Inhibition of p53 Tumor Suppressor Function by HTLV-I

Brady, John

Abstract

Inhibition of p53 function, either through mutation or inhibition by viral transforming proteins, correlates strongly with the oncogenic potential of the cell. In the following report, we describe a unique mechanism of p53 inactivation that involves the interaction of p53 with the p65 subunit of NF-kB. The inactive p53 complex is induced in HTLV-1-transformed and ATL leukemic cells. This mechanism of p53 inhibition may occur in other human cancers. We initially demonstrated that wild-type p53 is stabilized and transcriptionally inactive in HTLV-transformed cells. The viral transcriptional activator Tax plays a role in both the stabilization and inactivation of p53. p53 is hyperphosphorylated at serines 15 and 392 in HTLV-1-transformed cells and phosphorylation of p53 at these specific residues inactivates p53 by blocking its interaction with basal transcription factors. In T-lymphocytes, Tax-induced p53 inactivation is dependent upon NF-kB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-kB. Further, the p65 subunit of NF-kB is critical and uniquely involved in the Tax-induced p53 inhibition pathway. Using chromatin immunoprecipitation assays we have determined that in HTLV-1-transformed cells, p53 and p65 form a complex on the inactive MDM2 promoter. Consistent with reduced transcription activity, TFIID binding is not observed. These studies provide evidence that the divergent NF-kkB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels which include post-translational modification of p53. Further studies determined that p53 inhibition by Tax required a unique IKKbeta complex that required AKT activation. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant resulted in increased Tax-mediated NF-kB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT siRNA prevented NF-kB activation and inhibition of p53. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kappaB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is """"""""druggable"""""""" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kappaB suggests a promising strategy for the treatment of HTLV-1-transformed cells. We further extended these studies to determine if the mechanism of p53 inactivation in HTLV-1 transformed cells could be more generalized in human cancers. To do this we examine the NCI-60 cells. Of the 60 cell lines, 8 had a wild type but impaired p53 protein. In all 8 cell lines which include renal carcinoma, melanoma, glioblastoma and ovarian carcinoma cells, the p53 protein was reactivated by treatment with 9AA. Transcriptional activation of p53 resulted in reduced cell viability and increased apoptosis. In contrast to HTLV-1-transformed cells, 9AA did not inhibit NF-kB activity in the NCI-60 cell lines tested. These results indicate that 9AA is effective at reactivating p53 function but may do this by various mechanisms depending on the cell type.