RESUMO
The tumor suppressor von Hippel-Lindau, pVHL, is a multifaceted protein. One function is to dock to the hypoxia-inducible transcription factor (HIF) and recruit a larger protein complex that destabilizes HIF via ubiquitination, preventing angiogenesis and tumor development. pVHL also binds to the tumor suppressor p53 to activate specific p53 target genes. The oncogene Mdm2 impairs the formation of the p53-pVHL complex and activation of downstream genes by conjugating nedd8 to pVHL. While Mdm2 can impact p53 and pVHL, how pVHL may impact Mdm2 is unclear. Like p53 somatic mutations, point mutations are evident in pVHL that are common in renal clear cell carcinomas (RCC). In patients with RCC, Mdm2 levels are elevated, and we examined whether there was a relationship between Mdm2 and pVHL. TCGA and DepMap analysis revealed that mdm2 gene expression was elevated in RCC with vhl point mutations or copy number loss. In pVHL reconstituted or deleted isogenetically match RCC or MEF cell lines, Mdm2 was decreased in the presence of pVHL. Furthermore, through analysis using genetic and pharmacological approaches, we show that pVHL represses Mdm2 gene expression by blocking the MAPK-Ets signaling pathway and blocks Akt-mediated phosphorylation and stabilization of Mdm2. Mdm2 inhibition results in an increase in the p53-p21 pathway to impede cell growth. This finding shows how pVHL can indirectly impact the function of Mdm2 by regulating signaling pathways to restrict cell growth.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Proteínas Proto-Oncogênicas c-mdm2 , Transdução de Sinais , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor Von Hippel-Lindau , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Humanos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53+/-pp5+/- or p53+/-pp5-/- mice revealed that complete loss of PP5 reduces tumorigenesis in the p53+/- mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.
Assuntos
Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Motivos de Aminoácidos , Animais , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Regulação para Baixo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Mouse double minute 2 (Mdm2) is a multifaceted oncoprotein that is highly regulated with distinct domains capable of cellular transformation. Loss of Mdm2 is embryonically lethal, making it difficult to study in a mouse model without additional genetic alterations. Global overexpression through increased Mdm2 gene copy number (Mdm2Tg ) results in the development of hematopoietic neoplasms and sarcomas in adult animals. In these mice, we found an increase in osteoblastogenesis, differentiation, and a high bone mass phenotype. Since it was difficult to discern the cell lineage that generated this phenotype, we generated osteoblast-specific Mdm2 overexpressing (Mdm2TgOb ) mice in 2 different strains, C57BL/6 and DBA. These mice did not develop malignancies; however, these animals and the MG63 human osteosarcoma cell line with high levels of Mdm2 showed an increase in bone mineralization. Importantly, overexpression of Mdm2 corrected age-related bone loss in mice, providing a role for the proto-oncogenic activity of Mdm2 in bone health of adult animals.
