Autophagy-dependent cancer cells circumvent loss of the upstream regulator RB1CC1/FIP200 and loss of LC3 conjugation by similar mechanisms

Macroautophagy/autophagy degrades proteins and organelles to generate macromolecular building blocks. As such, some cancer cells are particularly dependent on autophagy. In a previous paper, we found that even highly autophagy-dependent cancer cells can adapt to circumvent autophagy inhibition. However, it remains unclear if autophagy-dependent cancer cells could survive the complete elimination of autophagosome formation. We extended our previous findings to show that knockout (KO) of both the upstream autophagy regulator <i>RB1CC1/FIP200</i> and the downstream regulator and mediator of LC3 conjugation, <i>ATG7</i>, strongly inhibits growth in highly autophagy-dependent cells within one week of editing. However, rare clones survived the loss of <i>ATG7</i> or <i>RB1CC1</i> and maintained growth even under autophagy-inducing conditions. Autophagy-dependent cells circumvent the complete loss of autophagy that is mediated by <i>RB1CC1</i> KO, similar to the loss of <i>ATG7</i>, by upregulating NFE2L2/NRF2 signaling. These results indicate that cancer cell lines could adapt to the complete loss of autophagy by changing their biology to adopt alternative ways of dealing with autophagy-mediated cellular functions. CGS: CRISPR growth score; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: Earl’s balanced salt solution; EEF2: eukaryotic translation elongation factor 2; FOXO3/FOXO3a: forkhead box O3; GFP: green fluorescent protein; KEAP1: kelch Like ECH associated protein 1; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NLS: nuclear localization signal; PCNA: proliferating cell nuclear antigen; PE: phosphatidylethanolamine; POLR2A: RNA polymerase II subunit A; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SNARE: soluble NSF attachment protein receptor; SQSTM1: sequestosome 1; STX17: syntaxin 17; TBHP: tert-butyl hydroperoxide; ULK1: unc-51 like autophagy activating kinase 1; ULK2: unc-51 like autophagy activating kinase 2; WT: wild type

巨自噬（macroautophagy，以下简称自噬）可降解蛋白质与细胞器，以生成大分子构筑单元。因此，部分癌细胞对自噬存在高度依赖。在既往研究中，我们发现即便是高度依赖自噬的癌细胞，也可通过适应性机制规避自噬抑制。然而目前仍不清楚，依赖自噬的癌细胞能否在自噬体（autophagosome）形成被完全阻断的情况下存活。
我们拓展了此前的研究结果，证实同时敲除（knockout，以下简称KO）上游自噬调控因子<i>RB1CC1/FIP200</i>，以及下游调控因子、微管相关蛋白1轻链3β（MAP1LC3B/LC3B）结合介导因子<i>ATG7</i>，可在基因编辑后一周内显著抑制高度依赖自噬的细胞的增殖。但极少数克隆可在<i>ATG7</i>或<i>RB1CC1</i>缺失后存活，且即便在自噬诱导条件下仍可维持增殖。
依赖自噬的细胞可通过上调核因子E2相关因子2（NFE2L2/NRF2）信号通路，来规避由<i>RB1CC1</i>敲除所介导的完全自噬缺失，这一机制与规避<i>ATG7</i>缺失的方式相似。上述结果表明，癌细胞系可通过改变自身生物学特性，采用替代途径以应对自噬介导的细胞功能，从而适应完全的自噬缺失。
CGS: CRISPR生长评分（CRISPR growth score）；CQ: 氯喹（chloroquine）；CRISPR: 成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats）；EBSS: 伊格尔平衡盐溶液（Earl’s balanced salt solution）；EEF2: 真核翻译延伸因子2（eukaryotic translation elongation factor 2）；FOXO3/FOXO3a: 叉头框蛋白O3（forkhead box O3）；GFP: 绿色荧光蛋白（green fluorescent protein）；KEAP1: Kelch样ECH相关蛋白1（kelch Like ECH associated protein 1）；KO: 敲除（knockout）；MAP1LC3B/LC3B: 微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta）；MEFs: 小鼠胚胎成纤维细胞（mouse embryonic fibroblasts）；NFE2L2/NRF2: 核因子红细胞2样2（nuclear factor, erythroid 2 like 2）；NLS: 核定位信号（nuclear localization signal）；PCNA: 增殖细胞核抗原（proliferating cell nuclear antigen）；PE: 磷脂酰乙醇胺（phosphatidylethanolamine）；POLR2A: RNA聚合酶II亚基A（RNA polymerase II subunit A）；PTEN: 张力蛋白同源缺失的磷酸酶（phosphatase and tensin homolog）；ROS: 活性氧（reactive oxygen species）；SNARE: 可溶性NSF附着蛋白受体（soluble NSF attachment protein receptor）；SQSTM1: 自噬衔接蛋白1（sequestosome 1）；STX17: 突触融合蛋白17（syntaxin 17）；TBHP: 叔丁基过氧化氢（tert-butyl hydroperoxide）；ULK1: UNC-51样自噬激活激酶1（unc-51 like autophagy activating kinase 1）；ULK2: UNC-51样自噬激活激酶2（unc-51 like autophagy activating kinase 2）；WT: 野生型（wild type）