RESUMO
BACKGROUND: BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms. METHODS: CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts. RESULTS: We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings. CONCLUSIONS: Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Fator de Iniciação de Tradução Eucariótico 5A , Melanoma , Mutação , Fatores de Iniciação de Peptídeos , Poliaminas , Proteínas Proto-Oncogênicas B-raf , Proteínas de Ligação a RNA , Vemurafenib , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Poliaminas/metabolismo , Camundongos , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Vemurafenib/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Sistemas CRISPR-Cas , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Lisina/análogos & derivadosRESUMO
BACKGROUND: CRISPR-based screens are revolutionizing drug discovery as tools to identify genes whose ablation induces a phenotype of interest. For instance, CRISPR-Cas9 screening has been successfully used to identify novel therapeutic targets in cancer where disruption of genes leads to decreased viability of malignant cells. However, low-activity guide RNAs may give rise to variable changes in phenotype, preventing easy identification of hits and leading to false negative results. Therefore, correcting the effects of bias due to differences in guide RNA efficiency in CRISPR screening data can improve the efficiency of prioritizing hits for further validation. Here, we developed an approach to identify hits from negative CRISPR screens by correcting the fold changes (FC) in gRNA frequency by the actual, observed frequency of indel mutations generated by gRNA. RESULTS: Each gRNA was coupled with the "reporter sequence" that can be targeted by the same gRNA so that the frequency of mutations in the reporter sequence can be used as a proxy for the endogenous target gene. The measured gRNA activity was used to correct the FC. We identified indel generation efficiency as the dominant factor contributing significant bias to screening results, and our method significantly removed such bias and was better at identifying essential genes when compared to conventional fold change analysis. We successfully applied our gRNA activity data to previously published gRNA screening data, and identified novel genes whose ablation could synergize with vemurafenib in the A375 melanoma cell line. Our method identified nicotinamide N-methyltransferase, lactate dehydrogenase B, and polypyrimidine tract-binding protein 1 as synergistic targets whose ablation sensitized A375 cells to vemurafenib. CONCLUSIONS: We identified the variations in target cleavage efficiency, even in optimized sgRNA libraries, that pose a strong bias in phenotype and developed an analysis method that corrects phenotype score by the measured differences in the targeting efficiency among sgRNAs. Collectively, we expect that our new analysis method will more accurately identify genes that confer the phenotype of interest.
Assuntos
Sistemas CRISPR-Cas , RNA , Vemurafenib , Mutação , Linhagem CelularRESUMO
Disruption of protein-protein interaction between transcriptional enhancer factor (TEA)-domain (TEAD; a transcription factor) and its co-activator Yes-associated protein (YAP)/ transcriptional co-activator with PDZ-binding motif (TAZ) is a potential therapeutic strategy against various types of solid tumors. Based on hit compound 8 and 9a, hydrazone derivatives with dioxo-benzo[d]isothiazole (9b-n) and oxime ester (10a-s) or amide derivatives (11a-r) with dioxo-benzo[b]thiophene were designed and synthesized as novel TEAD-YAP interaction inhibitors. Amide derivative 11q exhibited a higher potency in inhibiting TEAD-YAP reporter expression activity (IC50 = 12.7 µM), endogenous target gene (e.g., CTGF and CYR61) expression, breast cancer cell growth (GI50 = 3.2 µM), and anchorage-independent growth in soft agar. Molecular docking analysis suggested that the newly synthesized compounds bound to interface 2 of TEAD had lower docking scores compared to the compounds that bind to interface 3; moreover, they were predicted to overlap with YAP. Therefore, we identified 11q as an attractive therapeutic agent for treating solid tumors overexpressing YAP/TAZ.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Simulação de Acoplamento Molecular , Fatores de Transcrição/metabolismo , AmidasRESUMO
Temporal modulation of the expression of multiple genes underlies complex complex biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. Here, we describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We use tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades that trigger the sequential accumulation of mutations. We build an all-in-one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within a gene expression cascade, and deletes itself for safety. Tandem GPCs offer a multi-tiered cellular programming tool for modeling multi-stage genetic changes, such as tumorigenesis and cellular differentiation.
