RESUMO
Therapeutic harnessing of adaptive immunity via checkpoint inhibition has transformed the treatment of many cancers. Despite unprecedented long-term responses, most patients do not respond to these therapies. Immunotherapy non-responders often harbor high levels of circulating myeloid-derived suppressor cells (MDSCs)-an immunosuppressive innate cell population. Through genetic and pharmacological approaches, we uncovered a pathway governing MDSC abundance in multiple cancer types. Therapeutic liver-X nuclear receptor (LXR) agonism reduced MDSC abundance in murine models and in patients treated in a first-in-human dose escalation phase 1 trial. MDSC depletion was associated with activation of cytotoxic T lymphocyte (CTL) responses in mice and patients. The LXR transcriptional target ApoE mediated these effects in mice, where LXR/ApoE activation therapy elicited robust anti-tumor responses and also enhanced T cell activation during various immune-based therapies. We implicate the LXR/ApoE axis in the regulation of innate immune suppression and as a target for enhancing the efficacy of cancer immunotherapy in patients.
Assuntos
Apolipoproteínas E/imunologia , Imunidade Inata , Receptores X do Fígado/imunologia , Células Supressoras Mieloides/imunologia , Neoplasias Experimentais/imunologia , Animais , Apolipoproteínas E/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Feminino , Receptores X do Fígado/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células Supressoras Mieloides/patologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cell cycle checkpoints are implemented to safeguard the genome, avoiding the accumulation of genetic errors. Checkpoint loss results in genomic instability and contributes to the evolution of cancer. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the role of an intact S-phase checkpoint in maintaining genome integrity. Although the basic framework of the S-phase checkpoint in multicellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome-11 band-q23 translocations disrupting the MLL gene lead to poor prognostic leukaemias. Here we assign MLL as a novel effector in the mammalian S-phase checkpoint network and identify checkpoint dysfunction as an underlying mechanism of MLL leukaemias. MLL is phosphorylated at serine 516 by ATR in response to genotoxic stress in the S phase, which disrupts its interaction with, and hence its degradation by, the SCF(Skp2) E3 ligase, leading to its accumulation. Stabilized MLL protein accumulates on chromatin, methylates histone H3 lysine 4 at late replication origins and inhibits the loading of CDC45 to delay DNA replication. Cells deficient in MLL showed radioresistant DNA synthesis and chromatid-type genomic abnormalities, indicative of S-phase checkpoint dysfunction. Reconstitution of Mll(-/-) (Mll also known as Mll1) mouse embryonic fibroblasts with wild-type but not S516A or ΔSET mutant MLL rescues the S-phase checkpoint defects. Moreover, murine myeloid progenitor cells carrying an Mll-CBP knock-in allele that mimics human t(11;16) leukaemia show a severe radioresistant DNA synthesis phenotype. MLL fusions function as dominant negative mutants that abrogate the ATR-mediated phosphorylation/stabilization of wild-type MLL on damage to DNA, and thus compromise the S-phase checkpoint. Together, our results identify MLL as a key constituent of the mammalian DNA damage response pathway and show that deregulation of the S-phase checkpoint incurred by MLL translocations probably contributes to the pathogenesis of human MLL leukaemias.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Cromatina/metabolismo , Dano ao DNA , Replicação do DNA/fisiologia , Genes Dominantes/genética , Instabilidade Genômica/fisiologia , Histona-Lisina N-Metiltransferase , Histonas/química , Histonas/metabolismo , Humanos , Leucemia/genética , Lisina/metabolismo , Metilação , Camundongos , Células Progenitoras Mieloides/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/deficiência , Proteína de Leucina Linfoide-Mieloide/genética , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Translocação Genética/genéticaRESUMO
Taspase1, a highly conserved threonine protease encoded by TASP1, cleaves nuclear histone-modifying factors and basal transcription regulators to orchestrate diverse transcription programs. Hereditary loss-of-function mutation of TASP1 has recently been reported in humans as resulting in an anomaly complex syndrome, which manifests with hematological, facial, and skeletal abnormalities. Here, we demonstrate that Taspase1-mediated cleavage of TFIIAα-ß, rather than of MLL1 or MLL2, in mouse embryos was required for proper fetal liver hematopoiesis and correct segmental identities of the axial skeleton. Homozygous genetic deletion of Taspase1 disrupted embryonic hematopoietic stem cell self-renewal and quiescence states and axial skeleton fates. Strikingly, mice carrying knockin noncleavable mutations of TFIIAα-ß, a well-characterized basal transcription factor, displayed more pronounced fetal liver and axial skeleton defects than those with noncleavable MLL1 and MLL2, 2 trithorax group histone H3 trimethyl transferases. Our study offers molecular insights into a syndrome in humans that results from loss of TASP1 and describes an unexpected role of TFIIAα-ß cleavage in embryonic cell fate decisions.
