线粒体AMPK抑制Warburg效应

AMP-activated protein kinase (AMPK) is a cellular fuel sensor that is activated under stress, leading to suppression of ATP- consuming anabolic processes and stimulation of catabolism and ATP generation. AMPK is currently a research focus of cancer biology. The metabolism of cancer cells is featured by glycolysis, a major energy generating mechanism even under aerobic circumstances. This phenomenon is called Warburg effect, which is nowadays regarded a therapeutic target for cancer. Only a few papers described the regulation of Warburg effect by AMPK, with opposing effects. Our preliminary data showed that AMPK was localized to mitochondria. We employed genetic engineering to target an active variant of AMPK to mitochondria and found that this AMPK variant inhibited glycolysis, stimulated mitochondrial fatty acid oxidation, and attenuated the growth of cancer cells in vitro and Xenograft tumor in vivo. Collectively, our data support a negative effect of AMPK on Warburg phenomenon. Therefore, in this grant application, we aim to carry out the studies in depth and width. We will (1) explore the physiological, pathological and pharmacological conditions for AMPK to migrate to mitochondria, (2) assess the effects of AMPK localization to mitochondria on cellular energy metabolism and mitochondrial function, (3) identify steps and partners/targets of AMPK action on mitochondrial respiratory chain and (4), determine the inhibitory effect of mitochondrial AMPK on tumorigenesis and tumor progression. Altogether, our studies would consolidate our argument that AMPK serves as a metabolic tumor suppressor and help to clarify the controversial role of AMPK in Warburg effect. In addition, since metformin, a pharmacological activator of AMPK, is being used for clinical trials of various cancers, the completion of our studies will provide guide and strategy for these trials and present new targets for cancer therapy.

AMPK 为细胞能量感受器，在肿瘤发生中的作用已成为当前肿瘤生物学的研究重点。肿瘤细胞的代谢特点是其能量主要来源于糖酵解，即使在有氧条件下，这一途径也占优势，该现象称为Warburg效应，被认为是肿瘤治疗的新靶点。仅少数几篇文献报道AMPK对Warburg效应的调节作用，但结论相反。我们的前期工作发现AMPK定位于细胞的线粒体，将AMPK的活性突变体靶向定位于肺癌细胞A549的线粒体能抑制糖酵解，刺激脂肪酸氧化，抑制细胞生长和异体移植肿瘤形成。因此，我们的初步结果支持AMPK对Warburg效应的抑制作用。本课题将进一步探索AMPK向线粒体迁移的生理、病理和药理学条件，研究AMPK与线粒体结合对细胞能量代谢和线粒体功能的影响，寻找AMPK在线粒体的作用位点和靶分子，确立AMPK线粒体定位对肿瘤发生发展的抑制作用。该研究课题有助于澄清关于AMPK对Warburg效应作用的争议。

肿瘤细胞的代谢特点是其能量来源于糖酵解，即使在有氧条件下，这一途径也占优势，该现象称为Warburg效应。而目前认为，阻断Warburg效应是肿瘤治疗的新策略。AMPK作为细胞能量感受器，其在肿瘤能量代谢中的作用备受关注，但是仅少数几篇文献报道AMPK对Warburg效应的调节作用，且结论相反。为了明确AMPK对Warburg效应的调节作用及其机制，本课题组开展了一系列的研究。首先，本课题组采用免疫荧光、免疫印迹证实了AMPKα和β亚基均定位于线粒体内，并通过构建一系列的AMPKα1变异体cDNA，明确了AMPKα在线粒体中的结合位点包涵于AMPKα1亚单位羧基段的AA313至548之间，且AMPKα与线粒体的结合不依赖于AMPKβ亚基。其次，构建了表达线粒体靶向定位的AMPK活性突变体的稳定细胞株，探究线粒体AMPK对肺癌生物行为学和能量代谢的影响。结果显示，线粒体靶向定位的AMPK活性突变体抑制肿瘤的生长、克隆形成和成瘤能力，抑制糖酵解和促进脂肪酸氧化。敲除AMPKβ亚基则完全抵消AMPK的效应，促进肿瘤生长、克隆形成和迁移能力，同时会导致线粒体DNA拷贝数降低，而表达AMPK活性突变体可以逆转β亚基敲除的效应。有趣的是，线粒体靶向定位的AMPK活性突变体并不能增加线粒体数目。进一步的研究发现，LKB1/AMPK可以通过上调PGC1α和Nrf-1的表达促进线粒体生物合成，而AMPKβ亚基敲除抑制线粒体生物合成。此外，本研究还发现，AMPKβ亚基敲除后，AMPKα亚基的表达下降，机制研究表明，AMPKβ亚基通过调控溶酶体自噬途径调控AMPKα的蛋白稳定性。该研究为肿瘤治疗提供新的位点和思路，也为AMPK激活剂(二甲双胍)治疗肿瘤提供了有力的理论和实验依据。