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 over 200 targeted genes leading to differences in p53-mediated biological consequences, due in part to variation in target response element (RE) sequence. We found that the target sequence motif differs considerably from the in vitro derived RE consensus target sequence previously described as 2 copies of RRRCA/TT/AGYYY 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. Recently, we identified super-transactivating sequences that provide high transactivation at low p53 levels. The yeast system has been used to establish the functional evolution of REs across species and was summarized in our piano model that describes functional variability within a transcriptional network. 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 by different agents in cancer cells. Binding often is not associated with transactivation. A de novo motif search for the in vivo consensus binding sequence revealed the p53 canonical motif is, in fact, 2 tandem RRRCWWGYYY decamers without spacer. Unexpectedly, 10% of p53 bound targets were 1/2-sites. Importantly, we showed that p53 can engage transcription also through recognition of half-sites across the genome and went on to define the minimal binding unit for p53-mediated transcription as a 1/2-site. INTERACTION OF p53 AND ER NETWORKS. We had identified a 1/2-site estrogen receptor RE that greatly increased p53 transactivation at the FLT1 1/2-site p53 RE, establishing a new dimension to the p53 network. Recently, we addressed the generality of synergistic transactivation by p53 and ER. p53 transactivation was greatly enhanced by ligand-activated ER acting in cis. The increase extends to several cancer-associated p53 mutants, suggesting ER-dependent mutant p53 activity for at least some REs and possibilities for reactivation of cancer mutants. We propose a general synergistic relationship between p53 and ER master regulators in transactivation of p53 target canonical and noncanonical REs which might be poorly responsive to p53 on their own. CANCER-ASSOCIATED P53 MUTANTS. Nearly all cancers have mutant or reduced expression of the p53 tumor suppressor gene. We found that p53 mutations can lead to considerable diversity in the spectrum of responses from REs including 1) decrease/loss-of-function;2) subtle changes;3) altered specificity;and 4) super-transactivation. 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. Having established that WT and p53 mutants identified in somatic and germline-associated tumors can modulate TLR expression differentially, we propose that chemotherapeutic manipulation of normal or mutant p53 responses along with immune challenges that include TLRs could enhance inflammatory/immune type responses to environmental factors. 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. Only 1/3 of the REs found in humans are predicted to be functionally conserved in rodents, although nearly 2/3 of the REs were conserved at the sequence level. Importantly, we found functional conservation of weakly responding REs including 1/2 sites. Among validated p53 REs conserved between rodents and humans, one third were comprised of 1/2- or 3/4-sites, each with a perfect consensus-site suggesting a selective advantage in retaining weak p53 REs. Similar to those observations, we identified in our recent ChIPseq cancer cell study that 60% of the p53 REs were conserved at the sequence level in rodents. Using Geneontology for gene function classification to identify those potential p53 target genes associated with DNA metabolism and repair functions, we found that only a few of the p53 RE sequences were conserved in rodents in agreement with our earlier findings. These results may indicate evolutionary selection on p53 dependent chromosomal stress responses. REGULATION OF THE IMMUNE RESPONSE. The immune system can impact tumor development. We found that among the 10 human TLRs, nine had canonical and noncanonical p53 REs. Using primary human cells, we examined expression of the entire TLR gene family following exposure to anti-cancer p53 inducing agents. Expressions of all TLR genes in blood lymphocytes and alveolar macrophages from healthy volunteers were inducible by DNA metabolic stressors. However, there is considerable inter-individual variability. Similarly, p53 dependent TLR expression is detected in human cancer cell lines. For some TLRs the p53 control seems to enhance the inflammatory responses, mediated by activation of TLRs in the presence of natural ligands. In particular, we found a p53-dependent increase in response to the TLR5 ligand flagellin as measured by both mRNA and protein production of the downstream cytokines IL-6 and IL-8 accompanied by a specific increase in phosphorylation of p38 MAP kinase, which is one of the mediators of TLR signaling in breast cancer cells. Global gene expression analysis revealed a group of 200 genes that exhibited this p53/ligand synergistic response including genes related to immune/inflammation processes. We also found that in the absence of TLR ligands, p53 that is induced by anticancer drugs can cooperate with Nuclear Factor-kappa beta (NFkappa-beta) to induce pro-inflammatory cytokines in primary human macrophages. Transcriptome analysis identified a global cooperative p53/NF-kappaB effect on expression of immune response genes, including several chemokines such as CXCL1, CXCL3, and CCL5. Recently, we established that the genome guardianship role of p53 extends to the innate immune system beyond the TLR genes. In our p53 ChIPseq studies in cancer cells, we identified 3 members of the APOBEC3 family, A3B, -C and H, that were directly regulated by p53 in a stress dependent manner. There are 7 members of the APOBEC3 family and their products are nucleic acid deaminases that can mutagenize and inactivate infecting RNA viral genomes, serving as warriors in the innate immune response. We found from our p53 cistrome studies that several members of the APOBEC3 family are directly regulated by p53 in human cancer and primary cells.

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20
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2013
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$1,306,411
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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
Lowe, Julie M; Menendez, Daniel; Fessler, Michael B (2014) A new inflammatory role for p53 in human macrophages. Cell Cycle 13:2983-4

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