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2.
Proc Natl Acad Sci U S A ; 121(21): e2318591121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38739802

RESUMO

The transcription factor p73, a member of the p53 tumor-suppressor family, regulates cell death and also supports tumorigenesis, although the mechanistic basis for the dichotomous functions is poorly understood. We report here the identification of an alternate transactivation domain (TAD) located at the extreme carboxyl (C) terminus of TAp73ß, a commonly expressed p73 isoform. Mutational disruption of this TAD significantly reduced TAp73ß's transactivation activity, to a level observed when the amino (N)-TAD that is similar to p53's TAD, is mutated. Mutation of both TADs almost completely abolished TAp73ß's transactivation activity. Expression profiling highlighted a unique set of targets involved in extracellular matrix-receptor interaction and focal adhesion regulated by the C-TAD, resulting in FAK phosphorylation, distinct from the N-TAD targets that are common to p53 and are involved in growth inhibition. Interestingly, the C-TAD targets are also regulated by the oncogenic, amino-terminal-deficient DNp73ß isoform. Consistently, mutation of C-TAD reduces cellular migration and proliferation. Mechanistically, selective binding of TAp73ß to DNAJA1 is required for the transactivation of C-TAD target genes, and silencing DNAJA1 expression abrogated all C-TAD-mediated effects. Taken together, our results provide a mechanistic basis for the dichotomous functions of TAp73 in the regulation of cellular growth through its distinct TADs.


Assuntos
Proliferação de Células , Domínios Proteicos , Ativação Transcricional , Proteína Tumoral p73 , Proteína Tumoral p73/metabolismo , Proteína Tumoral p73/genética , Humanos , Movimento Celular/genética , Mutação , Linhagem Celular Tumoral , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Fosforilação , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética
3.
BMC Cancer ; 24(1): 587, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38741073

RESUMO

YAP and TAZ, the Hippo pathway terminal transcriptional activators, are frequently upregulated in cancers. In tumor cells, they have been mainly associated with increased tumorigenesis controlling different aspects from cell cycle regulation, stemness, or resistance to chemotherapies. In fewer cases, they have also been shown to oppose cancer progression, including by promoting cell death through the action of the p73/YAP transcriptional complex, in particular after chemotherapeutic drug exposure. Using HCT116 cells, we show here that oxaliplatin treatment led to core Hippo pathway down-regulation and nuclear accumulation of TAZ. We further show that TAZ was required for the increased sensitivity of HCT116 cells to oxaliplatin, an effect that appeared independent of p73, but which required the nuclear relocalization of TAZ. Accordingly, Verteporfin and CA3, two drugs affecting the activity of YAP and TAZ, showed antagonistic effects with oxaliplatin in co-treatments. Importantly, using several colorectal cell lines, we show that the sensitizing action of TAZ to oxaliplatin is dependent on the p53 status of the cells. Our results support thus an early action of TAZ to sensitize cells to oxaliplatin, consistent with a model in which nuclear TAZ in the context of DNA damage and p53 activity pushes cells towards apoptosis.


Assuntos
Antineoplásicos , Neoplasias do Colo , Via de Sinalização Hippo , Oxaliplatina , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteína Supressora de Tumor p53 , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Via de Sinalização Hippo/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina/farmacologia , Porfirinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteína Tumoral p73/metabolismo , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Verteporfina/farmacologia , Verteporfina/uso terapêutico , Proteínas de Sinalização YAP/metabolismo
4.
Sci Rep ; 14(1): 9894, 2024 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-38688978