Assuntos
Calcificação Fisiológica/fisiologia , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proto-Oncogenes/fisiologia , Análise de Variância , Animais , Densidade Óssea/fisiologia , Remodelação Óssea/fisiologia , Osso Esponjoso/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Proto-Oncogene MasRESUMO
Murine double minute-2 protein (Mdm2) is a multifaceted phosphorylated protein that plays a role in regulating numerous proteins including the tumor suppressor protein p53. Mdm2 binds to and is involved in conjugating either ubiquitin or Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8) to p53. Although regulation of the E3 ubiquitin activity of Mdm2 has been investigated, regulation of the neddylating activity of Mdm2 remains to be defined. Here we show that activated c-Src kinase phosphorylates Y281 and Y302 of Mdm2, resulting in an increase in Mdm2 stability and its association with Ubc12, the E2 enzyme of the neddylating complex. Mdm2-dependent Nedd8 conjugation of p53 results in transcriptionally inactive p53, a process that is reversed with a small molecule inhibitor to either Src or Ubc12. Thus, our studies reveal how Mdm2 may neutralize and elevate p53 in actively proliferating cells and also provides a rationale for using therapies that target the Nedd8 pathway in wild-type p53 tumors.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinas/metabolismo , Quinases da Família src/metabolismo , Animais , Western Blotting , Linhagem Celular , Humanos , Imunoprecipitação , Espectrometria de Massas , Camundongos , Proteína NEDD8 , Fosforilação , UbiquitinaçãoRESUMO
Since its discovery nearly 40 years ago, p53 has ascended to the forefront of investigated genes and proteins across diverse research disciplines and is recognized most exclusively for its role in cancer as a tumor suppressor. Levine and Oren (2009) reviewed the evolution of p53 detailing the significant discoveries of each decade since its first report in 1979. In this review, we will highlight the emerging non-canonical functions and regulation of p53 in stem cells. We will focus on general themes shared among p53's functions in non-malignant stem cells and cancer stem-like cells (CSCs) and the influence of p53 on the microenvironment and CSC niche. We will also examine p53 gain of function (GOF) roles in stemness. Mutant p53 (mutp53) GOFs that lead to survival, drug resistance and colonization are reviewed in the context of the acquisition of advantageous transformation processes, such as differentiation and dedifferentiation, epithelial-to-mesenchymal transition (EMT) and stem cell senescence and quiescence. Finally, we will conclude with therapeutic strategies that restore wild-type p53 (wtp53) function in cancer and CSCs, including RING finger E3 ligases and CSC maintenance. The mechanisms by which wtp53 and mutp53 influence stemness in non-malignant stem cells and CSCs or tumor-initiating cells (TICs) are poorly understood thus far. Further elucidation of p53's effects on stemness could lead to novel therapeutic strategies in cancer research.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Lennert et al., 1975; Thiele et al., 1999; Chagraoui et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell-cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb-mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK-to-OB signaling ultimately results in significant increases in the expression of c-fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin-mediated signaling mechanism, activating a novel response axis that de-represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone-related diseases.
Assuntos
Diferenciação Celular/genética , Megacariócitos/citologia , Osteoblastos/citologia , Transdução de Sinais/genética , Ciclo Celular/genética , Linhagem da Célula , Proliferação de Células/genética , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases/genética , Megacariócitos/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair to ensure genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions (RPA-DBi) and RPA protein-protein interactions (RPA-PPIi); both activities are required for ATR activation. Here, we biochemically reconstitute the ATR kinase signaling pathway and demonstrate that RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of target proteins with selectivity advantages over active site ATR inhibitors. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation does not affect ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway-targeted cancer therapeutics.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , DNA de Cadeia Simples , Processamento de Proteína Pós-Traducional , Proteína de Replicação A , Transdução de Sinais , Proteína de Replicação A/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Humanos , Fosforilação , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Proteínas de Transporte , Proteínas NuclearesRESUMO
The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair towards ensuring genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions, termed RPA-DBi, and RPA protein-protein interactions, termed RPA-PPIi; both activities are required for ATR activation. Here, we employ a biochemically reconstituted ATR kinase signaling pathway and demonstrate that both RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of downstream target proteins. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation has no effect on ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway targeted cancer therapeutics.
RESUMO
The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton.
