Role of the Y-Located TSPY Gene in Human Oncogenesis

描述(由申请人提供)： 睾丸特异蛋白Y编码(TSPY)基因是位于人类Y染色体短臂上的一种重复序列基因。其2.8kb的结构基因嵌入在一个20kb的高度同源单位中，该单位重复多达次，构成了人类基因组中最大的功能序列串联阵列。TSPY重复序列是基因组不稳定和Y染色体表观遗传失调的热点。TSPY是Y染色体性腺母细胞瘤基因座(GBY)的原癌基因，是该男性特有染色体上唯一的致癌基因。我们的研究表明，TSPY含有一个保守的蛋白质相互作用结构域，称为SET/NAP。TSPX是X染色体上的单拷贝TSPY同源物。TSPY和TSPX在基因组织和蛋白质结构上都有显著的同源性。当异位表达时，TSPY和TSPX在细胞周期调节方面表现出相反的特性。TSPY异位表达，在性腺母细胞瘤、睾丸生殖细胞肿瘤、前列腺癌和性二型癌(如肝细胞癌)的发生和发展中起关键作用。TSPY可促进裸鼠细胞增殖，加速细胞过渡，破坏G2/M检查点，结合细胞周期蛋白B，增强细胞周期蛋白B-CDK1激酶活性，促进裸鼠肿瘤生长。TSPY在转基因小鼠的卵巢中诱导性母细胞瘤样结构。TSPX是一种肿瘤抑制因子。它结合细胞周期蛋白B，但抑制细胞周期蛋白B-CDK1的活性。异位表达TSPX可抑制裸鼠肿瘤生长，使细胞滞留于G2/M期。我们推测，TSPX在维持细胞有序增殖和生长方面发挥正常功能，而TSPY在异位表达时，抵消了这一功能(S)，并促进了易感细胞的肿瘤发生。最近，我们发现TSPY与雄激素受体(AR)相互作用，并加剧其对反应基因的配体依赖的反式激活，而TSPX相互作用但抑制AR功能。因此，TSPY和TSPX可能参与雄激素依赖的前列腺癌发生所介导的转录程序。当TSPY转基因基因被引入前列腺癌转基因小鼠(Lady)模型中时，在双转基因小鼠的前列腺癌形成开始时，它被异位激活，导致比没有TSPY的动物产生更多的结节肿瘤。这些结果为研究TSPY在前列腺癌发生中的作用机制提供了重要的线索和极好的资源。我们将在三个具体目标下对这些问题进行研究。首先，我们计划从肿瘤发病机制、转基因表达、TSPY启动子和外显子1的DNA甲基化状态以及受这种表观遗传变化影响的转录因子和抑制因子的鉴定等方面阐明TSPY转基因激活与TSPY-Lady双转基因小鼠前列腺癌发生相关的表观遗传学变化。其次，我们将确定负责TSPY和TSPX在AR反式激活中的不同功能的结构域，并用前列腺组织重组策略阐明结构与功能的关系。第三，我们计划使用先进的染色质免疫沉淀和启动子平铺微阵列(CHIP-Chip)方法来鉴定和确认TSPY-AR转录复合体的靶基因。我们将测定TSPY-Lady和Lady转基因小鼠在正常和激素消融条件下的肿瘤转录，从而推断在前列腺癌发生中受异位表达的TSPY转基因影响最大的基因和途径。我们将详细描述那些差异表达的基因，其启动子也被TSPY-AR转录复合体结合。这项拟议的研究将为TSPY和TSPX在前列腺癌发生中的作用提供重要的见解，并为前列腺癌的治疗策略开发诊断生物标记物和靶点提供翻译机会。 公共卫生相关性： 前列腺癌是退伍军人中的一种常见癌症。这种仅限于男性的癌症的病因尚不清楚。在过去的几年里，我们研究了男性特有染色体(即Y染色体)上的一个基因的功能，并发现它含有一个癌基因，可以促进包括前列腺癌在内的各种癌症的肿瘤发展。它在X染色体上有一个类似的(同源)基因，与Y癌基因的功能相反。因此，X基因是一种肿瘤抑制基因。我们证明，这两个性染色体基因可以影响睾酮(男性激素)相关的生理，同样是在相反的方向。由于睾酮对前列腺癌的发生和发展非常重要，我们建议在前列腺癌的动物模型中检查这两种性染色体基因的差异。这个项目将对Y癌基因和X肿瘤抑制基因如何运作以及如何干预或促进它们各自治疗前列腺癌的作用提供重要的见解。

DESCRIPTION (provided by applicant): The testis-specific protein Y-encoded (TSPY) gene is a tandemly repeated gene on the short arm of the human Y chromosome. Its 2.8-kb structural gene is embedded within a 20-kb highly homologous unit that is tandemly repeated up to 64 times, constituting the largest tandem array of functional sequences in the human genome. The TSPY repeats are hot spots for both genomic instability and epigenetic dysregulation on the Y chromosome. TSPY is the proto-oncogene for the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Our research shows that TSPY harbors a conserved protein interacting domain, termed SET/NAP. TSPX is a single-copy TSPY homologue on the X chromosome. Both TSPY and TSPX share significant homologies at both gene organization and protein structure. When ectopically expressed, TSPY and TSPX show contrasting properties in cell cycle regulation. TSPY is ectopically expressed and plays key roles in the initiation and/or progression of gonadoblastoma, testicular germ cell tumors, prostate cancer, and sexual dimorphic cancers, such as hepatocellular carcinoma. TSPY potentiates cell proliferation, accelerates the transition and disrupts the G2/M checkpoints, binds cyclin B and enhances cyclin B-CDK1 kinase activities, and promotes tumor growth in athymic mice. TSPY induces gonadoblastoma-like structure in the ovaries of transgenic mice. TSPX is a tumor suppressor. It binds cyclin B, but represses the cyclin B-CDK1 kinase activity. Ectopic expression of TSPX arrests cells at G2/M stage, and represses tumor growth in athymic mice. We postulate that TSPX serve a normal function in maintaining an orderly cell proliferation and growth while TSPY, when ectopically