PO.MCB06.01 · 分子与细胞生物学
表观遗传CRISPR筛选鉴定出卵巢癌球体形成所必需的染色质调控因子
Epigenetic CRISPR screen identifies chromatin regulators essential for ovarian cancer spheroid formation
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：癌症干样细胞（CSC）促成高级别浆液性卵巢癌（HGSOC）的复发和化疗耐药。超低黏附（ULA）培养中的球体形成可模拟CSC的自我更新，然而维持这一三维表型的表观遗传调控因子和分子依赖性仍大部分未明确。
方法：在OVCAR-3球体中进行了一项靶向800个表观遗传和染色质调控因子的聚焦CRISPR敲除筛选。三维选择后的sgRNA富集鉴定出球体生长所必需的基因。通过评估球体形态、致密度、活力和增殖行为，在BRCA1野生型、BRCA1-LOH和BRCA1突变型细胞系中对最优候选基因进行功能验证。
结果：该筛选鉴定出一组高置信度的球体形成所需调控因子，包括DNA损伤反应基因（BRCA1、PARG）、多梳复合物成员（SUZ12、BMI1）、RNA结合蛋白（NCL、SYNCRIP）和细胞周期调控因子（CDK2、PBRM1）。这些命中基因汇聚于染色质重塑、组蛋白甲基化和转录调控通路，将表观遗传调控与DDR信号传导联系起来。功能实验揭示了显著的BRCA1依赖性：BRCA1-WT和LOH细胞系形成致密、有活力的球体，而BRCA1突变型细胞产生杂乱、松散聚集、过度增殖且结构完整性差的结构。这表明BRCA1对建立和维持三维干样组织是必需的，而非增殖本身。将CRISPR命中基因与表型结果整合，提示DDR因子、RNA结合调控因子和染色质修饰因子之间存在协作网络，共同维持CSC样行为。
结论：我们的发现表明，BRCA1功能支撑了球体结构的表观遗传和染色质调控，揭示了DNA修复保真度、染色质完整性和癌症干性之间的机制联系。所鉴定的弱点突显了BRCA1缺陷型卵巢癌和乳腺癌的潜在治疗靶点。
查看英文原文 English abstract
Background: Cancer stem-like cells (CSCs) contribute to recurrence and chemoresistance in high-grade serous ovarian cancer (HGSOC). Spheroid formation in ultra-low attachment (ULA) cultures models CSC self-renewal, yet the epigenetic regulators and molecular dependencies sustaining this 3D phenotype remain largely undefined.
Methods: A focused CRISPR knockout screen targeting 800 epigenetic and chromatin regulators was performed in OVCAR-3 spheroids. sgRNA enrichment after 3D selection identified genes essential for spheroid growth. Top candidates were functionally validated in BRCA1-wildtype, BRCA1-LOH, and BRCA1-mutant cell lines by assessing spheroid morphology, compaction, viability, and proliferative behavior.
Results: The screen identified a high-confidence set of regulators required for spheroid formation, including DNA damage response genes (BRCA1, PARG), Polycomb complex members (SUZ12, BMI1), RNA-binding proteins (NCL, SYNCRIP), and cell cycle regulators (CDK2, PBRM1). These hits converge on chromatin remodeling, histone methylation, and transcriptional control pathways, linking epigenetic regulation with DDR signaling.Functional assays revealed a striking BRCA1 dependency: while BRCA1-WT and LOH cell lines formed compact, viable spheroids, BRCA1-mutant cells produced disorganized, loosely aggregated, hyperproliferative structures with poor architectural integrity. This indicates that BRCA1 is essential not for proliferation per se, but for establishing and maintaining 3D stem-like organization. Integration of the CRISPR hits with phenotypic outcomes suggests a cooperative network between DDR factors, RNA-binding regulators, and chromatin modifiers in sustaining CSC-like behavior.
Conclusions: Our findings demonstrate that BRCA1 function underpins the epigenetic and chromatin-based control of spheroid architecture, revealing a mechanistic link between DNA repair fidelity, chromatin integrity, and cancer stemness. The identified vulnerabilities highlight potential therapeutic targets for BRCA1-deficient ovarian and breast cancers.
利益披露 Disclosure
D. Demirel Yavuz, None..
I. Durmaz Sahin, None.