hCINAP在肿瘤细胞代谢和增殖中的调节作用及分子机制研究

Warburg effect or aerobic glycosis is known as the most common metabolic phenotype of cancer cells. Even under sufficient oxygen condition, cancer cells prefer to use glycolysis instead of oxidative phosphorylation. A significant amount of evidence demonstrates that upregulated glycosis is correlated to activated oncogenes, and which stimulate the proliferation of cancer cells. Therefore, Warburg effect is considered as a hallmark of cancer. Despite the fact that increased glycolysis is correlated with upregulated biosynthetic metabolism, the detailed molecular mechanisms how increased glycosis is regulated are still not well understood. Therefore, identification of novel regulators involved in Warburg effect and study the molecular mechanism attract great attenuations for scientists and has important biological significance. Our previous study showed that ATPase/AK activity containing hCINAP is highly expressed in cancers, which promotes tumorigenesis and correlates with poor cancer patient survival. Both in vitro and in vivo experiments revealed that depletion of hCINAP significant inhibited tumorigenesis. And our preliminary work has shown that hCINAP may interact with some critical enzymes in aerobic glycolysis and is essential to the enzyme activities. Moreover, homozygous CINAP-/- mice show embryonic lethality. Based on our new findings, by using hCINAP liver-specific knockout mice together with various techniques of molecular biology, biochemistry, cell biology and structural biology, we aim to identify more partners of hCINAP, investigate the regulatory functions and underlying mechanisms of hCINAP in Warburg effect and tumor cell proliferation, and understand the correlation to tumorigenesis. These studies will contribute to the overall understanding of aerobic glycosis and tumorigenesis, and provide effective target for cancer therapy and theoretical basis for drug design.

Warburg效应是肿瘤细胞代谢的标志性特征，肿瘤细胞代谢的异常调控显著影响肿瘤细胞的增殖。但有关肿瘤细胞代谢调控的分子机制有待于深入研究。因此，鉴定肿瘤细胞代谢新的调控因子，揭示其对肿瘤细胞增殖的影响及分子机制具有重要意义。我们的前期研究发现，具有ATPase/AK活性的蛋白hCINAP在肿瘤组织中高表达，与癌症病人的预后密切相关，敲低hCINAP能够明显抑制裸鼠肿瘤的生长。并且，hCINAP与乳酸脱氢酶等相互作用并明显影响其酶的活性。该基因的全敲除导致小鼠胚胎死亡。在此基础上，本课题将利用hCINAP肝脏组织特异性敲除和hCINAP+/-小鼠观察hCINAP的缺失所引起的代谢表型和成瘤的变化。同时利用生化、分子、细胞和结构生物学等方法，鉴定更多受hCINAP调控的蛋白，深入地研究hCINAP在肿瘤细胞代谢、细胞增殖和肿瘤发生发展中的调控作用和分子机理，为寻找新的肿瘤治疗靶点提供有用信息

Warburg效应是肿瘤细胞代谢的标志性特征，肿瘤细胞代谢的异常调控显著影响肿瘤细胞的增殖。但有关肿瘤细胞代谢调控的分子机制有待于深入研究。因此，鉴定肿瘤细胞代谢新的调控因子，揭示其对肿瘤细胞增殖的影响及分子机制具有重要意义。本项目的研究发现，具有ATPase/AK活性的蛋白hCINAP在肿瘤组织中高表达，与癌症病人的预后密切相关，敲低hCINAP能够明显抑制裸鼠肿瘤的生长。我们揭示了hCINAP调控肿瘤发生发展的分子机理。通过co-IP及质谱分析，我们发现hCINAP与Warburg效应中关键酶乳酸脱氢酶（LDHA）相互作用，能够促进FGFR1催化的LDHA的第10位酪氨酸的磷酸化，进而影响LDHA酶的活性和肿瘤代谢效率。hCINAP的敲低抑制了结直肠癌干细胞的成瘤，使CD133和LGR5双阳性的结直肠癌干细胞减少，使结直肠癌干细胞通过自我更新形成肿瘤克隆的能力降低，抑制了结直肠癌干细胞标志物基因CD133等的表达，增强了表皮化基因的表达，减弱了间充质化基因的表达，抑制了肿瘤转移相关的基质金属蛋白酶基因的表达，减弱了结直肠癌干细胞对5-氟尿嘧啶或奥沙利铂等化疗药物的抗药性。说明hCINAP在肿瘤中的过表达可以促进结直肠癌干细胞的干性维持。hCINAP能够促进结直肠癌干细胞中的Warburg效应，提高结直肠癌干细胞对代谢压力的抵抗性，进而抑制结直肠癌细胞的凋亡。但hCINAP的敲低不会引起正常的结肠干细胞的凋亡，暗示靶向hCINAP的结直肠癌治疗中产生的副作用会较小。而且，我们构建了hCINAP肝脏特异性敲除小鼠，发现其在高脂饲养条件下总胆固醇水平下降明显，肝脏结构异常，核糖体合成受到抑制。通过免疫共沉淀-质谱的方法鉴定hCINAP与影响胆固醇代谢的酶具有相互作用。目前正在对其分子机制进行深入研究。此外，我们还鉴定了PKM2的去泛素化酶，对其调控PKM2活性以及肿瘤细胞代谢以及肿瘤发生发展的分子机制开展了深入研究。这些研究揭示了hCINAP及Warburg效应重要调控因子在肿瘤细胞代谢、细胞增殖和肿瘤发生发展中的调控作用和分子机理，为寻找新的肿瘤治疗靶点提供了有用信息。