Nuclear lamin A/C phosphorylation by loss of Androgen Receptor is a global determinant of cancer-associated fibroblast activation [ChIP-Seq2]. Nuclear lamin A/C phosphorylation by loss of Androgen Receptor is a global determinant of cancer-associated fibroblast activation [ChIP-Seq2]

Alterations of nuclear structure and function, and associated impact on gene transcription, are a hallmark of cancer cells. Little is known of these alterations in Cancer-Associated Fibroblasts (CAFs), a key component of the tumor stroma. Here we show that loss of androgen receptor (AR), which triggers early steps of CAF activation in human dermal fibroblasts (HDFs), leads to nuclear membrane alterations with increased micronuclei formation and frequent ruptures, with similar alterations occurring in fully established CAFs. AR associates with nuclear lamin A/C and loss of AR results in a substantially increased lamin A/C nucleoplasmic redistribution. Mechanistically, AR functions as a bridge between lamin A/C with the protein phosphatase PPP1. In parallel with a decreased lamin-PPP1 association, AR loss results in a marked increase of lamin A/C phosphorylation at Ser 301, which is also a feature of CAFs. Phospho-Ser301 Lamin A/C binds to the transcription promoter regulatory region of multiple CAF marker genes upregulated by AR loss, and expression of a lamin A/CSer301 phosphomimetic mutant is sufficient for conversion of normal fibroblasts into tumor-promoting CAFs. The findings point to an AR - lamin A/C - PPP1 axis and lamin A/C phosphorylation at ser 301 as critical determinants of CAF activation. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) of total Lamin A/C in HDF cells with and without AR silencing.

细胞核结构与功能的改变及其对基因转录的影响，是癌细胞的标志性特征。而作为肿瘤基质关键组分的癌症相关成纤维细胞（Cancer-Associated Fibroblasts, CAFs），其相关改变却鲜有研究。本研究证实，雄激素受体（androgen receptor, AR）缺失可触发人类真皮成纤维细胞（human dermal fibroblasts, HDFs）中CAF激活的早期进程，同时引发核膜异常：微核形成增多且核膜频繁破裂，这一表型在完全活化的CAFs中同样存在。AR可与核纤层蛋白A/C（nuclear lamin A/C）结合，AR缺失则会显著促进核纤层蛋白A/C在核质中的重新分布。机制层面，AR充当了核纤层蛋白A/C与蛋白磷酸酶PPP1（protein phosphatase PPP1）之间的桥梁。伴随核纤层蛋白与PPP1结合水平的下降，AR缺失会导致核纤层蛋白A/C在Ser301位点的磷酸化水平显著升高，这一特征同样见于CAFs中。磷酸化Ser301的核纤层蛋白A/C可结合AR缺失后上调的多个CAF标志物基因的转录启动子调控区域，而表达lamin A/C Ser301磷酸化模拟突变体，足以将正常成纤维细胞转化为促肿瘤型CAFs。本研究结果揭示，AR-核纤层蛋白A/C-PPP1轴以及核纤层蛋白A/C的Ser301位点磷酸化，是CAF激活的关键决定因素。整体实验设计：对经AR沉默处理与未处理的HDF细胞，开展总核纤层蛋白A/C的染色质免疫沉淀测序（Chromatin immunoprecipitation DNA-sequencing, ChIP-seq）。