expressed, counteracts such function(s) and promotes tumorigenesis in susceptible cells. Recently, we have showed that TSPY interacts with androgen receptor (AR) and exacerbates its ligand-dependent transactivation of responsive genes while TSPX interacts but represses AR functions. Hence, TSPY and TSPX could participate in the transcriptional programs mediated by androgen-dependent prostatic oncogenesis. When a TSPY transgene is introduced into the transgenic mouse (LADY) model of prostate cancer, it is ectopically activated at the onset of prostatic oncogenesis in the bi-transgenic mice, resulting in qualitatively more nodular tumors than those from animals without TSPY. These results have provided some important clues and excellent resources for investigating the mechanisms by which TSPY could contribute to prostatic oncogenesis. We will study these problems under three specific aims. First, we plan to elucidate the epigenetic changes associated with TSPY transgene activation during prostatic oncogenesis in the TSPY-LADY bi-transgenic mice, in terms of tumor pathogenesis, transgene expression, DNA methylation status of the TSPY promoter and exon 1, and identification of transcription factors and repressors affected by such epigenetic changes. Second, we will determine the domains responsible for the contrasting functions of TSPY and TSPX on AR transactivation and elucidate the structure-function relationship with a prostatic tissue recombination strategy. Third, we plan to identify and confirm the target genes of a TSPY-AR transcription complex using advanced chromatin immunoprecipitation and promoter tiling microarray (ChIP-Chip) approach. We will determine the transcriptomes of tumors from TSPY-LADY and LADY transgenic mice under normal and hormone-ablated conditions, thereby deducing the genes and pathways mostly affected by an ectopically expressed TSPY transgene in prostatic oncogenesis. We will characterize in details those differentially expressed genes whose promoters are also bound by a TSPY-AR transcriptional complex. The proposed research will provide critical insights on the roles of TSPY and TSPX on prostatic oncogenesis, and translational opportunities in developing diagnostic biomarkers and targets for therapeutic strategies for prostate cancer. PUBLIC HEALTH RELEVANCE: Prostate cancer is a prevalent cancer among our veterans. The etiology of this man-only cancer is uncertain. For the past few years, we have examined the functions of a gene on the male-specific chromosome (i.e. Y chromosome) and showed that it contains an oncogene that could promote tumor development in various cancers, including prostate cancer. It has a similar (homologous) gene on the X chromosome that has opposite functions against those of the Y-located oncogene. Hence, the X-gene is a tumor suppressor gene. We show that these two sex chromosome genes can affect testosterone (male-hormone) associated physiology, again in opposing directions. Since testosterone is very important for prostate cancer development and progression, we propose to examine the differences of these two sex chromosome genes in an animal model of prostate cancer. This project will shed important insights on how the Y-oncogene and X-tumor suppressor operate and how one can interfere or boost their actions respectively to treat prostate cancer.