The Role of the Y Chromosome in Bladder Tumor Development, Growth And Progression

项目2--摘要 Y染色体丢失(LOY)与男性癌症发展和死亡风险增加相关 有几种癌症类型。在与烟草有关的恶性肿瘤-膀胱癌(BC)中，LOY出现在约30%的肿瘤中， 然而，这一现象的生物学相关性尚不清楚。在这里，我们将解决这一知识鸿沟，目标是 确定LOY驱动生长和进展的分子机制，并通过以下方式产生影响 将这些转化为对膀胱癌和其他癌症类型的有效干预。初步数据：我们检查了 使用我们开发的LOY基因签名的TCGA BC RNAseq数据集，发现签名分数较低 与手术后患者存活率降低有关。为了确定Loy是否推动了卑诗省的增长，我们开发了 具有(Y+)或w/o(Y-)Y染色体的谱系相关雄性小鼠细胞系。Y-肿瘤生长速度快于Y+肿瘤 在免疫活性小鼠和细胞图谱中发现前者有更强的免疫抑制作用 肿瘤相关巨噬细胞、CD8+T细胞表型耗竭，而后者NK细胞增多。在……里面 缺乏T、B和NK细胞、Y+和Y-肿瘤的小鼠(Rag2-/-；IL2RG-/-)生长相似。转录特征分析 发现四个Y染色体基因在这些类型的肿瘤中差异表达，并带有染色质组织基因 UTY和Kdm5d是唯一在TCGA BC患者中表达分层预后的基因。因此，我们 Y+细胞中的这些基因被基因删除，并发现只有免疫能力强的小鼠肿瘤生长增强 而它们在Y细胞中的过度表达抑制了它们的生长。因此，我们检验了UTY和Kdm5d的假设 缺失通过建立免疫抑制的肿瘤微环境促进BC的生长和进展 三个具体目标。在目标1中，我们确定了UTY和Kdm5d在肿瘤发生发展中的作用 使用完全和尿路上皮特异性条件性Kdm5d或UTY缺失小鼠暴露于化学致癌物BBN。 在项目3中，我们检验了UTY和它的X染色体对偶UTX(又名Kdm6a)具有 完全和尿路上皮特异性杂交对BC形成和发展的独立抑制作用 有条件的UTY和UTX缺失小鼠，并将它们暴露于BBN。在目标2中，我们定义了Kdm5d和UTY如何抑制 BC和免疫系统在这一过程中的作用使用定义的LOF功能突变来定义区域 这些对肿瘤生长有抑制作用的蛋白质。在项目1中，我们使用空间蛋白质组学来 检测免疫浸润物与突变型和WT Kdm5d及UTY癌细胞表达的关系 各种基因敲除小鼠或抗体耗尽策略，以确定免疫系统的哪些细胞成分 导致肿瘤生长差异。在目标3中，我们检测了ANPEP，它是Y-肿瘤中表达最高的基因，即 在BC患者中也是一个预后不佳的因素，也是一个可用药的靶点，作为一个候选的常染色体效应基因，驱动Y- 肿瘤生长。我们发现Y-肿瘤比Y+肿瘤对抗PD-1和ANPEP耗竭的反应更强。 细胞使肿瘤对抗PD-1更加敏感。在这里，我们将研究ANPEP删除或 药物抑制对抗PD-1免疫检查点阻断治疗效果的影响。

PROJECT 2 – SUMMARY Loss of the Y chromosome (LOY) is associated with increased risk of cancer development and death in men with several cancer types. In bladder cancer (BC), a tobacco related malignancy, LOY is seen in ~30% of tumors, yet the biological relevance of this is unknown. Here we address this Knowledge Gap with the Objective to define the molecular mechanisms responsible for LOY driven growth and progression and have Impact by translating these into effective interventions for bladder and other cancer types. Preliminary Data: We examined TCGA BC RNAseq dataset using an LOY gene signature we developed, and found low signature scores associated with reduced patient survival following surgery. To determine if LOY drives BC growth, we developed lineage related male murine cell lines with (Y+) or w/o (Y-) Y chromosome. Y- tumors grew faster than Y+ tumors in immunocompetent mice and cytometric profiling found the former to have increased immunosuppressive tumor-associated macrophages, CD8+ T cells with exhausted phenotype while latter had increased NK cells. In mice (Rag2-/-;Il2rg-/-) that are devoid of T, B and NK cells, Y+ and Y- tumors grew similarly. Transcriptional profiling found four Y chromosome genes differentially expressed in these tumor types, with chromatin organization genes Uty and Kdm5d being the only ones whose expression stratified outcome in TCGA BC patients. Thus, we genetically deleted these genes in Y+ cells and found enhanced tumor growth only in immunocompetent mice while their overexpression in Y- cells reduced their growth. Hence, we test the Hypothesis that Uty and Kdm5d loss enhance BC growth and progression via creation of an immunosuppressive tumor microenvironment in three Specific Aims. In Aim 1 we determine role of Uty and Kdm5d in tumor development and progression using full and urothelial specific conditional Kdm5d or Uty null mice be exposed to the chemical carcinogen BBN. With Project 3 we test the hypothesis that Uty and its X chromosome paralog Utx (aka, Kdm6a) have independent suppressive effects on BC formation and progression by crossing full and urothelial specific conditional Uty and Utx null mice and exposing them to BBN. In Aim 2 we define how Kdm5d and Uty suppress BC and the role of the immune system in this process using defined LOF functional mutants to define the regions on these proteins responsible for tumor growth suppression. With Project 1, we use spatial proteomics to examine immunological infiltrates in relation to mutant and WT Kdm5d and Uty cancer cell expression and various knockout mice or antibody depletion strategy to define which cellular components of the immune system drive tumor growth differences. In Aim 3 we examine ANPEP, the most upregulated gene in Y- tumors that is also a poor prognostic in BC patients and druggable target, as a candidate autosomal effector gene driving Y- tumor growth. We found Y- tumors are more responsive than Y+ tumors to anti-PD-1 and ANPEP depletion in Y- cells made tumors more sensitive to anti-PD-1. Here we will examine the impact of ANPEP deletion or pharmacologic inhibition on the efficacy of anti-PD-1 immune checkpoint blockade therapy.