RESUMO

This study aims to decipher crucial biomarkers regulated by p73 for the early detection of colorectal cancer (CRC) by employing a combination of integrative bioinformatics and expression profiling techniques. The transcriptome profile of HCT116 cell line p53 - / - p73 + / + and p53 - / - p73 knockdown was performed to identify differentially expressed genes (DEGs). This was corroborated with three CRC tissue expression datasets available in Gene Expression Omnibus. Further analysis involved KEGG and Gene ontology to elucidate the functional roles of DEGs. The protein-protein interaction (PPI) network was constructed using Cytoscape to identify hub genes. Kaplan-Meier (KM) plots along with GEPIA and UALCAN database analysis provided the insights into the prognostic and diagnostic significance of these hub genes. Machine/deep learning algorithms were employed to perform TNM-stage classification. Transcriptome profiling revealed 1289 upregulated and 1897 downregulated genes. When intersected with employed CRC datasets, 284 DEGs were obtained. Comprehensive analysis using gene ontology and KEGG revealed enrichment of the DEGs in metabolic process, fatty acid biosynthesis, etc. The PPI network constructed using these 284 genes assisted in identifying 20 hub genes. Kaplan-Meier, GEPIA, and UALCAN analyses uncovered the clinicopathological relevance of these hub genes. Conclusively, the deep learning model achieved TNM-stage classification accuracy of 0.78 and 0.75 using 284 DEGs and 20 hub genes, respectively. The study represents a pioneer endeavor amalgamating transcriptomics, publicly available tissue datasets, and machine learning to unveil key CRC-associated genes. These genes are found relevant regarding the patients' prognosis and diagnosis. The unveiled biomarkers exhibit robustness in TNM-stage prediction, thereby laying the foundation for future clinical applications and therapeutic interventions in CRC management.


Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mapas de Interação de Proteínas , Proteína Tumoral p73 , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biologia Computacional/métodos , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Mapas de Interação de Proteínas/genética , Prognóstico , Células HCT116 , Transcriptoma , Estimativa de Kaplan-Meier
5.
J Pathol ; 263(3): 328-337, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38629257

RESUMO

Peritoneal metastasis of colorectal origin appears in ~10-15% of patients at the time of diagnosis and in 30-40% of cases with disease progression. Locoregional spread through the peritoneum is considered stage IVc and is associated with a poor prognosis. The development of a regional therapeutic strategy based on cytoreductive surgery, and hyperthermic intra-abdominal chemotherapy has significantly altered the course of the disease. Although recent evidence supports the benefits of cytoreductive surgery, the benefits of hyperthermic intra-abdominal chemotherapy are, however, still a matter of debate. Understanding the molecular alterations underlying the disease is crucial for developing new therapeutic strategies. Here, we evaluated the involvement in peritoneal dissemination of the oncogenic isoform of TP73, ΔNp73, and its effector targets in in vitro and mouse models, and in 30 patients diagnosed with colorectal peritoneal metastasis. In an orthotopic mouse model, we observed that tumor cells overexpressing ΔNp73 present a higher avidity for the peritoneum and that extracellular vesicles secreted by ΔNp73-upregulating tumor cells enhance their dissemination. In addition, we identified that tumor cells overexpressing ΔNp73 present with dysregulation of genes associated with an epithelial/mesothelial-to-mesenchymal transition (MMT) and that mesothelial cells exposed to the conditioned medium of tumor cells with upregulated ΔNp73 present a mesenchymal phenotype. Lastly, ΔNp73 and its effector target RNAs were dysregulated in our patient series, there were positive correlations between ΔNp73 and its effector targets, and MSN and ITGB4 (ΔNp73 effectors) predicted patient survival. In conclusion, ΔNp73 and its effector targets are involved in the peritoneal dissemination of colorectal cancer and predict patient survival. The promotion of the EMT/MMT and modulation of the adhesion capacity in colorectal cancer cells might be the mechanisms triggered by ΔNp73. Remarkably, ΔNp73 protein is a druggable protein and should be the focus of future studies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Neoplasias Colorretais , Transição Epitelial-Mesenquimal , Neoplasias Peritoneais , Proteína Tumoral p73 , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/patologia , Animais , Masculino , Feminino , Proteína Tumoral p73/metabolismo , Proteína Tumoral p73/genética , Pessoa de Meia-Idade , Idoso , Camundongos , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral
6.
PLoS One ; 18(10): e0292434, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37796859

