APOBEC3G Is a p53-Dependent Restriction Factor in Respiratory Syncytial Virus Infection of Human Cells Included in the p53/Immune Axis.

Identifying and understanding genetic factors that influence the propagation of the human respiratory syncytial virus (RSV) can lead to health benefits and possibly augment recent vaccine approaches. We previously identified a p53/immune axis in which the tumor suppressor p53 directly regulates the expression of immune system genes, including the seven members of the APOBEC3 family of DNA cytidine deaminases (A3), which are innate immune sentinels against viral infections. Here, we examined the potential p53 and A3 influence in RSV infection, as well as the overall p53-dependent cellular and p53/immune axis responses to infection. Using a paired p53 model system of p53+ and p53- human lung tumor cells, we found that RSV infection activates p53, leading to the altered p53-dependent expression of A3D, A3F, and A3G, along with p53 site-specific binding. Focusing on A3G because of its 10-fold-greater p53 responsiveness to RSV, the overexpression of A3G can reduce RSV viral replication and syncytial formation. We also observed that RSV-infected cells undergo p53-dependent apoptosis. The study was expanded to globally address at the transcriptional level the p53/immune axis response to RSV. Nearly 100 genes can be directly targeted by the p53/immune axis during RSV infection based on our p53BAER analysis (Binding And Expression Resource). Overall, we identify A3G as a potential p53-responsive restriction factor in RSV infection. These findings have significant implications for RSV clinical and therapeutic studies and other p53-influenced viral infections, including using p53 adjuvants to boost the response of A3 genes.

Etoposide-induced DNA damage is increased in p53 mutants: identification of ATR and other genes that influence effects of p53 mutations on Top2-induced cytotoxicity.

The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.

p53-responsive TLR8 SNP enhances human innate immune response to respiratory syncytial virus.

The Toll-like receptor 8 (TLR8) has an important role in innate immune responses to RNA viral infections, including respiratory syncytial virus (RSV). We previously reported that TLR8 expression was increased directly by the tumor suppressor and transcription factor p53 via a single nucleotide polymorphism (SNP) (rs3761624) in the TLR8 promoter, thereby placing TLR8 in the p53/immune axis. Because this SNP is in linkage disequilibrium with other SNPs associated with several infectious diseases, we addressed the combined influence of p53 and the SNP on downstream inflammatory signaling in response to a TLR8 cognate ssRNA ligand. Using human primary lymphocytes, p53 induction by chemotherapeutic agents such as ionizing radiation caused SNP-dependent synergistic increases in IL-6 following incubation with an ssRNA ligand, as well as TLR8 RNA and protein expression along with p53 binding at the TLR-p53 SNP site. Because TLR8 is X-linked, the increases were generally reduced in heterozygous females. We found a corresponding association of the p53-responsive allele with RSV disease severity in infants hospitalized with RSV infection. We conclude that p53 can strongly influence TLR8-mediated immune responses and that knowledge of the p53-responsive SNP can inform diagnosis and prognosis of RSV disease and other diseases that might have a TLR8 component, including cancer.

Revealing a human p53 universe.

p53 transcriptional networks are well-characterized in many organisms. However, a global understanding of requirements for in vivo p53 interactions with DNA and relationships with transcription across human biological systems in response to various p53 activating situations remains limited. Using a common analysis pipeline, we analyzed 41 data sets from genome-wide ChIP-seq studies of which 16 have associated gene expression data, including our recent primary data with normal human lymphocytes. The resulting extensive analysis, accessible at p53 BAER hub via the UCSC browser, provides a robust platform to characterize p53 binding throughout the human genome including direct influence on gene expression and underlying mechanisms. We establish the impact of spacers and mismatches from consensus on p53 binding in vivo and propose that once bound, neither significantly influences the likelihood of expression. Our rigorous approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53. Importantly, we identify a core cistrome signature composed of genes appearing in over half the data sets, and we identify signatures that are treatment- or cell-specific, demonstrating new functions for p53 in cell biology. Our analysis reveals a broad homeostatic role for human p53 that is relevant to both basic and translational studies.

ETV7-Mediated DNAJC15 Repression Leads to Doxorubicin Resistance in Breast Cancer Cells.