Assuntos
Anormalidades Múltiplas/genética , Endopeptidases , Desenvolvimento Fetal/fisiologia , Fator de Transcrição TFIIA/genética , Animais , Embrião de Mamíferos , Endopeptidases/genética , Endopeptidases/metabolismo , Células-Tronco Hematopoéticas , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Camundongos Knockout , Mutação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Clivagem do RNA , Transplante de Células-TroncoRESUMO
Colorectal cancer (CRC) is a leading cause of cancer mortality. Creatine metabolism was previously shown to critically regulate colon cancer progression. We report that RGX-202, an oral small-molecule SLC6A8 transporter inhibitor, robustly inhibits creatine import in vitro and in vivo, reduces intracellular phosphocreatine and ATP levels, and induces tumor apoptosis. RGX-202 suppressed CRC growth across KRAS wild-type and KRAS mutant xenograft, syngeneic, and patient-derived xenograft (PDX) tumors. Antitumor efficacy correlated with tumoral expression of creatine kinase B. Combining RGX-202 with 5-fluorouracil or the DHODH inhibitor leflunomide caused regressions of multiple colorectal xenograft and PDX tumors of distinct mutational backgrounds. RGX-202 also perturbed creatine metabolism in patients with metastatic CRC in a phase 1 trial, mirroring pharmacodynamic effects on creatine metabolism observed in mice. This is, to our knowledge, the first demonstration of preclinical and human pharmacodynamic activity for creatine metabolism targeting in oncology, thus revealing a critical therapeutic target.
Assuntos
Antineoplásicos , Neoplasias do Colo , Neoplasias Colorretais , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias Colorretais/patologia , Creatina/metabolismo , Creatina/farmacologia , Creatina/uso terapêutico , Humanos , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Nus , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
The BCL-2 family proteins are central regulators of apoptosis. However, cells deficient for BAX and BAK or overexpressing BCL-2 still succumb to oxidative stress upon DNA damage or matrix detachment. Here, we show that ΔNp63α overexpression protects cells from oxidative stress induced by oxidants, DNA damage, anoikis, or ferroptosis-inducing agents. Conversely, ΔNp63α deficiency increases oxidative stress. Mechanistically, ΔNp63α orchestrates redox homeostasis through transcriptional control of glutathione biogenesis, utilization, and regeneration. Analysis of a lung squamous cell carcinoma dataset from The Cancer Genome Atlas (TCGA) reveals that TP63 amplification/overexpression upregulates the glutathione metabolism pathway in primary human tumors. Strikingly, overexpression of ΔNp63α promotes clonogenic survival of p53-/-Bax-/-Bak-/- cells against DNA damage. Furthermore, co-expression of BCL-2 and ΔNp63α confers clonogenic survival against matrix detachment, disrupts the luminal clearance of mammary acini, and promotes cancer metastasis. Our findings highlight the need for a simultaneous blockade of apoptosis and oxidative stress to promote long-term cellular well-being.
Assuntos
Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/fisiologia , Morte Celular , Linhagem Celular , Citometria de Fluxo , Glutationa/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
PBRM1 is the second most commonly mutated gene after VHL in clear cell renal cell carcinoma (ccRCC). However, the biological consequences of PBRM1 mutations for kidney tumorigenesis are unknown. Here, we find that kidney-specific deletion of Vhl and Pbrm1, but not either gene alone, results in bilateral, multifocal, transplantable clear cell kidney cancers. PBRM1 loss amplified the transcriptional outputs of HIF1 and STAT3 incurred by Vhl deficiency. Analysis of mouse and human ccRCC revealed convergence on mTOR activation, representing the third driver event after genetic inactivation of VHL and PBRM1. Our study reports a physiological preclinical ccRCC mouse model that recapitulates somatic mutations in human ccRCC and provides mechanistic and therapeutic insights into PBRM1 mutated subtypes of human ccRCC.
