Mattia Lion

p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

Background The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. Methodology/Principal Findings Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i) variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1. Conclusions/Significance We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.

Interaction between p53 and estradiol pathways in transcriptional responses to chemotherapeutics.

Estrogen receptors (ERs) and p53 can interact via cis-elements to regulate the angiogenesis-related VEGFR-1 (FLT1) gene, as we reported previously. Here, we address cooperation between these transcription factors on a global scale. Human breast adenocarcinoma MCF7 cells were exposed to single or combinatorial treatments with the chemotherapeutic agent doxorubicin and the ER ligand 17β-estradiol (E2). Whole-genome transcriptome changes were measured by expression microarrays. Nearly 200 differentially expressed genes were identified that showed limited responsiveness to either doxorubicin treatment or ER ligand alone but were upregulated in a greater than additive manner following combined treatment. Based on exposure to 5-fuorouracil and nutlin-3a, the combined responses were treatment-specific. Among 16 genes chosen for validation using quantitative real-time PCR, seven (INPP5D, TLR5, KRT15, EPHA2, GDNF, NOTCH1, SOX9) were confirmed to be novel direct targets of p53, based on responses in MCF7 cells silenced for p53 or cooperative targets of p53 and ER. Promoter pattern searches and chromatin IP experiments for the INPP5D, TLR5, KRT15 genes supported direct, cis-mediated p53 and/or ER regulation through canonical and noncanonical p53 and ER response elements. Collectively, we establish that combinatorial activation of p53 and ER can induce novel gene expression programs that have implications for cell-cell communications, adhesion, cell differentiation, development and inflammatory responses as well as cancer treatments.

Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay.

Co-evolution of transcription factors (TFs) with their respective cis-regulatory network enhances functional diversity in the course of evolution. We present a new approach to investigate transactivation capacity of sequence-specific TFs in evolutionary studies. Saccharomyces cerevisiae was used as an in vivo test tube and p53 proteins derived from human and five commonly used animal models were chosen as proof of concept. p53 is a highly conserved master regulator of environmental stress responses. Previous reports indicated conserved p53 DNA binding specificity in vitro, even for evolutionary distant species. We used isogenic yeast strains where p53-dependent transactivation was measured towards chromosomally integrated p53 response elements (REs). Ten REs were chosen to sample a wide range of DNA binding affinity and transactivation capacity for human p53 and proteins were expressed at two levels using an inducible expression system. We showed that the assay is amenable to study thermo-sensitivity of frog p53, and that chimeric constructs containing an ectopic transactivation domain could be rapidly developed to enhance the activity of proteins, such as fruit fly p53, that are poorly effective in engaging the yeast transcriptional machinery. Changes in the profile of relative transactivation towards the ten REs were measured for each p53 protein and compared to the profile obtained with human p53. These results, which are largely independent from relative p53 protein levels, revealed widespread evolutionary divergence of p53 transactivation specificity, even between human and mouse p53. Fruit fly and human p53 exhibited the largest discrimination among REs while zebrafish p53 was the least selective.

Reactivation of TAp73 tumor suppressor by protoporphyrin IX, a metabolite of aminolevulinic acid, induces apoptosis in TP53-deficient cancer cells

The p73 protein is a tumor suppressor that shares structural and functional similarity with p53. p73 is expressed in two major isoforms; the TA isoform that interacts with p53 pathway, thus acting as tumor suppressor and the N-terminal truncated ΔN isoform that inhibits TAp73 and p53 and thus, acts as an oncogene. By employing a drug repurposing approach, we found that protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid (ALA) applied in photodynamic therapy of cancer, stabilizes TAp73 and activates TAp73-dependent apoptosis in cancer cells lacking p53. The mechanism of TAp73 activation is via disruption of TAp73/MDM2 and TAp73/MDMX interactions and inhibition of TAp73 degradation by ubiquitin ligase Itch. Our findings may in future contribute to the successful repurposing of PpIX into clinical practice.