Breast cancer treatment often includes Doxorubicin as adjuvant as well as neoadjuvant chemotherapy. Despite its cytotoxicity, cells can develop drug resistance to Doxorubicin. Uncovering pathways and mechanisms involved in drug resistance is an urgent and critical aim for breast cancer research oriented to improve treatment efficacy. Here we show that Doxorubicin and other chemotherapeutic drugs induce the expression of ETV7, a transcriptional repressor member of ETS family of transcription factors. The ETV7 expression led to DNAJC15 down-regulation, a co-chaperone protein whose low expression was previously associated with drug resistance in breast and ovarian cancer. There was a corresponding reduction in Doxorubicin sensitivity of MCF7 and MDA-MB-231 breast cancer cells. We identified the binding site for ETV7 within DNAJC15 promoter and we also found that DNA methylation may be a factor in ETV7-mediated DNAJC15 transcriptional repression. These findings of an inverse correlation between ETV7 and DNAJC15 expression in MCF7 cells in terms of Doxorubicin resistance, correlated well with treatment responses of breast cancer patients with recurrent disease, based on our analyses of reported genome-wide expression arrays. Moreover, we demonstrated that ETV7-mediated Doxorubicin-resistance involves increased Doxorubicin efflux via nuclear pumps, which could be rescued in part by DNAJC15 up-regulation. With this study, we propose a novel role for ETV7 in breast cancer, and we identify DNAJC15 as a new target gene responsible for ETV7-mediated Doxorubicin-resistance. A better understanding of the opposing impacts of Doxorubicin could improve the design of combinatorial adjuvant regimens with the aim of avoiding resistance and relapse.

Dioxomorpholines and Derivatives from a Marine-Facultative Aspergillus Species.

Two new dioxomorpholines, 1 and 2, three new derivatives, 3-5, and the known compound PF1233 B (6) were isolated from a marine-facultative Aspergillus sp. MEXU 27854. Their structures were established by 1D and 2D NMR and HRESIMS data analysis. The absolute configuration of 1 and 2 was elucidated by comparison of experimental and DFT-calculated vibrational circular dichroism spectra. Compounds 3, 5, and 6 were noncytotoxic to a panel of human cancer cell lines with different functional status for the tumor-suppressor protein p53, but were inhibitors of P-glycoprotein-reversing multidrug resistance in a doxorubicin-resistant cell line.

The Cytidine Deaminase APOBEC3 Family Is Subject to Transcriptional Regulation by p53.

The APOBEC3 (A3) family of proteins are DNA cytidine deaminases that act as sentinels in the innate immune response against retroviral infections and are responsive to IFN. Recently, a few A3 genes were identified as potent enzymatic sources of mutations in several human cancers. Using human cancer cells and lymphocytes, we show that under stress conditions and immune challenges, all A3 genes are direct transcriptional targets of the tumor suppressor p53. Although the expression of most A3 genes (including A3C and A3H) was stimulated by the activation of p53, treatment with the DNA-damaging agent doxorubicin or the p53 stabilizer Nutlin led to repression of the A3B gene. Furthermore, p53 could enhance IFN type-I induction of A3 genes. Interestingly, overexpression of a group of tumor-associated p53 mutants in TP53-null cancer cells promoted A3B expression. These findings establish that the "guardian of the genome" role ascribed to p53 also extends to a unique component of the immune system, the A3 genes, thereby integrating human immune and chromosomal stress responses into an A3/p53 immune axis.Implications: Activated p53 can integrate chromosomal stresses and immune responses through its influence on expression of APOBEC3 genes, which are key components of the innate immune system that also influence genomic stability. Mol Cancer Res; 15(6); 735-44. ©2017 AACR.

The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a stress-response gene that has been associated with cancer, but no studies have differentiated among or defined the regulation or function of any of its several recently described expression variants. We found that TNFAIP8 variant 2 (v2) is overexpressed in multiple human cancers, whereas other variants are commonly downregulated in cancer (v1) or minimally expressed in cancer or normal tissue (v3-v6). Silencing v2 in cancer cells induces p53-independent inhibition of DNA synthesis, widespread binding of p53, and induction of target genes and p53-dependent cell cycle arrest and DNA damage sensitization. Cell cycle arrest induced by v2 silencing requires p53-dependent induction of p21. In response to the chemotherapeutic agent doxorubicin, p53 regulates v2 through binding to an intragenic enhancer, together indicating that p53 and v2 engage in complex reciprocal regulation. We propose that TNFAIP8 v2 promotes human cancer by broadly repressing p53 function, in essence offsetting p53-dependent tumor suppression.

Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells.

Diversity within the p53 transcriptional network can arise from a matrix of changes that include target response element sequences and p53 expression level variations. We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses. Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors. While many mutants retained the ability to drive TLR expression at WT levels, others exhibited null, limited, or change-of-spectrum transactivation of TLR genes. Using TLR3 signaling as a model, we show that some cancer-associated p53 mutants amplify cytokine, chemokine and apoptotic responses after stimulation by the cognate ligand poly(I:C). Furthermore, restoration of WT p53 activity for loss-of-function p53 mutants by the p53 reactivating drug RITA restored p53 regulation of TLR3 gene expression and enhanced DNA damage-induced apoptosis via TLR3 signaling. Overall, our findings have many implications for understanding the impact of WT and mutant p53 in immunological responses and cancer therapy.

Developing a Gene Biomarker at the Tipping Point of Adaptive and Adverse Responses in Human Bronchial Epithelial Cells.

Determining mechanism-based biomarkers that distinguish adaptive and adverse cellular processes is critical to understanding the health effects of environmental exposures. Shifting from in vivo, low-throughput toxicity studies to high-throughput screening (HTS) paradigms and risk assessment based on in vitro and in silico testing requires utilizing toxicity pathway information to distinguish adverse outcomes from recoverable adaptive events. Little work has focused on oxidative stresses in human airway for the purposes of predicting adverse responses. We hypothesize that early gene expression-mediated molecular changes could be used to delineate adaptive and adverse responses to environmentally-based perturbations. Here, we examined cellular responses of the tracheobronchial airway to zinc (Zn) exposure, a model oxidant. Airway derived BEAS-2B cells exposed to 2-10 µM Zn2+ elicited concentration- and time-dependent cytotoxicity. Normal, adaptive, and cytotoxic Zn2+ exposure conditions were determined with traditional apical endpoints, and differences in global gene expression around the tipping point of the responses were used to delineate underlying molecular mechanisms. Bioinformatic analyses of differentially expressed genes indicate early enrichment of stress signaling pathways, including those mediated by the transcription factors p53 and NRF2. After 4 h, 154 genes were differentially expressed (p < 0.01) between the adaptive and cytotoxic Zn2+ concentrations. Nearly 40% of the biomarker genes were related to the p53 signaling pathway with 30 genes identified as likely direct targets using a database of p53 ChIP-seq studies. Despite similar p53 activation profiles, these data revealed widespread dampening of p53 and NRF2-related genes as early as 4 h after exposure at higher, unrecoverable Zn2+ exposures. Thus, in our model early increased activation of stress response pathways indicated a recoverable adaptive event. Overall, this study highlights the importance of characterizing molecular mechanisms around the tipping point of adverse responses to better inform HTS paradigms.

p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways.

The p53 tumor suppressor regulates transcription of genes associated with diverse cellular functions including apoptosis, growth arrest, DNA repair and differentiation. Recently, we established that p53 can modulate expression of Toll-like receptor (TLR) innate immunity genes but the degree of cross-talk between p53 and TLR pathways remained unclear. Here, using gene expression profiling we characterize the global effect of p53 on the TLR5-mediated transcription in MCF7 cells. We found that combined activation of p53 and TLR5 pathways synergistically increases expression of over 200 genes, mostly associated with immunity and inflammation. The synergy was observed in several human cancer cells and primary lymphocytes. The p53-dependent amplification of transcriptional response to TLR5 activation required expression of NFκB subunit p65 and was mediated by several molecular mechanisms including increased phosphorylation of p38 MAP kinase, PI3K and STAT3 signaling. Additionally, p53 induction increased cytokine expression in response to TNFα, another activator of NFκB and MAP kinase pathways, suggesting a broad interaction between p53 and these signaling pathways. The expression of many synergistically induced genes is elevated in breast cancer patients responsive to chemotherapy. We suggest that p53's capacity to enhance immune response could be exploited to increase antitumor immunity and to improve cancer treatment.

p53 and NF-κB coregulate proinflammatory gene responses in human macrophages.