Assuntos
Actinas/metabolismo , Caspases/metabolismo , Quinase 2 de Adesão Focal/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Megacariócitos/metabolismo , Osteoblastos/metabolismo , Processamento Alternativo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Adesões Focais/metabolismo , Isoenzimas/biossíntese , Megacariócitos/citologia , Camundongos , Osteoblastos/citologiaRESUMO
The most common gynecological cancer in Europe and the United States is endometrial. Like most cancers, early-stage endometrial cancer has a more favorable prognosis, while high-grade, including endometrioid and nonendometrioid, has the worst prognosis. In endometrioid human tumors, the tumor suppressor genes PTEN and p53 (Trp53) are frequently altered or lost, as identified in datasets from The Cancer Genome Atlas. These suppressors' somatic mutations or loss of gene expression can lead to neoplastic development, tumor progression, and therapeutic resistance. In addition, somatic missense mutations are prevalent in another tumor suppressor, the F-box and WD repeats containing 7 (FBXW7). FBXW7 is part of the SCF-ßTrCP ubiquitin complex that signals protein destruction. Specifically, FBXW7 is responsible for binding and facilitating the destabilization of proteins involved in proliferation and migration. Losing the function of multiple tumor suppressors could activate pathways involved in neoplastic progression, malignancy, therapeutic resistance, and formation of different tumor subtypes. The study by Brown et al in this issue of EMBO Mol Med (Brown et al, 2023) provides insight into the complexity of tumor suppressor mutations in malignant endometrial cancer.
Assuntos
Neoplasias do Endométrio , Proteína Supressora de Tumor p53 , Feminino , Humanos , Proteína 7 com Repetições F-Box-WD/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Proteína Supressora de Tumor p53/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Mutação , Europa (Continente)RESUMO
Mdm2 and Mdmx are oncoproteins that have essential yet nonredundant roles in development and function as part of a multicomponent ubiquitinating complex that targets p53 for proteasomal degradation. However, in response to DNA damage, Mdm2 and Mdmx are phosphorylated and protect p53 through various mechanisms. It has been predicted that Mdm2-Mdmx complex formation modulates Mdm2 ligase activity, yet the mechanism that promotes formation of Mdm2-Mdmx complexes is unknown. Here, we show that optimal Mdm2-Mdmx complex formation requires c-Abl phosphorylation of Mdm2 both in vitro and in vivo. In addition, Abl phosphorylation of Mdm2 is required for efficient ubiquitination of Mdmx in vitro, and eliminating c-Abl signaling, using c-Abl(-/-) knock-out murine embryonic fibroblasts, led to a decrease in Mdmx ubiquitination. Further, p53 levels are not induced as efficiently in c-Abl(-/-) murine embryonic fibroblasts following DNA damage. Overall, these results define a direct link between genotoxic stress-activated c-Abl kinase signaling and Mdm2-Mdmx complex formation. Our results add an important regulatory mechanism for the activation of p53 in response to DNA damage.
Assuntos
Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Animais , Benzamidas , Linhagem Celular Tumoral , Dano ao DNA , Técnicas de Inativação de Genes , Humanos , Mesilato de Imatinib , Camundongos , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/deficiência , Proteínas Proto-Oncogênicas c-abl/genética , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The p53 family member, p73, has been characterized as a tumor suppressor and functions in a similar manner as p53 to induce cellular death. The phosphatase and tensin homolog (PTEN) can function as a dual specificity lipid/protein phosphatase. However, recent data have described multiple roles for nuclear PTEN independent of its lipid phosphatase activity. PTEN can directly or indirectly activate p53 to promote apoptosis. We examined whether PTEN would interact and regulate p73 independent of p53. Co-localization in the nucleus and complex formation of p73/PTEN were observed after DNA damage. Furthermore, we also demonstrate that p73α/PTEN proteins directly bind one another. Both overexpressed and endogenous p73-PTEN interactions were determined to be the strongest in the nuclear fraction after DNA damage, which suggested formation of a transcriptional complex. We employed chromatin immunoprecipitation (ChIP) and found that p73 and PTEN were associated with the PUMA promoter after genotoxic stress in TP53-null cells. We found that another p73 target, BAX, had an increased expression in the presence of p73 and PTEN. In addition, in virus-transduced cell lines stably expressing p73, PTEN, or both p73/PTEN, we found that the p73/PTEN cells were more sensitive to genotoxic stress and cellular death as measured by increased poly(ADP-ribose) polymerase cleavage and PUMA/Bax induction. Conversely, knockdown of PTEN dramatically reduced Bax and PUMA levels. Thus, a p73-PTEN protein complex is engaged to induce apoptosis independent of p53 in response to DNA damage.
