Study on identification and application of a novel cancer-associated gene PCA-1 in human prostate carcinoma

最近日本前列腺癌的发病率有所上升。尽管有很多关于前列腺癌病因的分子事件的信息，但这些遗传改变在肿瘤发生过程中是如何发生的仍不清楚。对于前列腺癌的潜在基因标记，我们使用荧光差异显示（FDD）分析从分子上鉴定出前列腺癌和正常组织之间表达差异的基因。我们发现了一个基因，命名为前列腺癌抗原-1 (PCA-1)，在前列腺癌中显示高mRNA表达。经数据库分析，PCA-1的氨基酸序列与大肠杆菌DNA烷基化损伤修复酶AlkB高度相似。通过免疫组化，PCA-1在前列腺癌样本和高级别前列腺上皮内瘤变的非典型细胞中大量表达，但在良性前列腺增生和正常邻近组织中不表达。在人前列腺癌细胞DU145中，发现FLIP样抑制蛋白（FLIP）的泛素化和降解增加，通过内源性PCA-1的siRNA基因沉默抑制FLIP依赖性Raf-1/细胞外应激调节激酶(ERK)/cyclin D1信号，从而抑制表皮生长因子（EGF）诱导的细胞生长。来自PCA-1的flip依赖性信号在紫杉醇刺激中也很明显，通过下调Raf-1/ERK， PCA-1被敲低可促进细胞凋亡。在内源性PCA-1和FLIP较差的LNCaP细胞中，过表达PCA-1可促进egf诱导的细胞生长，同时通过增强FLIP/Raf-1信号通路，减弱紫杉醇诱导的细胞凋亡。用siRNA沉默FLIP可以消除这两种效应。综上所述，这些结果表明，PCA-1是flip依赖性Raf-1信号传导的重要上游元件，在前列腺癌细胞的存活中起着关键作用。

The incidence of prostate cancer has increased recently in Japan. Although there is much information relating to molecular events underlying the etiology of prostate cancer, it is still unclear as to how these genetic alterations occur in the tumorigenesis. For potential gene markers for prostate carcinoma, we have molecularly identified genes showing differential expression between prostate cancers and normal tissues using fluorescent differential display (FDD) analysis. We identified a gene, designated prostate cancer antigen-1 (PCA-1), which shows high mRNA expression in prostate cancer. Database analysis of the deduced amino acid sequence of PCA-1 indicated high similarity to Escherichia coli AlkB, a DNA alkylation damage repair enzyme. By immunohistochemistry, PCA-1 was expressed in a high number of both prostate cancer samples and in the atypical cells within high-grade prostatic intraepithelial neoplasias, but not in benign prostatic hyperplasia or normal adjacent tissues.Ubiquitylation and degradation of FLICE-like inhibitory protein (FLIP) was found to be increased and FLIP-dependent Raf-1/extracellular stress-regulated kinase (ERK)/cyclin D1 signal was inhibited by siRNA gene silencing of endogenous PCA-1, resulting in suppression of epidermal growth factor (EGF)-induced cell growth in the human prostate cancer cell line, DU145. FLIP-dependent signaling from PCA-1 was also apparent on paclitaxel stimulation, and apoptosis was enhanced by PCA-1 knock down through down-regulation of Raf-1/ERK. In LNCaP cells with poor endogenous PCA-1 and FLIP, overexpression of PCA-1 promoted EGF-induced cell growth, while attenuating paclitaxel-induced apoptosis through enhanced FLIP/Raf-1 signaling. Both effects were canceled by silencing of FLIP with siRNA. Taken together, the results indicate that PCA-1 is an essential upstream element in FLIP-dependent Raf-1 signaling, playing a critical role in survival of prostate cancer cells.