Daniel Menendez

The expanding universe of p53 targets

The p53 tumour suppressor is modified through mutation or changes in expression in most cancers, leading to the altered regulation of hundreds of genes that are directly influenced by this sequence-specific transcription factor. Central to the p53 master regulatory network are the target response element (RE) sequences. The extent of p53 transactivation and transcriptional repression is influenced by many factors, including p53 levels, cofactors and the specific RE sequences, all of which contribute to the role that p53 has in the aetiology of cancer. This Review describes the identification and functionality of REs and highlights the inclusion of non-canonical REs that expand the universe of genes and regulation mechanisms in the p53 tumour suppressor network.

Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin (DXR) or Nutlin-3 (Nutlin) led to strikingly different p53 gene binding patterns based on chromatin immunoprecipitation with high-throughput sequencing experiments. Although two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR. Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network.

The Toll-Like Receptor Gene Family Is Integrated into Human DNA Damage and p53 Networks

In recent years the functions that the p53 tumor suppressor plays in human biology have been greatly extended beyond “guardian of the genome.” Our studies of promoter response element sequences targeted by the p53 master regulatory transcription factor suggest a general role for this DNA damage and stress-responsive regulator in the control of human Toll-like receptor (TLR) gene expression. The TLR gene family mediates innate immunity to a wide variety of pathogenic threats through recognition of conserved pathogen-associated molecular motifs. Using primary human immune cells, we have examined expression of the entire TLR gene family following exposure to anti-cancer agents that induce the p53 network. Expression of all TLR genes, TLR1 to TLR10, in blood lymphocytes and alveolar macrophages from healthy volunteers can be induced by DNA metabolic stressors. However, there is considerable inter-individual variability. Most of the TLR genes respond to p53 via canonical as well as noncanonical promoter binding sites. Importantly, the integration of the TLR gene family into the p53 network is unique to primates, a recurrent theme raised for other gene families in our previous studies. Furthermore, a polymorphism in a TLR8 response element provides the first human example of a p53 target sequence specifically responsible for endogenous gene induction. These findings—demonstrating that the human innate immune system, including downstream induction of cytokines, can be modulated by DNA metabolic stress—have many implications for health and disease, as well as for understanding the evolution of damage and p53 responsive networks.

Noncanonical DNA motifs as transactivation targets by wild type and mutant p53.

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network.

A SNP in the flt-1 promoter integrates the VEGF system into the p53 transcriptional network

The VEGF system is essential for angiogenesis. VEGF overexpression frequently correlates with increased microvascularity and metastasis and decreased spontaneous apoptosis. Although a precise mechanism has not been established, studies suggest that VEGF expression is negatively regulated by p53, a master regulator and tumor suppressor. There are no reports of additional components of the VEGF signal transduction pathway being part of the p53 transcriptional network. A target of VEGF, the VEGF receptor 1/flt-1, can regulate growth and migration of endothelial cells and modulate angiogenesis. VEGF appears to be up-regulated in various cancers in which flt-1 may have a role in tumor progression and metastasis. We identified a C-to-T SNP upstream of the transcriptional start site in ≈6% of the people examined. The SNP is located within a putative p53 response element. Only the promoter with the T SNP (FLT1-T) was responsive to p53 when examined with reporter assays or by endogenous gene expression analysis in cell lines with different SNP status. In response to doxorubicin-induced DNA damage, there was clear allele discrimination based on p53 binding at the FLT1-T but not FLT1-C promoters as well as p53-dependent induction of flt-1 mRNA, which required the presence of FLT1-T. Our results establish that p53 can differentially stimulate transcription at a polymorphic variant of the flt-1 promoter and directly places the VEGF system in the p53 stress-response network via flt-1 in a significant fraction of the human population. We suggest that the p53-VEGF-flt-1 interaction is relevant to risks in angiogenesis-associated diseases, including cancer.

The Human TLR Innate Immune Gene Family Is Differentially Influenced by DNA Stress and p53 Status in Cancer Cells

The transcription factor p53 regulates genes associated with a wide range of functions, including the Toll-like receptor (TLR) set of innate immunity genes, suggesting that p53 also modulates the human immune response. The TLR family comprises membrane glycoproteins that recognize pathogen-associated molecular patterns (PAMP) and mediate innate immune responses, and TLR agonists are being used as adjuvants in cancer treatments. Here, we show that doxorubicin, 5-fluorouracil, and UV and ionizing radiation elicit changes in TLR expression that are cell line- and damage-specific. Specifically, treatment-induced expression changes led to increased downstream cytokine expression in response to ligand stimulation. The effect of DNA stressors on TLR expression was mainly mediated by p53, and several p53 cancer-associated mutants dramatically altered the pattern of TLR gene expression. In all cell lines tested, TLR3 induction was p53-dependent, whereas induction of TLR9, the most stress-responsive family member, was less dependent on status of p53. In addition, each of the 10 members of the innate immune TLR gene family tested was differentially inducible. Our findings therefore show that the matrix of p53 status, chromosome stress, and responsiveness of individual TLRs should be considered in TLR-based cancer therapies.

