U19/EAF2 is a newly identified tumor suppressor that is frequently down-regulated in human prostate cancer and has been shown to induce tumorigenesis in multiple tissues in the mouse model. However, the mechanism of U19/EAF2 tumor suppression remains unclear. In the present study, we have generated evidence for a link between U19/EAF2 and the p53 pathway and shown that U19/EAF2 is required for normal expression of TSP-1, providing a molecular mechanism involved in U19/EAF2 action.
The effect of U19/EAF2 knockout on TSP-1 expression is unlikely to be secondary to the phenotype changes in the knockout prostate. The TSP-1 down-regulation was identified in 3 month old U19/EAF2 knock out mice, which did not develop obvious phenotype. The modulation of TSP-1 promoter activity by U19/EAF2, particularly in the presence of p53, in cultured cells suggested that U19/EAF2 regulation of TSP-1 expression in vivo also occurs at the promoter, not secondary to phenotype changes. The ability of U19/EAF2 to increase the activity of TSP-1 promoter, particularly in the presence of p53, in cultured cells is in agreement with the in vivo observation that U19/EAF2 knockout caused TSP-1 down-regulation.
Studies on p53 regulation of TSP-1 expression are conflicting. While some studies have shown that p53 expression levels corresponds with TSP-1 expression and that the loss of p53 and resulting reduction in TSP-1 expression contributes to angiogenesis, others have failed to demonstrate a correlation between p53, TSP-1 and tumor progression (reviewed in (Ren et al., 2006
). The effect of tumor suppressors and oncogenes, including p53, on TSP-1 expression seems to vary and may be cell type- or tissue type-specific. Our studies showed that p53 inhibited the TSP-1 promoter activity in PC-3 cells. The inhibition was also observed in LNCaP, H1299 and 293 cell lines, and p53 suppression of TSP-1 expression has also been reported in HA1E cells (Zhou et al., 2009
), suggesting that p53 repression of TSP-1 promoter activity is likely to occur in many different types of cells. However, several other studies have reported that p53 has no effect on TSP-1 promoter activity (Cinatl et al., 1999
; Reiher et al., 2001
; Zhou et al., 2009
). We have searched for and were unable to identify potential p53 binding sites in the TSP-1 promoter using the basic local alignment search tool (BLAST) (Altschul et al., 1990
), suggesting that p53 may not directly bind to the TSP-1 promoter. The repression of TSP-1 promoter by p53 may be mediated through other co-factors or co-repressors that bind to the TSP-1 promoter. Further studies will be needed to elucidate the mechanisms by which p53 regulates TSP-1 expression and the mechanisms by which U19/EAF2 modulates p53 regulation of the TSP-1 promoter.
Down-regulation of TSP-1 is likely to contribute to the elevated CD31-positive blood vessel density in the U19/EAF2 knock out liver. TSP-1 is a well established anti-angiogenesis factor. Significant TSP-1 down-regulation at both mRNA and protein levels is likely to have functional consequences, causing elevated blood vessel density. VEGF is another important factor that regulates angiogenesis. Since VEGF expression was not altered in U19/EAF2 knockout mice, U19/EAF2 affect on CD31-positive blood vessel density is unlikely mediated through VEGF. Taken together, our observations suggested that TSP-1 down-regulation represents a major mechanism mediating the influence of U19/EAF2 knockout CD31 positive blood vessel density.
Our studies argue that U19/EAF2 could suppress tumorigenesis via multiple pathways. U19/EAF2 could inhibit cell proliferation directly, indicating that this loss of growth inhibition would represent another mechanism of tumorigenesis in U19/EAF2 knockout mice. In this study, we found increased CD31-positive large vessel density in the U19/EAF2 knockout liver, suggesting a potential role of U19/EAF2 in the regulation of angiogenesis via TSP-1 down-regulation. Given the established importance of angiogenesis in carcinogenesis, down-regulated TSP-1 expression may also contribute to the increased tumor formation in U19/EAF2 knockout mice.
The functional interactions between U19/EAF2 and p53 may be indirect. Although U19/EAF2 could alleviate p53 repression of TSP-1 promoter activity, U19/EAF2 did not block p53 inhibition of colony formation in prostate cancer cells. This observation suggests that U19/EAF2 does not counteract the influence of p53 on cell growth and/or survival in culture. Both U19/EAF2 and p53 are growth suppressive and our results suggest that the inhibition of colony formation by U19/EAF2 and p53 together is greater than the inhibition by either of them individually. Thus, these two tumor suppressors could work together in inhibiting carcinogenesis under certain circumstances, while antagonizing the regulation of TSP-1 expression.
Previous studies showed that androgen receptor (AR) can regulate TSP-1 expression in the prostate via direct binding to androgen response elements (AREs) in the TSP-1 promoter. Since U19/Eaf2 expression is regulated by AR in the prostate, androgens could influence TSP-1 expression in the prostate both directly, via AR, and indirectly, via U19/Eaf2. These direct and indirect mechanisms may allow androgens to more dramatically modulate TSP-1 expression in the prostate.
The biochemical nature of U19/EAF2’s association with p53 remains to be elucidated. They may be brought together via their mutual binding partners, such as ELL and/or pVHL. Whether U19/Eaf2 can make contact with p53 directly remains to be investigated. We were also unable to show whether endogenous U19/EAF2 could co-immunoprecipitate with endogenous p53 at this point. Existing anti-U19/EAF2 antibodies are insufficient for protein analysis because they are non-specific. Additionally, because U19/EAF2 is growth suppressive and frequently down-regulated in prostate cancer cells, endogenous U19/EAF2 protein expression level is very low. The association between endogenous U19/EAF2 and p53 also may not be stable. The co-immunoprecipitation studies to address these issues may be feasible when high quality anti-U19/EAF2 antibodies are developed and available.
Our studies showed that TSP-1 down-regulation by U19/EAF2 knockout was tissue-specific in the mouse model. For example, we did not detect significant differences in TSP-1 expression between wild-type and U19/EAF2 knockout lung (data not shown). This indicates the tissue-specificity of U19/EAF2 regulation of TSP-1 expression and the influence of cellular context on the regulation TSP-1 by U19/EAF2. One potential explanation may be that EAF1, the U19/EAF2 homolog, may be able to compensate for the loss of U19/EAF2 in TSP-1 regulation in some tissues, such as the lung, but not in other tissues, such as the liver and prostate. Alternatively, U19/EAF2 may not interact with p53 or p53 may affect TSP-1 expression in an organ-specific manner.
In conclusion, our studies provide evidence for functional interactions between a well established tumor suppressor, p53, and a newly identified tumor suppressor, U19/EAF2. Mouse knockout studies revealed that U19/EAF2 is required for the normal expression of TSP-1 and blood vessel formation in a tissue-specific manner. We found that U19/EAF2 co-immunoprecipitated and co-localized with p53 in transient transfected cells suggesting functional interaction. Co-transfection studies suggested that U19/EAF2 regulation of TSP-1 expression may involve alleviation of p53 repression of the TSP-1 promoter activity. Taken together, our studies indicate that U19/EAF2 can modulate blood vessel formation in part via alleviating p53 repression of TSP-1 expression.