Forward genetics piggyBac transposon Colorectal cancer Ras pathway
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摘要

Abstract Background The Ras pathway genes KRAS, BRAF, or ERBBs have somatic mutations in ~ 60% of human colorectal carcinomas. At present, it is unknown whether the remaining cases lack mutations activating the Ras pathway or whether they have acquired mutations in genes hitherto unknown to belong to the pathway. Methods To address the second possibility and extend the compendium of Ras pathway genes, we used genome-wide transposon mutagenesis of two human colorectal cancer cell systems deprived of their activating KRAS or BRAF allele to identify genes enabling growth in low glucose, a Ras pathway phenotype, when targeted. Results Of the 163 recurrently targeted genes in the two different genetic backgrounds, one-third were known cancer genes and one-fifth had links to the EGFR/Ras/MAPK pathway. When compared to cancer genome sequencing datasets, nine genes also mutated in human colorectal cancers were identified. Among these, stable knockdown of FOXO3, NCOA3, and TCF7L2 restored growth in low glucose but reduced MEK/MAPK phosphorylation, reduced anchorage-independent growth, and modulated expressions of GLUT1 and Ras pathway related proteins. Knockdown of NCOA3 and FOXO3 significantly decreased the sensitivity to cetuximab of KRAS mutant but not wild-type cells. Conclusions This work establishes a proof-of-concept that human cell-based genome-wide forward genetic screens can assign genes to pathways with clinical importance in human colorectal cancer.

译文

背景 Ras 通路基因 KRAS 、 BRAF 或 ERBBs 在 ~ ~ 的人结直肠癌中存在体细胞突变。目前,尚不清楚其余病例是否缺乏激活 Ras 通路的突变,或者它们是否获得了迄今未知的属于该通路的基因突变。方法解决第二种可能性并扩展 Ras 通路基因的概要, 我们使用了两个人类结直肠癌细胞系统的全基因组转座子诱变,剥夺了它们激活的 KRAS 或 BRAF 等位基因,以鉴定在靶向时能够在低葡萄糖 (一种 Ras 通路表型) 中生长的基因。结果在两种不同遗传背景下的 163 个反复靶向基因中,3分之1 是已知的癌症基因,5分之1 与 EGFR/Ras/MAPK 通路有关。与癌症基因组测序数据集相比,人类结直肠癌中也发现了 9 个突变基因。其中,稳定击倒 FOXO3 、 NCOA3 和 TCF7L2 恢复了在低糖中的生长,但减少了 MEK/MAPK 磷酸化,减少了非锚定生长, 以及调节 GLUT1 和 Ras 通路相关蛋白的表达。敲除 NCOA3 和 FOXO3 显著降低 KRAS 突变体对西妥昔单抗的敏感性,但不降低野生型细胞的敏感性。结论这项工作建立了一个概念验证,即基于人类细胞的全基因组正向遗传筛选可以将基因分配到在人类结直肠癌中具有临床重要性的通路。

FOXO3

肿瘤 转录因子 临床研究术语
概述  :  

FOXO3是由FOXO3基因编码的人类蛋白质,也称Forkhead box O3或 FOXO3a。叉头转录蛋白O(FOXO)家族是于2000年发现的一个新蛋白质家族,它在大肠杆菌及人类中均有表达,对细胞增殖、分化、机体生长发育、细胞周期、凋亡等具有重要作用。该家族通过对转录过程的特异性激活参与调节细胞周期进程、能量代谢及肿瘤发生,FOXO功能失调将导致细胞增殖失控和DNA损伤积累,从而产生致癌作用。   FOXO3是FOXO亚家族的成员,亦称为

FOXO3

释    义   n. FOXO3转录因子

例    句   HepG2 cells were transfected with small interfering RNA to mediate FoxO3 knockdown, or adenovirus and plasmid to mediate FoxO3 overexpression. 以小干扰RNA体外转染HepG2细胞,介导FoxO3敲除,或以腺病毒和质粒介导FoxO3过表达。

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