Ankrd22靶向Smad2抑制结直肠癌干细胞上皮间质转化的分子机制研究

Cancer stem cells (CSCs) contribute greatly to the invasion, metastasis and recurrence of colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is one of the main traits of colorectal cancer stem cells (CCSCs), and EMT also promotes CRC cells to de-differentiate and regain CSCs-like properties. It is of great importance to inhibit the characteristic of EMT for targeted eradicating CCSCs, yet the regulatory mechanisms need to be further elucidated. We previously identified a new molecule named Ankrd22 from CCSCs using retrovirus library. Knockdown of Ankrd22 resulted in significantly increased proportion of CCSCs，while overexpression of Ankrd22 down-regulated TGF-β signaling activity by inhibiting nuclear translocation of Smad2. Moreover, Ankrd22 was decreased in advanced CRC, which is associated with poor prognosis. These results indicate that Ankrd22 plays an inhibitory effect on EMT of CCSCs, but the molecular mechanisms remain unclear. Based on previous results, our current study is aimed to screen and identify the critical candidate targeting molecules of Ankrd22 by binary tagging tandem affinity chromatography mass spectrometry and to clarify the molecular mechanisms of Ankrd22 suppressing CCSCs' stemness and EMT. This study will lay a theoretical basis for the development of new molecular targeted drugs to eradicate CSCs in the future.

肿瘤干细胞是结直肠癌浸润转移和复发的主要原因之一。上皮间质转化（EMT）是结直肠癌干细胞主要特性之一，EMT还促进结直肠癌细胞去分化和重新获得干细胞特性。抑制结直肠癌干细胞EMT能力是靶向清除结直肠癌干细胞的重要手段，然其调控机制有待明了。我们先前用逆转录病毒文库从结直肠癌干细胞中鉴定了一个新分子Ankrd22，发现沉默Ankrd22可显著增加结直肠癌干细胞比例，表达Ankrd22则通过抑制Smad2入核而下调TGF-β通路活性，进展期结直肠癌Ankrd22表达明显降低，提示Ankrd22是一个新的结直肠癌干细胞EMT抑制因子，然其抑制EMT分子机制尚不明了。本研究拟在先前基础上用双标签亲和串联质谱方法筛选鉴定Ankrd22靶向Smad2抑制EMT的关键作用靶蛋白，以明确Ankrd22通过EMT抑制结直肠癌干细胞特性的分子机制。本研究将为今后研制清除肿瘤干细胞新的分子靶向药物打下基础。

肿瘤干细胞是结直肠癌浸润转移和复发的主要原因之一。我们先前用逆转录病毒文库从结直肠癌干细胞中鉴定了一个新分子ANKRD22，先前初步研究提示ANKRD22可能是结直肠癌干细胞上皮间质转化抑制因子。在本研究过程中我们发现ANKRD22是一个核编码的线粒体定位膜蛋白，在结直肠癌干细胞中也可入核，受多种肿瘤微环境因素诱导经p38/MAX通路调控在结直肠癌干细胞中高表达。ANKRD22通过与有氧糖酵解和ATP合成通路中PDK1等多个靶点相互作用促进结直肠癌细胞有氧糖酵解和ATP产生减少；也可与E-Syt1协同将过量脂质转运到线粒体导致结直肠癌干细胞线粒体脂质异常堆积和数量减少；最后我们发现p53是核内ANKRD22的作用靶点。上述研究结果提示ANKRD22介导的结直肠癌干细胞葡萄糖和脂质代谢重编程可能是肿瘤微环境促进结直肠癌干细胞发生存留的一个新机制，E-Syt1作为ANKRD22将脂质转运到线粒体的关键分子，可能是一个结直肠癌干细胞干预的潜在作用靶点。