Assuntos
Apoptose , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Transformada , Fragmentação do DNA , Proteínas de Ligação a DNA/genética , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The alteration of tumorigenic pathways leading to cancer is a degenerative disease process typically involving inactivation of tumor suppressor proteins and hyperactivation of oncogenes. One such oncogenic protein product is the murine double-minute 2, or Mdm2. While, Mdm2 has been primarily associated as the negative regulator of the p53 tumor suppressor protein there are many p53-independent roles demonstrated for this oncogene. DNA damage and chemotherapeutic agents are known to activate Mdm2 and DNA repair pathways. There are five primary DNA repair pathways involved in the maintenance of genomic integrity: Nucleotide excision repair (NER), Base excision repair (BER), Mismatch repair (MMR), Non-homologous end joining (NHEJ) and homologous recombination (HR). In this review, we will briefly describe these pathways and also delineate the functional interaction of Mdm2 with multiple DNA repair proteins. We will illustrate the importance of these interactions with Mdm2 and discuss how this is important for tumor progression, cellular proliferation in cancer.
Assuntos
Carcinogênese/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Animais , Reparo de Erro de Pareamento de DNA , Recombinação Homóloga , Humanos , Transdução de Sinais/genéticaRESUMO
Mutations in the tumor suppressor TP53 are rare in renal cell carcinomas. p53 is a key factor for inducing antiangiogenic genes and RCC are highly vascularized, which suggests that p53 is inactive in these tumors. One regulator of p53 is the Mdm2 oncogene, which is correlated with high-grade, metastatic tumors. However, the sole activity of Mdm2 is not just to regulate p53, but it can also function independent of p53 to regulate the early stages of metastasis. Here, we report that the oncoprotein Mdm2 can bind directly to the tumor suppressor VHL, and conjugate nedd8 to VHL within a region that is important for the p53-VHL interaction. Nedd8 conjugated VHL is unable to bind to p53 thereby preventing the induction of antiangiogenic factors. These results highlight a previously unknown oncogenic function of Mdm2 during the progression of cancer to promote angiogenesis through the regulation of VHL. Thus, the Mdm2-VHL interaction represents a pathway that impacts tumor angiogenesis.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , TransfecçãoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Hdm2 is elevated in numerous types of malignancies and is thought to impede the function of wild-type p53. Reactivation of p53 by disrupting the association with Hdm2 was the impetus for the development of Nutlin3. Although regulation of p53 has been the central focus of Hdm2 activity, it also binds other proteins through its p53-binding domain. Here, we show that hypoxia-inducible factor 1alpha (HIF1alpha) binds to Hdm2 in the domain designated to bind p53. HIF1alpha and p53 share a conserved motif that is required to bind Hdm2. Distinct complexes form between Hdm2-HIF1alpha and Hdm2-p53 as determined by immunoprecipitation of nuclear extracts and in vitro. The Hdm2 antagonist Nutlin3 prevents the association between Hdm2 and HIF1alpha. The vascular endothelial growth factor (VEGF) gene is a transcriptional target of HIF1alpha, and under normoxic or hypoxic conditions, Hdm2 increases HIF1alpha activity to induce VEGF production. Blocking the association of Hdm2 and HIF1alpha by Nutlin3, or ablating Hdm2 expression, diminished the level of VEGF under conditions of normoxia or hypoxia. Our findings establish a unique role for Nutlin3 in attenuating VEGF induction by preventing the association of Hdm2 with HIF1alpha.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Interferente Pequeno/genética , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Oncoproteins and tumor-suppressor proteins regulate cell growth and viability. Recent observations show that phosphoinositide 3-kinase (PtdIns 3-kinase)-Akt signaling promotes the phosphorylation and movement of the Mdm2 oncoprotein into the nucleus, where it downregulates the p53 tumor-suppressor protein. The PTEN tumor suppressor protein inhibits activation of Akt and this restricts Mdm2 to the cytoplasm. Restriction of Mdm2 to the cytoplasm promotes p53 function and thereby sustains the sensitivity of cancer cells to chemotherapy. p53 acutely induces Mdm2, providing damaged cells the opportunity for repair, but subsequently induces PTEN, favoring the death of mutated or irrevocably damaged cells. Thus, oncoproteins and tumor suppressor proteins are networked to promote normal cell function and eliminate mutated cells.
