PO.ET06.05 · 实验与分子治疗
ID4在侵袭性三阴性乳腺癌中维持未分化和免疫逃逸状态，代表一种新型可成药易感靶点
ID4 maintains undifferentiated and immune-evasive states in aggressive triple-negative breast cancer and represents a novel druggable vulnerability
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：分化抑制因子4（ID4）在三阴性乳腺癌（TNBC）中过表达，对MDA-MB-231细胞的增殖、非锚定依赖性生长和致瘤性至关重要。这些发现将ID4确立为未分化、侵袭性TNBC表型的候选驱动因素。然而，其在分子异质性TNBC亚型中的表达模式及其治疗潜力仍未明确。
方法：我们将对临床注释TNBC队列的计算机分析与ID4高表达TNBC模型的功能研究相结合。使用bc-GenExMiner v5.2评估ID4在TNBC各亚型中的表达和共表达模式。在三种代表性TNBC细胞系中通过CRISPR-Cas9或shRNA沉默ID4：MDA-MB-231（间质干样，MSL）、MDA-MB-468（基底样1）和BT-549（间质样）。使用首创的泛ID降解剂AGX51实现ID4的药理学降解。通过MTT法和IncuCyte活细胞成像监测增殖。在五种患者来源的基底样乳腺癌干细胞（BCSC）培养物中进一步评估药物反应，其中五种均评估增殖，两种评估乳腺球形成。
结果：计算机分析显示，ID4表达在两个最具侵袭性、免疫抑制的TNBC亚型中显著富集：基底样免疫抑制型（BLIS）和间质样免疫改变/免疫调节型（MLIA；原称间质干样/MSL）。在MLIA肿瘤中，ID4与间质、肌上皮和干性程序（YAP/TAZ-Hippo、FOXC1、SOX10、ACTA2和基底细胞角蛋白）强烈共表达。ID4的基因沉默和药理学降解均显著损害了所有受测细胞系和BCSC培养物的增殖。值得注意的是，AGX51在所检测的两个BCSC模型中完全破坏了乳腺球形成和3D结构完整性。成功建立了稳定的ID4敲除和敲低TNBC细胞系，用于正在进行的RNA-seq和DNA甲基化分析。
结论：这些数据将ID4确立为最具侵袭性和免疫抑制TNBC亚群中一种亚型富集的可成药易感靶点。正在进行的转录组学和表观基因组学研究将阐明ID4所调控的分子回路，并确定针对BLIS、MLIA和间质干样TNBC的合理联合策略。本摘要的部分文本借助人工智能进行了修改，以提高语言清晰度和简洁性。
查看英文原文 English abstract
Background: Inhibitor of Differentiation 4 (ID4) is overexpressed in triple-negative breast cancer (TNBC) and is essential for proliferation, anchorage-independent growth, and tumorigenicity of MDA-MB-231 cells. These findings position ID4 as a candidate driver of undifferentiated, aggressive TNBC phenotypes. However, its expression pattern across molecularly heterogeneous TNBC subtypes and its therapeutic potential remain undefined.
Methods: We integrated in silico analyses of clinically annotated TNBC cohorts with functional studies in ID4-high TNBC models. ID4 expression and co-expression patterns across TNBC subtypes were evaluated using bc-GenExMiner v5.2. ID4 was silenced by CRISPR-Cas9 or shRNA in three representative TNBC cell lines: MDA-MB-231 (mesenchymal stem-like, MSL), MDA-MB-468 (basal-like 1), and BT-549 (mesenchymal-like). Pharmacologic ID4 degradation was achieved using AGX51, a first-in-class pan-ID degrader. Proliferation was monitored by MTT assay and IncuCyte live-cell imaging. Drug response was further assessed in five patient-derived basal-like breast cancer stem cell (BCSC) cultures, with proliferation evaluated in all five and mammosphere formation in two.
Results: In silico analysis revealed that ID4 expression is significantly enriched in the two most aggressive, immune-suppressed TNBC subtypes: basal-like immune-suppressed (BLIS) and mesenchymal-like immune-altered/immunomodulatory (MLIA; formerly mesenchymal stem-like/MSL). In MLIA tumors, ID4 strongly co-expresses with mesenchymal, myoepithelial, and stemness programs (YAP/TAZ-Hippo, FOXC1, SOX10, ACTA2, and basal cytokeratins). Both genetic silencing and pharmacologic degradation of ID4 markedly impaired proliferation across all tested cell lines and BCSC cultures. Notably, AGX51 completely disrupted mammosphere formation and 3D structural integrity in the two BCSC models examined. Stable ID4-knockout and knockdown TNBC cell lines were successfully generated for ongoing RNA-seq and DNA-methylation profiling.
Conclusions: These data establish ID4 as a subtype-enriched, druggable vulnerability in the most aggressive and immune-suppressed TNBC subsets. Ongoing transcriptomic and epigenomic studies will elucidate the molecular circuits governed by ID4 and identify rational combination strategies for BLIS, MLIA, and mesenchymal stem-like TNBC. This abstract incorporates text revised with the assistance of artificial intelligence for language clarity and conciseness
利益披露 Disclosure
L. Rivera, None..
C. Toro, None..
J. Fernandez, None..
M. T. Branham, None.