Abstract 1208: Stable EP4 receptor knockdown inhibits prostate cancer growth in vivo
Authors: Huang Hosea , Shu Ping , Murphy Thomas , Jordan Mark 
Published: August 2012

INTRODUCTION AND OBJECTIVES: Prostaglandin E2 (PGE2) has been implicated in prostate cancer (CaP) growth. The cellular effects of PGE2 are mediated through the G protein-coupled receptors EPR1-4. We have previously shown increased EP4 expression in CaP clinical radical prostatectomy specimens compared with benign prostate tissue. In order to determine whether EP4 plays a functional role in CaP growth, we derived stable EP4 knockdown (KD) clones from the murine RM1 cell line and evaluated in vitro growth and in vivo tumorigenesis. METHODS: Stable knockdown of the EP4 gene in mouse CaP RM1 cells was achieved by lentiviral infection with EP4 shRNA particles (Santa Cruz Biotech). Of the 5 randomly selected clones derived from puromycin resistant single cells, EP4 knockdown was confirmed in 2 clones (2B3 and 1F5) by RT-PCR. These 2 clones were further assessed for in vitro cell growth by direct cell counting, soft agar colony formation and in vitro directed cell migration. In vivo tumorigenesis was assessed by injecting 25 x 103 cells suspended in 0.1 ml of Matrigel into the flank region of syngeneic B6 mice. Tumor size was measured twice weekly, and the mice were sacrificed at the end of the 3rd week. Controls were wild type (wt) and non-targeting shRNA infected (nTG) RM1 cells. RESULTS: Clones 2B3 and 1F5 had selectively reduced EP4 mRNA (RT-PCR) with preserved EP1-3 mRNA levels and hence were used for further study. EP4 KD cells (clones 1F5 and 2B3) exhibited significantly (p&lt;0.01) reduced in vitro growth, colony formation (p&lt;0.01) and in vitro directed migration (p&lt;0.05) compared with that of wt RM1 and nTG controls. Mean tumor weights (gm) from a representative experiment of 4 (n=8 animals per group): 1F5: 0.03 ± 0.04; 2B3: 0.80 ± 0.1 gm; both p&lt;0.001 vs wt RM1: 1.27 ± 0.31 and nTG: 1.25±0.26). Reduced EP4 expression and normal expression of EP1, EP2 and EP3 in tumor tissue derived from 2B3 and 1F5 cells were further confirmed by IHC. The proportion of animals developing tumors after in vivo implantation of stable EP4 knockdown cells from clones 2B3 and 1F5 were also reduced (55% and 27%, respectively) compared with WT (100%) and nTG (87%) controls. IHC of in tumors derived from 1F5 and 2B3 cells also revealed reduced expression of MMP9 and VEGF compared with WT and nTG controls. CONCLUSIONS: shRNA knockdown of the EP4 gene in murine CaP RM1 cells significantly reduced subsequent in vivo tumorigenesis with reduced tumor number and tumor weight, colony formation, migration, and expression of the metastasis-related genes MMP9 and VEGF. These findings show that EP4 knockdown inhibits in vivo CaP growth associated with reduced metastasis-related gene expression and suggests a novel therapeutic target for CaP.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1208. doi:1538-7445.AM2012-1208