogenes in the intestine. The expected results will allow to link specific oncogenic signals to activation levels of target genes, to changes in cell fate, and finally to experimental therapeutic intervention. The proposed research will thus aid our understanding of how the clinically relevant subtypes of colon cancer form and will help to identify novel vulnerabilities of cancer cells.

结肠癌细胞含有改变主要信号传导途径活性的重复突变组，其中包括调节肠上皮中干细胞性状、增殖、分化和凋亡的Wnt/β-连环蛋白、MAPK和PI 3 K途径。一个特定的癌蛋白对癌症表型的贡献是难以剖析的，因为许多突变的蛋白质同时有助于异常信号传导。因此，我们在过去几年中建立了一系列转基因小鼠，其允许使用四环素控制的转基因表达单独或组合诱导致癌形式的β-连环蛋白（基因符号CTNNB 1）、KRAS、BRAF或PIK 3CA。通过这种方法，癌基因可以在肠道中以普遍的方式诱导，从而可以在几天内研究早期癌蛋白驱动的信号传导事件和对细胞命运的影响。对所有模型的初步分析以及对CTNNB 1和BRAF模型的深入分析表明，在癌基因诱导后，小鼠肠道迅速发展出不同亚型结肠肿瘤的特征。我们建议在这里系统地分析细胞信号和细胞组成的转基因小鼠的肠道。首先，我们建议使用免疫组织化学（IHC），基因表达和荧光激活细胞分选（FACS）分析的组合，以时间和空间分辨的方式（即干细胞，增殖细胞，分化细胞，凋亡和衰老细胞的数量和定位），评估由每个癌基因引起的肠细胞层次结构的变化。同时，我们将使用IHC和Phospho-Western分析评估中央信号转导物CTNNB 1、MEK、ERK和AKT的定位、数量和磷酸化状态。重要的信号传导途径的功能作用将随后使用干扰小分子抑制剂在致癌基因诱导的器官型原代细胞cultures来自mice.Second，我们建议确定全面的基因组，其表达失调后，在肠干细胞和分化的细胞中，使用RNA测序的FACS分选的细胞群体的致癌基因激活。重要的是，以前没有在肠干细胞中进行过这样的分析。因此，这些实验适合于揭示新的和相关的癌蛋白诱导的癌细胞转录组的变化。总之，该提案旨在解开和分离肠中复发的CTNNB 1，KRAS，BRAF和PIK 3CA癌基因的影响。预期的结果将允许将特定的致癌信号与靶基因的激活水平、细胞命运的变化以及最终的实验性治疗干预联系起来。因此，拟议的研究将有助于我们了解临床相关的结肠癌亚型是如何形成的，并将有助于识别癌细胞的新弱点。