Assuntos
Carcinoma de Células Renais/metabolismo , Proteínas HMGB/metabolismo , Neoplasias Renais/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Proteínas de Ligação a DNA , Regulação para Baixo/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas HMGB/deficiência , Humanos , Hidronefrose/genética , Hidronefrose/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Integrases/metabolismo , Rim/metabolismo , Rim/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Fosforilação Oxidativa , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Sexual development in Dictyostelium discoideum is initiated by the fusion of opposite mating type cells to form zygote giant cells, which subsequently gather and phagocytose surrounding cells for nutrition to form macrocysts. Here we performed the targeting of 24 highly gamete-enriched genes we previously isolated, and successfully generated knockout mutants for 16 genes and RNAi mutants for 20 genes including 6 genes without disruptants. In the knockout mutants of two genes, cell aggregation toward the giant cells was much less extensive and many cells remained around poorly formed macrocysts. We named these genes tmcB and tmcC. Although macrocyst formation of wild type cells was suppressed by the addition of exogenous cAMP, that of knockout mutants of tmcB was much less sensitive. The mRNA level of phosphodiesterase (pde) was higher and that of its inhibitor (pdi) was lower in the latter cells compared to the parental strains during sexual development. Thus, tmcB appeared to be a novel regulator of the cAMP signaling pathway specific to sexual development. Knockout mutants of tmcC were indistinguishable from the wild type cells with respect to the cAMP response, suggesting that this gene is relevant to other processes.
Assuntos
AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Técnicas Genéticas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Relação Dose-Resposta a Droga , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese , Mutação , RNA/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de TempoRESUMO
Head morphogenesis requires complex signal relays to enable precisely coordinated proliferation, migration, and patterning. Here, we demonstrate that, during mouse head formation, taspase1-mediated (TASP1-mediated) cleavage of the general transcription factor TFIIA ensures proper coordination of rapid cell proliferation and morphogenesis by maintaining limited transcription of the negative cell cycle regulators p16Ink4a and p19Arf from the Cdkn2a locus. In mice, loss of TASP1 function led to catastrophic craniofacial malformations that were associated with inadequate cell proliferation. Compound deficiency of Cdkn2a, especially p16Ink4a deficiency, markedly reduced the craniofacial anomalies of TASP1-deficent mice. Furthermore, evaluation of mice expressing noncleavable TASP1 targets revealed that TFIIA is the principal TASP1 substrate that orchestrates craniofacial morphogenesis. ChIP analyses determined that noncleaved TFIIA accumulates at the p16Ink4a and p19Arf promoters to drive transcription of these negative regulators. In summary, our study elucidates a regulatory circuit comprising proteolysis, transcription, and proliferation that is pivotal for construction of the mammalian head.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Endopeptidases/fisiologia , Fator de Transcrição TFIIA/metabolismo , Transcrição Gênica , Animais , Encéfalo/embriologia , Encéfalo/patologia , Proliferação de Células , Células Cultivadas , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Ossos Faciais/anormalidades , Ossos Faciais/embriologia , Loci Gênicos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Crânio/anormalidades , Crânio/embriologiaRESUMO
Multidomain pro-apoptotic BAX and BAK, once activated, permeabilize mitochondria to trigger apoptosis, whereas anti-apoptotic BCL-2 members preserve mitochondrial integrity. The BH3-only molecules (BH3s) promote apoptosis by either activating BAX-BAK or inactivating anti-apoptotic members. Here, we present biochemical and genetic evidence that NOXA is a bona fide activator BH3. Using combinatorial gain-of-function and loss-of-function approaches in Bid(-/-)Bim(-/-)Puma(-/-)Noxa(-/-) and Bax(-/-)Bak(-/-) cells, we have constructed an interconnected hierarchical model that accommodates and explains how the intricate interplays between the BCL-2 members dictate cellular survival versus death. BID, BIM, PUMA and NOXA directly induce stepwise, bimodal activation of BAX-BAK. BCL-2, BCL-XL and MCL-1 inhibit both modes of BAX-BAK activation by sequestering activator BH3s and 'BH3-exposed' monomers of BAX-BAK, respectively. Furthermore, autoactivation of BAX and BAK can occur independently of activator BH3s through downregulation of BCL-2, BCL-XL and MCL-1. Our studies lay a foundation for targeting the BCL-2 family for treating diseases with dysregulated apoptosis.