Abstract 746: Functional crosstalk between the p53 and NF-kB transcription factors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The tumor suppressor p53 and NF-kB are sequence-specific transcription factors (TFs) playing crucial roles in controlling cell proliferation and cell survival with an undeniable impact on cancer progression. In response to cellular stresses they induce the expression of a large variety of genes. Synergistic or antagonistic interactions between the p53 and NF-kB proteins may modulate the expression of common targets and potentially lead to a modified cellular response. To specifically investigate cell-autonomous, cooperative interactions between NF-kB and p53 on gene expression changes, we performed a genome-wide transcriptome analysis in the breast-cancer derived MCF-7 cells following single or combinatorial treatments with the chemotherapeutic drug doxorubicin (DXR) and the NF-kB inducer TNF-alpha (TNF). The microarray results revealed 720 upregulated (log2FC>1.5) and 926 repressed genes for the DXR+TNF treatment (log2FC 0.1} and 188 repressed genes {log2[FCDXR+TNF] - (log2[FCDXR] + log2[FCTNF]) < -0.1}. Notably, gene ontology analysis applied to these gene groups indicated an apparent enrichment for epithelial mesenchymal transition and cell migration functions. We focused the validation experiments on 15 up-regulated genes to investigate the direct involvement of p53 and NF-kB on their expression using RT-qPCR assays coupled to p53 activation by DXR, 5-fluorouracil or Nutlin, p53 RNAi and/or NF-kB chemical inhibition. In silico analysis of the potential target genes’ promoter sequence identified both p53 and NF-kB responsive regions where cis-mediated interactions of these TFs may occur, a result that has been followed up by mining of available ChIP-seq data. Among the 15 chosen targets, we uncovered five (LAMP3, ETV7, UNC5B, NTN1 and PLK3) that were induced synergistically after DXR + TNF treatment in MCF-7 breast cancer cells. Over-expression of LAMP3 has already been associated with an increased metastatic potential for particular cancers, and ETV7 can act as a regulator of cell proliferation. To study the migratory phenotype associated with the DXR+TNF combinatorial treatment, we performed migration assays using three different approaches: trans-well migration assay, Xcelligence Migration Assay and wound healing. The results consistently showed an increase in migration potential upon DXR+TNF compared to mock or to single treatments. We propose that the functional crosstalk between the p53 and NF-kB TFs can lead to the activation of specific gene expression programs that may impact on cancer phenotypes. Given that most tumors carry an activated NF-kB pathway, and a significant proportion of breast cancers, particularly the luminal subtype, maintains wild type p53, the uncovered crosstalk could potentially modify the efficiency of cancer therapy. Citation Format: Alessandra Bisio, Judit Zamborszky, Sara Zaccara, Mattia Lion, Toma Tebaldi, Yari Ciribilli, Alberto Inga. Functional crosstalk between the p53 and NF-kB transcription factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2013-746

Abstract 2286: p53-miR-dependent post-transcriptional circuits: mechanisms, targets and inter-individual variation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Using bioinformatics approaches, we identified a group of candidate microRNAs (miRs) for direct p53 transcriptional control. To validate p53 family-mediated control of the newly predicted target miRs we evaluated the potential for wild type p53, p63α and p73α to transactivate from p53 response elements (REs) mapped at the miR promoters. Results obtained with an established yeast-based assay indicated that the REs found at miR-10b, -23b, -106a, -151, -191, -198, -202, -221, -320, -1204, -1026 genes were responsive to p53 of which 8 were also responsive to p63α or p73α. ChIP assays in doxorubicin-treated HCT116 cells -p53+/+ vs p53-/– revealed moderate p53 occupancy at the miR-202, -1206 sites, and weak occupancy at miR-10b, -191. RT-qPCR analyses in HCT116 and MCF7 cells showed modest doxorubicin- and/or p53-dependent regulation for miR-23b, -151, -191. We then focused on miR-191 and its potential impact on MDM4, an important modulator of p53. The A>C SNP (rs4245739) in the proximal portion of the MDM4 3′UTR occurs in a predicted binding site for miR-191, with the A allele resulting in a mismatch at the predicted miR-191 seed. This SNP is in strong association with the tag-SNP (rs2369244) within the MDM4 haplotype that was reported to impact on cancer risk. Using MCF7 and HCT116 cells that are heterozygous for rs4245739, we examined at the endogenous MDM4 gene level the impact of varying the expression of miR-191. A slight allelic imbalance was observed by allele-specific qPCR when miR-191 was over-expressed, and the co-transfection of miR-34a, for which seed sequences are predicted in the distal portion of the MDM4-3′UTR, had an additive effect, leading to ∼20% reduction of the C-allele. Western blot analyses indicated that steady-state MDM4 protein levels were reduced by miR-191 and/or miR-34a overexpression. Our study reveals additional miRs that could be directly regulated by the p53-family of transcription factors and could contribute to the tuning of p53-induced responses with the possibility of inter-individual variations due to functional SNPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2286. doi:1538-7445.AM2012-2286