Characterization of FOXA1 mutations in breast cancer (ChIP-seq). Characterization of FOXA1 mutations in breast cancer (ChIP-seq)

Invasive Lobular Carcinoma (ILC) is the second most frequent breast cancer (BCa) type and encompasses 10-15% of BCa cases. FOXA1 specific mutations are enriched in this subtype of BCa, however their role in breast cancer pathogenesis is still ill-defined. FOXA1, together with estrogen receptor (ER), is a key transcription factor for the correct activation of estrogen-dependent gene expression and, consequently, for mammary gland development and BCa identity. FOXA1 has the capability to bind to and de-compact heterochromatin to render it accessible for other nuclear proteins, such as ER, and allow activation of their transcriptional programs upon estrogen stimulation. In this project, we aim to elucidate the role of FOXA1 missense mutations in altering its binding to DNA, chromatin accessibility, ER-dependent transcription and their implication in limiting the sensitivity to standard-of-care anti-hormonal therapy, commonly used in ER-positive BCa patients. To this end, we have generated BCa cell lines expressing these FOXA1 mutations and we will employ ATAC-Seq, RIME assays, ChIP-Seq and RNA-Seq to ascertain their effect on DNA accessibility, DNA binding capability, as well as binding of transcriptional coregulators, such as ER, to chromatin. We will extend our analyses to our internal BCa patient datasets with detailed clinical annotation to study the correlation between presence of FOXA1 mutations and response to anti-hormonal therapy of ER-positive BCa patients. The results of this project will allow to understand how the different mutations in the Forkhead domain can alter FOXA1 and ER function, transcriptional regulation and response to anti-hormonal therapy in ER-positive BCa patients. Overall design: ChIP-Seq analysis of MCF7 cells overexpressing either empty vector (EV), FOXA1 wild-type (WTNS) or FOXA1 mutant (SY242CS, H247Y, S250F, F266L) constructs, in duplicates.

浸润性小叶癌（Invasive Lobular Carcinoma, ILC）是第二高发的乳腺癌（Breast Cancer, BCa）亚型，约占所有乳腺癌病例的10%~15%。该亚型乳腺癌中富集FOXA1（Forkhead Box A1）特异性突变，但此类突变在乳腺癌发病机制中的作用仍未明确。
FOXA1与雌激素受体（Estrogen Receptor, ER）同为调控雌激素依赖基因表达的关键转录因子，进而对乳腺发育及乳腺癌表型维持发挥核心作用。FOXA1能够结合并松解异染色质，使其可被ER等其他核蛋白所访问，并在雌激素刺激下激活其转录程序。
本研究旨在阐明FOXA1错义突变如何改变其与DNA的结合能力、染色质开放性以及ER依赖的转录调控，并揭示其对ER阳性乳腺癌患者临床标准抗激素治疗敏感性的影响。为此，我们已构建携带此类FOXA1突变的乳腺癌细胞系，并将采用ATAC测序（ATAC-Seq）、RIME实验、染色质免疫共沉淀测序（ChIP-Seq）及RNA测序（RNA-Seq），以明确其对DNA开放性、DNA结合能力以及ER等转录辅因子与染色质结合的影响。
我们还将结合带有详细临床注释的内部乳腺癌患者数据集，分析FOXA1突变状态与ER阳性乳腺癌患者抗激素治疗应答之间的相关性。本项目的研究结果将有助于阐明叉头框结构域内的不同突变如何改变FOXA1与ER的功能、转录调控模式以及ER阳性乳腺癌患者对抗激素治疗的应答反应。
总体实验设计：对过表达空载体（Empty Vector, EV）、FOXA1野生型（WTNS）或FOXA1突变体（SY242CS、H247Y、S250F、F266L）构建体的MCF7细胞进行ChIP-Seq分析，每组设置双生物学重复。