RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies. Advanced PDAC is considered incurable. Nearly 90% of pancreatic cancers are caused by oncogenic KRAS mutations. The mechanisms of primary or acquired resistance to KRAS inhibition are currently unknown. Here, we propose that oncogenic dependency, rather than KRAS mutation per se, plays a dominant role in the immune response to cancer, including late-stage PDAC. Classifying tumor samples according to KRAS activity scores allows accurate prediction of tumor immune composition and therapy response. Dual RAS/MAPK pathway blockade combining KRAS and MEK inhibitors is more effective than the selective KRAS inhibitor alone in attenuating MAPK activation and unblocking the influx of T cells into the tumor. Lowering KRAS activity in established tumors promotes immune infiltration, but with a limited antitumor effect, whereas combining KRAS/MEK inhibition with immune checkpoint blockade achieves durable regression in preclinical models. The results are directly applicable to stratifying human PDAC based on KRAS dependency values and immune cell composition to improve therapeutic design.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Mutação , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , ImunidadeRESUMO
A dichotomy exists regarding the role of signal transducer and activator of transcription 3 (STAT3) in cancer. Functional and genetic studies demonstrate either an intrinsic requirement for STAT3 or a suppressive effect on common types of cancer. These contrasting actions of STAT3 imply context dependency. To examine mechanisms that underlie STAT3 function in cancer, we evaluated the impact of STAT3 activity in KRAS-driven lung and pancreatic cancer. Our study defines a fundamental and previously unrecognized function of STAT3 in the maintenance of epithelial cell identity and differentiation. Loss of STAT3 preferentially associates with the acquisition of mesenchymal-like phenotypes and more aggressive tumor behavior. In contrast, persistent STAT3 activation through Tyr705 phosphorylation confers a differentiated epithelial morphology that impacts tumorigenic potential. Our results imply a mechanism in which quantitative differences of STAT3 Tyr705 phosphorylation, as compared with other activation modes, direct discrete outcomes in tumor progression.
Assuntos
Neoplasias Pulmonares/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator de Transcrição STAT3/metabolismo , Adenocarcinoma/genética , Animais , Carcinogênese , Diferenciação Celular , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/citologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/fisiologia , Fator de Transcrição STAT3/química , Transativadores/fisiologia , Peixe-ZebraRESUMO
Pancreatic cancer is one of the deadliest human malignancies. A striking feature of pancreatic cancer is that activating Kras mutations are found in â¼90% of cases. However, apart from a restricted population of cells expressing pancreatic and duodenal homeobox 1 (PDX1), most pancreatic cells are refractory to Kras-driven transformation. In the present study, we sought to determine which subsets of PDX1+ cells may be responsible for tumor growth. Using the Lox-Stop-Lox-KrasG12D genetic mouse model of pancreatic carcinogenesis, we isolated a population of KrasG12D-expressing PDX1+ cells with an inherent capacity to metastasize. This population of cells bears the surface phenotype of EpCAM+CD24+CD44+CD133-SCA1- and is closer in its properties to stem-like cells than to more mature cell types. We further demonstrate that the tumorigenic capacity of PDX1+ cells is limited, becoming progressively lost as the cells acquire a mature phenotype. These data are consistent with the hypothesis that the adult pancreas harbors a dormant progenitor cell population that is capable of initiating tumor growth under conditions of oncogenic stimulation. We present evidence that constitutive activation of the mitogen-activated protein kinase (MAPK/ERK) signaling and stabilization of the MYC protein are the two main driving forces behind the development of pancreatic cancer cells with stem-cell-like properties and high metastatic potential. Our results suggest that pancreatic cells bearing Kras mutation can be induced to differentiate into quasi-normal cells with suppressed tumorigenicity by selective inhibition of the MAPK/ERK/MYC signaling cascade.
