Abstract 4194: The superoxide dismutase 1 (SOD1) 3′-UTR maintains high expression of the SOD1 gene in cancer cells

Abstract

Abstract Background: Superoxide dismutase 1 (SOD1), the ubiquitously expressed and predominant superoxide dismutase, catalyzes the conversion of superoxide ions into hydrogen peroxide which will be further detoxified by catalase or glutathione peroxidase. The SOD1 gene has been reported to be over-expressed in human cancer cells, and targeting SOD1 has been proposed as a new strategy for cancer therapy. While the transcriptional regulation of the SOD1 gene has been well-characterized, the contribution of the SOD1 3′-UTR to SOD1 gene expression has not been previously examined in any model systems. Methods: The full length (325 bases) and different fragments of the SOD1 3′UTR were amplified from human cancer cells. Each of the amplified products was inserted into a pGL3-promoter reporter vector downstream of the luciferase cDNA. The reporter constructs were transfected into Panc-1 (pancreatic cancer), A2780 (ovarian cancer), and HepG2 (liver cancer) cells and luciferase activity was analyzed. The wild type pGL3-promoter vector and the antisense orientation of the SOD1 3′-UTR reporter construct served as controls. Luciferase mRNA stability was examined by reverse transcriptase real-time PCR. Western blot was performed to examine endogenous SOD1 protein expression levels. Results: The luciferase activity was 10 (HepG2) and 70 (A2780 and Panc-1) fold higher in cells transfected with the full-length SOD1 3′-UTR compared with those in control cells. The dramatically increased luciferase activity was likely attributed to enhanced luciferase mRNA stability as analyzed by real-time RT-PCR. Significantly reduced luciferase activities were found in cells transfected with the reporter constructs bearing different fragments of the SOD1 3′-UTR. Most noticeably, transfection with the reporter construct bearing a deletion of the first 200 bases of the SOD1 3′-UTR brought the reporter activity down to a level similar to that in controls. Further deletion analysis showed that the secondary structure of the SOD1 3′-UTR may also play a key role in SOD1 3′-UTR-mediated gene expression and the putative AU-rich elements are unlikely to confer the sustained SOD1 gene expression. Moreover, we found that the full-length SOD1 3′-UTR can serve as a decoy or competitor to attenuate endogenous SOD1 protein expression, indicating the involvement of trans-acting RNA binding factors in maintaining high expression of the SOD1 gene. Summary: The SOD1 3′-UTR maintains the high expression of the SOD1 gene in human cancer cells likely through increasing SOD1 mRNA stability. The interaction of the SOD1 3′-UTR with the trans-acting RNA binding proteins is required to maintain SOD1 gene expression. We are actively working on identifying these RNA binding proteins and their interacting cis-elements in the SOD1 3′-UTR in our model systems. 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 4194. doi:1538-7445.AM2012-4194