The p53 tumor suppressor is central to human DNA repair, damage checkpoints and many aspects of human biology. Importantly, most cancers are altered for p53 function. Although the guardian of the genome p53 has been extensively studied, much about its function and its targets remains unknown. IDENTIFYING p53 TARGETS. Because of its importance as a human tumor suppressor and its other recently discovered functions, identifying the universe of p53 direct transcriptional targets is clinically relevant, especially since many of these may have therapeutic value. There is considerable variation in p53 dependent expression across targeted genes leading to differences in p53-mediated biological consequences, due in part to variation in target response element (RE) sequence. We have focused on RE functionality, i.e., the ability of REs to support transactivation by p53 in human cells in culture and ex vivo. Several studies have used genome-wide, next-generation sequencing approaches to identify p53 binding sites in the human genome. We developed a rigorous method for reanalyzing all the raw data from the individual ChIP-seq studies and associated gene expression using a single analysis workflow, then combining information using a common set of criteria. Our approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53, including the identification of new potential p53 transcriptional targets involved both in the classical roles of p53 function (DNA repair) as well as in processes not immediately related to classical p53 outcomes, for example, the immune response. INFLUENCE OF P53 TRANSCRIPTIONAL NETWORK IN DNA DAMAGE RESPONSE. Using our p53 cistrome studies we have identified nearly 120 potential human target genes associated with DNA Damage Response (DDR). We identified around 50 putative novel p53 transcriptional targets under stressed and unstressed conditions with various human cancer cells that vary in p53 functional status. We validated by RT-qPCR the p53-dependency and expression profiles of nine of these genes, APTX, ATR, ATRIP, DDX5, LIG1, MSH4, POLD1, POLH and REV3L after the treatment with three p53-inducing drugs: Doxorubicin, Etoposide, and Nutlin. By ChIP analysis we confirmed the binding of p53 to the regulatory regions of 8 of these genes. THE p53 IMMUNE CISTROME. We have established that p53 plays important physiologic roles in the immune system. p53 upregulates most members of the pathogen sensor TLR family in human cells to consequently enhance TLR-dependent production of proinflammatory cytokines in response to cognate ligands. Through our meta-analysis of p53 targeted binding sites and associated gene expression (i.e., cistrome genes) following stresses in many cell types, including human lymphocytes treated with p53 activating drugs, we identified 100 genes targeted by p53 involved in cellular immune and inflammatory processes including several viral/bacterial restriction factors. Currently we are validating the p53 responsiveness of these genes in two subsets of primary immune cells: lymphocytes and monocytes. Our studies emphasize the influence of p53 in modulating the immune system, which defends against external and internal threats as well as tumorigenesis. Among these genes we found that several of the human cytidine deaminase APOBEC3 gene family (A3) involved in the innate immune system that deal with RNA virus infections are also subject to p53 transcriptional control by WT and mutant p53 in different directions. THE INFLUENCE OF p53 ON HUMAN IMMUNE RESPONSES DURING VIRAL INFECTIONS. We observed that RSV, the major cause of respiratory problems in young children worldwide, induced p53 protein activation, resulting in the expression of several A3 genes in a p53-dependent manner and influencing negatively the virus replication. A transcriptome analysis confirmed that p53 responsive cells have a higher number of immune related genes differentially expressed than deficient p53 cell lines in response to RSV infection. A SNP in TLR8 regulatory region creates a p53 responsive sequence that results in responsiveness and transcriptional control of this gene by p53. We found that this SNP influences RSV disease severity in infants infected by this virus. In association with this project, we addressed the influence of lactoferrin. Lactoferrin (LF) is an iron-binding glycoprotein present in human milk and a key component of the innate immune system with well-known antimicrobial effects. We observed that (LF) has a protective effect in response to RSV infection in human lung cells, potentially blocking the entry of the virus into the cells and increasing innate immune gene expression. In a project supported by the Office of AIDS Research (OAR), currently we are investigating in an in vitro model ( CD4+ T lymphocytes) the immune role that p53, its polymorphism in codon 72 and SNPs in TLR8 including the p53RE SNP (rs3761624), might have during HIV-1 infection. A difference in HIV titers and HIV-LRT expression in cell cultures of TLR8 p53RE SNP lymphocytes donors pretreated with the p53 activating drug Nutlin have been observed. Donor that show induction of TLR8 after p53 activation, exhibit a 30-50% reduction in HIV infectivity when compared to the donors with no TLR8 induction. We confirmed in H9 leukemia T cells (p53 null) cells and CD4+ T lymphocytes of healthy people, that activation of p53 results in upregulation of subset of HIV restriction factors genes including A3G, A3H, SAMHD1, TRIM5, HERC5, TRIM5, MARCH2, MX1, TSG101 PRK and ZAP. In collaboration with Johns Hopkins School of Public Health, we identified 263 HIV-1 seroconverts in the Multicenter AIDS Cohort Study (MACS) previously genotyped for TP53 SNP rs1042522 and TLR8 SNP rs37646880, which is in high linkage disequilibrium with TLR8-p53RE SNP. A preliminary analysis showed that especially TP53 codon 72 variant, may be associated with a lower risk of developing AIDS but the presence of both protective alleles (TP53 rs1042522=GG and TLR8 rs37646880=A) might convey an overall 65% reduction in the risk of AIDS. CANCER-ASSOCIATED p53 MUTANTS. With inclusion of immune response-related TLR genes into the p53 network, we evaluated the effect of 25 tumor-associated p53 mutants on TLR gene family expression after transient transfection in p53-null cancer cell. Changes in TLR transactivation patterns, including change-of-spectrum, were observed, suggesting that p53 tumor status might be an important factor in adjuvant therapy employing TLR pathways to treat cancer. Furthermore, several p53 mutants also altered the expression of the A3 gene family, particularly, several mutants promoted expression of A3B, which normally is repressed by WT p53, suggesting a clear gain of function phenotype for these p53 mutants. Since TP53 gene mutations occur in many human cancers, it is important to identify anticancer drugs that specifically target p53 mutant tumor cells. We are pursuing the identification of synthetic lethal (SL) genes with genome-wide siRNA-based screens, that when reduced in expression in p53 mutant cells cause death or reduced growth in cells treated (or untreated) with anticancer agents. The SLs are expected to lead to potential targets and provide opportunities for anticancer drug development. We have identified several SL targets for two of the most frequent tumor-associated p53 mutants (R175H and R273H) in the presence of anticancer drug etoposide as well as for the WT p53 and p53 null conditions. Among the SL genes identified, several are related to the DNA damage response including ATR, BRCA2, SOD1, TOP1, ZNF45. We have found that drug mediated inhibition of ATR in combination with etoposide is an SL target in a p53 mutant specific manner (R273H and p53 null) and impacts Topoisomerase-mediated enzymatic activities.
| 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 |
Showing the most recent 10 out of 32 publications