Assuntos
Proteínas Nucleares , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Humanos , PTEN Fosfo-Hidrolase , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-mdm2 , Células Tumorais CultivadasRESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. Here we discover that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Furthermore, genetic and pharmacological inhibition of EZH2 decreases the repopulating potential of p53 mutant HSPCs. Thus, we uncover an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.
Assuntos
Epigênese Genética , Doenças Hematológicas/metabolismo , Hematopoese , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Doenças Hematológicas/genética , Doenças Hematológicas/fisiopatologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Mutação , Ligação ProteicaRESUMO
Hdm2 and HdmX coordinately regulate the stability and function of p53. Each is overexpressed in subsets of many different types of malignancy, and most of these subsets maintain wild-type p53. Nutlins, newly discovered small-molecule inhibitors of the Hdm2-p53 interaction, offer a novel strategy for therapy of tumors with wild-type p53. We now show that Nutlin-3 efficiently induces apoptosis and diminishes long-term survival of human fibroblasts transformed in vitro by Hdm2 but not HdmX. The resistance of cells overexpressing HdmX to Nutlin-3 is due to its inability to disrupt the p53-HdmX interaction, resulting in continued suppression of p53 activity. Although HdmX overexpression yielded cells resistant to Nutlin-3, ablation of HdmX expression by short hairpin RNA sensitized tumor cells to Nutlin-3-mediated cell death or arrest. Furthermore, deletion of the COOH-terminal RING finger domain of HdmX completely reversed the resistance to Nutlin-3, probably reflecting the requirement of the RING finger for interaction with Hdm2. Thus, the relative abundance of Hdm2 and HdmX and the specificity of Nutlin-3 for Hdm2 influence the sensitivity of cells to p53-dependent apoptosis or arrest in response to Nutlin-3. Our findings establish Hdm2 and HdmX as independent therapeutic targets with respect to reactivating wild-type p53 as a means for cancer therapy.
Assuntos
Imidazóis/farmacologia , Proteínas Nucleares/biossíntese , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Ciclo Celular , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Doxorrubicina/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HCT116 , Humanos , Imidazóis/administração & dosagem , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/administração & dosagem , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The transcription factors p53 and p73 are critical to the induction of apoptotic cell death, particularly in response to cell stress that activates c-Jun N-terminal kinase (JNK). Mutations in the DNA-binding domain of p53, which are commonly seen in cancers, result in conformational changes that enable p53 to interact with and inhibit p73, thereby suppressing apoptosis. In contrast, wild-type p53 reportedly does not interact with p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53, as well as mutant p53, to form a complex with p73. Structural algorithms predicted that phosphorylation of Thr81 exposes the DNA-binding domain in p53 to enable its binding to p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes [such as those encoding p53-up-regulated modulator of apoptosis (PUMA) and Bcl-2-associated X protein (BAX)] and, subsequently, the induction of apoptosis in response to JNK activation by cell stress in various cells. Thus, JNK phosphorylation of mutant and wild-type p53 promotes the formation of a p53/p73 complex that determines cell fate: apoptosis in the context of wild-type p53 or cell survival in the context of the mutant. These findings refine our current understanding of both the mechanistic links between p53 and p73 and the functional role for Thr81 phosphorylation.