FUNCTIONAL CHARACTERIZATION OF PSEUDOGENES AS NEW BIOMARKER IN PROSTATE CANCER

描述(申请人提供)：前列腺癌仍然是美国男性中最常见的诊断癌症和第二大癌症相关死亡病例，2011年新增诊断240,890例，死亡约33,720例(美国癌症协会)。准确的诊断和早期干预将减少转移性前列腺癌的死亡人数。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记将减少良性疾病患者不必要的活检，并指导侵袭性疾病的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像差而分为几组。约50-60%的前列腺癌患者存在E26转化特异性(ETS)基因重排，而在5-10%的ETS阴性前列腺癌中发现SPINK1过表达。最近，我们报道了在1-2%的ETS阴性前列腺癌中发现了涉及RAF激酶基因(SLC45A3-BRAF和ESRP1-RAF1)的“可药物”基因融合。其余30-40%的ETS阴性前列腺癌的遗传学基础仍不清楚。在这项提案中，我们将利用下一代测序技术在全基因组范围内研究前列腺癌假基因表达的转录图景。在我们对89个前列腺癌样本的初步研究中，我们确定了8个候选假基因和一个新的融合基因，该融合基因涉及一个仅在前列腺癌中表达的假基因。具体目标1：我们将验证8个候选假基因在前列腺癌大队列中的表达，以及每个候选基因是否基于相互排斥的表达模式识别出不同的分子亚型，或者在前列腺癌的发生发展中发挥协同作用。在我们的初步验证研究中，我们在8个候选基因中选择了CXADR假基因(CXADR-伪基因)，因为同源野生型基因具有肿瘤抑制作用。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，我们意外地发现了一个融合基因，涉及KLK4和KRSP1，KLK4是一个蛋白质编码基因，KRSP1是一个只在前列腺癌中表达的非编码假基因。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。在特定目标2：我们将在体外和体内实验中检测CXADR-假基因和KLK4-KRSP1融合的功能作用。此外，我们还将探索CXADR-假基因和KLK4-KRSP1作为生物标志物的可能性，并将开发基于聚合酶链式反应、免疫组织化学和RNA原位杂交的诊断试剂，用于针刺活检、前列腺切除术和DRE后尿样的可靠检测。我们预计，这些新的分子标志物的验证将增加前列腺癌诊断的宝库，以识别将发展为转移性疾病的患者，这些患者需要早期积极的治疗策略。

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.