Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 fusion OE iHep RNA-seq]. Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 fusion OE iHep RNA-seq]

Cell fate plasticity enables development, yet unlocked plasticity is a cancer hallmark. While transcription master regulators induce lineage-specific genes to restrict plasticity, it remains unclear whether plasticity is actively suppressed by lineage-specific repressors. Here, we computationally predict so-called safeguard repressors for 18 cell types that block phenotypic plasticity lifelong. We validated hepatocyte-specific candidates using reprogramming, revealing that Prospero homeobox protein 1 (PROX1) enhanced hepatocyte identity by direct repression of alternative fate master regulators. In mice, Prox1 was required for efficient hepatocyte regeneration after injury and was sufficient to prevent liver tumorigenesis. In line with patient data, Prox1 depletion caused hepatocyte fate loss in vivo and enabled the transition of hepatocellular carcinoma to cholangiocarcinoma. Conversely, overexpression promoted cholangiocarcinoma to hepatocellular carcinoma transdifferentiation. Our findings provide evidence for PROX1 as a hepatocyte-specific safeguard and support a model where cell type-specific repressors actively suppress plasticity throughout life to safeguard lineage identity and thus prevent disease. Overall design: RNA-seq of MEFs with overexpression of various Prox1 fusion proteins or controls, harvested after one week of 4in1-driven reprogramming to induced hepatocytes.

细胞命运可塑性（cell fate plasticity）是发育过程的核心基础，而解除抑制的细胞可塑性则是癌症的典型标志之一。尽管转录主调控因子可通过诱导谱系特异性基因表达以限制细胞可塑性，但目前仍不清楚细胞可塑性是否由谱系特异性阻遏因子主动抑制。本研究通过计算预测了18种细胞类型中可终身阻断表型可塑性的所谓“保护性阻遏因子”（safeguard repressors）。我们通过细胞重编程实验验证了肝细胞特异性候选保护性阻遏因子，结果发现Prospero同源盒蛋白1（Prospero homeobox protein 1, PROX1）可通过直接抑制其他命运的主调控因子，从而增强肝细胞身份特性。在小鼠模型中，Prox1是损伤后高效肝细胞再生所必需的，同时足以抑制肝脏肿瘤发生。结合患者样本数据，体内敲除Prox1会导致肝细胞命运丧失，并可促使肝细胞癌向胆管癌转变。反之，过表达Prox1则可促进胆管癌向肝细胞癌转分化。本研究结果证实PROX1是肝细胞特异性的保护性阻遏因子，并支持如下模型：细胞类型特异性阻遏因子可在生命全程中主动抑制细胞可塑性，以维持谱系身份特性，从而预防疾病发生。实验整体设计：将经4因子重编程诱导为诱导性肝细胞1周后的小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs），分别过表达不同Prox1融合蛋白或对照载体，随后进行RNA测序（RNA-seq）。