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最常见的诊断癌症和第二大癌症相关死亡病例，2013年新增诊断238,590例，死亡约29,720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，我们利用下一代测序技术在全基因组范围内研究了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。基于关于候选假基因功能作用的令人鼓舞的初步结果，我们将把重点放在CXADRP2和KLK4-KLKP1的功能特征上。具体目标1：我们将扩大候选假基因在大量前列腺癌队列中的有效性，以了解它们在前列腺癌中的独特作用。在我们的初步验证研究中，我们证实了CXADRP2在ETS融合阴性前列腺癌中的表达，以及KLK4-KLKP1在ETS融合阳性前列腺癌中的表达。RNA原位杂交显示KLK4-KLKP1在高Gleason评分(GS-7或以上)前列腺癌中的表达显着增加，提示其在侵袭性前列腺癌中可能起作用。我们还提供了这两个假基因在DRE后尿样中表达的初步数据，表明通过非侵入性方法进行筛查的可能性。由于CXADRP2和KLK4-KLKP1分别与Ets阳性和Ets阴性前列腺癌有很强的相关性，我们将其表达与已知的前列腺特异性非编码RNA PCA3和PCAT-1相关联，以了解假基因在前列腺癌亚群中的相互排斥作用。鉴于KLKP1中唯一的54个氨基酸，我们将制备KLKP1的特异性抗体，通过免疫印迹分析和免疫组织化学方法进行筛选。在特定目标2：我们使用体外和体内研究显示了强大的功能数据。我们还表明，这两种基因中的任何一种都对另一种基因有任何影响，这表明前列腺癌存在独立的机制。我们将在过度表达的细胞中进行基因表达微阵列分析，以了解假基因所涉及的分子机制和潜在的信号通路。此外，基于体内CAM试验令人鼓舞的结果，我们将在小鼠模型中验证致癌特性。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 238,590 new diagnosis and ~29,720 deaths in 2013 (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 utilized 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. Based on the encouraging preliminary results on the functional role of the candidate pseudogenes, we will prioritize our studies with a focus on the functional characterization of CXADRP2 and KLK4-KLKP1. In specific aim 1: We will expand the validation of candidate pseudogenes in a large cohort of prostate cancer to understand their unique role in prostate cancer. In our preliminary validation studies, we confirmed the expression of CXADRP2 in ETS fusion-negative prostate cancer and KLK4-KLKP1 in ETS fusion positive prostate cancer. Further using RNA in situ hybridization assay we showed significant increase in the expression of KLK4-KLKP1 in high Gleason score (GS-7 or above) prostate cancer indicating its potential role in aggressive prostate cancer. We also presented preliminary data on the expression of these two pseudogenes in post-DRE urine samples, indicating the potential to screen by non-invasive methods. With the strong correlation with ETS -positive and ETS -negative prostate cancer for CXADRP2 and KLK4-KLKP1; respectively, we will correlate the expression with known prostate specific non-coding RNA PCA3 and PCAT-1 to understand the mutually exclusive function of pseudogenes in subsets of prostate cancer. Given the unique 54 amino acid peptide from KLKP1, we will develop KLKP1 specific antibody to screen by western blot analysis and immunohistochemistry. In specific aim 2: We showed strong functional data using in vitro and in vivo studies. We also show that either of the gene has any effect on the other, suggesting an independent mechanism in prostate cancer. We will perform gene expression microarray analysis in cells with overexpression to understand the molecular mechanisms involved and potential signaling pathways affected by the pseudogenes. Further, based on the encouraging results from the in vivo CAM assay, we will validate the oncogenic properties in murine models. 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.