The p53 tumor suppressor and master regulator is central to human DNA repair, damage checkpoints and many aspects of human biology. It controls cell fate in response to many types of internal and environmental stress. Importantly, most cancers are altered for p53 function. We have discovered a greatly expanded universe of p53 targets and human diversity as well as variations in responses to specific stresses. The DNA binding and transactivation of p53 is critical for tumor suppression. There is considerable variation in p53 dependent expression across 100s of targeted genes leading to differences in p53-mediated biological consequences. Much of that is due to target RE sequence. As described below the universe of genes subject to direct p53 control is much larger than originally anticipated based simply on previously established response element (RE) consensus target sequence, consisting of 2 copies of the sequenceRRRCA/TT/AGYYY (R, purine;Y, pyrimidine) separated by a spacer of up to 13 bases. There is considerable flexibility in what constitutes a functional RE. We focus on RE functionality, i.e., the ability of REs to support transactivation by p53. To directly assess transactivation responsiveness of human REs, we developed promoter systems in budding yeast for variable p53 expression (i.e., rheostatable). This has been used to establish the functional evolution of REs across species (discussed below) and has been summarized in our piano model that describes functional variability within a transcriptional network. We have translated many of the findings in yeast to p53 in human cells in culture and ex vivo. Recently, we established functionality of and sites. Also, increases in spacer length of only a few bases in a target RE greatly reduces functionality in terms of p53 transactivation. With Gilbert Schoenfelder (Berlin), we identified a novel mechanism of p53 transcriptional control of genes in studies of a SNP located in the promoter of the Flt1 gene. This C to T SNP results in generation of a perfect p53 -site RE and is present in 5% of the population. Our findings provided the first report of a functional p53 -site. Endogenous p53-dependent induction of FLT-1 mRNA was only observed in cell lines containing the FLT1-T allele in response to several DNA damaging agents. These results established that p53 can differentially stimulate transcription at a polymorphic variant of the FLT1 promoter and placed the VEGF system directly in the p53 stress-response transcriptional network via FLT1. We recently examined transactivation capacity towards a panel of 15 p53 -sites in p53-null human osterosarcoma cells (SaOS2) transiently co-transfected with a WT p53 vector. Some 1/2 site REs supported p53 transactivation to levels similar to a weakly responding full-site RE. This finding greatly expands the potential p53 master regulatory network. We are currently using p53 ChIP-seq approaches to identify targeted genes and networks, investigate crosstalk between regulatory networks especially other stress and DNA response networks. INTERACTION OF p53 AND ER REGULATORY NETWORKS. We identified a -site estrogen receptor RE (ERE) that greatly increased p53 induced transactivation at the FLT1 -site p53 RE, thereby establishing a new dimension to the p53 master regulatory network. Recently, we addressed the generality of synergistic transactivation by p53 and ER acting in cis. The 1/2 site in the FLT1 promoter was replaced with various 1/2 sites as well as canonical weak and strong human p53 REs. p53 transactivation was greatly enhanced by ligand-activated ER acting in cis. Enhanced transactivation extends to several cancer-associated p53 mutants with altered function, suggesting ER-dependent mutant p53 activity for at least some REs and possibilities for reactivation of cancer mutants. We propose a general synergistic relationship between the p53 family and ER master regulators in transactivation of p53 target canonical and noncanonical REs which might be poorly responsive to p53 on their own. We are developing a functional matrix as a tool for genome-wide searches for putative p53 target genes via noncanonical sites and augmenting transcriptional factors. In a limited search to identify motifs containing both strong p53 noncanonical RE sequences (including decamers with a CATG core) and associated ER responsive sequences (FLT1-like motifs), we identified the human RAP80 gene and Toll-like receptors (TLRs) that determine innate immunity. The identification of p53 target REs associated with the TLR genes has led to exciting new findings. Among the 10 human TLRs, nine