Assuntos
Apoptose , Fibroblastos/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína 11 Semelhante a Bcl-2 , Células Cultivadas , Citocromos c/genética , Citocromos c/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Immunoblotting , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
PURPOSE: Rapalogs are allosteric mTOR inhibitors and approved agents for advanced kidney cancer. Reports of clonal heterogeneity in this disease challenge the concept of targeted monotherapy, yet a small subset of patients derives extended benefit. Our aim was to analyze such outliers and explore the genomic background of extreme rapalog sensitivity in the context of intratumor heterogeneity. EXPERIMENTAL DESIGN: We analyzed archived tumor tissue of 5 patients with renal cell carcinoma, who previously achieved durable disease control with rapalogs (median duration, 28 months). DNA was extracted from spatially separate areas of primary tumors and metastases. Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Whole-exome sequence analysis was added to investigate genes beyond this original target list. RESULTS: Five long-term responders contributed 14 specimens to explore clonal heterogeneity. Genomic alterations with activating effect on mTOR signaling were detected in 11 of 14 specimens, offering plausible explanation for exceptional treatment response through alterations in two genes (TSC1 and MTOR). In two subjects, distinct yet functionally convergent alterations activated the mTOR pathway in spatially separate sites. In 1 patient, concurrent genomic events occurred in two separate pathway components across different tumor regions. CONCLUSIONS: Analysis of outlier cases can facilitate identification of potential biomarkers for targeted agents, and we implicate two genes as candidates for further study in this class of drugs. The previously reported phenomenon of clonal convergence can occur within a targetable pathway which might have implications for biomarker development beyond this disease and this class of agents.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Inibidores de Proteínas Quinases/administração & dosagem , Serina-Treonina Quinases TOR/biossíntese , Idoso , Carcinoma de Células Renais/patologia , Exoma , Genoma Humano , Humanos , Indóis/administração & dosagem , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Pirróis/administração & dosagem , Transdução de Sinais/genética , Sunitinibe , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/biossínteseRESUMO
The evolution of tissue-specific general transcription factors (GTFs), such as testis-specific TBP-related factor 2 (TRF2), enables the spatiotemporal expression of highly specialized genetic programs. Taspase1 is a protease that cleaves nuclear factors MLL1, MLL2, TFIIAα-ß, and ALFα-ß (TFIIAτ). Here, we demonstrate that Taspase1-mediated processing of TFIIAα-ß drives mammalian spermatogenesis. Both Taspase1(-/-) and noncleavable TFIIAα-ßnc/nc testes release immature germ cells with impaired transcription of Transition proteins (Tnp) and Protamines (Prm), exhibiting chromatin compaction defects and recapitulating those observed with TRF2(-/-) testes. Although the unprocessed TFIIA still complexes with TRF2, this complex is impaired in targeting and thus activating Tnp1 and Prm1 promoters. The current study presents a paradigm in which a protease (Taspase1) cleaves a ubiquitously expressed GTF (TFIIA) to enable tissue-specific (testis) transcription, meeting the demand for sophisticated regulation of distinct subsets of genes in higher organisms.
Assuntos
Endopeptidases/metabolismo , Espermatogênese/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Fator de Transcrição TFIIA/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Endopeptidases/genética , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/metabolismo , Regiões Promotoras Genéticas , Protaminas/genética , Espermatozoides , Proteína 2 de Ligação a Repetições Teloméricas/genéticaRESUMO
HGF signals through its cognate receptor, MET, to orchestrate diverse biological processes, including cell proliferation, cell fate specification, organogenesis, and epithelial-mesenchymal transition. Mixed-lineage leukemia (MLL), an epigenetic regulator, plays critical roles in cell fate, stem cell, and cell cycle decisions. Here, we describe a role for MLL in the HGF-MET signaling pathway. We found a shared phenotype among Mll(-/-), Hgf(-/-), and Met(-/-) mice with common cranial nerve XII (CNXII) outgrowth and myoblast migration defects. Phenotypic analysis demonstrated that MLL was required for HGF-induced invasion and metastatic growth of hepatocellular carcinoma cell lines. HGF-MET signaling resulted in the accumulation of ETS2, which interacted with MLL to transactivate MMP1 and MMP3. ChIP assays demonstrated that activation of the HGF-MET pathway resulted in increased occupancy of the MLL-ETS2 complex on MMP1 and MMP3 promoters, where MLL trimethylated histone H3 lysine 4 (H3K4), activating transcription. Our results present an epigenetic link between MLL and the HGF-MET signaling pathway, which may suggest new strategies for therapeutic intervention.
