FUNCTIONAL CHARACTERIZATION OF PSEUDOGENES AS NEW BIOMARKER IN PROSTATE CANCER

描述（由申请人提供）：前列腺癌仍然是美国男性中最常诊断的癌症和第二大癌症相关死亡，2011年有240，890例新诊断和约33，720例死亡（ACS）。准确的诊断和早期干预将减少因转移性前列腺癌而死亡的人数。前列腺癌的诊断最初是基于有争议的血清前列腺特异性抗原（PSA）水平。为了克服与检测PSA水平相关的问题，识别准确可靠的癌症特异性分子标志物将减少良性疾病患者不必要的活检，并指导侵袭性疾病患者进行适当的治疗。前列腺癌的遗传基础可以根据特定的驱动分子畸变分为几组。已知约50-60%的前列腺癌患者具有E26转化特异性（ETS）基因重排，并且在5-10%的ETS阴性前列腺癌中鉴定出SPINK 1的过表达。最近，我们报道了在1-2%的ETS阴性前列腺癌中鉴定出涉及RAF激酶基因（SLC 45 A3-BRAF和ESRP 1-RAF 1）的“可药物化”基因融合。其余30-40%的ETS阴性前列腺癌的遗传基础仍然未知。在这项提案中，我们将利用下一代测序技术研究前列腺癌的转录景观在全基因组范围内的假基因表达。在我们使用89个前列腺癌样本的初步研究中，我们鉴定了8个候选假基因和一个新的融合基因，该融合基因涉及仅在前列腺癌中表达的假基因。具体目标1：我们将验证8个候选假基因在一个大型前列腺癌队列中的表达，以及每个候选假基因是否基于相互排斥的表达模式识别不同的分子亚型或在前列腺癌发展中发挥协同作用。在我们的初步验证研究中，在8个候选基因中，我们选择了CXADR假基因（CXADR-假基因），因为同源野生型基因的肿瘤抑制作用。我们证实了CXADR-假基因在ETS融合阴性前列腺癌中的表达。此外，我们意外地鉴定了涉及KLK 4（蛋白质编码基因）和KRSP 1（仅在前列腺癌中表达的非编码假基因）的融合基因。在这里，我们建议在DRE后尿液中验证这些标志物，以开发非侵入性诊断/预后工具。具体目标2：我们将在体外和体内实验中检查CXADR假基因和KLK 4-KRSP 1融合的功能作用。此外，我们将探索CXADR-假基因和KLK 4-KRSP 1作为生物标志物的潜力，我们将开发基于PCR、免疫组织化学和RNA原位杂交的诊断试剂，用于针吸活检、膀胱切除术和DRE后尿液样本中的可靠检测。我们预计，这些新的分子标记物的验证将添加到前列腺癌诊断的医疗设备，以确定将发展为转移性疾病的患者，这些患者需要早期和积极的治疗策略。

DESCRIPTION (provided by applicant): Prostate cancer remains the most commonly diagnosed cancer and second leading cancer-related death in men in the United States with 240,890 new diagnosis and ~33,720 deaths in 2011 (ACS). Accurate diagnosis and early intervention will reduce the number of deaths due to metastatic prostate cancer. Prostate cancer diagnosis is based initially on the controversial serum prostate specific antigen (PSA) level. To overcome the problems associated with testing PSA level, identification of accurate and reliable cancer-specific molecular markers will reduce unnecessary biopsies for those with benign conditions and direct patients with aggressive disease for appropriate treatment. The genetic basis of prostate cancer can be classified into groups based on specific driving molecular aberrations. About 50-60% of prostate cancer patients are known to have E26 transformation specific (ETS) gene rearrangements and overexpression of SPINK1 was identified in 5-10% of ETS-negative prostate cancer. Recently, we reported the identification of "druggable" gene fusions involving RAF kinase genes (SLC45A3-BRAF and ESRP1-RAF1) in 1-2% of ETS negative prostate cancer. The genetic basis of the remaining 30-40% of ETS-negative prostate cancer remains unknown. In this proposal we will utilize next generation sequencing technology to study the transcriptional landscape of prostate cancer on a genome-wide scale for pseudogene expression. In our pilot study using 89 prostate cancer samples we identified 8 candidate pseudogenes and a novel fusion gene involving a pseudogene expressed only in prostate cancer. In specific aim 1: We will validate the expression of 8 candidate pseudogenes in a large cohort of prostate cancer and whether each candidate identifies a distinct molecular subtype based on mutually exclusive expression pattern or play a cooperative role in prostate cancer development. In our preliminary validation studies, of the 8 candidate genes we selected CXADR pseudogene (CXADR-pseudogene) because of the tumor suppressor role of the cognate wild-type gene. We confirmed the expression of CXADR- pseudogene in ETS fusion-negative prostate cancer. Further, we unexpectedly identified a fusion gene involving KLK4, a protein coding gene and KRSP1, a non-coding pseudogene expressed only in prostate cancer. Here, we propose to validate these markers in post-DRE urine to develop non-invasive diagnostic/prognostic tools. In specific aim 2: We will examine the functional role of CXADR- pseudogene and KLK4-KRSP1 fusion in in vitro and in vivo experiments. Additionally we will explore the potential for CXADR- pseudogene and KLK4-KRSP1 to serve as biomarkers and we will develop diagnostic reagents based on PCR, immunohistochemistry and RNA in situ hybridization for reliable detection in needle biopsy, prostatectomy and in post-DRE urine samples. We anticipate that validation of these new molecular markers will add into the armamentarium of prostate cancer diagnostics to identify patients that will develop metastatic disease who require early and aggressive treatment strategies.