ER-phagy and proteostasis defects create a pre-malignant injury state in Kras-mediated tumourigenesis (RNA-Seq)

Pancreatic acinar cells are surprisingly resistant to pre-malignant transformation by the mutant Kras oncogene. Here, reporter mice show that an early, transcriptionally-mediated effect of Kras activation is downregulation of ER-phagy. Subsequently, stochastic failure of ER-phagy correlates with pre-malignant transdifferentiation into acinar-ductal metaplasia (ADM). Genetic perturbation of ER-phagy, combined with proteomics and transcriptomics, reveals endogenous injury, micro-inflammation and ADM across the acinar epithelium. Phenotypically, this occurs alongside intra-acinar formation of distinctive insoluble aggregates of ER proteins, including the injury marker REG3B. Mechanistically, REG3B is shown to bind to the ER-phagy receptor CCPG1 to facilitate degradation. Strikingly, spatial transcriptomics shows that oncogenic Kras also drives this cell state in a highly stochastic fashion, again characterised by protein aggregation, partial ductal identity, evidence of injury and propensity for ADM. Importantly, expression of insoluble mutant REG3B shows that protein aggregate formation directly engenders this state. Pancreatic cancer can thus arise from stochastic proteostatic defects that are both made probable by, and co-operate with, oncogenic Kras. To investigate the transcriptional effect of loss of ER-phagy in murine pancreatic acinar cells, Ccpg1 was floxed in KC mice (LSL-KrasG12D; Pdx1-Cre). RNA was extracted from pancreata of 10-week-old mice, 8 KC Ccpg1+/+ an 8 KC Ccpg1Panc. Differential gene expression analyses were performed using data from bulk mRNA-Seq.胰腺腺泡细胞对突变型Kras癌基因诱导的癌前转化具有出人意料的抵抗性。本研究通过报告基因小鼠实验证实，Kras激活的早期转录介导效应为内质网自噬(ER-phagy)的下调。后续研究发现，内质网自噬的随机失活与癌前转分化为腺泡-导管化生(ADM)密切相关。通过内质网自噬的遗传扰动结合蛋白质组学与转录组学分析，研究人员在整个腺泡上皮中观察到内源性损伤、微炎症反应及腺泡-导管化生的发生。表型层面，该过程伴随腺泡内特征性内质网蛋白不溶性聚集体的形成，其中包括损伤标志物REG3B。机制层面上，研究证实REG3B可结合内质网自噬受体CCPG1以促进其降解。值得注意的是，空间转录组学分析显示，致癌性Kras同样以高度随机的方式诱导该细胞状态，该状态同样以蛋白质聚集、部分导管表型、损伤证据及腺泡-导管化生易感性为特征。尤为重要的是，不溶性突变型REG3B的表达实验证实，蛋白质聚集体的形成可直接诱导该细胞状态的产生。由此可见，胰腺癌可源于随机的蛋白稳态缺陷，而该缺陷既可由致癌性Kras诱发，又可与其协同发挥作用。为探究内质网自噬缺失对小鼠胰腺腺泡细胞的转录调控效应，研究人员在KC小鼠（LSL-KrasG12D；Pdx1-Cre）中构建了Ccpg1条件性敲除模型。研究人员从10周龄小鼠的胰腺组织中提取RNA，其中包括8只KC Ccpg1+/+小鼠与8只KC Ccpg1Panc小鼠。研究人员基于批量mRNA测序(bulk mRNA-Seq)数据开展了差异基因表达分析。