RESUMO
The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl-/- mice, which lack the NH2-amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport to the membrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P. aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy.
Assuntos
Membrana Celular/imunologia , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Fibrose Cística/imunologia , PTEN Fosfo-Hidrolase/imunologia , Infecções por Pseudomonas/imunologia , Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Animais , Benzodioxóis/farmacologia , Membrana Celular/efeitos dos fármacos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/microbiologia , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Ligação Proteica , Conformação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Quinolonas/farmacologia , Transdução de SinaisRESUMO
With advances in chemically induced proximity technologies, heterobifunctional modalities such as proteolysis targeting chimeras (PROTACs) have been successfully advanced to clinics for treating cancer. However, pharmacologic activation of tumor-suppressor proteins for cancer treatment remains a major challenge. Here, we present a novel Acetylation Targeting Chimera (AceTAC) strategy to acetylate the p53 tumor suppressor protein. We discovered and characterized the first p53Y220C AceTAC, MS78, which recruits histone acetyltransferase p300/CBP to acetylate the p53Y220C mutant. MS78 effectively acetylated p53Y220C lysine 382 (K382) in a concentration-, time-, and p300-dependent manner and suppressed proliferation and clonogenicity of cancer cells harboring the p53Y220C mutation with little toxicity in cancer cells with wild-type p53. RNA-seq studies revealed novel p53Y220C-dependent upregulation of TRAIL apoptotic genes and downregulation of DNA damage response pathways upon acetylation induced by MS78. Altogether, the AceTAC strategy could provide a generalizable platform for targeting proteins, such as tumor suppressors, via acetylation.
Assuntos
Proteína Supressora de Tumor p53 , Acetilação , Humanos , Linhagem Celular Tumoral , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Mutação , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
The enhancer of zeste homolog 2 (EZH2) is the main enzymatic subunit of the PRC2 complex, which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to promote transcriptional silencing. EZH2 is overexpressed in multiple types of cancer including triple-negative breast cancer (TNBC), and high expression levels correlate with poor prognosis. Several EZH2 inhibitors, which inhibit the methyltransferase activity of EZH2, have shown promise in treating sarcoma and follicular lymphoma in clinics. However, EZH2 inhibitors are ineffective at blocking proliferation of TNBC cells, even though they effectively reduce the H3K27me3 mark. Using a hydrophobic tagging approach, we generated MS1943, a first-in-class EZH2 selective degrader that effectively reduces EZH2 levels in cells. Importantly, MS1943 has a profound cytotoxic effect in multiple TNBC cells, while sparing normal cells, and is efficacious in vivo, suggesting that pharmacologic degradation of EZH2 can be advantageous for treating the cancers that are dependent on EZH2.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Piperazinas/farmacologia , Piridinas/farmacologia , Animais , Antineoplásicos/farmacocinética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Técnicas de Inativação de Genes , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Proteólise/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND AIMS: The pattern of genetic alterations in cancer driver genes in patients with hepatocellular carcinoma (HCC) is highly diverse, which partially explains the low efficacy of available therapies. In spite of this, the existing mouse models only recapitulate a small portion of HCC inter-tumor heterogeneity, limiting the understanding of the disease and the nomination of personalized therapies. Here, we aimed at establishing a novel collection of HCC mouse models that captured human HCC diversity. METHODS: By performing hydrodynamic tail-vein injections, we tested the impact of altering a well-established HCC oncogene (either MYC or ß-catenin) in combination with an additional alteration in one of eleven other genes frequently mutated in HCC. Of the 23 unique pairs of genetic alterations that we interrogated, 9 were able to induce HCC. The established HCC mouse models were characterized at histopathological, immune, and transcriptomic level to identify the unique features of each model. Murine HCC cell lines were generated from each tumor model, characterized transcriptionally, and used to identify specific therapies that were validated in vivo. RESULTS: Cooperation between pairs of driver genes produced HCCs with diverse histopathology, immune microenvironments, transcriptomes, and drug responses. Interestingly, MYC expression levels strongly influenced ß-catenin activity, indicating that inter-tumor heterogeneity emerges not only from specific combinations of genetic alterations but also from the acquisition of expression-dependent phenotypes. CONCLUSIONS: This novel collection of murine HCC models and corresponding cell lines establishes the role of driver genes in diverse contexts and enables mechanistic and translational studies.
