ANKLE1 digests mitochondrial DNA and contributes to cancer risk by driving the Warburg effect and apoptosis resistance

Alleles within the chr19p13.1 locus are associated with increased risk of both ovarian and breast cancer and increased expression of the ANKLE1 gene. ANKLE1 is molecularly characterized as an endonuclease that shuttles between the nucleus and cytoplasm and efficiently cuts branched DNA. However, the role of ANKLE1 in mammalian development and homeostasis remains unknown. In normal development ANKLE1 expression is limited to the erythroblast lineage and we found that ANKLE1's role is to cleave the mitochondrial genome during erythropoiesis. We show that ectopic expression of ANKLE1 in breast epithelial-derived cells leads to genome instability and mitochondrial DNA (mtDNA) digestion. mtDNA cleavage then leads to mitophagy and causes a shift from oxidative phosphorylation to glycolysis (Warburg effect). Moreover, mtDNA degradation activates STAT1 and expression of epithelial-mesenchymal transition (EMT) genes. Reduction in mitochondrial content contributes to apoptosis resistance, which may allow precancerous cells to avoid apoptotic checkpoints and proliferate. These findings provide evidence that ANKLE1 is the causative gene in the chr19p13.1 locus and provides mechanisms by which higher ANKLE1 expression promotes cancer risk. Overall design: RNA-seq on 22 samples; control pEGFP-C1 or pEGFP-C1-ANKLE1 overexpressing HEK293T cells; 1, 3 and 7 days after transfection; 4 biological replicates each for Day 1; 4 biological replicates each for Day 3; 3 biological replicates each for Day 7

19号染色体短臂1区3带1亚区（chr19p13.1）位点内的等位基因与卵巢癌、乳腺癌的发病风险升高及ANKLE1基因（ANKLE1）的表达上调显著相关。ANKLE1在分子层面被鉴定为一种可在细胞核与细胞质间穿梭转运的核酸内切酶，能够高效切割分支型DNA。然而，其在哺乳动物发育与稳态维持中的具体功能仍未明确。在正常生理发育过程中，ANKLE1的表达仅局限于成红血细胞谱系；本研究发现，ANKLE1在红细胞生成过程中的功能为切割线粒体基因组。
研究证实，在乳腺上皮来源的细胞中异位表达ANKLE1，会引发基因组不稳定及线粒体DNA（mtDNA）降解。mtDNA的切割可进一步诱导线粒体自噬，并使细胞代谢模式从氧化磷酸化转向糖酵解（瓦伯格效应，Warburg effect）。此外，mtDNA降解会激活信号转导与转录激活因子1（STAT1），并上调上皮-间质转化（EMT）相关基因的表达。线粒体含量的降低会使细胞获得凋亡抵抗能力，这或许可使癌前细胞规避凋亡检查点并持续增殖。
上述研究结果证实，ANKLE1正是chr19p13.1位点的致病基因，并阐明了ANKLE1高表达促进癌症发病风险的潜在分子机制。
整体实验设计：对22份样本进行RNA测序；实验分组为转染空载体pEGFP-C1的对照组，以及转染过表达载体pEGFP-C1-ANKLE1的HEK293T细胞；分别于转染后第1、3、7天收集样本；其中第1天、第3天各组均设4次生物学重复，第7天各组均设3次生物学重复。