背景:肝内胆管癌(ICCA)是一类异质性恶性肿瘤,其特点是复发率高,预后差。异染色质蛋白1α(HP1α)是通过异染色质形成和结构维持参与转录沉默的最重要的非组蛋白染色体蛋白之一。HP1α对ICCA进展的影响尚不清楚。
方法:通过在两种细胞系和两种ICCA小鼠模型中的实验来检测对ICCA增殖的影响。使用电喷雾电离质谱(ESI-MS)确定HP1α与组蛋白脱乙酰酶1(HDAC1)之间的相互作用,并使用免疫沉淀测定(co-IP)研究结合机制。通过RNA测序(RNA-seq)筛选出目标基因。通过生物信息学方法预测DNA结合蛋白和组蛋白修饰的占用,并通过靶标和标签下的裂解(CUT&Tag)和染色质免疫沉淀(ChIP)进行评估。
结果:HP1α在肝内胆管癌(ICCA)组织中上调,并通过抑制干扰素途径,以信号转导和转录激活因子1(STAT1)依赖性方式调节ICCA细胞的增殖。机械上,STAT1由HP1α-HDAC1复合物直接和表观遗传地通过启动子结合和不同组蛋白修饰的变化进行转录调节,通过高通量测序验证。广谱HDAC抑制剂(HDACi)通过下调HP1α和靶向异源二聚体激活干扰素途径并抑制ICCA细胞的增殖。发现广谱HDACi加干扰素制备方案可改善体内和体外的抗增殖作用并延迟ICCA的发展,它利用了基础激活以及干扰素途径的直接激活。HP1α参与介导对两种试剂的细胞抗性。
结论:HP1α-HDAC1复合物通过在转录水平上直接和表观调节STAT1影响干扰素途径的激活。广谱HDACi加干扰素制剂方案抑制ICCA发展,为ICCA治疗提供可行的策略。靶向HP1α-HDAC1-STAT1轴是治疗ICCA的可能策略,尤其是HP1α阳性病例。
BACKGROUND: Intrahepatic cholangiocarcinoma (ICCA) is a heterogeneous group of malignant tumors characterized by high recurrence rate and poor prognosis. Heterochromatin Protein 1α (
HP1α) is one of the most important nonhistone chromosomal proteins involved in transcriptional silencing via heterochromatin formation and structural maintenance. The effect of
HP1α on the progression of ICCA remained unclear.
METHODS: The effect on the proliferation of ICCA was detected by experiments in two cell lines and two ICCA mouse models. The interaction between HP1α and Histone Deacetylase 1 (HDAC1) was determined using Electrospray Ionization Mass Spectrometry (ESI-MS) and the binding mechanism was studied using immunoprecipitation assays (co-IP). The target gene was screened out by RNA sequencing (RNA-seq). The occupation of DNA binding proteins and histone modifications were predicted by bioinformatic methods and evaluated by Cleavage Under Targets and Tagmentation (CUT & Tag) and Chromatin immunoprecipitation (ChIP).
RESULTS: HP1α was upregulated in intrahepatic cholangiocarcinoma (ICCA) tissues and regulated the proliferation of ICCA cells by inhibiting the interferon pathway in a Signal Transducer and Activator of Transcription 1 (STAT1)-dependent manner. Mechanistically, STAT1 is transcriptionally regulated by the HP1α-HDAC1 complex directly and epigenetically via promoter binding and changes in different histone modifications, as validated by high-throughput sequencing. Broad-spectrum HDAC inhibitor (HDACi) activates the interferon pathway and inhibits the proliferation of ICCA cells by downregulating HP1α and targeting the heterodimer. Broad-spectrum HDACi plus interferon preparation regimen was found to improve the antiproliferative effects and delay ICCA development in vivo and in vitro, which took advantage of basal activation as well as direct activation of the interferon pathway.
HP1α participates in mediating the cellular resistance to both agents.
CONCLUSIONS: HP1α-HDAC1 complex influences interferon pathway activation by directly and epigenetically regulating STAT1 in transcriptional level. The broad-spectrum HDACi plus interferon preparation regimen inhibits ICCA development, providing feasible strategies for ICCA treatment. Targeting the
HP1α-HDAC1-STAT1 axis is a possible strategy for treating ICCA, especially
HP1α-positive cases.