Assuntos
Carcinoma Hepatocelular/metabolismo , Fator de Crescimento de Hepatócito/fisiologia , Neoplasias Hepáticas Experimentais/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína Proto-Oncogênica c-ets-2/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Carcinoma Hepatocelular/secundário , Embrião de Mamíferos/anormalidades , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Neoplasias Hepáticas Experimentais/patologia , Masculino , Metaloproteinases da Matriz Secretadas/genética , Metaloproteinases da Matriz Secretadas/metabolismo , Metilação , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Invasividade Neoplásica , Transplante de Neoplasias , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteína Proto-Oncogênica c-ets-2/química , Transdução de Sinais , Ativação TranscricionalRESUMO
The clinical efficacy of tyrosine kinase inhibitors supports the dependence of distinct subsets of cancers on specific driver mutations for survival, a phenomenon called "oncogene addiction." We demonstrate that PUMA and BIM are the key apoptotic effectors of tyrosine kinase inhibitors in breast cancers with amplification of the gene encoding human epidermal growth factor receptor 2 (HER2) and lung cancers with epidermal growth factor receptor (EGFR) mutants. The BH3 domain containing proteins BIM and PUMA can directly activate the proapoptotic proteins BAX and BAK to permeabilize mitochondria, leading to caspase activation and apoptosis. We delineated the signal transduction pathways leading to the induction of BIM and PUMA by tyrosine kinase inhibitors. Inhibition of the mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway caused increased abundance of BIM, whereas antagonizing the phosphoinositide 3-kinase (PI3K)-AKT pathway triggered nuclear translocation of the FOXO transcription factors, which directly activated the PUMA promoter. In a mouse breast tumor model, the abundance of PUMA and BIM was increased after inactivation of HER2. Moreover, deficiency of Bim or Puma impaired caspase activation and reduced tumor regression caused by inactivation of HER2. Similarly, deficiency of Puma impeded the regression of EGFR(L858R)-driven mouse lung tumors upon inactivation of the EGFR-activating mutant. Overall, our study identified PUMA and BIM as the sentinels that interconnect kinase signaling networks and the mitochondrion-dependent apoptotic program, which offers therapeutic insights for designing novel cell death mechanism-based anticancer strategies.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/genética , Neoplasias da Mama/metabolismo , Inativação Gênica/fisiologia , Proteínas de Membrana/metabolismo , Oncogenes/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Compostos de Bifenilo/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Receptores ErbB/metabolismo , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Lapatinib , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/genética , Camundongos , Nitrofenóis/farmacologia , Oncogenes/genética , Piperazinas/farmacologia , Plasmídeos/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Quinazolinas , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Sulfonamidas/farmacologiaRESUMO
PURPOSE: To investigate the impact of newly identified chromosome 3p21 epigenetic tumor suppressors PBRM1, SETD2, and BAP1 on cancer-specific survival (CSS) of 609 patients with clear cell renal cell carcinoma (ccRCC) from 2 distinct cohorts. EXPERIMENTAL DESIGN: Select sequencing on 3p tumor suppressors of 188 patients who underwent resection of primary ccRCC at the Memorial Sloan-Kettering Cancer Center (MSKCC) was conducted to interrogate the genotype-phenotype associations. These findings were compared with analyses of the genomic and clinical dataset from our nonoverlapping The Cancer Genome Atlas (TCGA) cohort of 421 patients with primary ccRCC. RESULTS: 3p21 tumor suppressors are frequently mutated in both the MSKCC (PBRM1, 30.3%; SETD2, 7.4%; BAP1, 6.4%) and the TCGA (PBRM1, 33.5%; SETD2, 11.6%; BAP1, 9.7%) cohorts. BAP1 mutations are associated with worse CSS in both cohorts [MSKCC, P = 0.002; HR 7.71; 95% confidence interval (CI)2.08-28.6; TCGA, P = 0.002; HR 2.21; 95% CI 1.35-3.63]. SETD2 are associated with worse CSS in the TCGA cohort (P = 0.036; HR 1.68; 95% CI 1.04-2.73). On the contrary, PBRM1 mutations, the second most common gene mutations of ccRCC, have no impact on CSS. CONCLUSION: The chromosome 3p21 locus harbors 3 frequently mutated ccRCC tumor suppressor genes. BAP1 and SETD2 mutations (6%-12%) are associated with worse CSS, suggesting their roles in disease progression. PBRM1 mutations (30%-34%) do not impact CSS, implicating its principal role in the tumor initiation. Future efforts should focus on therapeutic interventions and further clinical, pathologic, and molecular interrogation of this novel class of tumor suppressors.
