Mechanisms of Progression in Oncogene-Incuded Prostate Cancer

描述（由申请人提供）： 我们最近鉴定了小鼠和人RTVP-1/GLIPR 1（分别为Glipr 1和GLIPR 1）作为直接p53靶基因，并表明GLIPR 1编码分泌蛋白，并且GLIPR 1表达在前列腺癌进展期间通过表观遗传机制下调。基于序列同源性，我们确定并表征了一个新的p53靶基因簇，该基因簇位于人类染色体12 q21上，包括GLIPR 1和两个GLIPR 1样基因（GLIPR 1 L），以及位于小鼠染色体10 D1上，包括Glipr 1和三个Glipr 1样基因（Glipr 1 l）。功能分析表明，GLIPR 1或GLIPR 1 L2基因过表达或用GLIPR 1蛋白处理在体外和体内诱导各种小鼠和人癌细胞系在G 0/G1期的生长停滞和/或凋亡。为了测试GLIPR 1的肿瘤抑制活性，我们产生了携带Glipr 1基因失活突变的小鼠。长期队列分析表明，Glipr 1功能的丧失导致了由独特的恶性肿瘤谱引起的无肿瘤生存率降低。机制研究表明，GLIPR 1表达导致c-myc mRNA的下调，这表明GLIPR 1介导的细胞周期控制中的关键控制点。有趣的是，GLIPR 1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，p27 Kip 1水平升高。我们鉴定并使用化学和分子抑制剂来确认与GLIPR 1表达相关的促凋亡途径，其涉及ROS的产生、ASK 1-MEK 4/7-JNK途径的激活、Bcl-2的抑制和广泛的半胱天冬酶激活。MEF的分析显示，与Glipr 1-/- MEF相比，用DNA损伤剂处理导致Glipr 1诱导，ROS水平和JNK活性升高，并且Glipr 1 +/+中的凋亡增加。此外，Glipr 1-/- MEF在体外对ras + myc诱导的转化的敏感性增加。我们的研究结果证实GLIPR 1是一种新型的广谱肿瘤抑制因子，通过产生ROS介导促凋亡活性 JNK信号。我们现建议：（2）分析GLIPR 1或GLIPR 1 L21表达后导致ROS-ASK 1-MEK 4/7-JNK-凋亡增加的分子途径;（3）鉴定GLIPR 1结合蛋白/膜受体并表征GLIPR 1蛋白摄取和（4）产生ARR 2 PB-c-myc转基因小鼠，将这些小鼠与Glipr 1-/-小鼠杂交，并使用双基因小鼠分析内源性Glipr 1抑制体内癌前病变和恶性前列腺病变发展的能力的遗传活性。项目叙述：本项目旨在了解GLIPR 1（一种新发现的肿瘤抑制基因）抑制前列腺癌和膀胱癌生长的机制。我们的研究结果可能会发现新的诊断方法和/或导致这些恶性肿瘤的新疗法。

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.