RESUMO

Cystic echinococcosis (CE) is a life-threatening helminthic disease caused by the Echinococcus granulosus sensulato complex. Previous evidence indicates that the host's innate immune responses against CE can combat and regulate the growth rate and mortality of hydatid cyst in the host's internal organs. However, the survival mechanisms of CE are not yet fully elucidated in the human body. In the present study, the apoptotic effects of fertile and infertile hydatid fluid (HF) were tested on murine peritoneal cells in vivo mice model. Mice were divided into five groups including; control group, fertile HF-treated peritoneal cells, infertile HF-treated peritoneal cells, protoscolices (PSCs)-treated peritoneal cells and HF+PSCs-treated peritoneal cells group. Mice groups were intraperitoneally inoculated with PBS, HF, and/or PSCs. Afterwards, peritoneal cells were isolated and mRNA expression of STAT3, caspase-3, p73 and Smac genes were evaluated by quantitative Real-time PCR. After 48 hours of exposure, the protein levels of Smac and STAT3 was determined by western blotting technique. After 6 hours of exposure, Caspase-3 activity was also measured by fluorometric assay. The intracellular reactive oxygen species (ROS) production was examined in all groups. The mRNA expression levels of p73, caspase-3 and also Caspase-3 activity in HF+PSCs-treated peritoneal cells were higher than in the test and control groups (Pv<0.05), while the mRNA expression level of anti-apoptotic STAT3 and Smac genes in HF+PSC-treated peritoneal cells were lower than in the other groups (Pv<0.05). As well, the level of intracellular ROS in the fertile HCF-treated peritoneal cells, infertile HCF-treated peritoneal cells, PSC-treated peritoneal cells and HF+PSC-treated peritoneal cells groups were significantly higher than in the control group (Pv<0.05).Current findings indicates that oxidative stress and p73 can trigger the apoptosis of murine peritoneal cells through modulator of HF-treated PSCs that is likely one of the hydatid cyst survival mechanisms in vivo mice model.


Assuntos
Apoptose , Equinococose , Echinococcus granulosus , Proteína Tumoral p73 , Animais , Camundongos , Caspase 3/metabolismo , Espécies Reativas de Oxigênio , RNA Mensageiro , Proteína Tumoral p73/metabolismo
7.
Cell Death Dis ; 14(10): 674, 2023 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-37828008

RESUMO

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p632/p732 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p632/p732 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.


Assuntos
Carcinoma de Células Escamosas , Fatores de Transcrição , Animais , Humanos , Camundongos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Proteínas de Repetição de Anquirina Projetadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo
8.
Cancer Discov ; 13(5): 1210-1229, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-36734633

RESUMO

Triple-negative breast cancers (TNBC) frequently inactivate p53, increasing their aggressiveness and therapy resistance. We identified an unexpected protein vulnerability in p53-inactivated TNBC and designed a new PROteolysis TArgeting Chimera (PROTAC) to target it. Our PROTAC selectively targets MDM2 for proteasome-mediated degradation with high-affinity binding and VHL recruitment. MDM2 loss in p53 mutant/deleted TNBC cells in two-dimensional/three-dimensional culture and TNBC patient explants, including relapsed tumors, causes apoptosis while sparing normal cells. Our MDM2-PROTAC is stable in vivo, and treatment of TNBC xenograft-bearing mice demonstrates tumor on-target efficacy with no toxicity to normal cells, significantly extending survival. Transcriptomic analyses revealed upregulation of p53 family target genes. Investigations showed activation and a required role for TAp73 to mediate MDM2-PROTAC-induced apoptosis. Our data, challenging the current MDM2/p53 paradigm, show MDM2 is required for p53-inactivated TNBC cell survival, and PROTAC-targeted MDM2 degradation is an innovative potential therapeutic strategy for TNBC and superior to existing MDM2 inhibitors. SIGNIFICANCE: p53-inactivated TNBC is an aggressive, therapy-resistant, and lethal breast cancer subtype. We designed a new compound targeting an unexpected vulnerability we identified in TNBC. Our MDM2-targeted degrader kills p53-inactivated TNBC cells, highlighting the requirement for MDM2 in TNBC cell survival and as a new therapeutic target for this disease. See related commentary by Peuget and Selivanova, p. 1043. This article is highlighted in the In This Issue feature, p. 1027.


Assuntos
Quimera de Direcionamento de Proteólise , Proteínas Proto-Oncogênicas c-mdm2 , Neoplasias de Mama Triplo Negativas , Proteína Supressora de Tumor p53 , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/fisiopatologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Quimera de Direcionamento de Proteólise/química , Quimera de Direcionamento de Proteólise/farmacologia , Quimera de Direcionamento de Proteólise/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Análise de Sobrevida , Apoptose/efeitos dos fármacos , Proteína Tumoral p73/metabolismo , Xenoenxertos , Proteólise/efeitos dos fármacos , Feminino
9.
Hum Cell ; 35(5): 1512-1520, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35896939

