GRHL2 is a key lineage determining factor which collaborates with FOXA1 to establish a targetable collateral pathway in the setting of endocrine therapy-resistant breast cancer [ChIP-seq]. GRHL2 is a key lineage determining factor which collaborates with FOXA1 to establish a targetable collateral pathway in the setting of endocrine therapy-resistant breast cancer [ChIP-seq]

The estrogen receptor (ER) is expressed in the majority of luminal breast cancers and inhibition of its transcriptional activity with selective estrogen receptor modulators, selective estrogen receptor degraders and/or aromatase inhibitors is a standard approach used in the management of this disease. Despite the positive clinical impact of these interventions, de novo and acquired resistance limits the therapeutic lifespan of these classes of drugs. Considering what is known about the complex mechanisms that contribute to the development of resistance it is likely that further development of ER-modulators will yield only incremental improvements. Thus, with the view that resistance is inevitable, we undertook the development of a new approach to treat ER-positive breast cancer by identifying and exploiting targetable vulnerabilities that emerge in endocrine therapy resistant disease. Genomic discovery platforms, including DNASeq, ChIPSeq and RNASeq were used to assess the epigenome, targeting global transcription factor binding profile, and transcriptome in cellular models of endocrine therapy sensitive and resistant disease. DNASeq was first used to identify the chromatin state, with a focus on differences, between these two models. Motif enrichment analysis indicated FOXA1 was a candidate transcription factor influencing the chromatin architecture, which was consistent with previously published studies. This led to the examination of the FOXA1 chromatin binding profile in these models. FOXA1 has previously been described to bind at enhancers. Furthermore, the relative transcription activity of specific enhancers has been shown to be indicated by the epigenomic marks on histones flanking transcription factor binding sites. For this reason, we assessed the specific pattern of histone 3 lysine 4 methylation to confirm enhancer status and histone 3 lysine 27 acetylation as an indicator of transcriptional activity. The specific patterns and distribution of FOXA1 binding was then integrated with this epigenomic information to reveal a subset of enhancers that became activated and another subset that gained enhanced activation in the tamoxifen resistant setting relative to the tamoxifen sensitive model from which it was derived. These results were integrated with the differential transcriptome, or genes shown to be differential expressed based on RNASeq, in the TAMR model as compared to its parental cell line, MCF7-WS8, and confirmed that the active enhancers were in fact associated with genes that were expressed more highly, on average, in the TAMR model. Motif enrichment at these two subgroups of enhancers indicated that another transcription factor, GRHL2, likely interacts with FOXA1 at these active sites. These results were again integrated with the “differential transcriptome” based on RNASeq and confirmed that the active enhancers, and indicated an even stronger enrichment of genes that were expressed more highly, on average, in the TAMR model relative to the MCF7-WS8 model. The GRHL2 transcriptome was then further defined by both GRHL2 ChIPSeq as well as RNASeq by comparing TAMR samples in which downregulation of GRHL2 expression had been achieved via siRNA as compared to control siRNA sequences. The collection of these data defined a subset of genes, the GRHL2 dependent transcriptome, that demonstrated increased expression in TAMR. The results of these cell lines studies were corroborated by assessing the transcriptome of xenograft mouse models of endocrine therapy sensitive and resistant disease. Integrative analysis of these data identified a collateral ER-independent signaling pathway in endocrine therapy resistant tumors that converges upon and modulates the FOXA1 and GRHL2 cistrome/transcriptome. Overall design: ChIP-Seq profiling of key factors in WS8 and TAMR cells. All samples have triplicate biological replicates.

雌激素受体（estrogen receptor，ER）在大多数腔面型乳腺癌中表达，采用选择性雌激素受体调节剂、选择性雌激素受体降解剂及/或芳香化酶抑制剂抑制其转录活性，是该疾病临床管理的标准方案。尽管此类干预手段具有积极的临床价值，但原发耐药与获得性耐药限制了这类药物的治疗有效期。鉴于目前已知的耐药发生机制十分复杂，ER调节剂的进一步开发或许仅能带来有限的疗效提升。因此，鉴于耐药性不可避免，我们着手开发治疗ER阳性乳腺癌的新策略：即识别并利用内分泌治疗耐药性疾病中出现的可靶向治疗弱点。

我们使用包括DNA测序（DNASeq）、染色质免疫共沉淀测序（ChIPSeq）与RNA测序（RNASeq）在内的基因组发现平台，对内分泌治疗敏感与耐药性细胞模型的表观基因组、全局转录因子结合谱以及转录组进行评估。首先通过DNASeq分析两种模型的染色质状态差异，重点关注其特征性区别。基序富集分析显示，叉头框蛋白A1（forkhead box A1，FOXA1）是影响染色质结构的候选转录因子，这与既往已发表的研究结果一致。由此，我们进一步检测了这两种模型中FOXA1的染色质结合谱。

既往研究表明，FOXA1可结合于增强子区域。此外，转录因子结合位点侧翼的组蛋白表观遗传标记，可反映特定增强子的相对转录活性。基于此，我们检测了组蛋白H3赖氨酸4甲基化模式以确认增强子状态，并以组蛋白H3赖氨酸27乙酰化作为转录活性的指示指标。

将FOXA1结合的特定模式与分布特征与上述表观基因组信息整合后，我们发现，相较于其亲本他莫昔芬敏感细胞模型，在他莫昔芬耐药（tamoxifen resistant，TAMR）模型中存在一组被激活的增强子，以及另一组激活程度进一步提升的增强子。我们将上述结果与基于RNASeq得到的差异转录组（即TAMR模型相较于其亲本细胞系MCF7-WS8的差异表达基因）进行整合，证实这些活性增强子确实与TAMR模型中平均表达水平更高的基因相关。

对这两组增强子进行基序富集分析发现，另一转录因子颗粒头样转录因子2（grainyhead-like transcription factor 2，GRHL2）可能与FOXA1在这些活性结合位点发生相互作用。我们再次将该结果与基于RNASeq的差异转录组进行整合，证实这些活性增强子所关联的基因，在TAMR模型相较于MCF7-WS8模型中平均表达水平更高，且富集程度更为显著。

我们通过对比经小干扰RNA（small interfering RNA，siRNA）下调GRHL2表达的TAMR样本与对照siRNA处理的样本，结合GRHL2的ChIPSeq与RNASeq数据，进一步明确了GRHL2调控的转录组。上述数据共同定义了一组在TAMR模型中表达上调的基因，即GRHL2依赖型转录组。

我们通过检测内分泌治疗敏感与耐药性异种移植小鼠模型的转录组，验证了上述细胞系研究的结果。对这些数据的整合分析发现，在内分泌治疗耐药性肿瘤中存在一条不依赖于ER的旁路信号通路，该通路可作用并调控FOXA1与GRHL2的顺反组/转录组。

整体实验设计：对WS8与TAMR细胞中的关键因子进行ChIP-Seq测序分析，所有样本均设置三次生物学重复。