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. There is considerable variation in p53 dependent expression across >400 targeted genes leading to differences in p53-mediated biological consequences, due in part to variation in target response element (RE) sequence. We found that RE motifs differ considerably from the in vitro derived RE consensus target sequence previously described as 2 copies of RRRCWWGYYY separated by a spacer of up to 13 bases. We have focused on RE functionality, i.e., the ability of REs to support transactivation by p53. To directly assess functionality of human REs, i.e., transactivation responsiveness, we developed promoter systems in budding yeast for variable human p53 expression and have translated many of the findings to human cells in culture and ex vivo. That led us to the identification of super-transactivating sequences (STARES) that provide high transactivation at low p53 levels. Remarkably, we observed that transactivation specificity is conserved among p53 family proteins. We found that p53 can transactivate through noncanonical binding sequences including half-sites, greatly expanding the p53 regulatory network. We extended these studies genome-wide using ChIP-Seq analysis to identify p53 binding sites and associated gene expression changes following p53 activation in cancer cells as well as in primary T-lymphocytes after treatment with anti-cancer drugs. Similar to cancer cells, we found that most p53 bound sites contained a canonical p53 response element (2 tandem RRRCWWGYYY decamers without spacer). Importantly, we showed that p53 can also engage transcription using half-sites across the genome and we went on to define the minimal binding unit for p53-mediated transcription as a 1/2-site. Among differentially expressed genes associated with p53 binding several are relevant to immunity and inflammation. Moreover, in a global approach we are determining the p53 network by examining p53 binding across the genome, induced gene expression and protein interactors using mass spectrometry methods, under diverse stress conditions. We identified 45 novel p53 protein interactors in response to the commonly-used chemotherapeutic drug Doxorubicin. We found in our ChIPseq studies that p53 bound the transcriptional regulatory regions of 97% of these interactors, revealing potential regulatory p53 loops and thereby provide a novel systems biology approach to understanding the p53 stress responsive network. 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, we demonstrated that tumor-associated 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 and also enhanced DNA damage induced-apoptosis via TLR3 signaling. Furthermore, several p53 mutants also altered the expression of the innate immune related APOBEC3 (A3) gene family. In particular, overexpression of a group of tumor-associated p53 mutants in p53 null cancer cells actually 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. Using a human cancer colorectal cell line as a model, 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 mediatied 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. 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. We identified in our recent ChIPseq cancer cell study that 60% of the p53 REs were conserved at the sequence level in rodents. Around 120 potential human target genes associated with DNA metabolism were found to be bound by p53. Most of them can also interact directly with p53 protein. Using a combination of gene expression/ transcriptomic and bioinformatic tools we have expanded the p53 transcriptome in DNA repair and nucleic acid metabolism transactions. We have validated the p53 transcriptional responsiveness of potential p53 DNA damage response target genes identified in our ChIPseq metanalysis under stressed and unstressed conditions using a Nanostring approach with various human cancer cells that vary in p53 functional status. 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 TLR family in human primary and cancer cells to consequently enhance TLR-dependent production of proinflammatory cytokines in response to cognate ligands. We found that the transcriptional cofactor and pro-apoptotic regulator ASPP1 and ASPP2 can enhance p53 mediated transactivation of several TLR genes. We also found that p53 can collaborate with NFkappaB to strongly alter several immune/inflammatory responses in primary lymphocytes and macrophages from healthy volunteers. Accordingly, we generated a p53 ChIP-seq dataset associated with global gene expression from primary human lymphocytes under non-stress conditions as well as after ex-vivo treatment with well-known p53 activators (Doxorubicin and Nutlin. Around 200 genes that exhibited a p53/ligand synergistic response to chemotherapeutic agents have been identified. Among these genes we found several of the human cytidine deaminase A3 gene family involved in the innate immune that deal with retrotransposons and RNA virus infection are also subject to p53 transcriptional control by WT and mutant p53 in different directions. We observed that Respiratory Syncytial Virus (RSV), the of 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. Currently we are investigating the immune role that p53 might have during HIV-1 infection.
| 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 |
| 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 |
| 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 |
| 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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