基于转录组测序与代谢组学分析探讨干扰“瓦博格效应”对乳腺癌的影响

Abnormal energy metabolism is one of the remarkable features of tumor, and glucose metabolism abnormalities are the worst. Normal cells live routinely: in the absence of oxygen, glucose is degraded by glycolysis and converted to lactate, whereas oxidative phosphorylation in aerobic condition, which inhibits the former. On the contrary, rebellious tumor cells utilize glucose by glycolysis even if oxygen is sufficient, which is famous as “Warburg effect”. Recently, “Warburg effect” has drawn great attention of researchers, and even some scholars believe that it may be the "Achilles' heel" of the tumor. M2 type of pyruvate kinase (PKM2) is one of the key enzymes in glycolysis. More and more studies show that it high expresses in tumor, and is closely related to the genesis and development of tumor, so PKM2 may be one of the important targets for cancer research. However, we found that blocking “Warburg effect” did not inhibit the occurrence and development of breast cancer completely, but induced autophagy to protect tumor cells. So, in addition to autophagy, whether did tumor cells also employ other ways to fight against the difficulties, such as accelerating the pentose phosphate bypass and serine synthesis pathway, changing the Krebs cycle, fatty acid, nucleic acid or amino acid metabolism? And what is the mechanism?In view of this, we want to investigate what and how does breast cancer cell employ to defend the blocking of Warburg effect, which is based on RNA-sequencing and metabonomics.

能量代谢异常是肿瘤的显著特征，而糖代谢异常又居其之首。正常细胞无氧行糖酵解，有氧则行有氧氧化，后者抑制前者。肿瘤细胞却我行我素：即使氧充足也糖酵解，即“瓦博格效应”。“瓦博格效应”近来引起研究者的极大关注，被看作是肿瘤的“阿基里斯之踵”。M2型丙酮酸激酶（PKM2）是“瓦博格效应”的关键酶之一，高表达于大多数恶性肿瘤，是抗肿瘤研究的重要靶点之一。然而，我们前一项目的研究发现干扰“瓦博格效应”并不能完全抑制乳腺癌的发生与发展，而且肿瘤细胞还可能启动自噬来保护自己。那么，此情此景下，除了启动自噬外，肿瘤细胞是否还雇佣了其他方式（加速磷酸戊糖旁路和丝氨酸合成通路，改变三羧酸循环、脂肪酸、核酸或氨基酸代谢水平等）以渡此劫？其分子机制又是什么？鉴于此，本项目拟通过转录组测序结合代谢组学来探讨“瓦博格效应”被阻断后，乳腺癌细胞启动的防御措施及其基因-代谢调控网络机制，以期为阐明肿瘤的发生发展提供依据。

近来，“瓦博格效应”引起研究者的极大关注，越来越多的学者认为肿瘤就是一种代谢疾病，而“瓦博格效应”可能就是肿瘤的“阿基里斯之踵”，擒获它便扼住了肿瘤的命门。因此，我们前一个项目以乳腺癌为研究对象，运用RNAi方法沉默PKM2，拟从切断肿瘤获取能量来源的角度去探讨肿瘤的发生发展问题。结果发现PKM2被敲低后，肿瘤细胞的增殖、凋亡、侵袭与转移能力和体内肿瘤生长能力等只有部分被削弱。原因何在？我们认为：肿瘤细胞虽以糖酵解作为主要能源供应，但一旦该途径被切断，有可能另辟途径以逃避死亡，如加速磷酸戊糖旁路、脂肪酸或氨基酸分解代谢以供能。为了证实这一想法便申请了本项目。本项目首先采用CRISPR/Cas9技术构建PKM2敲除的稳定细胞株，并观察其表型变化，结果与之前的一致。为了探讨其中可能的机制，我们进行了细胞转录组测序，结果发现PKM2被敲除后，细胞基因表达谱出现很大变化，对这些差异基因进行KEGG pathway聚类，发现它们主要聚集在细胞生长和死亡的细胞过程、癌症疾病以及物质代谢过程；后续的细胞代谢组测序结果发现差异代谢物富集在丙酮酸代谢、脂类代谢、糖异生、糖酵解和胰岛素调控的代谢通路，除了影响糖代谢途径外，还影响了氨基酸代谢通路。其中，令我们最感兴趣的是PKM2敲除后，细胞脂质代谢出现较大的变化，脂肪酸β氧化过程中重要的酶ACADVL上调，而脂类合成代谢相关的酶MVD、IDI1等却下调。这表明PKM2敲除后，细胞可能通过上调脂肪酸β氧化水平来弥补细胞能量的短缺。