The PML1-WDR5 axis regulates H3K4me3 marks and promotes stemness of estrogen receptor-positive breast cancer

The alternative splicing of PML precursor mRNA gives rise to various PML isoforms, yet their expression profile in breast cancer cells remains uncharted. We discovered that PML1 is the most abundant isoform in all breast cancer subtypes, and its expression is associated with unfavorable prognosis in estrogen receptor-positive (ER+) breast cancers. PML depletion reduces cell proliferation, invasion, and stemness, while heterologous PML1 expression augments these processes and fuels tumor growth and resistance to fulvestrant, an FDA-approved drug for ER+ breast cancer, in a mouse model. Moreover, PML1, rather than the well-known tumor suppressor isoform PML4, rescues the proliferation of PML knockdown cells. ChIP-seq analysis reveals significant overlap between PML-, ER-, and Myc-bound promoters, suggesting their coordinated regulation of target gene expression, including genes involved in breast cancer stem cells (BCSCs), such as JAG1, KLF4, YAP1, SNAI1, and MYC. Loss of PML reduces BCSC-related gene expression, and exogenous PML1 expression elevates their expression. Consistently, PML1 restores the association of PML with these promoters in PML-depleted cells. We identified a novel association between PML1 and WDR5, a key component of H3K4 methyltransferase (HMTs) complexes that catalyze H3K4me1 and H3K4me3. ChIP-seq analyses showed that the loss of PML1 reduces H3K4me3 in numerous loci, including BCSC-associated gene promoters. Additionally, PML1, not PML4, re-establishes the H3K4me3 mark on these promoters in PML-depleted cells. Significantly, PML1 is essential for recruiting WDR5, MLL1, and MLL2 to these gene promoters. Inactivating WDR5 by knockdown or inhibitors phenocopies the effects of PML1 loss, reducing BCSC-related gene expression and tumorsphere formation and enhancing fulvestrant’s anticancer activity. Our findings challenge the conventional understanding of PML as a tumor suppressor, redefine its role as a promoter of tumor growth in breast cancer and offer new insights into the unique roles of PML isoforms in breast cancer. ChIP-seq was conducted on H3K4me3 in control and PML knockdown MCF-7 and ZR-75-1 cells, along with PML ChIP-seq in ZR-75-1 cells.

早幼粒细胞白血病蛋白（PML）前体mRNA的可变剪接可产生多种PML亚型，但目前乳腺癌细胞中这些亚型的表达谱仍未明确。我们发现，PML1是所有乳腺癌亚型中丰度最高的亚型，且其表达与雌激素受体阳性（estrogen receptor-positive, ER+）乳腺癌患者的不良预后相关。敲低PML会降低细胞增殖、侵袭能力及干细胞干性；而异源过表达PML1则可增强这些过程，并在小鼠模型中促进肿瘤生长以及对氟维司群（fulvestrant，美国食品药品监督管理局（Food and Drug Administration, FDA）批准的ER+乳腺癌治疗药物）的耐药性。此外，PML1而非经典的抑癌亚型PML4，能够挽救PML敲低细胞的增殖能力。染色质免疫共沉淀测序（ChIP-seq）分析显示，PML、ER以及Myc结合的启动子存在显著重叠，提示三者协同调控靶基因的表达，其中包括参与乳腺癌干细胞（breast cancer stem cells, BCSCs）相关进程的基因，如JAG1、KLF4、YAP1、SNAI1及MYC。PML功能缺失会降低BCSC相关基因的表达，而外源性过表达PML1则可上调这些基因的表达。与之一致的是，在PML敲低的细胞中，PML1能够恢复PML与这些启动子的结合。我们还发现了PML1与WDR5的全新关联：WDR5是催化组蛋白H3赖氨酸4单甲基化（H3K4me1）和三甲基化（H3K4me3）的组蛋白H3K4甲基转移酶（H3K4 methyltransferase, HMTs）复合物的关键组分。ChIP-seq分析表明，敲除PML1会降低众多位点的H3K4me3水平，其中就包括BCSC相关基因的启动子区域。此外，在PML敲低的细胞中，PML1而非PML4能够重新建立这些启动子区域的H3K4me3修饰。尤为重要的是，PML1对于将WDR5、MLL1及MLL2招募至这些基因启动子处是必需的。通过敲低或抑制剂使WDR5失活，可模拟PML1缺失的效应：降低BCSC相关基因的表达、减少肿瘤球形成，并增强氟维司群的抗癌活性。本研究的发现挑战了将PML视为抑癌基因的传统认知，重新定义了其在乳腺癌中作为肿瘤生长促进因子的角色，并为解析PML亚型在乳腺癌中的独特功能提供了全新视角。本研究针对对照组及PML敲低的MCF-7、ZR-75-1细胞开展了H3K4me3的ChIP-seq实验，同时在ZR-75-1细胞中完成了PML的ChIP-seq分析。