Macrophages are sentinel immune cells that survey the tissue microenvironment, releasing cytokines in response to both exogenous insults and endogenous events such as tumorigenesis. Macrophages mediate tumor surveillance and therapy-induced tumor regression; however, tumor-associated macrophages (TAM) and their products may also promote tumor progression. Whereas NF-κB is prominent in macrophage-initiated inflammatory responses, little is known about the role of p53 in macrophage responses to environmental challenge, including chemotherapy or in TAMs. Here, we report that NF-κB and p53, which generally have opposing effects in cancer cells, coregulate induction of proinflammatory genes in primary human monocytes and macrophages. Using Nutlin-3 as a tool, we demonstrate that p53 and NF-κB rapidly and highly induce interleukin (IL)-6 by binding to its promoter. Transcriptome analysis revealed global p53/NF-κB co-regulation of immune response genes, including several chemokines, which effectively induced human neutrophil migration. In addition, we show that p53, activated by tumor cell paracrine factors, induces high basal levels of macrophage IL-6 in a TAM model system [tumor-conditioned macrophages (TCM)]. Compared with normal macrophages, TCMs exhibited higher p53 levels, enhanced p53 binding to the IL-6 promoter, and reduced IL-6 levels upon p53 inhibition. Taken together, we describe a mechanism by which human macrophages integrate signals through p53 and NF-κB to drive proinflammatory cytokine induction. Our results implicate a novel role for macrophage p53 in conditioning the tumor microenvironment and suggest a potential mechanism by which p53-activating chemotherapeutics, acting upon p53-sufficient macrophages and precursor monocytes, may indirectly impact tumors lacking functional p53.

Mutant TP53 posttranslational modifications: challenges and opportunities.

The wild-type (WT) human p53 (TP53) tumor suppressor can be posttranslationally modified at over 60 of its 393 residues. These modifications contribute to changes in TP53 stability and in its activity as a transcription factor in response to a wide variety of intrinsic and extrinsic stresses in part through regulation of protein-protein and protein-DNA interactions. The TP53 gene frequently is mutated in cancers, and in contrast to most other tumor suppressors, the mutations are mostly missense often resulting in the accumulation of mutant (MUT) protein, which may have novel or altered functions. Most MUT TP53s can be posttranslationally modified at the same residues as in WT TP53. Strikingly, however, codons for modified residues are rarely mutated in human tumors, suggesting that TP53 modifications are not essential for tumor suppression activity. Nevertheless, these modifications might alter MUT TP53 activity and contribute to a gain-of-function leading to increased metastasis and tumor progression. Furthermore, many of the signal transduction pathways that result in TP53 modifications are altered or disrupted in cancers. Understanding the signaling pathways that result in TP53 modification and the functions of these modifications in both WT TP53 and its many MUT forms may contribute to more effective cancer therapies.

Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code.

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein-DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.

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.

p53 Integrates host defense and cell fate during bacterial pneumonia.

Cancer and infection are predominant causes of human mortality and derive, respectively, from inadequate genomic and host defenses against environmental agents. The transcription factor p53 plays a central role in human tumor suppression. Despite its expression in immune cells and broad responsiveness to stressors, it is virtually unknown whether p53 regulates host defense against infection. We report that the lungs of naive p53(-/-) mice display genome-wide induction of NF-κB response element-enriched proinflammatory genes, suggestive of type 1 immune priming. p53-null and p53 inhibitor-treated mice clear Gram-negative and -positive bacteria more effectively than controls after intrapulmonary infection. This is caused, at least in part, by cytokines produced by an expanded population of apoptosis-resistant, TLR-hyperresponsive alveolar macrophages that enhance airway neutrophilia. p53(-/-) neutrophils, in turn, display heightened phagocytosis, Nox-dependent oxidant generation, degranulation, and bacterial killing. p53 inhibition boosts bacterial killing by mouse neutrophils and oxidant generation by human neutrophils. Despite enhanced bacterial clearance, infected p53(-/-) mice suffer increased mortality associated with aggravated lung injury. p53 thus modulates host defense through regulating microbicidal function and fate of phagocytes, revealing a fundamental link between defense of genome and host during environmental insult.

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 17ß-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.

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.