Assuntos
Edição de Genes/métodos , Redes Reguladoras de Genes , Engenharia Genética/métodos , Genoma Humano , Plasmídeos/metabolismo , Transposases/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Carcinogênese/genética , Carcinogênese/metabolismo , Diferenciação Celular , Loci Gênicos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Plasmídeos/química , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Transcrição Gênica , Transposases/metabolismoRESUMO
Since its first demonstration for mammalian gene editing, CRISPR/Cas technology has been widely adopted in research, industry, and medicine. Beyond indel mutations induced by Cas9 activity, recent advances in CRISPR/Cas have enabled DNA or RNA base editing. In addition, multiple orthogonal methods for the spatiotemporal regulation of CRISPR/Cas activity and repurposed Cas proteins for the visualization and relocation of specific genomic loci in living cells have been described. By harnessing the versatility of CRISPR/Cas-based devices and gene circuits, synthetic biologists are developing memory devices for lineage tracing and technologies for unbiased, high-throughput interrogation of combinatorial gene perturbations. We envision that such approaches will enable researchers to gain deeper insights into the translation of genotypes to phenotypes in healthy and diseased states.
Assuntos
Sistemas CRISPR-Cas , Mamíferos , Biologia Sintética , Animais , DNA/genética , Redes Reguladoras de Genes , Edição de RNARESUMO
Mechanical tensions are usually generated during development at spatially defined regions within tissues. Such physical cues dictate the cellular decisions of proliferation or cell cycle arrest. Yet, the mechanisms by which mechanical stress controls the cell cycle are not yet fully understood. Here, we report that mechanical cues function upstream of Skp2 transcription in human breast cancer cells. We found that YAP, the mechano-responsive oncogenic Hippo signaling effector, directly promotes Skp2 transcription. YAP inactivation induces cell cycle exit (G0) by down-regulating Skp2, causing p21/p27 to accumulate. Both Skp2 reconstitution and p21/p27 depletion can rescue the observed defect in cell cycle progression. In the context of a tissue-mimicking 3D culture system, Skp2 inactivation effectively suppresses YAP-driven oncogenesis and aberrant stiff 3D matrix-evoked epithelial tissue behaviors. Finally, we also found that the expression of Skp2 and YAP is positively correlated in breast cancer patients. Our results not only reveal the molecular mechanism by which mechanical cues induce Skp2 transcription, but also uncover a role for YAP-Skp2 oncogenic signaling in the relationship between tissue rigidity and cancer progression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Estresse Mecânico , Ciclo Celular , Linhagem Celular Tumoral , Feminino , Humanos , Transdução de Sinais , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
YAP (Yes-associated protein) and TAZ (transcription activator with PDZ binding motif) are important in tissue regeneration and cancer development, highlighting the importance of discovering partners that regulate their oncogenicity. SGK1 (serum/glucocorticoid regulated kinase 1), initially identified as a homolog of Akt in phosphoinositide 3-kinase signaling, acts as a serine/threonine protein kinase in multiple oncogenic pathways. However, possible links between SGK1 and Hippo-YAP/TAZ signaling remain unexplored. Here, we reveal that SGK1 is a potential positive feedback regulator of YAP and TAZ, showing that the TEAD-YAP/TAZ complex directly activates SGK1 transcription by binding to the distal enhancer of SGK1, and SGK1, in turn, stabilizes YAP/TAZ. Moreover, we demonstrate that expression of YAP/TAZ target genes is positively regulated by SGK1. Mechanistically, SGK1 inhibits ubiquitin-mediated degradation of TAZ by inhibiting GSK3ß activity. These findings expand our understanding of YAP/TAZ regulation to include the novel downstream target of YAP, SGK1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular , Humanos , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