Assuntos
Carcinoma Hepatocelular/genética , Heterogeneidade Genética , Proto-Oncogenes/genética , Animais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Biologia Computacional , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Transgênicos , Evasão Tumoral/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Regulation of RNA polymerase II (RNAPII)-mediated transcription controls cellular phenotypes such as cancer. Phosphatase and tensin homologue deleted on chromosome ten (PTEN), one of the most commonly altered tumor suppressors in cancer, affects transcription via its role in antagonizing the PI3K/AKT signaling pathway. Using co-immunoprecipitations and proximal ligation assays we provide evidence that PTEN interacts with AFF4, RNAPII, CDK9, cyclin T1, XPB and CDK7. Using ChIP-seq, we show that PTEN co-localizes with RNAPII and binds to chromatin in promoter and putative enhancer regions identified by histone modifications. Furthermore, we show that loss of PTEN affects RNAPII occupancy in gene bodies and further correlates with gene expression changes. Interestingly, PTEN binds to promoters and negatively regulates the expression of genes involved in transcription including AFF4 and POL2RA, which encodes a subunit of RNAPII. Loss of PTEN also increased cells' sensitivity to transcription inhibition via small molecules, which could provide a strategy to target PTEN-deficient cancers. Overall, our work describes a previously unappreciated role of nuclear PTEN, which by interacting with the transcription machinery in the context of chromatin exerts an additional layer of regulatory control on RNAPII-mediated transcription.
Assuntos
Cromatina/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Células Cultivadas , Cromatina/genética , Células HEK293 , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Polimerase II/genética , Transdução de Sinais/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Depending on the circumstance, FOXO (Forkhead O) (FOXO1, FOXO3, and FOXO4) transcription factors activate the expression of markedly different sets of genes to produce different phenotypic effects. For example, distinct FOXO-regulated transcriptional programs stimulate cell death or enhance organism life span. To gain insight into how FOXOs select specific genes for regulation, we performed a screen for genes that modify FOXO activation of TRAIL, a death receptor ligand capable of inducing extrinsic apoptosis. We discovered that the bZIP transcriptional repressor NFIL3 (nuclear factor interleukin 3-regulated) hindered FOXO transcription factor access to chromatin at the TRAIL promoter by binding to nearby DNA and recruiting histone deacetylase-2 (HDAC2) to reduce histone acetylation. In the same manner, NFIL3 repressed expression of certain FOXO targets--e.g., FAS, GADD45α (growth arrest and DNA damage-inducible, α), and GADD45ß--but not others. NFIL3, which we found to be overexpressed in different cancers, supported tumor cell survival largely through repression of TRAIL and antagonized hydrogen peroxide-induced cell death. Moreover, its expression in cancer was associated with lower patient survival. Therefore, NFIL3 alters cancer cell behavior and FOXO function by acting on chromatin to restrict the menu of FOXO target genes. Targeting of NFIL3 could be of therapeutic benefit for cancer patients.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteína Forkhead Box O1 , Células HEK293 , Histona Desacetilases/metabolismo , Humanos , Estimativa de Kaplan-Meier , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a phosphatase that is frequently altered in cancer. PTEN has phosphatase-dependent and -independent roles, and genetic alterations in PTEN lead to deregulation of protein synthesis, the cell cycle, migration, growth, DNA repair, and survival signaling. PTEN localization, stability, conformation, and phosphatase activity are controlled by an array of protein-protein interactions and post-translational modifications. Thus, PTEN-interacting and -modifying proteins have profound effects on the tumor suppressive functions of PTEN. Moreover, recent studies identified mechanisms by which PTEN can exit cells, via either exosomal export or secretion, and act on neighboring cells. This review focuses on modes of PTEN protein regulation and ways in which perturbations in this regulation may lead to disease.
Assuntos
PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Animais , Ciclo Celular , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , PTEN Fosfo-Hidrolase/química , Processamento de Proteína Pós-TraducionalRESUMO
While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gßγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gßγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.
