p53 Functional Interactions: the Study of a New Crosstalk with Estradiol Pathway in Transcriptional Responses to Chemotherapeutics

BACKGROUND: Objective of this thesis has been the analysis of the sequence specific transcription factor p53, a critical tumor suppressor protein, specifically, the crosstalk (or functional interactions) with other transcription factors, namely, the estrogen receptors, and the modeling in reconstituted assays of the interaction of p53 with positive and negative cofactors (e.g. MDM4 and 53BP1) and the impact of small molecules, including chemotherapeutic drugs, on such interactions. Previous reports have revealed a complex, often negative, crosstalk between p53 and estrogen receptors (ERs) related in part to the physical interaction between the two proteins. An example of transcriptional cooperation mediated by cognate, non-canonical cis-elements was instead discovered for the angiogenesis related VEGFR1, FLT1 promoter. MAIN TASK: Transcriptional cooperation between p53 and ERs was sought out on a global scale using the human breast adenocarcinoma MCF7 cells as a model and transcriptome analyses. Cells were subjected to single or combinatorial treatments with the chemotherapeutic agent doxorubicin (able to induce p53 protein stabilization) and the ER ligand 17β-estradiol (E2). 201 differentially expressed genes, that showed limited responsiveness to either doxorubicin treatment or ER ligand alone, but were up-regulated in a greater than additive manner following combined treatment were identified. Among sixteen genes chosen for validation using quantitative real-time PCR (qPCR), seven (INPP5D, TLR5, KRT15, EPHA2, GDNF, NOTCH1, SOX9) were confirmed to be novel direct targets of p53, based on responses in stable MCF7 clone cells silenced for p53, or cooperative targets of p53 and ER. Based on exposure to 5-fuorouracil (another genotoxic drug) and nutlin-3a (a non-genotoxic p53-specific activator), the combined response identified genes that were consistently regulated, although with different kinetics (e.g. INPP5D, CDH26, KRT15), while others (e.g. TLR5, SOX9) were treatment selective. Promoter pattern searches and chromatin IP experiments for the INPP5D, TLR5, KRT15 genes were also performed to interrogate a direct, cis-mediated p53 and ERs regulation. While these analyses confirmed the identification of novel direct p53 targets, the important contribution of ER in their transcriptional modulation and the role of non-canonical response elements, the correlation between occupancy levels and gene expression varied. SECONDARY TASK: Using a newly developed miniaturized yeast-based assay, functional interactions between p53 and its regulators MDM4 and 53BP1 was investigated. MDM4 was confirmed as a p53 negative regulator and the impact of nutlin-3a or RITA (apoptosis inducer through p53 binding) on the p53-MDM4 interaction was explored. Instead, no stimulatory effect of the p53 co-activator 53BP1 was detected. CONCLUSIONS: Collectively, the results indicate that combinatorial activation of p53 and ER can induce novel gene expression programs which have implications for cell-cell communications, adhesion, cell differentiation, development and inflammatory responses as well as cancer treatments. The yeast-based assay represents a versatile tool to study p53 interactions with cofactors.