Assuntos
Proteínas de Homeodomínio/metabolismo , Neoplasias Pulmonares/secundário , Sistema de Sinalização das MAP Quinases/fisiologia , Metástase Neoplásica/fisiopatologia , Neoplasias Pancreáticas/fisiopatologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Transativadores/metabolismo , Animais , Western Blotting , Citometria de Fluxo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Mutação de Sentido Incorreto/genética , Neoplasias Pancreáticas/metabolismo , Reação em Cadeia da Polimerase , Proteína Supressora de Tumor p53/genéticaRESUMO
Genetically or epigenetically defined reprogramming is a hallmark of cancer cells. However, a causal association between genome reprogramming and cancer has not yet been conclusively established. In particular, little is known about the mechanisms that underlie metastasis of cancer, and even less is known about the identity of metastasizing cancer cells. In this study, we used a model of conditional expression of oncogenic KrasG12D allele in primary mouse cells to show that reprogramming and dedifferentiation is a fundamental early step in malignant transformation and cancer initiation. Our data indicate that stable expression of activated KrasG12D confers on cells a large degree of phenotypic plasticity that predisposes them to neoplastic transformation and acquisition of stem cell characteristics. We have developed a genetically tractable model system to investigate the origins and evolution of metastatic pancreatic cancer cells. We show that metastatic conversion of KrasG12D-expressing cells that exhibit different degrees of differentiation and malignancy can be reconstructed in cell culture, and that the proto-oncogene c-Myc controls the generation of self-renewing metastatic cancer cells. Collectively, our results support a model wherein non-stem cancer cells have the potential to dedifferentiate and acquire stem cell properties as a direct consequence of oncogene-induced plasticity. Moreover, the disturbance in the normally existing dynamic equilibrium between cancer stem cells and non-stem cancer cells allows the formation of cancer stem cells with high metastatic capacity at any time during cancer progression.
Assuntos
Transdiferenciação Celular/genética , Transformação Celular Neoplásica/genética , Genes myc , Genes ras , Animais , Transformação Celular Neoplásica/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Genes p53 , Camundongos , Camundongos Knockout , Modelos Genéticos , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Mutação PuntualRESUMO
Oncogenic mutations in KRAS are among the most common in cancer. Classical models suggest that loss of epithelial characteristics and the acquisition of mesenchymal traits are associated with cancer aggressiveness and therapy resistance. However, the mechanistic link between these phenotypes and mutant KRAS biology remains to be established. Here, we identify STAT3 as a genetic modifier of TGF-beta-induced epithelial to mesenchymal transition. Gene expression profiling of pancreatic cancer cells identifies more than 200 genes commonly regulated by STAT3 and oncogenic KRAS. Functional classification of the STAT3-responsive program reveals its major role in tumor maintenance and epithelial homeostasis. The signatures of STAT3-activated cell states can be projected onto human KRAS mutant tumors, suggesting that they faithfully reflect characteristics of human disease. These observations have implications for therapeutic intervention and tumor aggressiveness.
Assuntos
Neoplasias Pancreáticas , Fator de Crescimento Transformador beta , Humanos , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Pâncreas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Oncogenic mutations in KRAS are among the most common in cancer. Classical models suggest that loss of epithelial characteristics and the acquisition of mesenchymal traits are associated with cancer aggressiveness and therapy resistance. However, the mechanistic link between these phenotypes and mutant KRAS biology remains to be established. Here we identify STAT3 as a genetic modifier of TGF-beta-induced epithelial to mesenchymal transition. Gene expression profiling of pancreatic cancer cells identifies more than 200 genes commonly regulated by STAT3 and oncogenic KRAS. Functional classification of STAT3 responsive program reveals its major role in tumor maintenance and epithelial homeostasis. The signatures of STAT3-activated cell states can be projected onto human KRAS mutant tumors, suggesting that they faithfully reflect characteristics of human disease. These observations have implications for therapeutic intervention and tumor aggressiveness.
RESUMO
Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the suppression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment.
