Molecular mechanisms of growth suppression by pharmacologically activated p53

Sammanfattning: The tumor suppressor p53 is a transcription factor that is crucial for protecting cells from cancer development. The importance of p53 tumor suppression function is highlighted by the fact that the p53 pathway is inactivated in most, if not all cancers. Mutation of the p53 gene occurs in about 50% of all tumors, whereas in the tumors which retain wild-type p53, the function of p53 is abolished due to deregulation of the p53 pathway. Due to the potency of p53 in suppressing tumors, p53 is considered to be an attractive therapeutic target. This is further strengthened by the in vivo mouse models which demonstrated regression of already established tumors upon reinstatement of p53. The aims of this thesis were to identify new p53 reactivating compounds and to investigate the mechanisms of action of the previously identified p53-reactivating molecule RITA. The NCI library of low molecular weight compounds was screened for molecules that suppressed cell growth in a p53-dependent manner. We identified the small molecule MITA, which induces p53-dependent cell death in a variety of human tumor cells. The p53/MDM2 interaction is blocked by MITA which results in accumulation of p53 in cells. The expression of p53 target genes MDM2, BAX, PUMA and GADD45 is induced upon activation of p53 by MITA. Importantly, MITA does not induce p53 or its target genes in normal human diploid fibroblasts (NHDF), which correlates with the absence of growth suppression. The low molecular weight compound RITA was previously identified in a cell-based screen. RITA has been shown to bind p53 and subsequently inhibit its interaction with MDM2. RITA induces p53-dependent apoptosis in cancer cells and in vivo in human tumor xenografts in mice. In the studies presented in this thesis we further elucidated the mechanisms of RITA action. Oligonucleotide microarray analysis of gene expression profiles revealed that RITA targets p53 with high specificity and that it mainly induces pro-apoptotic targets of p53. In line with these results, p53 wild-type expressing cell lines of different origin revealed a predominant apoptotic response. We demonstrate that MDM2 released from p53 by RITA promotes degradation of p21 and the p53 cofactor hnRNP K, required for p21 transcription. This leads to downregulation of p21 on both transcriptional and protein level. Consequently, MDM2 acts as a switch between growth arrest and apoptosis upon p53 activation. The preferential induction of apoptotic response by RITA is further facilitated by the targeting of the p53 regulator HDMX. RITA inhibits the p53/HDMX interaction and induces p53-dependent downregulation of HDMX on protein and on mRNA level. This ensures a robust activation of p53 leading to induction of apoptosis. Additionally, a new cellular target of RITA was identified, namely the redox protein TrxR1. We demonstrated that RITA binds purified TrxR1 protein directly and inhibits its activity in vitro and in cells. Notably, the inhibition of TrxR by RITA appeared to be p53-depedent and correlated with the induction of a covalently linked TrxR1 dimer and induction of ROS. We believe that inhibition of TrxR1 in a tumor-dependent and p53-dependent manner might contribute to the tumor-specific cell killing properties of RITA. In conclusion, we believe that compounds such as RITA and MITA may not only be used as lead compounds for anti-cancer therapy, but may also be useful tools to study of p53 functional activity.