RESUMO
The marsupial Tasmanian devil (Sarcophilus harrisii) faces extinction due to transmissible devil facial tumor disease (DFTD). To unveil the molecular underpinnings of this transmissible cancer, we combined pharmacological screens with an integrated systems-biology characterization. Sensitivity to inhibitors of ERBB tyrosine kinases correlated with their overexpression. Proteomic and DNA methylation analyses revealed tumor-specific signatures linked to the evolutionary conserved oncogenic STAT3. ERBB inhibition blocked phosphorylation of STAT3 and arrested cancer cells. Pharmacological blockade of ERBB or STAT3 prevented tumor growth in xenograft models and restored MHC class I expression. This link between the hyperactive ERBB-STAT3 axis and major histocompatibility complex class I-mediated tumor immunosurveillance provides mechanistic insights into horizontal transmissibility and puts forward a dual chemo-immunotherapeutic strategy to save Tasmanian devils from DFTD. VIDEO ABSTRACT.
Assuntos
Receptores ErbB/metabolismo , Neoplasias Faciais/tratamento farmacológico , Neoplasias Faciais/veterinária , Proteômica/métodos , Fator de Transcrição STAT3/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Metilação de DNA , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Faciais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/metabolismo , Marsupiais , Camundongos , Fosforilação , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transcriptional networks control the differentiation of the hepatocyte and cholangiocyte lineages from embryonic liver progenitor cells and their subsequent maturation to the adult phenotype. However, how relative levels of hepatocyte and cholangiocyte gene expression are determined during differentiation remains poorly understood. Here, we identify microRNA (miR)-337-3p as a regulator of liver development. miR-337-3p stimulates expression of cholangiocyte genes and represses hepatocyte genes in undifferentiated progenitor cells in vitro and in embryonic mouse livers. Beyond the stage of lineage segregation, miR-337-3p controls the transcriptional network dynamics of developing hepatocytes and balances both cholangiocyte populations that constitute the ductal plate. miR-337-3p requires Notch and transforming growth factor-ß signaling and exerts a biphasic control on the hepatocyte transcription factor hepatocyte nuclear factor 4α by modulating its activation and repression. With the help of an experimentally validated mathematical model, we show that this biphasic control results from an incoherent feedforward loop between miR-337-3p and hepatocyte nuclear factor 4α. CONCLUSION: Our results identify miR-337-3p as a regulator of liver development and highlight how tight quantitative control of hepatic cell differentiation is exerted through specific gene regulatory network motifs. (Hepatology 2018;67:313-327).
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Fator 1-alfa Nuclear de Hepatócito/genética , Hepatócitos/metabolismo , MicroRNAs/genética , Animais , Western Blotting , Células Cultivadas , Camundongos , Transdução de Sinais/genética , Estatísticas não Paramétricas , Fatores de TranscriçãoRESUMO
Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This "ying-yang" effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient's breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxirredução , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pharmacologic blockade of the activation of signal transducer and activator of transcriptionâ 3 (STAT3) in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) cell lines characterized by kinase-independent resistance was shown to re-sensitize CML cells to TKI therapy, suggesting that STAT3 inhibitors in combination with TKIs are an effective combinatorial therapeutic for the treatment of CML. Benzoic acid- and hydroxamic acid-based STAT3 inhibitors SH-4-054 and SH-5-007, developed previously in our laboratory, demonstrated promising activity against these resistant CML cell lines. However, pharmacokinetic studies in murine models (CD-1 mice) revealed that both SH-4-054 and SH-5-007 are susceptible to glutathione conjugation at the para position of the pentafluorophenyl group via nucleophilic aromatic substitution (SN Ar). To determine whether the electrophilicity of the pentafluorophenyl sulfonamide could be tempered, an in-depth structure-activity relationship (SAR) study of the SH-4-054 scaffold was conducted. These studies revealed that AM-1-124, possessing a 2,3,5,6-tetrafluorophenylsulfonamide group, retained STAT3 protein affinity (Ki =15â µm), as well as selectivity over STAT1 (Ki >250â µm). Moreover, in both hepatocytes and in inâ vivo pharmacokinetic studies (CD-1 mice), AM-1-124 was found to be dramatically more stable than SH-4-054 (t1/2 =1.42â h cf. 10â min, respectively). AM-1-124 is a promising STAT3-targeting inhibitor with demonstrated bioavailability, suitable for evaluation in preclinical cancer models.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Sulfonamidas/farmacologia , para-Aminobenzoatos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Disponibilidade Biológica , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , para-Aminobenzoatos/síntese química , para-Aminobenzoatos/químicaRESUMO