RESUMO

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. The aim of the present study was to explore the expression level of tumor protein 73 (TP73) in highly malignant CRC tumors and how the long non-coding RNA tumor protein 73 antisense RNA 1 (TP73-AS1) influences that transcription. We found that TP73-AS1 was highly expressed in malignant CRC samples in The Cancer Genome Atlas (TCGA) database. We also demonstrated TP73-AS1 was expressed in thirty samples of CRC tissues collected from China Medical University patients as well as in HCT116, RKO and SW480 CRC cell lines but not in HCoEpiC or CCD-18Co normal colon cells. Only wild-type TP73-AS1, but not any of its alternate splicing isoforms, was positively correlated with tumor malignancy. TP73-AS1 transcripts were shown to be located in cell nuclei especially in close proximity to the TP73 promoter in CRC cells, but not in normal colon cells. In addition, an interaction between lysine demethylase 5A (KDM5A) and TP73-AS1 in CRC cells, but not normal colon cells, and KDM5A localization on the TP73 promoter were influenced by TP73-AS1. Interestingly, the H3K4me3 level on the TP73 promoter was reduced, but was elevated by TP73-AS1 knockdown in CRC cells. In conclusion, these results suggest a novel epigenetic role of TP73-AS1 on histone demethylation that influences TP73 transcription, and shed light on malignancy in CRC.


Assuntos
Neoplasias Colorretais , MicroRNAs , RNA Longo não Codificante/metabolismo , Proteína Tumoral p73/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Lisina/metabolismo , MicroRNAs/genética , Proteínas de Neoplasias/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo
10.
Cell Death Differ ; 29(12): 2445-2458, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35717504

RESUMO

The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.


Assuntos
Proteínas de Repetição de Anquirina Projetadas , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Proteína Tumoral p73/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , DNA/química
11.
Mol Carcinog ; 61(7): 629-642, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35560453

RESUMO

Members of the p53 family of transcription factors-p53, p63, and p73-share a high degree of homology; however, members can be activated in response to different stimuli, perform distinct (sometimes opposing) roles and are expressed in different tissues. The level of complexity is increased further by the transcription of multiple isoforms of each homolog, which may interact or interfere with each other and can impact cellular outcome. Proteins perform their functions through interacting with other proteins (and/or with nucleic acids). Therefore, identification of the interactors of a protein and how they interact in 3D is essential to fully comprehend their roles. By utilizing an in silico protein-protein interaction prediction method-HMI-PRED-we predicted interaction partners of p53 family members and modeled 3D structures of these protein interaction complexes. This method recovered experimentally known interactions while identifying many novel candidate partners. We analyzed the similarities and differences observed among the interaction partners to elucidate distinct functions of p53 family members and provide examples of how this information may yield mechanistic insight to explain their overlapping versus distinct/opposing outcomes in certain contexts. While some interaction partners are common to p53, p63, and p73, the majority are unique to each member. Nevertheless, most of the enriched pathways associated with these partners are common to all members, indicating that the members target the same biological pathways but through unique mediators. p63 and p73 have more common enriched pathways compared to p53, supporting their similar developmental roles in different tissues.


Assuntos
Fatores de Transcrição , Proteína Supressora de Tumor p53 , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
12.
Proc Natl Acad Sci U S A ; 119(22): e2123202119, 2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35617425

RESUMO

p73, a p53 family member, undergoes alternative splicing at the 3' end to produce multiple isoforms, but their expression and activity are largely unknown. Thus, CRISPR was used to knock out exon 12 (E12) in human cancer cell lines and mice, leading to isoform switch from p73α to isoform p73α1. We found that p73α1 is naturally expressed and induced by DNA damage. We also found that knockout of E12 suppresses cell growth and migration in H1299 and MIA PaCa-2 cells and promotes cellular senescence in mouse embryonic fibroblasts. Similarly, ectopic expression of p73α1 suppresses cell proliferation, whereas knockdown of p73α1 restores the cell proliferative and migratory capacities of E12−/− cells. Consistently, we found that E12+/− mice are not prone to spontaneous tumors. Instead, E12+/− mice are prone to systemic inflammation and exhibit elevated TNFα expression in inflamed tissues. Moreover, we found that Notch1, a master regulator of the inflammatory response, is regulated by p73α1 and highly expressed in E12−/− cells and inflamed E12+/− mouse tissues. Furthermore, through knockdown of p73α1 and/or Notch1 in E12−/− cells, we found that Notch1 is necessary for p73α1-mediated growth suppression. Together, these data suggest that p73α1 plays a critical role in tumor suppression and the inflammatory response via Notch1.


