Dickkopf-Related Protein 2 is Epigenetically Inactivated and Suppresses Colorectal Cancer Growth and Tumor Metastasis by Antagonizing Wnt/β-Catenin Signaling
Dickkopf-Related Protein 2 is Epigenetically Inactivated and Suppresses Colorectal Cancer Growth and Tumor Metastasis by Antagonizing Wnt/β-Catenin Signaling
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Dickkopf 相关蛋白 2 表观遗传失活，通过拮抗 Wnt/β-Catenin 信号传导抑制结直肠癌生长和肿瘤转移
DOI：
10.1159/000471861
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发表时间：
2017-01-01
影响因子：
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通讯作者：
Ren, Guosheng
中科院分区：
文献类型：
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作者：
Wang, Can;Yue, Yujuan;Ren, Guosheng
背景/目的：Wnt/ β -连环蛋白信号通路的异常激活在包括消化道肿瘤在内的多种肿瘤的发病机制中起关键作用。最近的研究报道dickkopf相关蛋白2 （DKK2）在许多类型的癌症中是表观遗传失活的，其基因产物表现出肿瘤抑制特性。然而，DKK2在结肠癌中的生物学功能和潜在的分子机制尚不清楚。方法：采用RT-PCR检测DKK2在结肠肿瘤细胞系中的表达，采用甲基化特异性PCR （methylation-specific PCR， MSP）检测DKK2启动子在结肠肿瘤细胞系和原发肿瘤中的甲基化状态。在其他实验之前，通过RT-PCR检测DKK2的异位表达。为了研究DKK2的功能，我们检测了集落形成和细胞增殖，利用流式细胞术分析细胞周期和吖啶橙/溴化乙啶（AO/EB）荧光染色检测细胞凋亡，并在体内检测了伤口愈合、跨井迁移和肿瘤生长。Western blot检测DKK2在上皮-间质转化和典型Wnt/ β -catenin信号传导中的作用机制。结果：我们在这里发现DKK2的下调或沉默与其启动子的高甲基化状态密切相关，并且DKK2的表达可以通过去甲基化处理恢复。DKK2启动子的甲基化在几乎所有肿瘤和肿瘤邻近组织中检测到，但在正常结肠组织中未检测到。DKK2在HCT116和HT-29结肠细胞系中异位表达，通过诱导细胞周期G0/G1阻滞和细胞凋亡，抑制了集落的形成和细胞活力，与对照组相比，DKK2感染的HCT116稳定细胞的生长减少。此外，DKK2通过部分逆转上皮细胞向间质细胞的转变以及下调几种干细胞标记物来抑制细胞迁移。我们的数据进一步表明，DKK2表达的恢复导致活性β -连环蛋白及其下游靶基因的下调。结论：DKK2可能是一种功能性肿瘤抑制因子，通过拮抗Wnt/ β -连环蛋白信号通路调节结直肠癌的肿瘤发生。(C) 2017作者：s . Karger AG，巴塞尔出版
Background/Aims: Aberrant activation of the Wnt/beta-catenin signaling pathway plays a key role in the pathogenesis of multiple tumors including digestive cancers. Recent studies have reported that Dickkopf-related protein 2 (DKK2) is epigenetically inactivated in numerous types of cancers and that its gene products exhibit tumor-suppressive properties. However, the biological functions and underlying molecular mechanisms of DKK2 in colon carcinoma remains obscure. Methods: We examined the expression of DKK2 in colon tumor cell lines by RT-PCR and its promoter methylation status in colon tumor cell lines and primary tumors by methylation-specific PCR (MSP). Ectopic expression of DKK2 was measured by RT-PCR prior to the other experiments. To investigate the function of DKK2, we assayed colony formation and cell proliferation, utilized flow cytometric analyses of the cell cycle and acridine orange/ethidium bromide (AO/EB) fluorescence staining for apoptosis, and examined wound healing, transwell migration and tumor growth in vivo. Western blots were used to explore the mechanisms of DKK2 in epithelial-mesenchymal transition and canonical Wnt/beta-catenin signaling. Results: We show here that downregulation or silencing of DKK2 was closely associated with the hypermethylation status of its promoter and that DKK2 expression could be restored by demethylation treatment. Methylation of the DKK2 promoter was detected in nearly all tumors and tumor-adjacent tissues, but not in normal colon tissues. Ectopic expression of DKK2 in colon cell lines HCT116 and HT-29 inhibited colony formation and cell viability by inducing cell cycle G0/G1 arrest and apoptosis, and growth of stable DKK2-infected HCT116 cells in nude mice was decreased compared to controls. Furthermore, DKK2 restrained cell migration through partial reversal of epithelial-to-mesenchymal transition and also by downregulating several stem cell markers. Our data further showed that restoration of DKK2 expression resulted in downregulation of active beta-catenin and its downstream target genes. Conclusion: DKK2 appears to be a functional tumor suppressor regulating tumorigenesis of colorectal cancer by antagonizing Wnt/beta-catenin signaling. (C) 2017 The Author(s) Published by S. Karger AG, Basel