had canonical and noncanonical p53 REs that may be functional. Using primary human cells obtained in a collaboration with the Clinical Research Unit we examined expression of the entire TLR gene family following exposure to anti-cancer p53 inducing agents. Expression of all TLR genes, TLR1-10, in blood lymphocytes and alveolar macrophages from healthy volunteers is inducible by DNA metabolic stressors. However, there is considerable inter-individual variability. Similarly p53 dependent TLR expression is detected in human cancer cell lines. For some TLRs the p53 control seems to enhance the inflammatory responses, mediated by activation of TLRs in the presence of natural ligands. Furthermore, a polymorphism in the TLR8 promoter provides the first human example of a p53 target RE sequence specifically responsible for endogenous gene induction. CANCER-ASSOCIATED P53 MUTANTS. Nearly all cancers have mutant or reduced expression of the p53 tumor suppressor gene. Using yeast-based and human cell systems, we show that functional p53 mutations can lead to considerable diversity in the spectrum of responses from REs including 1) decrease/loss-of-function;2) subtle changes;3) altered specificity;and 4) super-transactivation all of which lead to variation in biological responses. Most of the functional mutants (8/9) were able to function at 1/2 sites. We extended our analysis of p53 mutants in yeast-based systems to a clinical study of breast cancer patients undergoing neoadjuvant treatment. Several transcriptionally active p53 mutants identified in breast had subtle defects in transcription at REs that can only be revealed at low expression levels achievable in the yeast-based system. It appears that nonfunctional as compared with functional missense mutants are more likely to exhibit stage III progression at diagnosis, high grade cancers and to relapse with cancers at distant sites. P53 NETWORK EVOLUTION. We are investigating evolution of REs in terms of responsiveness to p53. Individual REs exhibited marked differences in potential transactivation as well as widespread turnover of functional REs during p53 network evolution. Only 1/3 of the REs found in humans are predicted to be functionally conserved in rodents. Surprisingly, the p53 responsiveness of the DNA repair/metabolism set of 15 p53 targeted genes in humans has evolved separately from mice suggesting differences in responsiveness to cancer-inducing agents between mice and humans. Importantly, we found functional conservation of weakly responding REs including 1/2 sites. Among validated p53 REs conserved between rodents and humans, one third were comprised of 1/2- or -sites, each with a perfect consensus -site suggesting a selective advantage in retaining weak p53 REs. Importantly, the integration of the TLR gene family into the p53 network also appears unique to primates.
| Alessandrini, Federica; Pezzè, Laura; Menendez, Daniel et al. (2018) ETV7-Mediated DNAJC15 Repression Leads to Doxorubicin Resistance in Breast Cancer Cells. Neoplasia 20:857-870 |
| Nguyen, Thuy-Ai T; Grimm, Sara A; Bushel, Pierre R et al. (2018) Revealing a human p53 universe. Nucleic Acids Res : |
| Aparicio-Cuevas, Manuel A; Rivero-Cruz, Isabel; Sánchez-Castellanos, Mariano et al. (2017) Dioxomorpholines and Derivatives from a Marine-Facultative Aspergillus Species. J Nat Prod 80:2311-2318 |
| Lowe, Julie M; Nguyen, Thuy-Ai; Grimm, Sara A et al. (2017) The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression. Cell Death Differ 24:181-191 |
| Menendez, Daniel; Nguyen, Thuy-Ai; Snipe, Joyce et al. (2017) The Cytidine Deaminase APOBEC3 Family Is Subject to Transcriptional Regulation by p53. Mol Cancer Res 15:735-743 |
| Currier, Jenna M; Cheng, Wan-Yun; Menendez, Daniel et al. (2016) Developing a Gene Biomarker at the Tipping Point of Adaptive and Adverse Responses in Human Bronchial Epithelial Cells. PLoS One 11:e0155875 |
| Menendez, Daniel; Lowe, Julie M; Snipe, Joyce et al. (2016) Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells. Oncotarget 7:61630-61642 |
| Shatz, Maria; Shats, Igor; Menendez, Daniel et al. (2015) p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways. Oncotarget 6:16963-80 |
| Sharma, Vasundhara; Jordan, Jennifer J; Ciribilli, Yari et al. (2015) Quantitative Analysis of NF-?B Transactivation Specificity Using a Yeast-Based Functional Assay. PLoS One 10:e0130170 |
| Nguyen, Thuy-Ai; Menendez, Daniel; Resnick, Michael A et al. (2014) Mutant TP53 posttranslational modifications: challenges and opportunities. Hum Mutat 35:738-55 |
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