DKK4调控HES1促进结直肠癌细胞静息的分子机制研究

Colorectal cancer quiescent cells (CoQCs) play a key role in theraputic resistance and recurrence of colorectal cancer. Mechanisms regulating colorectal cancer cells into quiescent status are still not clearly defined. Based on builted irinotecan enriching CoQCs model in nude mice using primary colorectal cancer cells, we find Dickkopf 4 (DKK4), an important node molecule, is significantly increased. Our results show that DKK4 can increase CoQCs ratio by upregulating Hairy and Enhancer of Split 1 (HES1) signaling, without augment cell apoptosis. Moreover, DKK4 is closely related with poorer patient survival. It represents a novel regulator for CoQCs, but its action mechanisn remains elusive. Accordingly, we propose that CoQCs is influenced by DKK4 molecule through regulating HES1 activity, which is non-dependent on canonical Wnt signaling. In this project, we plan to identify the forming mechanisms of CoQCs and corresponding pathways using DKK4 overexpression, knock down system, Co-IP, protein chip and Western blot techniques. We will focus on identification of essential signaling pathway for DKK4 involved in HES1 networks, and also evaluate their clinical value in chemotherapy and the prognosis. Thus completion of the proposed study would provide new angels for regulating CoQCs, which has important scientific significance and potential clinical transformation prospects.

结直肠癌静息细胞（CoQCs）是结直肠癌耐药与复发的根源，而细胞静息发生的分子机制尚不清。课题组前期利用培养的原代结直肠癌细胞构建了伊立替康裸鼠体内富集CoQCs模型，鉴定出CoQCs重要节点分子DKK4；预实验显示DKK4能显著上调静息细胞关键因子HES1的表达，提高CoQCs细胞比率，且不增加细胞凋亡，是一个新的调控CoQCs分子，证实与结直肠癌的预后不良有关，但其具体分子机制国内外尚无报导。因此，我们推测DKK4可能通过非依赖于经典Wnt通路的HES1分子网络来调控结直肠癌细胞的静息状态。本项目拟利用过表达或敲减DKK4、免疫共沉淀、蛋白芯片、Western blot等方法阐明DKK4对CoQCs静息的影响和发挥功能的机制，鉴定DKK4调控HES1的分子网络，评价其在结直肠癌治疗中的价值。本课题将揭示DKK4影响CoQCs的新机制，具有重要科学意义和潜在临床转化前景。

结直肠癌静息细胞（CoQCs）是结直肠癌发生复发与转移的根源，是肿瘤治疗的关键。课题组通过PKH26和细胞周期特性来分选、鉴定结肠癌静息细胞，并模拟化疗药物Irinotecan杀伤快速增殖的肿瘤细胞而导致静息细胞的富集，细胞周期分析显示该群细胞富含G0期；通过基因芯片和生物信息学鉴定出CoQCs重要节点分子DKK4，进一步研究发现DKK4能上调静息细胞关键调控因子HES1表达，并显著增加G0期细胞数量，而不增加细胞的凋亡；同时上调P16及P21等细胞周期蛋白，影响AMPK磷酸化水平，提示DKK4可通过HES1分子网络途径来影响结直肠癌静息状态，从而参与结直肠癌细胞的耐药。利用COX多因素分析显示，DKK4与结直肠癌的总生存率呈负相关。本课题首次揭示DKK4影响细胞静息的新机制，具有重要科学意义和潜在临床转化前景。