Assuntos
Evasão da Resposta Imune/fisiologia , Modelos Genéticos , Neoplasias Pancreáticas/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Edição de Genes , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Transcriptoma , Neoplasias PancreáticasRESUMO
The inflammatory cytokine IL-6 is known to play a causal role in the promotion of cancer, although the underlying mechanisms remain to be completely understood. Interplay between endogenous and environmental cues determines the fate of cancer development. The Eµ-myc transgenic mouse expresses elevated levels of c-Myc in the B cell lineage and develops B cell lymphomas with associated mutations in p53 or other genes linked to apoptosis. We generated Eµ-myc mice that either lacked the IL-6 gene, or lacked the STAT3 gene specifically in B cells to determine the role of the IL-6/JAK/STAT3 pathway in tumor development. Using the Eµ-myc lymphoma mouse model, we demonstrate that IL-6 is a critical tumor promoter during early stages of B cell lymphomagenesis. IL-6 is shown to inhibit the expression of tumor suppressors, notably BIM and PTEN, and this may contribute to advancing MYC-driven B cell tumorigenesis. Several miRNAs known to target BIM and PTEN are upregulated by IL-6 and likely lead to the stable suppression of pro-apoptotic pathways early during the tumorigenic process. STAT3, a classical downstream effector of IL-6, appears dispensable for Eµ-myc driven lymphomagenesis. We conclude that the growth-promoting and anti-apoptotic mechanisms activated by IL-6 are critically involved in Eµ-myc driven tumor initiation and progression, but the B cell intrinsic expression of STAT3 is not required.
Assuntos
Interleucina-6/metabolismo , Linfoma de Células B/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose/genética , Linfócitos B/metabolismo , Morte Celular/genética , Genes myc , Interleucina-6/imunologia , Janus Quinases/metabolismo , Linfoma/patologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/fisiologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
The presence of tumor-infiltrating T cells is associated with favorable patient outcomes, yet most pancreatic cancers are immunologically silent and resistant to currently available immunotherapies. Here we show using a syngeneic orthotopic implantation model of pancreatic cancer that Pik3ca regulates tumor immunogenicity. Genetic silencing of Pik3ca in KrasG12D/Trp53R172H-driven pancreatic tumors resulted in infiltration of T cells, complete tumor regression, and 100% survival of immunocompetent host mice. By contrast, Pik3ca-null tumors implanted in T cell-deficient mice progressed and killed all of the animals. Adoptive transfer of tumor antigen-experienced T cells eliminated Pik3ca-null tumors in immunodeficient mice. Loss of PIK3CA or inhibition of its effector, AKT, increased the expression of MHC Class I and CD80 on tumor cells. These changes contributed to the increased susceptibility of Pik3ca-null tumors to T cell surveillance. Our results indicate that tumor cell PIK3CA-AKT signaling limits T cell recognition and clearance of pancreatic cancer cells. Strategies that target this pathway may yield an effective immunotherapy for this cancer.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Pancreáticas/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Knockout , Camundongos SCID , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/genética , Linfócitos T/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
TP73, despite significant homology to TP53, is not a classic tumor suppressor gene, since it exhibits upregulation of nonmutated products in human tumors and lacks a tumor phenotype in p73-deficient mice. We recently reported that an N-terminally truncated isoform, DeltaNp73, is upregulated in breast and gynecological cancers. We further showed that DeltaNp73 is a potent transdominant inhibitor of wild-type p53 and TAp73 in cultured human tumor cells by efficiently counteracting their target gene transactivations, apoptosis, and growth suppression functions (A. I. Zaika et al., J. Exp. Med. 6:765-780, 2002). Although these data strongly suggest oncogenic properties of DeltaNp73, this can only be directly shown in primary cells. We report here that DeltaNp73 confers resistance to spontaneous replicative senescence of primary mouse embryo fibroblasts (MEFs) and immortalizes MEFs at a 1,000-fold-higher frequency than occurs spontaneously. DeltaNp73 cooperates with cMyc and E1A in promoting primary cell proliferation and colony formation and compromises p53-dependent MEF apoptosis. Importantly, DeltaNp73 rescues Ras-induced senescence. Moreover, DeltaNp73 cooperates with oncogenic Ras in transforming primary fibroblasts in vitro and in inducing MEF-derived fibrosarcomas in vivo in nude mice. Wild-type p53 is likely a major target of DeltaNp73 inhibition in primary fibroblasts since deletion of p53 or its requisite upstream activator ARF abrogates the growth-promoting effect of DeltaNp73. Taken together, DeltaNp73 behaves as an oncogene that targets p53 that might explain why DeltaNp73 upregulation may be selected for during tumorigenesis of human cancers.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas ras/fisiologia , Animais , Apoptose , Divisão Celular , Transformação Celular Neoplásica , Células Cultivadas , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/química , Fibroblastos/metabolismo , Genes Supressores de Tumor , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Cinética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias/metabolismo , Proteínas Nucleares/química , Fenótipo , Testes de Precipitina , Isoformas de Proteínas , Estrutura Terciária de Proteína , Retroviridae/genética , Fatores de Tempo , Ativação Transcricional , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor , Regulação para Cima , Proteínas ras/metabolismoRESUMO
