前列腺癌由静默状态向临床侵袭性转变过程中胰岛素的诱导作用及ZFX基因调控的分子机制

Prostate cancer has two biological forms, the latent form which has histological features without the invasive behaviors, and the clinical evident form with aggressive behaviors. The unique heterogeneity of prostate cancer is the high proportion of the latent form but a relatively low proportion of the aggressive form. The alteration of micro-environment plays a significant role in the transition of the biological behaviors of prostate cancer. Our preliminary investigations showed that obese patients with high insulin level had relatively poor prognosis. The insulin receptors were observed in the tissues of prostate cancer as well as the high expressions of ZFX gene. The high level of insulin accumulatively promoted the proliferation and aggressiveness of the cancer cells and increased the expression of ZFX protein in the cancer cells which were once at the level of low proliferation; high fat diet was likely to induce the insulin resistance and the transition from the highly differentiated tumor to the poorly differentiated tumor in TRAMP models. ZFX is a transcriptional factor which can regulate the cell self-renewal. We hypothesize that insulin may induce the transition from the latent form of prostate cancer to the clinical aggressive form through the regulation of the cell self-renewal. This protocol aims to evaluate the regulating function of ZFX gene, the related downstream effectors, and the interaction between ZFX and insulin/IGF-1 pathway in the process of the insulin-induced acquisition of invasive ability in the low-proliferative potential type of prostate cancer in the model of TRAMP and prostate cancer cell lines, in order to pave the way for the clinical translational research for the identification of the latent prostate cancer and the prevention of clinical aggressive cancer.

前列腺癌有2种生物学形式：组织学上是癌但不发生临床侵袭的静默期癌和具有侵袭转移能力的临床侵袭性癌。高比例的静默期癌和相对低发的临床侵袭性癌的异质性现象形成其特点。微环境的变化是前列腺癌生物学行为改变的重要因素。我们的前期工作发现胰岛素水平较高的肥胖患者的前列腺癌预后较差，前列腺癌组织上存在胰岛素受体和ZFX蛋白的高表达；高水平胰岛素的累积刺激可促使低增殖状态的癌细胞增殖加快、侵袭力增强和ZFX蛋白表达增强；高脂饮食可以诱导转基因前列腺癌小鼠(TRAMP)出现胰岛素抵抗及高分化癌向低分化癌的转变。ZFX是具有调控细胞自我更新功能的转录因子，由此推测胰岛素可能通过细胞自我更新诱导静默状态的前列腺癌获得侵袭能力。本项目拟在前列腺癌细胞和TRAMP小鼠上研究胰岛素诱导低增殖状态前列腺癌获得侵袭能力的转变中ZFX基因调控的下游效应分子以及ZFX与胰岛素信号通路的关系，为预防临床侵袭性前列腺癌做基础。

临床实践提示肥胖患者的前列腺癌预后较差，但缺乏循证依据。我们课题组经大样本荟萃分析证实肥胖与前列腺穿刺阳性率和前列腺癌根治术后生化复发呈正相关的结果作为重要临床证据收录入《美国肿瘤学会前列腺癌监测指南》(J Clin. 2014, 64(4): 225-249)。前期工作发现前列腺癌存在胰岛素受体和ZFX蛋白高表达，ZFX是有调控细胞自我更新功能的转录因子，ZFX与胰岛素类因子及前列腺癌侵袭性变化的关系和效应分子是本项目的主要研究内容。我们建立了高脂饮食诱导的肥胖前列腺癌TRAMP转基因小鼠模型（已申请发明专利），在此模型上观察到高脂饲料喂养28周的肥胖小鼠血清胰岛素和IGF-2变化不明显，但IGF-1水平明显升高（P＜0.05）。肥胖小鼠肿瘤形成和体积明显高于普通饮食组（P＜0.05），IGF-1水平与小鼠肿瘤大小形成的变化趋势一致。随着IGF-1升高、小鼠肿瘤的ZFX表达亦明显增高（P＜0.05），提示体内IGF-1促使ZFX表达使肿瘤增大。.针对前列腺癌细胞（PC-3和DU145）实验，在不同浓度IGF-1（100和200ng/ml）组观察到IGF-1可增强PC-3和DU145的增殖和迁移能力。用qPCR和Western Blot方法发现100ng/ml的IGF-1可促进PC-3中ZFX转录和蛋白表达。构建干扰序列shZFX和对照序列shCON慢病毒荧光载体，通过病毒感染方式敲减细胞ZFX，抑制转录和表达，对感染shZFX和shCON慢病毒的前列腺癌细胞通过CCK-8、流式细胞、克隆形成、划痕和Transwell实验发现敲减ZFX后细胞的增殖、迁移侵袭、克隆形成和细胞周期等均受到抑制。.我们尝试了中药提取物的干预实验，发现黄芩素（Baicalein）和淫羊藿素可以抑制前列腺癌细胞的增殖、迁移侵袭、细胞周期和成瘤能力，Baicalein通过下调ZFX表达抑制前列腺癌的进展；淫羊藿素通过PEA3/HER2/AR通路抑制肿瘤，同时下调血清促炎症细胞因子（IL-1α、IL-1β、IL-6 和TNF-α）水平，延长TRAMP小鼠的生存。.更有趣的发现是：高脂饲料喂养的肥胖孕母鼠可以促进子代TRAMP鼠前列腺癌的发生。拟申请新一轮资助，做深入的研究。高脂饮食和肥胖是现代社会突出的健康问题，肥胖前列腺癌的IGF-1升高促使ZFX表达，促进肿瘤侵袭，通过传统中药下调ZFX水平