Peptidylarginine deiminase IV (PADI4) has a novel tumor cell-autonomous suppressor role in regulating breast cancer stem cells [RNA-Seq_1]. Peptidylarginine deiminase IV (PADI4) has a novel tumor cell-autonomous suppressor role in regulating breast cancer stem cells [RNA-Seq_1]

Peptidylarginine deiminases (PADIs) catalyze post-translational modification of many target proteins and have been suggested to play a role in carcinogenesis. Since citrullination of histones by PADI4 was recently implicated in regulating embryonic stem and hematopoietic progenitor cells, here we investigated a possible role for PADI4 in regulating breast cancer stem cells. We showed by genetic and pharmacologic approaches that PADI4 activity limits the number of cancer stem cells (CSCs) in vitro and in vivo in multiple breast cancer models. A gene signature reflecting tumor cell-autonomous PADI4 inhibition is associated with poor outcome in human breast cancer datasets, consistent with a tumor suppressive role for PADI4. Mechanistically, PADI4 inhibition resulted in a global redistribution of histone H3 with accumulation around transcriptional start sites. Interestingly, epigenetic effects of PADI4 on the bulk tumor cell population did not explain the CSC phenotype. However, in sorted tumor cell populations, PADI4 down-regulated expression of the master transcription factors of stemness, NANOG and POU5F1, specifically in the cancer stem cell compartment, by reducing the transcriptionally activating H3R17me2a histone mark at those loci. This effect was not seen in the non-stem cells. Our findings reveal a novel role for PADI4 as a tumor suppressor in regulating breast cancer stem cells, and provide insights into context-specific effects of PADI4 in epigenetic modulation. Overall design: Cells were cultured in DMEM/F12 media supplemented with 5% horse serum. Cells were treated with PADI4 inhibitor (GSK484) or control compound (GSK107) for 72 hours.

肽基精氨酸脱亚胺酶（Peptidylarginine deiminases, PADIs）可催化多种靶蛋白的翻译后修饰，且被认为在肿瘤发生过程中发挥作用。鉴于近期研究表明肽基精氨酸脱亚胺酶4（Peptidylarginine deiminase 4, PADI4）介导的组蛋白瓜氨酸化修饰参与调控胚胎干细胞与造血祖细胞，本研究探讨了PADI4在乳腺癌干细胞调控中的潜在作用。本研究通过遗传学与药理学手段证实，在多种乳腺癌模型中，PADI4的活性可在体外及体内限制癌症干细胞（Cancer Stem Cells, CSCs）的数量。

反映肿瘤细胞自主性PADI4抑制状态的基因特征，与人类乳腺癌数据集所对应的不良预后显著相关，这与PADI4的肿瘤抑制作用相一致。从机制层面来看，PADI4抑制可导致组蛋白H3发生全局重分布，并在转录起始位点周围出现富集。值得注意的是，PADI4对整体肿瘤细胞群体的表观遗传调控效应，无法解释癌症干细胞的表型特征。然而，在分选获得的肿瘤细胞亚群中，PADI4仅在癌症干细胞区间内，通过降低这些基因座上的转录激活型组蛋白标记H3R17me2a，下调干性核心转录因子NANOG与POU5F1的表达；该效应在非干细胞亚群中并未观察到。

本研究结果揭示了PADI4作为肿瘤抑制因子调控乳腺癌干细胞的全新功能，为理解PADI4在表观遗传调控中的情境特异性效应提供了新的研究视角。
实验整体设计：将细胞培养于添加5%马血清的DMEM/F12培养基中，使用PADI4抑制剂GSK484或对照化合物GSK107处理72小时。