Molecular mechanism of involvement of homeobox transcription factors Cdx2 and Cdxl in intestinal tumors

尾型同源盒转录因子Cdx2和Cdx1参与肠上皮细胞的分化和肿瘤发生，但其分子机制尚不清楚。在本研究中，我们通过改进版的染色质免疫沉淀（ChIP）筛选，确定了SLC5A8和PLEKHG1作为Cdx2和Cdx1的新靶基因。SLC5A8编码短链脂肪添加剂和单羧酸盐（包括丁酸盐和丙酮酸盐）的钠偶联转运体。SLC5A8的表达在结肠癌中经常下调，SLC5A8的高表达与结肠癌患者更长的无病生存期相关。使用SLC5A8启动子和qRT-PCR分析SLC5A8在人结肠癌细胞中被迫表达或敲低CDX2和/或CDX1后的报告基因分析表明，SLC5A8确实是CDX2和CDX1的直接转录靶点。另一方面，PLEKHG1编码一个含有1385个氨基酸的蛋白，携带dl -homology （DH）域和pleckstrin homology （PH）域。虽然ft被认为是Rho/Rac/Cdc42家族小g蛋白的GTP/GDP交换因子（GEF），但尚未有关于该分子的报道。我们发现PLEKHG1在正常肠组织和一部分结肠癌细胞系中均有表达，其表达受CDX2和CDX1调控。对这些新的Cdx靶基因的进一步表征有望揭示它们在肠上皮细胞分化和/或肿瘤发生中的作用。我们还发现PKC ζ，一个参与细胞极性控制的非典型PKC，可以磷酸化位于Cdx2同位域的一个保守的苏氨酸，并且该残基的磷酸化可能调节Cdx2的二聚化。进一步分析PKC信号与Cdx2之间可能的联系，有助于理解Cdx2控制PKC信号分化和转化的机制。

The caudal-type homeobox transcription factors Cdx2 and Cdx1 ate involved in differentiation and tumorigenesis of intestinal epthelial cells, yet the underlying molecular mechanisms remain unclear. In this study, we have identified SLC5A8 and PLEKHG1 as novel target genes of Cdx2 and Cdx1, by using an improved version of chromatin immunoprecipitation (ChIP) screen. SLC5A8 codes for a sodium-coupled transporter for short chain fatty adds and monocarboxylates, including butyrate and pyruvate. Expression of SLC5A8 is frequently down regulated in colon cancer, and higher expression of SLC5A8 correlates with longer disease-free survival in colon cancer patients. Reporter gene assays using SLC5A8 promoter and qRT-PCR analysis of SLC5A8 in human colon cancer cell fines following forced expression or knockdown of CDX2 and/or CDX1 have shown that SLC5A8 is indeed a direct transcriptional target of Cdx2 and Cdx1. On the other hand, PLEKHG1 encodes a protein with 1385 amino acids, carrying Dbl-homology (DH) domain and pleckstrin homology (PH) domain. Although ft is expected to function as a GTP/GDP exchange factor (GEF) for Rho/Rac/Cdc42 family small G-proteins, no reports have been published of this molecule. We have found that PLEKHG1 is expressed in the normal intestinal tissues as well as in a subset of colon cancer cell lines, and that its expression is regulated by CDX2 and CDX1. Further characterization of these novel target genes of Cdx is expected to reveal their roles in differentiation and/or tumorigenesis of the intestinal epithelial cells. We have also shown that PKC ζ, an atypical PKC involved in cell polarity control, can phosphorylate a well-conserved threonine reside in the homeodomain of Cdx2, and that phosphorylation of this residue may regulate dimerization of Cdx2. Further analysis of the possible link between PKC signaling and Cdx2 may he understand the mechanism by which Cdx2 controls their differentiation and transformation.