Genome-wide location analysis in breast cancer cell-lines. Homo sapiens

Recurrent mutations in histone modifying enzymes in multiple cancer types imply key roles in tumorigenesis. However, the functional relevance of these mutations remains unknown. Here we show that the JARID1B histone H3 lysine 4 demethylase is frequently amplified and overexpressed in luminal breast tumors and a somatic point mutation of JARID1B leads to the gain of luminal-specific gene expression programs. Downregulation of JARID1B in luminal breast cancer cells induces the expression of basal cell-specific genes and growth arrest, which is partially rescued by the inhibition of TGFBR thereby indicating a key role for TGFb signaling. Integrated genome-wide analysis of JARID1B chromatin binding, histone H3 lysine trimethyl (H3K4me3) and dimethyl (H3K4me2) patterns, and gene expression profiles in luminal and basal-like breast cancer cells suggest a key role for JARID1B in luminal cell-specific gene expression programs. A significant fraction of JARID1B binding-sites overlaps with CTCF in both luminal and basal-like breast cancer cells. CTCF also co-immunoprecipitates with JARID1B and it may influence its histone demethylase (HDM) activity as the H3K4me3/me2 ratio is lower at the CTCF-overlapping compared to JARID1B-unique sites. Additionally, a heterozygous JARID1B missense mutation (K1435R) in the HCC2157 basal-like breast cancer cell line is associated with unique JARID1B chromatin-binding and gene expression patterns implying gain of luminal features. In line with this, exogenous expression of this mutant in basal-like breast cancer cells leads to a gain of JARID1B binding at many luminal-specific genes. A PARADIGM score reflecting JARID1B activity in luminal breast cancer cells is associated with poor clinical outcome in patients with luminal breast tumors. Together, our data imply that JARID1B is a luminal lineage-driving oncogene and that its therapeutic targeting may represent a novel therapeutic strategy in treatment-resistant luminal breast tumors. Overall design: ChIP-Seq on JARID1B, H3K4me2, H3K4me3, CTFC, and input on breast cancer cell-lines. 50 cycles of sequencing on Illumina platform in 6 cell-lines.

多种癌症类型中组蛋白修饰酶（histone modifying enzymes）的复发性突变（recurrent mutation）提示其在肿瘤发生（tumorigenesis）中发挥关键作用。然而，此类突变的功能相关性仍未明确。
本研究显示，组蛋白H3赖氨酸4去甲基化酶（histone H3 lysine 4 demethylase）JARID1B在腔面型乳腺肿瘤（luminal breast tumors）中频繁发生扩增与过表达，且JARID1B的体细胞点突变（somatic point mutation）可诱导腔面特异性基因表达程序（luminal-specific gene expression programs）的激活。在腔面型乳腺癌细胞中下调JARID1B的表达，会诱导基底细胞特异性基因（basal cell-specific genes）的表达与细胞生长停滞（growth arrest），而该表型可通过抑制转化生长因子β受体（transforming growth factor beta receptor, TGFBR）得到部分挽救，这提示转化生长因子β（transforming growth factor β, TGFβ）信号通路发挥关键作用。
通过对腔面型与基底样乳腺癌细胞（basal-like breast cancer cells）中JARID1B染色质结合（chromatin binding）情况、组蛋白H3赖氨酸4三甲基化（H3K4me3）与二甲基化（H3K4me2）修饰模式，以及基因表达谱（gene expression profiles）进行整合性全基因组分析（genome-wide analysis），本研究揭示JARID1B在腔面细胞特异性基因表达程序中扮演关键角色。在腔面型与基底样乳腺癌细胞中，JARID1B的结合位点（binding site）有相当比例与CCCTC结合因子（CCCTC-binding factor, CTCF）的结合位点重叠。CTCF可与JARID1B发生免疫共沉淀（co-immunoprecipitation），且可能调控其组蛋白去甲基化酶（histone demethylase, HDM）活性：相较于JARID1B独有的结合位点，在与CTCF重叠的结合位点处，H3K4me3/me2的比值更低。
此外，在HCC2157基底样乳腺癌细胞系（cell line）中，JARID1B存在一处杂合错义突变（heterozygous missense mutation）（K1435R），该突变与独特的JARID1B染色质结合模式及基因表达谱相关，提示该突变可赋予细胞腔面表型（luminal features）。
与此一致，在基底样乳腺癌细胞中外源表达（exogenous expression）该突变体（mutant），可使JARID1B在众多腔面特异性基因处的结合增加。
反映腔面型乳腺癌细胞中JARID1B活性的PARADIGM评分（PARADIGM score），与腔面型乳腺肿瘤患者的不良临床结局（clinical outcome）显著相关。
综上，本研究数据表明JARID1B是一种腔面谱系驱动致癌基因（lineage-driving oncogene），靶向JARID1B或可成为治疗耐药性（treatment-resistant）腔面型乳腺肿瘤的全新治疗策略。
实验整体设计：对6株乳腺癌细胞系开展JARID1B、H3K4me2、H3K4me3、CTCF（原文笔误为CTFC）及Input对照样本的染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-Seq），并在Illumina测序平台（Illumina platform）上进行50个循环的测序。