p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. We recently showed that wild-type p53 rapidly translocates to mitochondria in response to multiple death stimuli in cultured cells. Mitochondrial p53 physically interacts with antiapoptotic Bcl proteins, induces Bak oligomerization, permeabilizes mitochondrial membranes, and rapidly induces cytochrome c release. Here we characterize the mitochondrial p53 response in vivo. Mice were subjected to gamma irradiation or intravenous etoposide administration, followed by cell fractionation and immunofluorescence studies of various organs. Mitochondrial p53 accumulation occurred in radiosensitive organs like thymus, spleen, testis, and brain but not in liver and kidney. Of note, mitochondrial p53 translocation was rapid (detectable at 30 min in thymus and spleen) and triggered an early wave of marked caspase 3 activation and apoptosis. This caspase 3-mediated apoptosis was entirely p53 dependent, as shown by p53 null mice, and preceded p53 target gene activation. The transcriptional p53 program had a longer lag phase than the rapid mitochondrial p53 program. In thymus, the earliest apoptotic target gene products PUMA, Noxa, and Bax appeared at 2, 4, and 8 h, respectively, while Bid, Killer/DR5, and p53DinP1 remained uninduced even after 20 h. Target gene induction then led to further increase in active caspase 3. Similar biphasic kinetics was seen in cultured human cells. Our results suggest that in sensitive organs mitochondrial p53 accumulation in vivo occurs soon after a death stimulus, triggering a rapid first wave of apoptosis that is transcription independent and may precede a second slower wave that is transcription dependent.
Assuntos
Mitocôndrias/patologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Western Blotting , Caspase 3 , Caspases/metabolismo , Morte Celular , Linhagem Celular , Células Cultivadas , Dano ao DNA , Vetores Genéticos , Homozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/metabolismo , Fatores de TempoRESUMO
A cDNA library enriched with Myc-responsive cDNAs but depleted of myc cDNAs was used in a functional screen for growth enhancement in c-myc-null cells. A cDNA clone for mitochondrial serine hydroxymethyltransferase (mSHMT) that was capable of partial complementation of the growth defects of c-myc-null cells was identified. Expression analysis and chromatin immunoprecipitation demonstrated that mSHMT is a direct Myc target gene. Furthermore, a separate gene encoding the cytoplasmic isoform of the same enzyme is also a direct target of Myc regulation. SHMT enzymes are the major source of the one-carbon unit required for folate metabolism and for the biosynthesis of nucleotides and amino acids. Our data establish a novel functional link between Myc and the regulation of cellular metabolism.
Assuntos
Carbono/metabolismo , Fenômenos Fisiológicos Celulares , Glicina Hidroximetiltransferase/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Separação Celular , Células Cultivadas , Fibroblastos/fisiologia , Citometria de Fluxo , Biblioteca Gênica , Genes myc , Glicina Hidroximetiltransferase/genética , Humanos , Mitocôndrias/enzimologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Ratos MutantesRESUMO
Complex proapoptotic functions are essential for the tumor suppressor activity of p53. We recently described a novel transcription-independent mechanism that involves a rapid proapoptotic action of p53 at the mitochondria and executes the shortest known circuitry of p53 death signaling. Here, we examine if this p53-dependent mitochondrial program could be exploited for tumor suppression in vivo. To test this, we engage Emu-Myc transgenic mice, a well-established model of p53-dependent lymphomagenesis. We show that exclusive delivery of p53 to the outer mitochondrial membrane confers a significant growth disadvantage on Emu-Myc-transformed B-cells of p53-deficient or alternate reading frame-deficient genotypes, resulting in efficient induction of apoptosis and impinged proliferation. Conversely, normal cells from thymus, spleen, and bone marrow showed poor infectivity with these viruses. This proof-of-principle experiment shows that exclusive reliance on the direct mitochondrial program exerts a significant tumor suppressor activity in vivo. Our in vivo data on the direct mitochondrial apoptotic p53 program lays the groundwork to further investigate its efficacy and safety and to address its possible therapeutic value in the future.