Assuntos
Neoplasias da Mama/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Oncogênicas v-erbB/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Células Tumorais CultivadasRESUMO
Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gßγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Quinases Ativadas por p21/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Isoproterenol/farmacologia , Células MCF-7 , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação/efeitos dos fármacos , Receptor de Insulina/genética , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Insulin activation of phosphoinositide 3-kinase (PI3K) signaling regulates glucose homeostasis through the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). The dual-specificity phosphatase and tensin homolog deleted on chromosome 10 (PTEN) blocks PI3K signaling by dephosphorylating PIP3, and is inhibited through its interaction with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (P-REX2). The mechanism of inhibition and its physiological significance are not known. Here, we report that P-REX2 interacts with PTEN via two interfaces. The pleckstrin homology (PH) domain of P-REX2 inhibits PTEN by interacting with the catalytic region of PTEN, and the inositol polyphosphate 4-phosphatase domain of P-REX2 provides high-affinity binding to the postsynaptic density-95/Discs large/zona occludens-1-binding domain of PTEN. P-REX2 inhibition of PTEN requires C-terminal phosphorylation of PTEN to release the P-REX2 PH domain from its neighboring diffuse B-cell lymphoma homology domain. Consistent with its function as a PTEN inhibitor, deletion of Prex2 in fibroblasts and mice results in increased Pten activity and decreased insulin signaling in liver and adipose tissue. Prex2 deletion also leads to reduced glucose uptake and insulin resistance. In human adipose tissue, P-REX2 protein expression is decreased and PTEN activity is increased in insulin-resistant human subjects. Taken together, these results indicate a functional role for P-REX2 PH-domain-mediated inhibition of PTEN in regulating insulin sensitivity and glucose homeostasis and suggest that loss of P-REX2 expression may cause insulin resistance.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Regulação Enzimológica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Resistência à Insulina , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Animais , Sítios de Ligação , Proteínas Sanguíneas/química , Domínio Catalítico , Proliferação de Células , Fibroblastos/metabolismo , Glucose/metabolismo , Células HEK293 , Homeostase , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/química , Fosforilação , Ligação ProteicaRESUMO
Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP-bound Rac1 then activates its downstream effectors, including p21-activated kinases (PAKs). PREX2 and Rac1 are frequently mutated in cancer and have key roles within the insulin-signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2-mediated activation of Rac1 by the second messengers PIP3 or Gßγ, we found that PREX2 was phosphorylated through a PAK-dependent mechanism. PAK-mediated phosphorylation of PREX2 reduced GEF activity toward Rac1 by inhibiting PREX2 binding to PIP3 and Gßγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Furthermore, the onset of insulin-induced phosphorylation of PREX2 was delayed compared with AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAKs phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Fosforilação/fisiologia , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: The basal-like breast cancer (BLBC) subtype is characterized by positive staining for basal mammary epithelial cytokeratin markers, lack of hormone receptor and HER2 expression, and poor prognosis with currently no approved molecularly-targeted therapies. The oncogenic signaling pathways driving basal-like tumorigenesis are not fully elucidated. METHODS: One hundred sixteen unselected breast tumors were subjected to integrated analysis of phosphoinositide 3-kinase (PI3K) pathway related molecular aberrations by immunohistochemistry, mutation analysis, and gene expression profiling. Incidence and relationships between molecular biomarkers were characterized. Findings for select biomarkers were validated in an independent series. Synergistic cell killing in vitro and in vivo tumor therapy was investigated in breast cancer cell lines and mouse xenograft models, respectively. RESULTS: Sixty-four % of cases had an oncogenic alteration to PIK3CA, PTEN, or INPP4B; when including upstream kinases HER2 and EGFR, 75 % of cases had one or more aberration including 97 % of estrogen receptor (ER)-negative tumors. PTEN-loss was significantly associated to stathmin and EGFR overexpression, positivity for the BLBC markers cytokeratin 5/14, and the BLBC molecular subtype by gene expression profiling, informing a potential therapeutic combination targeting these pathways in BLBC. Combination treatment of BLBC cell lines with the EGFR-inhibitor gefitinib plus the PI3K pathway inhibitor LY294002 was synergistic, and correspondingly, in an in vivo BLBC xenograft mouse model, gefitinib plus PI3K-inhibitor PWT-458 was more effective than either monotherapy and caused tumor regression. CONCLUSIONS: Our study emphasizes the importance of PI3K/PTEN pathway activity in ER-negative and basal-like breast cancer and supports the future clinical evaluation of combining EGFR and PI3K pathway inhibitors for the treatment of BLBC.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Redes Reguladoras de Genes , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Androstadienos/administração & dosagem , Androstadienos/farmacologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cromonas/administração & dosagem , Cromonas/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Sinergismo Farmacológico , Receptores ErbB/genética , Feminino , Gefitinibe , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Camundongos , Pessoa de Meia-Idade , Morfolinas/administração & dosagem , Morfolinas/farmacologia , PTEN Fosfo-Hidrolase/genética , Monoéster Fosfórico Hidrolases/genética , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise Serial de Tecidos/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The tumor suppressor PTEN dephosphorylates PIP3 to inhibit PI3K signaling in cells. Altering PTEN intracellular signaling can therefore significantly affect cell behavior. Two novel mechanisms of PTEN regulation including the secretion and entry of the translational variant PTEN-L, and enzymatic inhibition by the interacting protein P-REX2, have been shown to modulate PI3K signaling, cellular proliferation and survival, and glucose metabolism. Here, we review the methods used to identify and validate the existence of both PTEN-L and the P-REX2-PTEN complex, to determine their effects on PTEN phosphatase activity, and to examine their role in cellular physiology.