The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

ABSTRACT Human tumor suppressor p53 is a sequence-specific master regulatory transcription factor that targets response elements (REs) in many genes. p53 missense mutations in the DNA-binding domain are often cancer associated. As shown with systems based on the yeast Saccharomyces cerevisiae, p53 mutants can alter the spectra and intensities of transactivation from individual REs. We address directly in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA-binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV or ionizing radiation, or spontaneously. These patterns reflected changes in the spectra and activities of target genes, as demonstrated for P21, MDM2, BAX, and MSH2. Thus, as originally proposed for “master genes of diversity,” p53 mutations in human cells can differentially influence target gene transactivation, resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy.

Interactions between the tumor suppressor p53 and immune responses.

Purpose of review The p53 tumor suppressor is a master regulator of antitumor defenses through its control of growth arrest, senescence and apoptosis. In recent years, p53 regulation was found to extend to a variety of biological processes including autophagy, fertility, metabolism and immune responses. Here, we focus on the role of p53 in the immune system. We explore the relationship between p53 and the innate immune response with particular emphasis on the Toll-like receptor (TLR) pathway and implications for cancer therapy. Recent findings Numerous studies have shown that the immune system, especially innate immunity, has a critical role in tumor development. It appears that p53 can influence innate immune responses as part of its tumor suppressor activities and recent work suggests that the complete set of innate immune TLR genes are responsive to chromosomal stress and the transcriptional network regulated by p53. Activation of p53 by common antitumor agents results in p53 dependent regulation of expression of most TLR genes in human primary and cancer cell lines, resulting in modulation of TLR downstream responses to cognate ligands. In addition several tumor-associated p53 mutants can also affect TLR gene expression. These observations together with the discovery of other immune-related p53 target genes provide new insights into the relationship between p53 and immunity and suggest approaches that might be useful in cancer therapies. Summary The tumor suppressor p53 can modulate innate immune gene responses in response to factors that can activate p53. This is expected to provide new opportunities in cancer diagnosis and in chemotherapeutic strategies that employ specific TLR agonists or antagonists that target the TLR pathway.

Functional evolution of the p53 regulatory network through its target response elements

Transcriptional network evolution is central to the development of complex biological systems. Networks can evolve through variation of master regulators and/or by changes in regulation of genes within networks. To gain insight into meaningful evolutionary differences in large networks, it is essential to address the functional consequences of sequence differences in response elements (REs) targeted by transcription factors. Using a combination of custom bioinformatics and multispecies alignment of promoter regions, we investigated the functional evolution of REs in terms of responsiveness to the sequence-specific transcription factor p53, a tumor suppressor and master regulator of stress responses. We identified REs orthologous to known p53 targets in human and rodent cells or alternatively REs related to the established p53 consensus. The orthologous REs were assigned p53 transactivation capabilities based on rules determined from model systems, and a functional heat map was developed to visually summarize conservation of sequence and relative level of responsiveness to p53 for 47 REs in 14 species. Individual REs exhibited marked differences in transactivation potentials and widespread evolutionary turnover. Functional differences were often not predicted from consensus sequence evaluations. Of the established human p53 REs analyzed, 91% had sequence conservation in at least one nonprimate species compared with 67.5% for functional conservation. Surprisingly, there was almost no conservation of functional REs for genes involved in DNA metabolism or repair between humans and rodents, suggesting important differences in p53 stress responses and cancer development.

A Single-Nucleotide Polymorphism in a Half-Binding Site Creates p53 and Estrogen Receptor Control of Vascular Endothelial Growth Factor Receptor 1†

ABSTRACT Interactions between master regulatory pathways provide higher-order controls for cellular regulation. Recently, we reported a C→T single-nucleotide polymorphism (SNP) in the vascular endothelial growth factor receptor 1 (VEGFR-1/Flt1) promoter that merges human VEGF and p53 pathways. This finding suggested a new layer in environmental controls of a pathway relevant to several diseases. The Flt1-T SNP created what appeared to be a half-site p53 target response element (RE). The absence of information about p53 gene responsiveness mediated by half-site REs led us to address how it influences Flt1 expression. We now identify a second regulatory sequence comprising a partial RE for estrogen receptors (ERs) upstream of the p53 binding site. Surprisingly, this provides for synergistic stimulation of transcription specifically at the Flt1-T allele through the combined action of ligand-bound ER and stress-induced p53. In addition to demonstrating direct control of Flt1 expression by ER and p53 proteins acting as sequence-specific transcription factors at half-site REs, we establish a new interaction between three master regulatory pathways, p53, ER, and VEGF. The mechanism of joint regulation through half-sites is likely relevant to transcriptional control of other targets and expands the number of genes that may be directly controlled in master regulatory networks.