干性因子Ascl2维持Lgr5+胃癌细胞有氧酵解的机制研究

Distinct metabolic modalities in cellular subpopulations may be determined by heterogeneous differentiation in tumor cells. We have previously identified a subset of poorly differentiated Lgr5+ gastric cancer cells（Lgr5+GCs）which exhibited significant aerobic glycolysis as compared with differentiated Lgr5-GCs. However, it remains unknown about the molecular mechanisms underlying glycolysis and high expression of pyruvate dehydrogenase kinases (PDKs) in Lgr5+GCs. Our preliminary data showed that knockdown of a stem cell transcription factor Ascl2 inhibited glycolysis and PDKs expression. Bioinformatic prediction revealed multiple binding sites for Ascl2 within promoter region of PDK genes. Therefore, the current project is to investigate the way by which "tumor metabolism" is regulated by "tumor stemness (Ascl2)". Our hypothesis is that Ascl2 may play a role in maintaining glycolysis via transcriptional activation of PDKs in Lgr5+GCs. Several molecular techniques such as CHIP、EMSA、Luciferase reporter assay will be applied to decipher the mechanisms by which Ascl2 might regulate expression of PDKs. We will also examine the functional consequences of PDKs knockdown on glycolysis and self-renewal in Lgr5+GCs. The novelty and significance of the current project is that cellular heterogeneity in tumor metabolism would be addressed by regulators of tumor cell stemness, thus leading to the identification of novel cellular and molecular targets to control aberrant tumor metabolism.

细胞能量代谢模式可能与细胞分化状态密切相关。我们发现低分化Lgr5+胃癌细胞（Lgr5+GCs）的一个独特表型- - -高有氧酵解活性，但Lgr5+GCs维持高有氧酵解活性及其关键限速酶丙酮酸脱氢酶激酶 (PDK1-4)表达的机制不详。初步结果显示下调干性因子Ascl2导致有氧酵解活性和PDKs表达降低， PDKs启动子含有多个Ascl2预测结合位点，因此本项目提出"肿瘤干性（Ascl2）"可能调控"肿瘤代谢"的假说：干性因子Ascl2可能转录激活PDKs表达，后者进而维持Lgr5+GCs 高有氧酵解活性。采用CHIP、EMSA、Luciferase等方法研究Ascl2激活PDKs的机制；分别下调PDK1-4表达，检测PDK各成员对Lgr5+GCs有氧酵解和自我更新的影响。本项目从"肿瘤干性"角度认识"细胞代谢异质性"的机制，为抗肿瘤代谢治疗提供新的靶细胞和靶分子。

肿瘤干细胞是恶性肿瘤发生发展的内在源动力，是导致肿瘤治疗抵抗与复发的种子细胞。肿瘤干细胞具有异常代谢特征和无限自我更新的生物学特性，但是维持其自我更新的分子机制不详。本项目聚焦干细胞因子 Ascl2对肿瘤干细胞特性等恶性生物学行为的调控作用，研究发现：1. 分析Lgr5+胃癌细胞具有干细胞特征，发现Ascl2不能直接调控PDK1表达，转录组分析发现Ascl2可能通过调控大量的下游靶基因影响肿瘤干细胞生物学特性；2. Lgr5+胃癌细胞和肝癌细胞具有肿瘤干细胞生物学特性，组蛋白去甲基化酶LSD1是维持Lgr5+肿瘤细胞的干性特征关键表观修饰酶，LSD1通过激活Wnt信号通路，维持Lgr5+肿瘤干细胞自我更新和耐药特性；3. ASCL2+胃癌干细胞一个对Wnt信号敏感的肿瘤干细胞亚群；4. 证明ASCL2高表达维持ASCL2+胃癌干细胞成瘤能力；5. Wnt信号通过上调SMYD3，依赖组蛋白表观修饰机制，促进ASCL2表达，进而维持胃癌干细胞自我更新等生物学特性。综上所述，本项目发现Lgr5+肿瘤细胞的干细胞特征，进一步阐明调控自我更新的分子机制及潜在干预靶点。我们还发现ASCL2高表达维持ASCL2+胃癌干细胞自我更新也非常重要，揭示了Wnt信号通过靶向SMYD3，从组蛋白表观修饰水平上调ASCL2表达和胃癌细胞的新机制。这些创新性发现为进一步阐明肿瘤干细胞生物学特性调控机理和潜在治疗价值。这些原创性发现为靶向肿瘤干细胞，从源头上根除肿瘤“种子细胞”提供了新的治疗策略，具有潜在的临床转化价值。