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.
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