Mechanisms of p53-mediated intrinsic and extrinsic tumor suppression

p53 is a promising target for cancer therapy. However, the molecular basis of the p53 tumor suppression function remains incompletely understood. Thus, in this thesis, we focused on studies of the molecular mechanisms of p53-mediated tumor suppression. Since p53 mainly functions as a transcription factor, we addressed whether it is the promoter binding pattern of p53 or its cooperation with different other transcription cofactors that determinates the transcription profile and the subsequent biological outcomes.

We explored the genome-wide binding sites of p53 with ChIP-seq. By comparing the p53-bound sites in chromatin in breast cancer cells upon p53 activating compounds inducing different outcomes (nutlin: cell cycle arrest; RITA: apoptosis; 5-FU: cell cycle arrest), we found that the major binding patterns of p53 are similar, regardless of the stimuli and biological outcomes. We identified 280 novel p53 target genes by parallel analysis of gene expression. Further investigation revealed that the repression of several genes, including oncogene AURKA, by p53 could be enhanced by STAT3 inhibition. We also found that Sp1 is a co-regulator of p53 transcriptional response and apoptosis upon RITA. Our results emphasized the importance of cofactors in p53-mediated transcriptional response.

In paper II, we performed genome-wide shRNA screen to identify genes essential for p53-mediated apoptosis. Integration of these data with gene expression analysis lead to the identification of Sp1 as a key cofactor indispensable for the initiation of p53-mediated pro-apoptotic transcriptional repression, required for the robust apoptosis. However, Sp1 had no effect on neither induction of pro-apoptotic genes nor p53-mediated cell cycle arrest. Using ChIP-seq data in combination with ChIP-PCR for p53 and Sp1, we uncovered that p53-mediated pro-apoptotic transcriptional repression required the co-binding of Sp1 to p53 target genes. Further study revealed that MDM2-medated degradation of Sp1 serves to counteract p53-mediated transcriptional repression and apoptosis. This study helps to promote our understanding of the mechanisms of p53-mediated apoptosis and provides new targets and strategy for p53/MDM2-based therapies.

Recent studies suggest that p53 plays a role in modulating the anti-tumor immune response. In paper III we focused on studies of the mechanisms by which p53 regulates immune surveillance. Our results show that reactivation of p53 by the small molecule RITA stimulated NK cell-mediated killing of primary human tumor cells derived from metastatic cancers of different origins via p53-dependent induction of ULPB2, a ligand of NK cell receptor NKG2D. We further identified ULBP2 as a direct transcriptional target gene of p53 with a p53 response element within its first intron, with which p53 regulates its transcription.

Interestingly, we found that, without p53 activation, this promoter region was methylated. The de-methylation of this region is required for ULPB2 induction by p53. Our studies provide a molecular evidence for the direct transcriptional control of immune surveillance upon pharmacological restoration of p53 function. This contributes to better understanding of the interaction between tumors and immune system, and opens up a possibility for novel approaches for p53-based anti-tumor immune therapy.

List of scientific papers

I. Nikulenkov F, Spinnler C*, Li H*, Tonelli C, Shi Y, Turunen M, Kivioja T, Ignatiev I, Kel A, Taipale J, Selivanova G. Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis. Cell Death Differ. 2012 Dec;19(12):1992-2002.
https://doi.org/10.1038/cdd.2012.89.

II. Hai Li, Yu Zhang, Anda Ströse, Yao Shi, Donato Tedesco, Katerina Gurova, Galina Selivanova. Integrated high throughput analysis identifies Sp1 as a crucial determinant of p53-mediated apoptosis. [Manuscript]

III. Li H, Lakshmikanth T, Garofalo C, Enge M, Spinnler C, Anichini A, Szekely L, Kärre K, Carbone E, Selivanova G. Pharmacological activation of p53 triggers anticancer innate immune response through induction of ULBP2. Cell Cycle. 2011 Oct 1;10(19):3346-58.
https://doi.org/10.4161/cc.10.19.17630