Abstract B47: Identification of novel E2F3 transcriptional targets expands the role of the RB/E2F axis beyond cell cycle control

Abstract

Abstract The RB/E2F axis is frequently disrupted in multiple tumors. Classical studies reported that the RB tumor suppressor exerts its transcriptional repressor function through an interaction with the E2F family of transcription factors (E2F1-3) to control the timely expression of genes required for DNA replication and cell cycle progression. The E2F3 locus encodes two E2F3 isoforms, a and b, which have unique N-terminal sequences. Multiple studies indicate that E2F3, rather than E2F1 or 2, is elevated in various cancers, including prostate. In prostate tumors elevated E2F3 expression is an independent prognostic factor of clinical outcome, but the direct transcriptional targets of E2F3a and E2F3b have not been characterized. We found that E2F3a and b expression was increased in prostate tumor derived cell lines when compared to non-transformed controls. An expression array analysis following knockdown (k/d) of total E2F3 identified several unexpected targets including Interleukin 6 receptor (IL-6R), a critical component of the IL6 signaling cascade and calpain 2, a protease that is essential in cellular migration and has been shown to be elevated during prostate tumorigenesis. Further studies showed that the two genes were regulated by different E2F3 isoforms- IL6R was a target of E2F3a, while calpain 2 was regulated by E2F3b. Expression of both genes was enhanced by RB ablation. E2F3a-dependent IL-6R regulation was apparent in PC3, LNCaP, CWR-R1, and 22Rv1 cells. Chromatin immunoprecipitation (ChIP) studies identified sequences in the IL-6R promoter that were bound by E2F3. Transient co-transfection studies using an E2F3a expression plasmid showed that E2F3a transactivated the IL-6R promoter in a dose dependent manner. The IL-6R initiated signaling cascade was perturbed following a k/d of E2F3a since the levels of ERK1/2 phosphorylation was reduced. An siRNA-mediated ablation of each isoform revealed that E2F3b, not E2Fa, was effective is reducing calpain 2 levels. ChIP studies indicated that E2F3 binds to the endogenous calpain 2 promoter, and in transient transfection studies E2F3b transactivated the calpain 2 promoter in a dose dependent manner. An analysis of the calpain 2 promoter identified an androgen receptor (AR) half site. Additional transfection studies showed that the AR can cooperate with E2F3b in transactivation of the calpain 2 promoter. Moreover, reduced expression of E2F3b impaired cellular migration in a wound assay. The expression of the E2F3 isoforms has not been previously analyzed in the TRAMP prostate tumor model. Western immunoblot studies of five TRAMP tumors and three age and strain matched prostates showed that TRAMP tumors had highly elevated expression of both E2F3 isoforms. These results indicate that increased expression of both E2F3 isoforms is a feature of human tumor-derived cell lines and the TRAMP mouse model of prostate tumorigenesis. Calpain 2, expression was readily detected in all of the TRAMP tumors, but not in the control tissue. Hence E2F3 overexpression and increased calpain levels are features of human and mouse tumors. This analysis broadens the role of E2F3 in prostate tumorigenesis beyond the regulation of cell cycle progression. E2F3a is a link between the E2F/RB and the IL-6 signaling cascade, while E2F3b regulates expression of a protease that is essential in cell adhesion and migration. Therefore E2F3 deregulation affects multiple signaling networks to promote tumorigenesis. Citation Format: Stephen J. Libertini, Maria Mudryj, Alan P. Lombard, Honglin Chen, Veronica Rodriguez, Carlos Perez-Stable, Bushra al-Bataina, Tilak Koilvaram, Michael George, Allen C. Gao. Identification of novel E2F3 transcriptional targets expands the role of the RB/E2F axis beyond cell cycle control [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B47.