Assuntos
Líquido Intracelular/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Humanos , Imunoprecipitação/métodosRESUMO
Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias and lymphomas (T-ALL), making this receptor a promising target for drugs such as gamma-secretase inhibitors, which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Here we show that NOTCH1 regulates the expression of PTEN (encoding phosphatase and tensin homolog) and the activity of the phosphoinositol-3 kinase (PI3K)-AKT signaling pathway in normal and leukemic T cells. Notch signaling and the PI3K-AKT pathway synergize in vivo in a Drosophila melanogaster model of Notch-induced tumorigenesis, and mutational loss of PTEN is associated with human T-ALL resistance to pharmacological inhibition of NOTCH1. Overall, these findings identify transcriptional control of PTEN and regulation of the PI3K-AKT pathway as key elements of the leukemogenic program activated by NOTCH1 and provide the basis for the design of new therapeutic strategies for T-ALL.
Assuntos
Proteínas de Drosophila/genética , Regulação Leucêmica da Expressão Gênica/genética , Leucemia de Células T/metabolismo , PTEN Fosfo-Hidrolase/genética , Receptor Notch1/antagonistas & inibidores , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Humanos , Leucemia de Células T/genética , Camundongos , Modelos Genéticos , Mutação , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Gravidez , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais , TransgenesRESUMO
Dedifferentiation or phenotype switching refers to the transition from a proliferative to an invasive cellular state. We previously identified a 122-gene epigenetic gene signature that classifies primary melanomas as low versus high risk (denoted as Epgn1 or Epgn3). We found that the transcriptomes of the Epgn1 low-risk and Epgn3 high-risk cells are similar to the proliferative and invasive cellular states, respectively. These signatures were further validated in melanoma tumor samples. Examination of the chromatin landscape revealed differential H3K27 acetylation in the Epgn1 low-risk versus Epgn3 high-risk cell lines that corroborated with a differential super-enhancer and enhancer landscape. Melanocytic lineage genes (MITF, its targets and regulators) were associated with super-enhancers in the Epgn1 low-risk state, whereas invasiveness genes were linked with Epgn3 high-risk status. We identified the ITGA3 gene as marked by a super-enhancer element in the Epgn3 invasive cells. Silencing of ITGA3 enhanced invasiveness in both in vitro and in vivo systems, suggesting it as a negative regulator of invasion. In conclusion, we define chromatin landscape changes associated with Epgn1/Epgn3 and phenotype switching during early steps of melanoma progression that regulate transcriptional reprogramming. This super-enhancer and enhancer-driven epigenetic regulatory mechanism resulting in major changes in the transcriptome could be important in future therapeutic targeting efforts.
Assuntos
Histonas , Melanoma , Humanos , Histonas/genética , Histonas/metabolismo , Melanoma/patologia , Desdiferenciação Celular/genética , Acetilação , Linhagem Celular Tumoral , Cromatina/genéticaRESUMO
Pten-/- cells display a partially defective checkpoint in response to ionizing radiation (IR). The checkpoint defect was traced to the ability of AKT to phosphorylate CHK1 at serine 280, since a nonphosphorylated mutant of CHK1 (S280A) complemented the checkpoint defect and restored CDC25A degradation. CHK1 phosphorylation at serine 280 led to covalent binding of 1 to 2 molecules of ubiquitin and cytoplasmic CHK1 localization. Primary breast carcinomas lacking PTEN expression and having elevated AKT phosphorylation had increased cytoplasmic CHK1 and displayed aneuploidy (p <0.005). We conclude that loss of PTEN and subsequent activation of AKT impair CHK1 through phosphorylation, ubiquitination, and reduced nuclear localization to promote genomic instability in tumor cells.