Assuntos
Carcinoma de Células Renais/metabolismo , Histona-Lisina N-Metiltransferase/biossíntese , Neoplasias Renais/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Ubiquitina Tiolesterase/biossíntese , Idoso , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Transformação Celular Neoplásica , Cromossomos Humanos Par 3 , Ensaios Clínicos como Assunto , Proteínas de Ligação a DNA , Intervalo Livre de Doença , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
BACKGROUND: Historically, VHL was the only frequently mutated gene in clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent sequencing efforts have identified several novel frequent mutations of histone modifying and chromatin remodeling genes in ccRCC including PBRM1, SETD2, BAP1, and KDM5C. PBRM1, SETD2, and BAP1 are located in close proximity to VHL within a commonly lost (approximately 90%) 3p locus. To date, the clinical and pathologic significance of mutations in these novel candidate tumor suppressors is unknown. OBJECTIVE: To determine the frequency of and render the first clinical and pathologic outcome associated with mutations of these novel candidate tumor suppressors in ccRCC. DESIGN, SETTING, AND PARTICIPANTS: Targeted sequencing was performed in 185 ccRCCs and matched normal tissues from a single institution. Pathologic features, baseline patient characteristics, and follow-up data were recorded. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The linkage between mutations and clinical and pathologic outcomes was interrogated with the Fisher exact test (for stage and Fuhrman nuclear grade) and the permutation log-rank test (for cancer-specific survival [CSS]). RESULTS AND LIMITATIONS: PBRM1, BAP1, SETD2, and KDM5C are mutated at 29%, 6%, 8%, and 8%, respectively. Tumors with mutations in PBRM1 or any of BAP1, SETD2, or KDM5C (19%) are more likely to present with stage III disease or higher (p = 0.01 and p = 0.001, respectively). Small tumors (<4 cm) with PBRM1 mutations are more likely to exhibit stage III pathologic features (odds ratio: 6.4; p = 0.001). BAP1 mutations tend to occur in Fuhrman grade III-IV tumors (p = 0.052) and are associated with worse CSS (p = 0.01). Clinical outcome data are limited by the number of events. CONCLUSIONS: Most mutations of chromatin modulators discovered in ccRCC are loss of function, associated with advanced stage, grade, and possibly worse CSS. Further studies validating the clinical impact of these novel mutations and future development of therapeutics remedying these tumor suppressors are warranted.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/secundário , Montagem e Desmontagem da Cromatina/genética , Genes Supressores de Tumor , Neoplasias Renais/genética , Neoplasias Renais/patologia , Mutação , Idoso , Carcinoma de Células Renais/mortalidade , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/mortalidade , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Razão de Chances , Fenótipo , Prognóstico , Medição de Risco , Fatores de Risco , Fatores de Tempo , Carga TumoralRESUMO
The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 µmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy.
