Mechanisms of Progression in Oncogene-Incuded Prostate Cancer

描述(申请人提供)：我们最近发现小鼠和人类RTVP-1/GLIPR1(分别为Glipr1和GLIPR1)是P53的直接靶基因，并表明GLIPR1编码一种分泌蛋白，并且GLIPR1的表达在前列腺癌进展过程中通过表观遗传机制下调。根据序列同源性，我们鉴定并鉴定了位于人类染色体12q21上的一个新的p53靶基因簇，它包括GLIPR1和两个GLIPR1L基因，以及小鼠染色体10d1上的Glipr1和三个Glipr1基因(Glipr1l)。功能分析表明，GLIPR1或GLIPR1L2基因过表达或GLIPR1蛋白处理可诱导多种小鼠和人癌细胞株在体内外发生G0/G1期生长停滞和/或细胞凋亡。为了测试GLIPR1的肿瘤抑制活性，我们培育了含有Glipr1基因失活突变的小鼠。长期队列分析表明，Glipr1功能的丧失导致了独特的恶性肿瘤谱导致的无瘤生存率降低。机制研究表明，GLIPR1的表达导致c-myc mRNA的下调，提示GLIPR1介导的细胞周期调控是一个关键控制点。有趣的是，GLIPR1过表达导致前列腺癌和膀胱癌细胞中细胞周期蛋白A和细胞周期蛋白D水平降低，p27Kip1水平升高。我们鉴定和使用了化学和分子抑制剂来确认与GLIPR1表达相关的促凋亡通路，该通路涉及ROS的产生，ASK1-MEK4/7-JNK通路的激活，Bcl2的抑制和广泛的caspase激活。MEF分析显示，与Glipr1-/-MEF相比，DNA损伤剂处理导致Glipr1诱导，ROS水平和JNK活性升高，Glipr1+/+细胞凋亡率增加。此外，Glipr1-/-MEF在体外对ras+myc诱导转化的敏感性增加。我们的结果证明GLIPR1是一种新的广谱肿瘤抑制因子，它通过产生ROS JNK信号来介导促凋亡活性。我们现在提议：(1)确定GLIPR1或GLIPR1L21介导的前列腺癌和膀胱癌细胞生长停滞的机制；(2)分析GLIPR1或GLIPR1L21表达后导致ROS-ASK1-MEK4/7-JNK凋亡增加的分子途径；(3)鉴定GLIPR1结合蛋白/膜受体并鉴定GLIPR1的蛋白摄取；(4)建立ARR2PB-c-myc转基因小鼠，将这些小鼠与Glipr1-/-小鼠杂交，并利用双基因小鼠分析内源性Glipr1抑制癌前病变和前列腺癌体内病变发展的遗传活性。项目简介：本项目旨在了解新近发现的肿瘤抑制基因GLIPR1GLIPR1抑制前列腺癌和膀胱癌生长的机制(S)。我们的研究结果可能会确定诊断这些恶性肿瘤的新方法和/或导致新的治疗方法。

DESCRIPTION (provided by applicant): We recently identified mouse and human RTVP-1/GLIPR1 (Glipr1 and GLIPR1, respectively) as direct p53 target genes and showed that GLIPR1 encodes a secreted protein and that GLIPR1 expression is down- regulated during prostate cancer progression through epigenetic mechanisms. Based on sequence homology we identified and characterized a novel p53 target gene cluster located on human chromosome 12q21 that includes GLIPR1 together with two GLIPR1-like (GLIPR1L) genes, and on mouse chromosome 10D1 that includes Glipr1 and three Glipr1-like (Glipr1l) genes. Functional analysis demonstrated that GLIPR1 or GLIPR1L2 gene overexpression or treatment with GLIPR1 protein induces growth arrest in G0/G1 and/or apoptosis in various mouse and human cancer cell lines in vitro and in vivo. To test the tumor suppressor activities of GLIPR1 we generated mice that harbored an inactivating mutation of the Glipr1 gene. Long-term cohort analysis demonstrated that loss of Glipr1 function led to reduced tumor free survival resulting from a unique spectrum of malignancies. Mechanistic studies demonstrated that GLIPR1 expression leads to down-regulation of c-myc mRNA, suggesting a critical control point in GLIPR1 mediated cell cycle control. Interestingly, GLIPR1 overexpression leads to reduced levels of cyclin A and cyclin D and increased levels of p27Kip1 in prostate and bladder cancer cells. We identified and used chemical and molecular inhibitors to confirm a pro-apoptotic pathway associated with GLIPR1 expression that involves generation of ROS, activation of ASK1-MEK4/7-JNK pathway, suppression of Bcl-2 and broad based caspase activation. Analysis of MEF revealed that treatment with DNA damaging agents resulted in Glipr1 induction, elevated ROS levels and JNK activities, and increased apoptosis in Glipr1+/+ compared to Glipr1-/- MEF. In addition, Glipr1-/- MEF had increased susceptibility to ras + myc induced transformation in vitro. Our results establish GLIPR1 as a novel, wide-spectrum tumor suppressor that mediates pro-apoptotic activities through generation of ROS JNK signaling. We now propose to: (1) Identify the mechanisms of GLIPR1 or GLIPR1L21 mediated growth arrest in prostate and bladder cancer cells; (2) Analyze the molecular pathways that lead to increased ROS-ASK1-MEK4/7-JNK-apoptosis following GLIPR1 or GLIPR1L21 expression; (3) Identify GLIPR1 binding proteins/membrane receptor and characterize GLIPR1 protein uptake and (4) Generate ARR2PB-c-myc transgenic mice, intercross these mice with Glipr1-/- mice and use the bigenic mice to analyze the genetic activities that underlie the capacity of endogenous Glipr1 to suppress the development of pre-malignant and malignant prostatic lesions in vivo. Project Narrative: This project seeks to understand the mechanism(s) through which GLIPR1, a newly identified tumor suppressor gene, inhibits the growth of prostate and bladder cancer. The results of our studies may identify new methods for diagnosing and/or lead to new therapies for these malignancies.