Assuntos
Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Citoplasma/metabolismo , Dano ao DNA , Embrião de Mamíferos/citologia , Fase G2 , Substâncias de Crescimento/metabolismo , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Mutação , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Radiação Ionizante , Serina/química , Transdução de Sinais , Células-Tronco/citologia , Fatores de Tempo , Ubiquitina/metabolismoRESUMO
Super-enhancers are large, densely concentrated swaths of enhancers that regulate genes critical for cell identity. Tumorigenesis is accompanied by changes in the super-enhancer landscape. These aberrant super-enhancers commonly form to activate proto-oncogenes, or other genes upon which cancer cells depend, that initiate tumorigenesis, promote tumor proliferation, and increase the fitness of cancer cells to survive in the tumor microenvironment. These include well-recognized master regulators of proliferation in the setting of cancer, such as the transcription factor MYC which is under the control of numerous super-enhancers gained in cancer compared to normal tissues. This Review will cover the expanding cell-intrinsic and cell-extrinsic etiology of these super-enhancer changes in cancer, including somatic mutations, copy number variation, fusion events, extrachromosomal DNA, and 3D chromatin architecture, as well as those activated by inflammation, extra-cellular signaling, and the tumor microenvironment.
Assuntos
Variações do Número de Cópias de DNA , Neoplasias , Humanos , Elementos Facilitadores Genéticos , Neoplasias/genética , Fatores de Transcrição/genética , Carcinogênese/genética , Microambiente TumoralRESUMO
Sustained chronic inflammation of the large intestine leads to tissue damage and repair, which is associated with an increased incidence of colitis-associated colorectal cancer (CAC). The genetic makeup of CAC is somewhat similar to sporadic colorectal carcinoma (sCRC), but there are differences in the sequence and timing of alterations in the carcinogenesis process. Several models have been developed to explain the development of CAC, particularly the "field cancerization" model, which proposes that chronic inflammation accelerates mutagenesis and selects for the clonal expansion of phenotypically normal, pro-tumorigenic cells. In contrast, the "Big Bang" model posits that tumorigenic clones with multiple driver gene mutations emerge spontaneously. The details of CAC tumorigenesis-and how they differ from sCRC-are not yet fully understood. In this Review, we discuss recent genetic, epigenetic, and environmental findings related to CAC pathogenesis in the past five years, with a focus on unbiased, high-resolution genetic profiling of non-dysplastic field cancerization in the context of inflammatory bowel disease (IBD).
RESUMO
Thymus and spleen, in contrast to liver, are radiosensitive tissues in which p53-dependent apoptosis is triggered after whole-body radiation in vivo. Combined RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analyses of radiation-treated mouse organs identifies both shared and tissue-specific p53 transcriptional responses. As expected, the p53 targets shared among thymus and spleen are enriched in apoptotic targets. The inability to upregulate these genes in the liver is not due to reduced gene occupancy. Use of an engineered mouse model shows that deletion of the C terminus of p53 can confer radiation-induced expression of p53 apoptotic targets in the liver with concomitant increased cell death. Global RNA-seq analysis reveals that an additional role of the C terminus is also needed for transcriptional activation of liver-specific p53 targets. It is hypothesized that both suppression of apoptotic gene expression combined with enhanced activation of liver-specific targets confers tissue-specific radio-resistance.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Proteína Supressora de Tumor p53 , Animais , Camundongos , Proteína Supressora de Tumor p53/metabolismo , RNA-Seq , Ativação Transcricional , Tolerância a RadiaçãoRESUMO
Here, we show that the tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) sensitizes cells to ferroptosis, an iron-dependent form of cell death, by restraining the expression and activity of the cystine/glutamate antiporter system Xc- (xCT). Loss of PTEN activates AKT kinase to inhibit GSK3ß, increasing NF-E2 p45-related factor 2 (NRF2) along with transcription of one of its known target genes encoding xCT. Elevated xCT in Pten-null mouse embryonic fibroblasts increases the flux of cystine transport and synthesis of glutathione, which enhances the steady-state levels of these metabolites. A pan-cancer analysis finds that loss of PTEN shows evidence of increased xCT, and PTEN-mutant cells are resistant to ferroptosis as a consequence of elevated xCT. These findings suggest that selection of PTEN mutation during tumor development may be due to its ability to confer resistance to ferroptosis in the setting of metabolic and oxidative stress that occurs during tumor initiation and progression.