Identification of beta-cetenin/TCF4 target genes in LS174T colorectal cancer cells using gene expression arrays

Aberrant activation of WNT signaling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumor stem cell phenotype and identify the zinc-finger transcription factor GATA6 as key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells while it restricts BMP signaling to differentiated tumor cells. Genetic deletion of Gata6 in mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumor stem cells. In human tumors, GATA6 competes with beta-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been previously linked to increased susceptibility to develop CRC. Hence, GATA6 creates a permissive environment for tumor stem cell expansion by controlling the major signaling pathways that influence CRC initiation. A 4-hydoxy tamoxifen inducible system was used to inducibly block beta-catenin/TCF activity for a period of 36h. Briefly, the ERT2 domain from the pCMV-CRE-ERT2 (Feil et al., 1996) was amplified by PCR and cloned into a modified FUGW lentiviral vector backbone (Lois et al., 2002). NTCF was then amplified byPCR from the pCDNA3.1-NTCF-NLS (van de Wetering et al., 2002) and cloned in frame upstream of the ERT2 from the FUW-CMV-ERT2 (ET) to create the FUW-CMV-NTCF-ERT2 (NET). Total RNA was extracted using the TRIzol® Plus RNA Purification Kit (Life Technologies).

WNT信号通路异常激活与BMP信号通路缺失是结直肠癌（colorectal cancer, CRC）发生的两大主要分子改变。本研究针对维持肿瘤干细胞表型所需的基因开展筛选，鉴定出锌指转录因子GATA6是结直肠癌中WNT与BMP通路的关键调控因子。GATA6可直接驱动腺瘤干细胞中LGR5的表达，同时将BMP信号通路限制于分化的肿瘤细胞内。在小鼠结肠腺瘤中特异性敲除Gata6会提升BMP因子的表达水平，而BMP信号可通过传导抑制肿瘤干细胞的自我更新能力。在人类肿瘤组织中，GATA6会与β-连环蛋白/TCF4竞争结合BMP4基因位点的远端调控区域——该区域此前已被证实与结直肠癌易感性升高相关。因此，GATA6通过调控影响结直肠癌发生的核心信号通路，为肿瘤干细胞的扩增营造了适宜的微环境。本研究采用4-羟基他莫昔芬诱导系统，对β-连环蛋白/TCF活性进行36小时的诱导性抑制。具体而言，通过PCR扩增pCMV-CRE-ERT2（Feil等，1996）中的ERT2结构域，并将其克隆至改造后的FUGW慢病毒载体骨架（Lois等，2002）中。随后，从pCDNA3.1-NTCF-NLS（van de Wetering等，2002）中扩增NTCF序列，并将其与FUW-CMV-ERT2（ET）中的ERT2结构域进行框内融合克隆，构建得到FUW-CMV-NTCF-ERT2（NET）载体。总RNA提取采用TRIzol® Plus RNA Purification Kit（Life Technologies）。