BACKGROUND & AIMS: Prostaglandin E2 (PGE2) is mediator of inflammation that regulates tissue regeneration, but its continual activation has been associated with carcinogenesis. Little is known about factors in the PGE2 signaling pathway that contribute to tumor formation. We investigated whether yes-associated protein 1 (YAP1), a transcriptional co-activator in the Hippo signaling pathway, mediates PGE2 function. METHODS: DLD-1 and SW480 colon cancer cell lines were transfected with vectors expressing transgenes or small hairpin RNAs and incubated with recombinant PGE2, with or without pharmacologic inhibitors of signaling proteins, and analyzed by immunoblot, immunofluorescence, quantitative reverse-transcription polymerase chain reaction, transcriptional reporter, and proliferation assays. Dextran sodium sulfate (DSS) was given to induce colitis in C57/BL6 (control) mice, as well as in mice with disruption of the hydroxyprostaglandin dehydrogenase 15 gene (15-PGDH-knockout mice), Yap1 gene (YAP-knockout mice), and double-knockout mice. Some mice also were given indomethacin to block PGE2 synthesis. 15-PGDH knockout mice were crossed with mice with intestine-specific disruption of the salvador family WW domain containing 1 gene (Sav1), which encodes an activator of Hippo signaling. We performed immunohistochemical analyses of colon biopsy samples from 26 patients with colitis-associated cancer and 51 age-and sex-matched patients with colorectal cancer (without colitis). RESULTS: Incubation of colon cancer cell lines with PGE2 led to phosphorylation of cyclic adenosine monophosphate-responsive element binding protein 1 and increased levels of YAP1 messenger RNA, protein, and YAP1 transcriptional activity. This led to increased transcription of the prostaglandin-endoperoxide synthase 2 gene (PTGS2 or cyclooxygenase 2) and prostaglandin E-receptor 4 gene (PTGER4 or EP4). Incubation with PGE2 promoted proliferation of colon cancer cell lines, but not cells with knockdown of YAP1. Control mice developed colitis after administration of DSS, but injection of PGE2 led to colon regeneration in these mice. However, YAP-knockout mice did not regenerate colon tissues and died soon after administration of DSS. 15-PGDH-knockout mice regenerated colon tissues more rapidly than control mice after withdrawal of DSS, and had faster recovery of body weight, colon length, and colitis histology scores. These effects were reversed by injection of indomethacin. SAV1-knockout or 15-PGDH-knockout mice did not develop spontaneous tumors after colitis induction, but SAV1/15-PGDH double-knockout mice developed polyps that eventually progressed to carcinoma in situ. Administration of indomethacin to these mice prevented spontaneous tumor formation. Levels of PGE2 correlated with those of YAP levels in human sporadic colorectal tumors and colitis-associated tumors. CONCLUSIONS: PGE2 signaling increases the expression and transcriptional activities of YAP1, leading to increased expression of cyclooxygenase 2 and EP4 to activate a positive signaling loop. This pathway promotes proliferation of colon cancer cell lines and colon tissue regeneration in mice with colitis. Constitutive activation of this pathway led to formation of polyps and colon tumors in mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colo/efeitos dos fármacos , Neoplasias Colorretais/genética , Dinoprostona/farmacologia , Fosfoproteínas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Colite/induzido quimicamente , Colo/metabolismo , Ciclo-Oxigenase 2 , Sulfato de Dextrana/toxicidade , Retroalimentação , Imunofluorescência , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Immunoblotting , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP4 , Regeneração/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF) play similar roles and exhibit significant crosstalk in directing transcriptional responses to chemical and physical extracellular cues. The mechanism underlying this crosstalk, however, remains unclear. Here, we show MRTF family proteins bind YAP via a conserved PPXY motif that interacts with the YAP WW domain. This interaction allows MRTF to recruit NcoA3 to the TEAD-YAP transcriptional complex and potentiate its transcriptional activity. We show this interaction of MRTF and YAP is critical for LPA-induced cancer cell invasion in vitro and breast cancer metastasis to the lung in vivo We also demonstrate the significance of MRTF-YAP binding in regulation of YAP activity upon acute actin cytoskeletal damage. Acute actin disruption induces nucleo-cytoplasmic shuttling of MRTF, and this process underlies the LATS-independent regulation of YAP activity. Our results provide clear evidence of crosstalk between MRTF and YAP independent of the LATS kinases that normally act upstream of YAP signaling. Our results also suggest a mechanism by which extracellular stimuli can coordinate physiological events downstream of YAP.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Coativador 3 de Receptor Nuclear/metabolismo , Ligação Proteica , Multimerização Proteica , Fatores de Transcrição de Domínio TEA , Proteínas de Sinalização YAPRESUMO
Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance.