Assuntos
Mitocôndrias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Núcleo Celular/metabolismo , Membranas Intracelulares/metabolismo , Linfoma/genética , Linfoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Células NIH 3T3 , Retroviridae/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
KRAS is the most commonly mutated oncogene, frequently associated with some of the deadliest forms of cancer. However, the need for potent and specific KRAS inhibitors remains unmet. Here, we evaluated the effects of selected cytotoxic agents on oncogenic KRAS signaling and drug response. The data provided new insights into the functional interaction between the KRAS and MYC pathways and revealed key differences between WT and mutant KRAS expressing cells. Systematic investigation of non-small cell lung cancer cell lines revealed that KRAS mutation can paradoxically increase the sensitivity of cells to cytotoxic agents. We identify MYC as a key regulator of the cellular stress responses and tumor cell viability as MYC expression was suppressed in drug-sensitive but not resistant cells. Furthermore, this suppression was driven by hyperactive KRAS/MAPK signaling. Our findings support a direct link between MYC and cancer cell viability, and raise the possibility that inactivation of MYC may be an effective therapeutic strategy for KRAS mutant tumors across various cancer types.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Antineoplásicos/farmacologia , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Citotoxinas/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pulmonares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Transformation of fibroblasts by V-SEA involves activation of the ERK and phosphatidylinositol 3-kinase (PI3K) pathways. Effector proteins that are key mediators of the ERK and PI3K pathways, namely Grb2, the tyrosine phosphatase, SHP2 and PI3K, interact with the two phosphotyrosines found in the bidentate motif in the carboxy-terminal region of V-SEA. Genetic analysis demonstrated that while Y557 was a primary binding site and thus activator of the PI3K-Akt pathway, Y564 also contributed to the activation of this pathway. Y564 was located within a Grb2-binding motif, this raised the possibility that a protein that associated with Grb2 might be important for this PI3K activation. The scaffolding proteins Gab1 and/or Gab2 were candidates for this role. In this report, we demonstrate that V-SEA preferentially interacts with Gab2. Furthermore by using Gab2 null fibroblasts, we demonstrate that Gab2 is essential for fibroblast transformation by V-SEA. Using mutant forms of Gab2, we show that activation of the PI3K-Akt pathway via Gab2 is required for V-SEA-induced transformation. However, efficient fibroblast transformation also requires the SHP2 interaction site on Gab2.
Assuntos
Transformação Celular Neoplásica/metabolismo , Fibroblastos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Camundongos , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
In p53-dependent apoptosis in response to genotoxic and hypoxic stress, a fraction of induced wild-type p53 rapidly translocates to mitochondria, triggering a rapid first wave of mitochondrial membrane permeabilization and apoptosis that is later fortified by the transcriptional program of p53. However, whether this direct mitochondrial program also occurs upon oncogenic stress is unknown. In normal cells, oncogenic signals can induce a p53-dependent fail-safe mechanism to counter uncontrolled proliferation by engaging p53-dependent apoptosis. To address whether mitochondrial p53 contributes to oncogene-induced fail-safe apoptosis, p53 translocation was determined in primary human epithelial and endothelial cells overexpressing c-Myc, E1A or E2F1. Serum starvation of these cells, but not of control cells, triggered rapid p53 accumulation at mitochondria, accompanied by cytochrome c and SMAC release and followed by apoptosis. Our data establishes the contribution of the transcription-independent mitochondrial p53 pathway to apoptosis of primary cells in response to deregulated oncogenes.
