Introduction: Regulation of AR mRNA stability and translation, two central processes that control AR expression, is little known. ErbB3 binding protein 1 (EBP1) overexpression decreases AR-target genes including AR itself at the mRNA and protein levels. Interestingly, AR protein levels were down regulated to a greater extent than mRNA levels. EBP1 has recently been demonstrated to be an RNA binding protein. We therefore examined the ability of EBP1 to regulate AR post-transcriptionally.

Experimental procedure: The stability of AR mRNA was analyzed in LNCaP cells engineered with EBP1 cDNA or shRNA at different times after treatment with actinomycin D by measuring the remaining levels of AR and GAPDH mRNAs by RT-qPCR. The steady-state levels of AR mRNA and protein were also detected by quantitative RT-PCR and immunoblotting analysis. To assess the association of endogenous EBP1 and AR mRNA, LNCaP cell lysates were immunoprecipitated with antibodies recognizing EBP1 or with control IgG, followed by PCR amplification using primers specific for AR. The interaction between the biotinylated transcripts spanning a 397-nt region of the 3’UTR containing the AR UC-rich element, or CAG polyglutamine repeat, which is predicted to form a stable stem-loop structure, at the 5’-end of the coding sequence in the AR mRNA, and recombinant His-EBP1 fusion proteins was assessed by biotin pull-down assays followed by Western blot analysis. In similar way, the domain of EBP1 responsible for the binding to CAG and UC-rich sequences was mapped. To determine if EBP1 affects the ability of the UC-rich region to accelerate AR mRNA decay, a pGL3-Luc-UC plasmid was constructed by fusing the UC-rich region in the 3’UTR of AR mRNA to the 3’ end of the Firefly-luciferase coding sequence and luciferase reporter assays performed in LNCaP cells engineered with EBP1 cDNA or shRNA. To test if EBP1 may modulate not only AR stability, but also its translation, linear sucrose gradient fractionation was performed to investigate the distribution of AR mRNA associated with polysomes in LNCaP cells engineered with EBP1 cDNA or shRNA.

A summary of the data: EBP1 promoted AR mRNA decay through physical interaction with a conserved UC-rich motif within the 3’-UTR of AR. The ability of EBP1 to accelerate AR mRNA decay was further enhanced by HRG treatment. EBP1 also bound to a CAG-formed stem-loop in the 5’ coding region of AR mRNA and reduced percentage of AR mRNA in a translationally active polysome pool.

Conclusion: Our current work indentified EBP1 as a novel AR mRNA-binding protein regulating AR mRNA stability and translation, and suggests that targeting EBP1 has potential mechanistic and functional significance in the therapeutic management of prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3163.