结肠癌前体细胞维持其自更新的重要分子--Ascl2在该细胞内过表达的调控机制

Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, is a downstream target of Wnt signaling that controls the fate of intestinal cryptic stem cells and colon cancer progenitor cells. Ascl2 is exclusively expressed in the intestinal mucosal cryptic stem cells and the colon cancer progenitor cells. Our previous works confirmed that Ascl2 decided the stemness and the self-renewal of conlon cancer progenitor cells via miRNA-302b, Ascl2 posttranscriptionally repressed miRNA-200 family members and further modulated the plasticity of EMT-MET in colon cancer cells and Ascl2 posttranscriptionally repressed CDX2 expression and modulated the differentiation of colon cancer cells towards goblet-cell phenotype (unpublished). However, the reason and the mechanism(s) of Ascl2 overexpression in colon cancer progenitor cells are largely unknown. The present project applying for financial support is to investigate the molecular mechanism(s) in which activates Ascl2 overexpression in colon cancer progenitor cells including (1) Crosstalk between Hippo/MST signaling and canonical WNT signaling leads to the overexpression of Yap1, phosphorylation of Yap1, nuclear translocation of phosphorylated Yap1 and complexed with β-catenin, and further activates Ascl2 overexpression; (2) Crosstalk between PI3K/AKT/mTOR/Sp1 signaling and canonical WNT signaling leads to the phosphorylation ofβ-catenin, and further activates Ascl2 overexpression, Sp1 expression and nuclear translocation of Sp1 and binding the cis-element of Sp1 in the Ascl2 proximal promoter, and further activates Ascl2 overexpression; (3) Ascl2 self-activated its expression in colon cancer progenitor cells through its binding the rich E-box present at its own proximal promoter, and transcriptinally activates its own expression and forms a positive feed-back aitivation loop, and further activates Ascl2 overexpression. These molecular mechanism(s) will help to find a possible target to inhibit Ascl2 expression in colon cancer progenitor cells and finally to inhibit the self-renewal of colon cancer progenitor cells.

Ascl2作为Wnt信号通路的靶基因，是控制肠粘膜内隐窝基底干细胞及结肠癌前体细胞自更新的重要转录因子，其独特地过表达在肠道的隐窝干细胞及结肠癌前体细胞。我们前期试验证明Ascl2通过miRNA-302b控制结肠癌前体细胞的干性，阻遏miRNA-200家族的表达调节结肠癌的EMT的可塑性，阻遏CDX2的表达抑制结肠癌细胞的分化。但是结肠癌前体细胞内Ascl2过表达的调控机制不清。本课题拟从Hippo/MST信号与经典的Wnt信号的Crosstalk和从PI3K/AKT/mTOR/Sp1与经典的Wnt信号的Crosstalk来诱导Ascl2的转录调控激活；从Ascl2自转录调节，形成结肠癌前体细胞内Ascl2的正反馈激活环三个分子机制，达到诱导Ascl2在结肠癌前体细胞内的过表达。我们通过上述研究揭示结肠癌前体内细胞Ascl2过表达调控机制，为将来针对结肠癌前体细胞的治疗提供新的靶位和思路。

Ascl2是控制肠粘膜内隐窝基底干细胞及结肠癌前体细胞自更新的重要转录因子，能通过miRNA-302b控制结肠癌前体细胞的干性，阻遏miRNA-200家族的表达调节结肠癌EMT的可塑性，阻遏CDX2表达抑制结肠癌细胞的分化。但是结肠癌细胞及结肠癌前体细胞内Ascl2过表达的调控机制不清。本课题研究发现①外源性Ascl2通过与自身启动子结合形成自调节环从而激活内源性Ascl2的表达；②在R-spondin1/Wnt激活下，Ascl2通过自调控机制增强自身表达，维持CD133+CD44+结肠癌细胞的干性。③Ascl2是HIF-1α的下游靶标，HIF-1α通过与Ascl2启动子的缺氧反应元件（HRE）结合，直接转录激活结肠癌细胞Ascl2的表达，进一步抑制miR-200b并诱导结肠癌细胞EMT的发生；④HIF-1α是miR-200b的直接靶标。MiR-200b可以与HIF-1α的3'-UTR结合，形成 HIF-1α/Ascl2/miR-200b反馈环并调节结肠癌细胞的EMT-MET可塑性。⑤Hippo信号通路效应子YAP1的干扰导致CD133+CD44+ HT-29或Caco-2细胞的“干性”减弱。⑥YAP1与KLF5结合共同作用于Ascl2启动子上的GC-box元件，从而诱导结肠癌前体细胞中Ascl2的转录。以上研究结果证实了Ascl2在结直肠癌高表达的三个主要机制：（1）Ascl2的自调控机制形成了转录开关，增强Ascl2自身表达。（2）缺氧及缺氧诱导因子HIF-1α直接转录诱导Ascl2，并经过HIF-1α/Ascl2/miR-200b反馈环维持结肠癌细胞Ascl2的表达；（3）Hippo信号通路关键分子YAP1与KLF5共同激活结肠癌癌前体细胞Ascl2的表达。我们通过上述研究揭示结肠癌细胞及其前体细胞内Ascl2过表达调控机制，为将来针对结直肠肿瘤的治疗提供新的靶位和思路。