Gene Expression profiles of colon from VhlF/F, VhlΔIE, VhlF/F/Apcmin/+, VhlΔIE/Apcmin/+

To identify the precise molecular mechanisms that could contribute to the increase in colon carcinogenesis, microarray gene expression analysis was performed on colon RNA isolated from 5-week-old VhlF/F and VhlΔIE, VhlΔIE/Apcmin/+ and VhlF/F/Apcmin/+ mice. Hypoxia-inducible factor (HIF) is a key modulator of the transcriptional response to hypoxia and is increased in colon cancer. However, the role of HIF in colon carcinogenesis in vivo remains unclear. Intestinal epithelium-specific disruption of the von Hippel-Lindau tumor suppressor protein (VHL) resulted in constitutive HIF signaling, and increased HIF expression augmented colon tumorigenesis in the Apcmin/+ intestinal tumor model. Intestine-specific disruption of Vhl increased colon tumor multiplicity and progression from adenomas to carcinomas. These effects were ameliorated in mice with double disruption of Vhl and Hif-2α. Activation of HIF signaling resulted in increased cell survival in normal colon tissue, however tumor apoptosis was not affected. Interestingly, a robust activation of cyclin D1 was observed in tumors of Apcmin/+ mice in which HIF-2α was activated in the intestine. Consistent with this result, BrdU incorporation indicated that cellular proliferation was increased in colon tumors following HIF activation. Further analysis demonstrated that dysregulation of the intestinal iron absorption transporter divalent metal transporter-1 (DMT-1) was a critical event in HIF-2α-mediated colon carcinogenesis. These data provide a mechanistic basis for the widely reported link between iron accumulation and colon cancer risk. Together, our findings demonstrate that a chronic increase in HIF-2α in the colon initiates pro-tumorigenic signaling which may have important implications in developing preventive and therapeutic strategies for colon cancer. Global gene expression profiling in colon RNAs isolated from 5-week-old VhlF/F (n=4, Shah 001), VhlF/F/Apcmin/+(n=3, Shah 003), VhlΔIE (n=3, Shah 002) and VhlΔIE/Apcmin/+ mice (n=5, Shah 004).

为明确可促进结肠癌发生的确切分子机制，本研究对从5周龄VhlF/F、VhlΔIE、VhlΔIE/Apcmin/+及VhlF/F/Apcmin/+小鼠中分离的结肠RNA开展了基因芯片表达谱分析（microarray gene expression analysis）。缺氧诱导因子（hypoxia-inducible factor, HIF）是介导缺氧转录应答的关键调节因子，在结肠癌中表达升高。然而，HIF在体内结肠癌发生发展中的具体作用仍不明确。肠上皮特异性敲除希佩尔-林道肿瘤抑制蛋白（von Hippel-Lindau tumor suppressor protein, VHL）可导致HIF信号通路持续激活，而在Apcmin/+肠道肿瘤模型中，HIF表达上调可促进结肠肿瘤发生。肠道特异性Vhl敲除可增加结肠肿瘤多发率，并促进腺瘤向腺癌的进展。在Vhl与Hif-2α双敲除小鼠中，上述促瘤效应可得到缓解。HIF信号通路激活可提升正常结肠组织的细胞存活率，但不影响肿瘤细胞的凋亡水平。有趣的是，在肠道特异性激活HIF-2α的Apcmin/+小鼠肿瘤中，可观察到细胞周期蛋白D1（cyclin D1）的显著激活。溴脱氧尿苷（Bromodeoxyuridine, BrdU）掺入实验结果显示，HIF激活后结肠肿瘤细胞的增殖能力显著提升，与上述发现一致。进一步分析表明，肠道铁吸收转运蛋白二价金属离子转运体1（divalent metal transporter-1, DMT-1）的表达失调，是HIF-2α介导结肠癌发生的关键事件。上述研究结果为学界广泛报道的铁蓄积与结肠癌发病风险之间的关联提供了分子机制层面的依据。综上，本研究结果表明，结肠组织中HIF-2α的慢性激活可启动促肿瘤发生的信号通路，这一发现对于开发结肠癌的预防与治疗策略具有重要指导意义。本研究对5周龄VhlF/F（n=4，Shah 001）、VhlF/F/Apcmin/+（n=3，Shah 003）、VhlΔIE（n=3，Shah 002）及VhlΔIE/Apcmin/+小鼠的结肠RNA开展了全基因组表达谱分析（global gene expression profiling）。