Assuntos
Proteínas Precoces de Adenovirus/metabolismo , Apoptose , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/metabolismo , Proteínas Precoces de Adenovirus/genética , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Humanos , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Activation of the p53 protein protects the organism against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. MDM2, a p53-specific E3 ubiquitin ligase, is the principal cellular antagonist of p53, acting to limit the p53 growth-suppressive function in unstressed cells. In unstressed cells, MDM2 constantly monoubiquitinates p53 and thus is the critical step in mediating its degradation by nuclear and cytoplasmic proteasomes. The interaction between p53 and MDM2 is conformation-based and is tightly regulated on multiple levels. Disruption of the p53-MDM2 complex by multiple routes is the pivotal event for p53 activation, leading to p53 induction and its biological response. Because the p53-MDM2 interaction is structurally and biologically well understood, the design of small lipophilic molecules that disrupt or prevent it has become an important target for cancer therapy.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação , Cisteína Endopeptidases/metabolismo , Humanos , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p14ARF/metabolismo , Ubiquitina/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive, metastatic disease with limited treatment options. Factors contributing to the metastatic predisposition and therapy resistance in pancreatic cancer are not well understood. Here, we used a mouse model of KRAS-driven pancreatic carcinogenesis to define distinct subtypes of PDAC metastasis: epithelial, mesenchymal and quasi-mesenchymal. We examined pro-survival signals in these cells and the therapeutic response differences between them. Our data indicate that the initiation and maintenance of the transformed state are separable, and that KRAS dependency is not a fundamental constant of KRAS-initiated tumors. Moreover, some cancer cells can shuttle between the KRAS dependent (drug-sensitive) and independent (drug-tolerant) states and thus escape extinction. We further demonstrate that inhibition of KRAS signaling alone via co-targeting the MAPK and PI3K pathways fails to induce extensive tumor cell death and, therefore, has limited efficacy against PDAC. However, the addition of histone deacetylase (HDAC) inhibitors greatly improves outcomes, reduces the self-renewal of cancer cells, and blocks cancer metastasis in vivo. Our results suggest that targeting HDACs in combination with KRAS or its effector pathways provides an effective strategy for the treatment of PDAC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Animais , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Genes ras , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Mutação , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Inibidores de Proteínas Quinases/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mice engineered to express c-Myc in B cells (Emu-myc mice) develop lethal lymphomas in which the gene encoding the p53 tumor suppressor is frequently mutated. Whether the p53 homolog p73 also functions as a tumor suppressor in vivo remains controversial. Here we have shown that p73 loss does not substantially affect disease onset and mortality in Emu-myc mice. However, it does alter the phenotype of the disease. Specifically, p73 loss decreased nodal disease and increased widespread extranodal dissemination. We further found that p53 acted as the dominant tumor suppressor during the onset of Emu-myc-driven B cell lymphomagenesis, while p73 modulated tumor dissemination and extranodal growth. Immunophenotyping and expression profiling suggested that p73 loss allowed increased maturation of malignant B cells and deregulated genes involved in lymphocyte homing and dissemination of human lymphomas. Consistent with this, p73 expression was frequently downregulated in a large cohort of human mature aggressive B cell lymphomas, and both the incidence and degree of p73 downregulation in these tumors correlated with their extranodal dissemination status. These data indicate that p73 is a modifier of Myc-driven lymphomas in mice, favoring tumor dissemination, and suggest that p73 could be a biomarker for human B cell lymphoma dissemination, a notion that can now be tested in clinicopathologic correlation studies.
Assuntos
Linfócitos B/patologia , Genes Supressores de Tumor , Linfoma de Células B/patologia , Linfoma/patologia , Proteína Supressora de Tumor p53/genética , Animais , Linfócitos B/metabolismo , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteína Supressora de Tumor p53/metabolismoRESUMO
Transcriptional silencing of the p73 gene through methylation has been demonstrated in human leukemias and lymphomas. However, the role of p73 in the malignant process remains to be explored. We show here that p73 acts as a T cell-specific tumor suppressor in a genetically defined mouse model, and that concomitant ablation of p53 and p73 predisposes mice to an increased incidence of thymic lymphomas compared to the loss of p53 alone. Our results demonstrate a causal role for loss of p73 in progression of T cell lymphomas to the stage of aggressive, disseminated disease. We provide evidence that tumorigenesis in mice lacking p53 and p73 proceeds through mechanisms involving altered patterns of gene expression, defects in early T cell development, impaired apoptosis, and the ensuing accumulation of chromosomal aberrations. Collectively, our data imply that tumor suppressive properties of p73 are highly dependent on cellular context, wherein p73 plays a major role in T cell development and neoplasia.