Assuntos
Arsenicais/farmacologia , Neoplasias Encefálicas/prevenção & controle , Neoplasias da Mama/prevenção & controle , Endopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Endopeptidases/genética , Células HEK293 , Humanos , Cinética , Masculino , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Bibliotecas de Moléculas Pequenas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Taspase1, the mixed lineage leukemia and TFIIAalpha-beta cleaving protease, enables cell proliferation and permits oncogenic initiation. Here, we show its critical role in cancer maintenance and thus offer a new anticancer target. Taspase1 is overexpressed in primary human cancers, and deficiency of Taspase1 in cancer cells not only disrupts proliferation but also enhances apoptosis. Mechanistically, loss of Taspase1 induces the levels of CDK inhibitors (CDKI: p16, p21, and p27) and reduces the level of antiapoptotic MCL-1. Therapeutically, deficiency of Taspase1 synergizes with chemotherapeutic agents and ABT-737, an inhibitor of BCL-2/BCL-X(L), to kill cancer cells. Taspase1 alone or in conjunction with MYC, RAS, or E1A fails to transform NIH/3T3 cells or primary mouse embryonic fibroblasts, respectively, but plays critical roles in cancer initiation and maintenance. Therefore, Taspase1 is better classified as a "non-oncogene addiction" protease, the inhibition of which may offer a novel anticancer therapeutic strategy. The reliance of oncogenes on subordinate non-oncogenes during tumorigenesis underscores the non-oncogene addiction hypothesis in which a large class of non-oncogenes functions to maintain cancer phenotypes and presents attractive anticancer therapeutic targets. The emergence of successful cancer therapeutics targeting non-oncogenes to which cancers are addicted supports the future development and potential application of small-molecule Taspase1 inhibitors for cancer therapy.
Assuntos
Endopeptidases/genética , Glioblastoma/genética , Melanoma/genética , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Compostos de Bifenilo/farmacologia , Processos de Crescimento Celular/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Endopeptidases/deficiência , Endopeptidases/metabolismo , Genes myc , Genes ras , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides , Células NIH 3T3 , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Transdução Genética , TransfecçãoRESUMO
Discovered in 1992 from cloning the gene involved in human leukemias carrying chromosome band 11q23 translocations, the MLL/HRX/ALL-1 gene has since attracted scientists from various disciplines by its diverse functions in normal physiological and pathological processes. MLL is the human orthologue of Drosophila trithorax (trx)-the founding member of trithorax group proteins, Trx-G. Leukemogenic11q23 translocations fuse the common MLL N-terminal 1400aa in-frame with a wide variety of fusion partners that share no structural or functional homology. The 500 kD precursor MLL undergoes evolutionarily conserved site-specific cleavage mediated by Taspase1, generating the mature MLL(N320/C180) heterodimer which methylates histone H3 at lysine 4 with its carboxy-terminal SET domain. Extensive biochemical and genetic studies on MLL/trx have established its critical role in maintaining the expression of Hox/homeotic genes. By contrast, the involvement of MLL in many other essential cellular processes remains unclear. Recent reports including ours began to elucidate the intricate interplay between MLL and the cell cycle machinery, which ensures proper cell cycle phase transitions. Thus, this review will focus on this novel activity of MLL and discuss the implications of its deregulation in MLL leukemias.
Assuntos
Ciclo Celular/fisiologia , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Leucemia/metabolismo , Modelos Biológicos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Transdução de Sinais/fisiologia , Humanos , Proteína de Leucina Linfoide-Mieloide/biossínteseRESUMO
Morphogens are secreted signaling molecules that form concentration gradients and control cell fate in developing tissues. During development, it is essential that morphogen range is strictly regulated in order for correct cell type specification to occur. One of the best characterized morphogens is Drosophila Decapentaplegic (Dpp), a BMP signaling molecule that patterns the dorsal ectoderm of the embryo by activating the Mad and Medea (Med) transcription factors. We demonstrate that there is a spatial and temporal expansion of the expression patterns of Dpp target genes in SUMO pathway mutant embryos. We identify Med as the primary SUMOylation target in the Dpp pathway, and show that failure to SUMOylate Med leads to the increased Dpp signaling range observed in the SUMO pathway mutant embryos. Med is SUMO modified in the nucleus, and we provide evidence that SUMOylation triggers Med nuclear export. Hence, Med SUMOylation provides a mechanism by which nuclei can continue to monitor the presence of extracellular Dpp signal to activate target gene expression for an appropriate duration. Overall, our results identify an unusual strategy for regulating morphogen range that, rather than impacting on the morphogen itself, targets an intracellular transducer.