Assuntos
Genes Supressores de Tumor , Inflamação , Neoplasias , Receptor Notch1 , Proteína Tumoral p73 , Animais , Linhagem Celular Tumoral , Dano ao DNA , Éxons/genética , Técnicas de Inativação de Genes , Humanos , Inflamação/genética , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo
13.
Cell Death Differ ; 29(5): 921-937, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35314772

RESUMO

The p53 protein family is the most studied protein family of all. Sequence analysis and structure determination have revealed a high similarity of crucial domains between p53, p63 and p73. Functional studies, however, have shown a wide variety of different tasks in tumor suppression, quality control and development. Here we review the structure and organization of the individual domains of p63 and p73, the interaction of these domains in the context of full-length proteins and discuss the evolutionary origin of this protein family. FACTS: Distinct physiological roles/functions are performed by specific isoforms. The non-divided transactivation domain of p63 has a constitutively high activity while the transactivation domains of p53/p73 are divided into two subdomains that are regulated by phosphorylation. Mdm2 binds to all three family members but ubiquitinates only p53. TAp63α forms an autoinhibited dimeric state while all other vertebrate p53 family isoforms are constitutively tetrameric. The oligomerization domain of p63 and p73 contain an additional helix that is necessary for stabilizing the tetrameric states. During evolution this helix got lost independently in different phylogenetic branches, while the DNA binding domain became destabilized and the transactivation domain split into two subdomains. OPEN QUESTIONS: Is the autoinhibitory mechanism of mammalian TAp63α conserved in p53 proteins of invertebrates that have the same function of genomic quality control in germ cells? What is the physiological function of the p63/p73 SAM domains? Do the short isoforms of p63 and p73 have physiological functions? What are the roles of the N-terminal elongated TAp63 isoforms, TA* and GTA?


Assuntos
Proteína Supressora de Tumor p53 , Proteínas Supressoras de Tumor , Animais , Proteínas de Ligação a DNA/metabolismo , Mamíferos/metabolismo , Proteínas Nucleares/metabolismo , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
14.
Biomolecules ; 12(3)2022 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-35327630

RESUMO

The p53 family has the following three members: p53, p63 and p73. p53 is a tumor suppressor gene that frequently exhibits mutation in head and neck cancer. Most p53 mutants are loss-of-function (LoF) mutants, but some acquire some oncogenic function, such as gain of function (GoF). It is known that the aggregation of mutant p53 can induce p53 GoF. The p73 activators RETRA and NSC59984 have an anti-cancer effect in p53 mutation cells, but we found that p73 activators were not effective in all head and neck squamous cell carcinoma (HNSCC) cell lines, with different p53 mutants. A comparison of the gene expression profiles of several regulator(s) in mutant HNSCC cells with or without aggregation of p53 revealed that nicotinamide phosphoribosyltransferase (NAMPT) is a key regulator of mutant p53 aggregation. An NAMPT inhibitor, to reduce abnormal aggregation of mutant p53, used in combination with a p73 activator, was able to effectively repress growth in HNSCC cells with p53 GoF mutants. This study, therefore, suggests a potential combination therapy approach for HNSCC with a p53 GoF mutation.


Assuntos
Neoplasias de Cabeça e Pescoço , Proteína Supressora de Tumor p53 , Proliferação de Células , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Int J Biol Macromol ; 206: 40-50, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35217090

RESUMO

p73 belongs to p53 family transcription factor activating more than 50% of cell fate p53 target genes involved in cell cycle, apoptosis, DNA damage response alongside neuronal system development and differentiation by binding to 20-bp response elements (REs) having sequence motif (PPPC-A/T-T/A-GYYY) where P-purines and Y-pyrimidines with each 10-bp separated by minimum 0 to 13-bp spacer. The promiscuous nature of recognizing both cell fate and development genes and the underlying RE selectivity mechanism by p73 is not well understood. Here, we report the molecular details of p73 recognizing the REs using the crystal structure of p73 DNA binding domain (DBD) in complex with 12 base pair DNA sequence 5'-cAGGCATGCCTg-3' and molecular dynamics simulations with six different p53 natural promoter sequences. Each 20-base pair natural promoter forms a different major/minor groove due to the presence of nucleotides A/T, A/C, G/G, T/T and G/T at positions 3, 8, 13, 18 uniquely recognized by p73 key residues Lys138 and Arg268. The loops L1 and L3 bearing these residues influence inter-and intra-dimer interfaces interactions and hence p73 forms a unique tetramer with each natural promoter sequence. Structural features of the DNA and the spacing between half-sites influence p73 tetramerization and its transactivation function.


