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, much about its function and its targets remains unknown. 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 to do that we developed reporter systems in budding yeast for variable human p53 expression and have translated many of the findings to 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. Recently, we took a rigorous method by 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 have validated the p53 transcriptional responsiveness of potential p53 DNA damage response target genes under stressed and unstressed conditions with various human cancer cells that vary in p53 functional status. We identified many putative novel p53 transcriptional targets. 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. THE INFLUENCE OF p53 ON HUMAN IMMUNE RESPONSES. 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. The transcriptional cofactors ASPP1 and ASPP2 can enhance p53 mediated transactivation of several TLR genes. Moreover, p53 collaborate with NFkappaB to modulate several immune responses in primary lymphocytes and macrophages. 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 target by p53 involved in cellular immune and inflammatory process including several viral/bacterial restriction factors. Our studies emphasized 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 several of the human cytidine deaminase APOBEC3 gene family (A3) involved in the innate immune that deal with RNA virus infections are also subject to p53 transcriptional control by WT and mutant p53 in different directions. 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 SNP in TLR8 regulatory region creates a p53 responsive sequence that renders 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 a project supported by the Office of AIDS Research (OAR), currently we are investigating 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. We recently 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. Furthermore, we have found that activation of p53 with Nutlin in CD4+ human T lymphocytes reduces HIV1 replication. In collaboration with Johns Hopkins School of Public Health, we identified 263 HIV-1 seroconverters 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. p53 mutants that induced expression of TLR3, enhanced cytokine and chemokine responses mediated by this receptor after exposing cells to TLR3 ligand poly-I:C alone or in presence of Doxorubicin. We also found that functional rescue of loss-of-function p53 mutants by the p53 reactivating drug RITA, restored TLR gene expression in a mutant p53 cell line, enhancing DNA damage induced-apoptosis via TLR3 signaling. Furthermore, several p53 mutants also altered the expression 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. 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 etoposide p53SL target ATR, impacts Topoisomerase-mediated enzymatic activities. In addition, we developed another SL screen using genome wide sRNAi approach to identify synthetic enhancement of lethality genes that alter responses of p53 mutant cells to ionizing radiation. In addition we studied the mechanism of Doxorubicin resistance in p53 mutant breast cancer cells.
| 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
