Loss of UTX/KDM6A and the activation of FGFR3 converge to regulate differentiation gene expression programs in bladder cancer [ChIP-seq]

Bladder cancer prognosis is closely linked to the underlying differentiation state of the tumor, ranging from the less aggressive and most differentiated luminal tumors to the more aggressive and least differentiated basal tumors. Sequencing of bladder cancer has revealed that loss-of-function mutations in chromatin regulators and mutations that activate receptor tyrosine kinase (RTK) signaling frequently occur in bladder cancer. However, little is known as to whether and how these two types of mutations functionally interact or cooperate to regulate tumor growth and differentiation state. Here, we focus on loss of the histone demethylase UTX (also known as KDM6A) and activation of the RTK FGFR3, two events that commonly co-occur in muscle invasive bladder tumors. We show that UTX loss and FGFR3 activation cooperate to disrupt the balance of luminal and basal gene expression in bladder cells. UTX localized to enhancers surrounding many genes that are important for luminal cell fate, and supported the transcription of these genes in a catalytic-independent manner. In contrast to UTX, FGFR3 activation was associated with lower expression of luminal genes in tumors and FGFR inhibition increased transcription of these same genes in cell culture models. This suggests an antagonistic relationship between UTX and FGFR3. In support of this model, UTX loss-of-function potentiated FGFR3-dependent transcriptional effects and the presence of UTX blocked an FGFR3-mediated increase in the colony formation of bladder cells. Taken together, our study reveals how mutations in UTX and FGFR3 converge to disrupt bladder differentiation programs that could serve as a therapeutic target. Overall design: ChIP-seq for UTX, MLL4 and various histone post-translational modifications in UMUC1 bladder cancer cells.

膀胱癌的预后与肿瘤的固有分化状态紧密相关，谱系跨度从侵袭性较弱、分化程度最高的腔面型（luminal）肿瘤，延伸至侵袭性较强、分化程度最低的基底型（basal）肿瘤。对膀胱癌的测序研究显示，染色质调控因子的功能丧失性突变，以及激活受体酪氨酸激酶（Receptor Tyrosine Kinase, RTK）信号通路的突变，在膀胱癌中频繁出现。然而，目前对于这两类突变是否、以及如何在功能上相互作用或协同调控肿瘤生长与分化状态，我们仍知之甚少。本研究聚焦于组蛋白去甲基化酶UTX（亦称KDM6A）的功能丧失，以及FGFR3（成纤维细胞生长因子受体3）的激活这两类在肌层浸润性膀胱肿瘤中常共同发生的事件。我们发现，UTX功能丧失与FGFR3激活可协同破坏膀胱细胞中腔面型与基底型基因表达的平衡。UTX定位至诸多对腔面细胞命运至关重要的基因的增强子区域，并以不依赖催化活性的方式支持这些基因的转录。与UTX不同，FGFR3激活与肿瘤中腔面型基因的低表达呈相关性；在细胞培养模型中，FGFR抑制剂可上调这些相同基因的转录水平。这提示UTX与FGFR3之间存在拮抗关系。为验证这一模型，我们发现UTX功能丧失会增强FGFR3依赖的转录调控效应，而UTX的存在则会阻断FGFR3介导的膀胱细胞集落形成能力的提升。综上，本研究揭示了UTX与FGFR3突变如何协同破坏膀胱分化程序，这一机制有望成为治疗靶点。总体实验设计：在UMUC1膀胱癌细胞系中开展UTX、MLL4以及多种组蛋白翻译后修饰的染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）实验。