Assuntos
Proteínas de Ligação a DNA , Proteína Supressora de Tumor p53 , DNA/química , Proteínas de Ligação a DNA/metabolismo , Genes Supressores de Tumor , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Elementos de Resposta/genética , Ativação Transcricional , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética
16.
Cancer Res ; 82(7): 1340-1352, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35149588

RESUMO

Targeting cyclin-dependent kinases 4 and 6 (CDK4/6) is a successful therapeutic approach against breast and other solid tumors. Inhibition of CDK4/6 halts cell cycle progression and promotes antitumor immunity. However, the mechanisms underlying the antitumor activity of CDK4/6 inhibitors are not fully understood. We found that CDK4/6 bind and phosphorylate the p53 family member p73 at threonine 86, which sequesters p73 in the cytoplasm. Inhibition of CDK4/6 led to dephosphorylation and nuclear translocation of p73, which transcriptionally activated death receptor 5 (DR5), a cytokine receptor and key component of the extrinsic apoptotic pathway. p73-mediated induction of DR5 by CDK4/6 inhibitors promoted immunogenic cell death of cancer cells. Deletion of DR5 in cancer cells in vitro and in vivo abrogated the potentiating effects of CDK4/6 inhibitors on immune cytokine TRAIL, 5-fluorouracil chemotherapy, and anti-PD-1 immunotherapy. Together, these results reveal a previously unrecognized consequence of CDK4/6 inhibition, which may be critical for potentiating the killing and immunogenic effects on cancer cells. SIGNIFICANCE: This work demonstrates how inhibition of CDK4/6 sensitizes cancer cells to chemotherapy and immune checkpoint blockade and may provide a new molecular marker for improving CDK4/6-targeted cancer therapies. See related commentary by Frank, p. 1170.


Assuntos
Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidores de Checkpoint Imunológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Ligante Indutor de Apoptose Relacionado a TNF , Proteína Tumoral p73 , Apoptose , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Fluoruracila/farmacologia , Humanos , Fosforilação , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína Tumoral p73/metabolismo
17.
Mol Biol Rep ; 49(7): 6859-6869, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35138524

RESUMO

BACKGROUND: Prostate cancer is a malignant disease that severely affects the health and comfort of the male population. The long non-coding RNA TP73-AS1 has been shown to be involved in the malignant transformation of various human cancers. However, whether TP73-AS1 contributes to prostate cancer progression has not been reported yet. Accordingly, here we aimed to report the role of TP73-AS1 in the development and progression of prostate cancer and determine its relationship with TP73. METHODS AND RESULTS: TP73-AS1-specific siRNA oligo duplexes were used to silence TP73-AS1 in DU-145 and PC-3 cells. Results indicated that TP73-AS1 was upregulated whereas TP73 was downregulated in prostate cancer cells compared to normal prostate cells and there was a negative correlation between them. Besides, loss of function experiments of TP73-AS1 in prostate cancer cells strongly induced cellular apoptosis, interfered with the cell cycle progression, and modulated related pro- and anti-apoptotic gene expression. Colony formation and migration capacities of TP73-AS1-silenced prostate cancer cells were also found to be dramatically reduced. CONCLUSIONS: Our findings provide novel evidence that suggests a chief regulatory role for the TP73-TP73-AS1 axis in prostate cancer development and progression, suggesting that the TP73/TP73-AS1 axis can be a promising diagnostic and therapeutic target for prostate cancer.


Assuntos
MicroRNAs , Neoplasias da Próstata , RNA Longo não Codificante , Proteína Tumoral p73/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , MicroRNAs/genética , Neoplasias da Próstata/genética , RNA Longo não Codificante/genética
18.
Food Funct ; 13(1): 316-326, 2022 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-34897340

