IFI16 is a novel WT1-associated protein identified by expression profiling in syndromic versus sporadic wilms tumors

Proc Amer Assoc Cancer Res, Volume 47, 2006 3966 The Wilms tumor gene ( WT1 ) is mutated in syndromic Wilms tumors (Denys-Drash, Frasier and WAGR syndromes) but such mutations are rare in sporadic Wilms tumors. Our prior comparison of gene profiles between syndromic and sporadic Wilms tumors identified sets of genes, including PAX8 , GPR39 and IFI16, that are differentially expressed in WT1-replete versus WT1-null cases (Li et al ., AJP 2004). Here, we focused on IFI16, a transcriptional modulator, which was over-expressed in 70 percent of the WT1-replete Wilms tumors but was not highly expressed in any of the WT1-null cases. To follow up the Wilms tumor data, we utilized tetracycline-repressible Saos-2 cell lines carrying either the wild-type WT1 (-KTS isoform) or a missense mutant of WT1, which is incapable of activating gene transcription. Using the HG-U95A microarrays and Northern blot analysis, we verified that IFI16 showed the strongest response to WT1. Immunohistochemical analysis demonstrated that nuclear IFI16 and WT1 were widely co-expressed in WT1-replete Wilms tumors, while IFI16 was undectable in WT1-null Wilms tumors and in normal human mid-gestation fetal kidneys. Chromatin immunoprecipitation assays performed in a Wilms tumor cell line and 293 kidney cells suggested that IFI16 is not a direct target gene of WT1. However, in transient reporter assays, WT1 activated a promoter sequence consisting of 1.6 kb of DNA upstream of the IFI16 transcriptional initiation site, suggesting that IFI16 represents an indirect transcriptional target of WT1. Endogenous IFI16 and WT1 interacted in vivo in two Wilms tumor cell lines, with the interaction mapped predominantly to the four C-terminal zinc fingers of WT1, while weak interaction was detected with the N-terminal effector domain of the protein. In transient reporter assays, IFI16 augmented WT1-mediated transcriptional activation of an artificial promoter containing consensus WT1-binding sites, as well as that of the Sprouty1 and IFI16 promoters. Interestingly, the augmentation of WT1-mediated transactivation by IFI16 correlated with a dose-dependent increase in the protein levels of WT1 in the presence of increasing amounts of IFI16. To determine the biological importance of IFI16 in Wilms tumors, we repressed expression of the protein using RNAi. Strikingly, knockdown of IFI16, as well as WT1, led to decreased growth of a Wilms tumor cell line. These data suggest that IFI16 and WT1, in the context of sporadic Wilms tumors, may represent oncoproteins that co-operate to contribute to tumor growth.