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Von Hippel-Lindau (VHL) disease results from the inactivation of the VHL gene and is characterized by highly vascular tumors. A consequence of VHL loss is the stabilization of hypoxia-inducible factor (HIF) alpha subunits and increased expression of HIF target genes, which include pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF). In mice, homozygous deletion of VHL is embryonic lethal due to vascular abnormalities in the placenta; and, VHL+/− mice develop proliferative vascular lesions in several major organs, most prominently the liver. Loss of ELL-associated factor (EAF2) in murine models has also been shown to induce increased vascular density in the liver as well as the prostate. Previously, EAF2 was determined to be a binding partner of VHL and loss of EAF2 induced a reduction in pVHL levels and an increase in hypoxia induced factor 1α (HIF1α) levels in vitro. Here we characterized the cooperative effects of VHL- and EAF2-deficiency on angiogenesis in the liver and prostate of male mice. VHL deficiency consistently increased the incidence of hepatic vascular lesions across three mouse strains. These vascular lesions where characterized by an increase in microvessel density, and staining intensity of VHL target proteins HIF1α and VEGF. EAF2−/−VHL+/− mice had increased incidence of proliferative hepatic vascular lesions (4/4) compared to VHL+/− (10/18) and EAF2−/− (0/5) mice. Prostates of EAF2−/−VHL+/− mice also displayed an increase in microvessel density, as well as stromal inflammation and prostatic intraepithelial neoplasia. These results suggest that cooperation of VHL and EAF2 may be critical for angiogenic regulation of the liver and prostate, and concurrent loss of these two tumor suppressors may result in a pro-angiogenic phenotype. Electronic supplementary material The online version of this article (doi:10.1007/s10456-011-9217-1) contains supplementary material, which is available to authorized users.

ELL-associated factor 2 (EAF2) is an androgen-responsive tumor suppressor frequently deleted in advanced prostate cancer that functions as a transcription elongation factor of RNA Pol II through interaction with the ELL family proteins. EAF2 knockout mice on a 129P2/OLA-C57BL/6J background developed late-onset lung adenocarcinoma, hepatocellular carcinoma, B-cell lymphoma and high-grade prostatic intraepithelial neoplasia. In order to further characterize the role of EAF2 in the development of prostatic defects, the effects of EAF2 loss were compared in different murine strains. In the current study, aged EAF2(-/-) mice on both the C57BL/6J and FVB/NJ backgrounds exhibited mPIN lesions as previously reported on a 129P2/OLA-C57BL/6J background. In contrast to the 129P2/OLA-C57BL/6J mixed genetic background, the mPIN lesions in C57BL/6J and FVB/NJ EAF2(-/-) mice were associated with stromal defects characteristic of a reactive stroma and a statistically significant increase in prostate microvessel density. Stromal inflammation and increased microvessel density was evident in EAF2-deficient mice on a pure C57BL/6J background at an early age and preceded the development of the histologic epithelial hyperplasia and neoplasia found in the prostates of older EAF2(-/-) animals. Mice deficient in EAF2 had an increased recovery rate and a decreased overall response to the effects of androgen deprivation. EAF2 expression in human cancer was significantly down-regulated and microvessel density was significantly increased compared to matched normal prostate tissue; furthermore EAF2 expression was negatively correlated with microvessel density. These results suggest that the EAF2 knockout mouse on the C57BL/6J and FVB/NJ genetic backgrounds provides a model of PIN lesions associated with an altered prostate microvasculature and reactive stromal compartment corresponding to that reported in human prostate tumors.

Introduction: Inactivation of EAF2 has been shown previously to lead to tumorigenesis in multiple organs; however the mechanism of EAF2 tumor suppression remains unclear. Demonstrating functional interactions between p53 and EAF2 would link the newly discovered tumor suppressor EAF2 to the well-established p53 pathway, providing new insights into the mechanisms of p53 and EAF2 action. We previously have shown that EAF2 can co-localize and co-immunoprecipitate with p53 in transfected cells. In a TSP-1 promoter-driven luciferase reporter assay, p53 transfection suppressed the TSP-1 promoter activity and EAF2 co-transfection blocked the p53 suppression of TSP-1 promoter. However, EAF2 transfection alone had little or no effect on the TSP-1 promoter. In a murine model, EAF2 knockout caused TSP-1 down-regulation, supporting an in vivo role for EAF2 in regulation of TSP-1 expression. Methods: Mouse knockout, transient transfection, co-localization, co-immunoprecipitation, and luciferase-based promoter assay were employed in this study. Results: Ongoing animal studies show that loss of EAF2 and p53 displayed much more dramatic phenotypes than loss of EAF2 or p53 individually and resulted in more severe splenic extramedullary hematopoiesis, and further increased vessel density in the prostate and liver. Furthermore, double knockout of EAF2 and p53, but not knockout of EAF2 or p53 alone, resulted in prostate tumorigenesis. Conclusion: The above observations together suggest that EAF2 and p53 cooperate in prostate tumorigenesis and increased vascularity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2011-2400