RESUMO

Maritime pine bark is a rich source of polyphenolic compounds and is commonly employed as a herbal supplement worldwide. This study was designed to check the potential of maritime pine tannin extract (MPTE) in anticancer therapy and to determine the underlying mechanism of action. Our results showed that MPTE, containing procyanidin oligomers and lanostane type terpenoids, has an inhibitory effect on cancer cell proliferation through cell cycle arrest in the G2/M phase. Treatment with MPTE also induced apoptosis in a concentration-dependent manner in human cancer cell lines (HeLa and U2OS), as evidenced by the enhanced activation of caspase 3 and the cleavage of PARP along with the downregulation of the antiapoptotic protein Bcl-2. Interestingly, human non-cancerous fibroblasts are much less sensitive to MPTE, suggesting that it preferentially targets cancer cells. MPTE played a pro-oxidant role in cancer cells and promoted the expression of the p73 tumor suppressor gene in p53-deficient cells. It also downregulated the protooncogenic proteins UHRF1 and DNMT1, mediators of the DNA methylation machinery, and reduced the global methylation levels in HeLa cells. Overall, our results show that maritime pine tannin extract can play a favorable role in cancer treatment, and can be further explored by the pharmaceutical industry.


Assuntos
Antineoplásicos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT , Epigênese Genética/efeitos dos fármacos , Pinus/química , Taninos/farmacologia , Ubiquitina-Proteína Ligases , Apoptose/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células HeLa , Humanos , Casca de Planta/química , Extratos Vegetais/farmacologia , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
19.
Sci Rep ; 11(1): 20974, 2021 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-34697383

RESUMO

Our lab has previously demonstrated Riluzole to be an effective drug in inhibiting proliferation and inducing apoptosis in both human and mouse osteosarcoma. Yes-associated protein is a transcription co-activator, known to be involved in cell proliferation or apoptosis depending on its protein partner. In the present study we investigated the role of YAP in apoptosis in osteosarcoma, we hypothesized that YAP may be activated by Riluzole to induce apoptosis in osteosarcoma. By knocking down the expression of YAP, we have demonstrated that Riluzole failed to induce apoptosis in YAP deficient osteosarcoma cells. Riluzole caused translocation of YAP from the cytoplasm to the nucleus, indicating YAP's role in apoptosis. Both Riluzole-induced phosphorylation of YAP at tyrosine 357 and Riluzole-induced apoptosis were blocked by inhibitors of c-Abl kinase. In addition, knockdown of c-Abl kinase prevented Riluzole-induced apoptosis in LM7 cells. We further demonstrated that Riluzole promoted interaction between YAP and p73, while c-Abl kinase inhibitors abolished the interaction. Subsequently, we demonstrated that Riluzole enhanced activity of the Bax promoter in a luciferase reporter assay and enhanced YAP/p73 binding on endogenous Bax promoter in a ChIP assay. Our data supports a novel mechanism in which Riluzole activates c-Abl kinase to regulate pro-apoptotic activity of YAP in osteosarcoma.


Assuntos
Neoplasias Ósseas/genética , Osteossarcoma/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Riluzol/farmacologia , Proteínas de Sinalização YAP/genética , Apoptose , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Osteossarcoma/metabolismo , Fosforilação , Transporte Proteico , Proteína Tumoral p73/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteína X Associada a bcl-2/genética
20.
Br J Cancer ; 125(11): 1523-1532, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34599296

RESUMO

BACKGROUND: Mutations in p53, identified in 90% of oesophageal squamous cell carcinoma (ESCC), are associated with unfavourable prognosis and chemo-resistance. APR-246 induces apoptosis by restoring transcriptional ability of mutant p53, and may be a promising therapeutic agent to overcome chemo-resistance in ESCC. METHODS: In ESCC cell lines differing in p53 status, we performed in vitro cell viability and apoptosis assays, evaluated reactive oxygen species (ROS) generation, and assessed signal changes by western blot after APR-246 administration with/without chemo-agent. Antitumour effects and signal changes were evaluated in in vivo experiments using xenograft and patient-derived xenograft (PDX) mouse models. RESULTS: APR-246 administration induced significant apoptosis by upregulating p73 and Noxa via ROS induction in ESCC cell lines harbouring p53 missense mutations. Moreover, APR-246 plus chemotherapy exerted combined antitumour effects in ESCC with p53 missense mutations. This effect was also mediated through enhanced ROS activity, leading to massive apoptosis via upregulation of p73 and Noxa. These findings were confirmed by xenograft and PDX models with p53 mutant ESCC. CONCLUSION: APR-246 strongly induced apoptosis by inducing ROS activity and p73-Noxa signalling, specifically in ESCC with p53 missense mutation. This antitumour effect was further enhanced by combination with 5-FU, which we first confirmed in ESCC preclinical model.


Assuntos
Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinuclidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Camundongos , Mutação de Sentido Incorreto , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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