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

结肠癌细胞包含一系列反复发生的突变，这些突变改变了主要信号通路的活性，其中包括调节肠上皮干细胞特性、增殖、分化和凋亡的Wnt/ β - catenin、MAPK和PI3K通路。一个特定的癌蛋白对癌症表型的贡献是很难解剖的，因为许多突变的蛋白质同时参与异常信号传导。因此，在过去几年中，我们建立了一系列转基因小鼠，使用四环素控制的转基因表达，可以诱导致癌形式的β -连环蛋白（基因符号CTNNB1）、KRAS、BRAF或PIK3CA单独或联合表达。通过这种方法，可以在肠道中以一种普遍的方式诱导癌基因，从而可以在几天内研究早期癌蛋白驱动的信号事件及其对细胞命运的影响。对所有模型的初步分析，以及对CTNNB1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。我们建议系统地分析转基因小鼠肠道的细胞信号和细胞组成。首先，我们建议使用免疫组织化学（IHC）、基因表达和荧光活化细胞分选（FACS）分析相结合的方法，以时间和空间分解的方式（即干细胞、增殖细胞、分化细胞、凋亡细胞和衰老细胞的数量和定位）评估每个癌基因引起的肠道细胞层次的变化。同时，我们将使用IHC和Phospho-Western分析来评估中枢信号转导CTNNB1、MEK、ERK和AKT的定位、数量和磷酸化状态。重要信号通路的功能作用随后将通过干扰来自小鼠的癌基因诱导的器官型原代细胞培养中的小分子抑制剂来询问。其次，我们建议使用facs分选细胞群的RNA-seq，确定肠道干细胞和分化细胞中癌基因激活后表达去调控的综合基因集。重要的是，之前还没有在肠道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。综上所述，本提案旨在解开和分离肠道中反复出现的CTNNB1、KRAS、BRAF和PIK3CA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结肠癌临床相关亚型的形成方式，并有助于识别癌细胞的新脆弱性。

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.