Hepatocyte-nuclear-factor-4a promotes gut neoplasia in mice and protects against the production of reactive oxygen species

Hepatocyte-nuclear-factor-4alpha (Hnf4a) is a transcription factor that controls epithelial cell polarity and maturation during embryogenesis. Hnf4a conditional deletion during post-natal development results in minor consequences on intestinal epithelium integrity but promotes activation of the Wnt/beta-catenin pathway. Here we show that Hnf4a does not act as a tumor suppressor gene but is crucial to promote gut tumorigenesis in mice. Polyp multiplicity in ApcMin mice that lacks Hnf4a is suppressed in comparison to littermate ApcMin controls. Analysis of microarray gene expression profiles from mice lacking Hnf4a in the intestinal epithelium identifies its novel function in regulating the expression of reactive oxygen species (ROS) detoxifying genes. This role is supported with the demonstration that HNF4a is functionally involved in the protection against spontaneous and 5-fluorouracil chemotherapy-induced production of intracellular ROS in colorectal cancer cell lines. The analysis of a colorectal cancer patient cohort establishes that HNF4a is significantly up-regulated at both gene transcript and protein levels in tumors relative to adjacent benign epithelial resections. Several genes involved in ROS neutralization are also up-regulated in correlation with HNF4a expression. All together, the findings point to the nuclear receptor HNF4a as a potential therapeutic target to eradicate aberrant epithelial cell resistance to ROS production during intestinal tumorigenesis. HNF4alpha was conditionally knockout in the mouse epithelial intestine with the 12.4-kb VillinCRE. A total of 3 control and 3 mutant littermates individuals were sacrificed at 7 months of age. The distal jejunum was harvested and Total RNA was isolated from each individuals. Each RNA sample was independently used to generate probes to screen affymetrix chips.肝细胞核因子4α（hepatocyte nuclear factor 4 alpha, Hnf4a）是一类转录因子，可调控胚胎发生过程中上皮细胞的极性建立与成熟过程。Hnf4a在出生后发育阶段的条件性敲除，会对肠道上皮完整性造成轻微影响，同时可激活Wnt/β-连环蛋白（Wnt/beta-catenin）通路。本研究表明，Hnf4a并非肿瘤抑制基因，反而是小鼠肠道肿瘤发生过程中的关键调控因子。与同窝ApcMin对照小鼠相比，缺失Hnf4a的ApcMin小鼠的息肉生成数量显著降低。对肠道上皮缺失Hnf4a的小鼠进行的微阵列基因表达谱分析，揭示了其在调控活性氧（reactive oxygen species, ROS）解毒基因表达方面的全新功能。这一功能得到实验验证：在结直肠癌细胞系中，HNF4α可有效对抗自发性及5-氟尿嘧啶（5-fluorouracil）化疗诱导的细胞内活性氧生成。对结直肠癌患者队列的分析显示，相较于邻近良性上皮切除组织，HNF4α在肿瘤组织中的基因转录水平与蛋白表达水平均显著上调。多项参与活性氧中和过程的基因的表达水平，同样随HNF4α的表达上调呈现正相关趋势。综上，本研究结果证实，核受体HNF4α可作为潜在治疗靶点，用于消除肠道肿瘤发生过程中异常上皮细胞对活性氧生成的耐药性。 本研究利用12.4 kb的VillinCRE元件，在小鼠肠道上皮细胞中条件性敲除HNF4α。共计3只对照小鼠与3只同窝突变型小鼠于7月龄时被处死。采集其远端空肠组织，并从每只小鼠体内分离总RNA。每份RNA样本均独立制备探针，用于Affymetrix基因芯片的杂交筛查。