Constitutively activated STAT3 protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study, PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low micromolar range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nmol/L by surface plasmon resonance, and showed no effect in a kinome screen (>100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized three-dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAF). In this coculture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D cocultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells. Mol Cancer Ther; 15(5); 794-805. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Ligantes , Masculino , Modelos Moleculares , Conformação Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Fosforilação , Ligação Proteica , Fator de Transcrição STAT3/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Domínios de Homologia de srcRESUMO
INTRODUCTION: The clinical utility of effective direct STAT inhibitors, particularly STAT3 and STAT5, for treating cancer and other diseases is well studied and known. AREAS COVERED: This review will highlight the STAT inhibitor patent literature from 2011 to 2015 inclusive. Emphasis will be placed on inhibitors of the STAT3, STAT5a/b, and STAT1 proteins for cancer treatment. The review will, where suitably investigated, describe the mode and the site of inhibition, list indications that were evaluated, and rank the inhibitor's relative potency among compounds in the same class. The reader will gain an understanding of the diverse set of approaches, used both in academia and industry, to target STAT proteins. EXPERT OPINION: There is still much work to be done to directly target the STAT3 and STAT5 proteins. As yet, there is still no direct STAT3 inhibitor in the clinic. While the SH2 domain remains a popular target for therapeutic intervention, the DNA-binding domain and N-terminal region are now attracting attention as possible sites for inhibition. Multiple putative STAT3 and STAT5 inhibitors have now been patented across a broad spectrum of chemotypes, each with their own advantages and limitations.
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Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT5/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Desenho de Fármacos , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Patentes como AssuntoRESUMO
Brain metastases (BM) represent the most common tumor to affect the adult central nervous system. Despite the increasing incidence of BM, likely due to consistently improving treatment of primary cancers, BM remain severely understudied. In this study, we utilized patient-derived stem cell lines from lung-to-brain metastases to examine the regulatory role of STAT3 in brain metastasis initiating cells (BMICs). Annotation of our previously described BMIC regulatory genes with protein-protein interaction network mapping identified STAT3 as a novel protein interactor. STAT3 knockdown showed a reduction in BMIC self-renewal and migration, and decreased tumor size in vivo. Screening of BMIC lines with a library of STAT3 inhibitors identified one inhibitor to significantly reduce tumor formation. Meta-analysis identified the oncomir microRNA-21 (miR-21) as a target of STAT3 activity. Inhibition of miR-21 displayed similar reductions in BMIC self-renewal and migration as STAT3 knockdown. Knockdown of STAT3 also reduced expression of known downstream targets of miR-21. Our studies have thus identified STAT3 and miR-21 as cooperative regulators of stemness, migration and tumor initiation in lung-derived BM. Therefore, STAT3 represents a potential therapeutic target in the treatment of lung-to-brain metastases.
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Neoplasias Encefálicas/secundário , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Genes Reguladores , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Mapeamento de Interação de Proteínas , Proteômica , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologiaRESUMO
Open burning for waste disposal is, in many countries, the dominant source of polychlorinated dibenzodioxins, dibenzofurans and biphenyls (PCDD/PCDF/PCB) release to the environment. To generate emission factors for open burning, experimental pile burns of about 100 kg of household waste were conducted with emissions sampling. From these experiments and others conducted by the same authors it is found that less compaction of waste or active mixing during the fire--"stirring"--promotes better combustion (as evidenced by lower CO/CO(2) ratio) and reduces emissions of PCDD/PCDF/PCB; an intuitive but previously undemonstrated result. These experiments also support previous results suggesting PCDD/PCDF/PCB generation in open burning - while still highly variable - tends to be greater in the later (smoldering) phases of burning when the CO/CO(2) ratio increases.