Loss of UTX/KDM6A and the activation of FGFR3 converge to regulate differentiation gene expression programs in bladder cancer [RNA-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: Two separate RNA-seq experiments analyzing the effect of UTX expression in UMUC1 bladder cancer cells, which have a UTX loss of function mutation: 1) UTX expression in full/regular media (10% FBS) and tumorsphere media (B27, FGF2, EGF), and 2) UTX-expression with co-expression of either wild type or S249C FGFR3. Both experiments were done in triplicate.

膀胱癌的预后与肿瘤的固有分化状态密切相关，涵盖侵袭性较弱、分化程度最高的腔面型（luminal）肿瘤，与侵袭性较强、分化程度最低的基底型（basal）肿瘤两大表型谱系。膀胱癌测序研究显示，染色质调控蛋白的功能缺失突变，以及激活受体酪氨酸激酶（receptor tyrosine kinase, RTK）信号通路的突变，在该病中频发。然而，目前对于这两类突变是否存在功能上的相互作用或协同效应，进而调控肿瘤生长与分化状态，仍所知甚少。本研究聚焦于组蛋白去甲基化酶UTX（亦称KDM6A）的缺失，以及RTK家族成员FGFR3的激活——这两类事件在肌层浸润性膀胱癌中常共同发生。我们发现，UTX缺失与FGFR3激活可协同破坏膀胱细胞内腔面型与基底型基因表达的平衡。UTX可定位至诸多调控腔面细胞命运的基因的增强子区域，并以不依赖其催化活性的方式维持这些基因的转录。与UTX的作用不同，FGFR3激活与肿瘤中腔面型基因的低表达呈相关性；而在细胞培养模型中，FGFR抑制剂可提升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功能缺失可增强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提升。综上，本研究揭示了UTX与FGFR3的突变如何协同破坏膀胱分化程序，这一机制可作为潜在治疗靶点。整体实验设计：开展两项独立的RNA测序（RNA-seq）实验，分析UTX表达在携带UTX功能缺失突变的UMUC1膀胱癌细胞中的影响：1）分别在完全常规培养基（含10%胎牛血清（FBS））及肿瘤球培养基（添加B27、成纤维细胞生长因子2（FGF2）与表皮生长因子（EGF））中过表达UTX；2）在过表达UTX的基础上，共表达野生型或S249C突变型FGFR3。两项实验均设置三次生物学重复。