Assuntos
Adenocarcinoma/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/genética , Receptores ErbB/genética , Feminino , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Ligação Proteica , Proteólise , Transplante Heterólogo , Carga Tumoral/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The Hippo pathway regulates the self-renewal and differentiation of various adult stem cells, but its role in cell fate determination and differentiation during liver development remains unclear. Here we report that the Hippo pathway controls liver cell lineage specification and proliferation separately from Notch signalling, using mice and primary hepatoblasts with liver-specific knockout of Lats1 and Lats2 kinase, the direct upstream regulators of YAP and TAZ. During and after liver development, the activation of YAP/TAZ induced by loss of Lats1/2 forces hepatoblasts or hepatocytes to commit to the biliary epithelial cell (BEC) lineage. It increases BEC and fibroblast proliferation by up-regulating TGFß signalling, but suppresses hepatoblast to hepatocyte differentiation by repressing Hnf4α expression. Notably, oncogenic YAP/TAZ activation in hepatocytes induces massive p53-dependent cell senescence/death. Together, our results reveal that YAP/TAZ activity levels govern liver cell differentiation and proliferation in a context-dependent manner.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fígado/embriologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Aciltransferases , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular , Diferenciação Celular , Proliferação de Células , Senescência Celular , Feminino , Fator 4 Nuclear de Hepatócito/metabolismo , Camundongos , Camundongos Knockout , Gravidez , Cultura Primária de Células , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Proteínas de Sinalização YAPAssuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Aciltransferases , Células-Tronco Adultas/citologia , Carcinogênese/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Interleucina-6/biossíntese , Proteínas de Sinalização YAPRESUMO
The efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is significantly limited by various resistance mechanisms to those drugs. The resistance to EGFR-TKI is largely divided by two classes; acquired resistance after EGFR-TKI treatment, and primary resistance marked by cancer cell's dependence on other oncogene, such as KRAS. YAP has emerged as critical oncogene in conferring drug resistance against targeted therapy. In this study, we evaluated the role of YAP in primary and acquired EGFR-TKI resistance using gefitinib-resistant A549 and PC9 cells and their parental cell lines. Our study revealed that EGFR-TKI resistance is associated with enhanced YAP activity. Notably, YAP activation was independent of the Hippo pathway. We confirmed that AXL is a downstream target of YAP that confers EGFR-TKI resistance. And our results showed that YAP can induce ERK activation in lung adenocarcinoma. The combination of YAP inhibition with EGFR-TKI overcomes primary and acquired EGFR-TKI resistance. We also found increased YAP expression in human lung cancer after acquiring EGFR-TKI resistance. Collectively, we suggest a novel EGFR-TKI resistance mechanism involving YAP activation and suggest targeting YAP and EGFR simultaneously may be a breakthrough treatment of primary and acquired EGFR-TKI resistant lung cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento Epidérmico/administração & dosagem , Via de Sinalização Hippo , Humanos , Fosfoproteínas/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição , Resultado do Tratamento , Proteínas de Sinalização YAPRESUMO
The Hippo pathway represses YAP oncoprotein activity through phosphorylation by LATS kinases. Although variety of upstream components has been found to participate in the Hippo pathway, the existence and function of negative feedback has remained uncertain. We found that activated YAP, together with TEAD transcription factors, directly induces transcription of LATS2, but not LATS1, to form a negative feedback loop. We also observed increased mRNA levels of Hippo upstream components upon YAP activation. To reveal the physiological role of this negative feedback regulation, we deleted Lats2 or Lats1 in the liver-specific Sav1-knockout mouse model which develops a YAP-induced tumor. Additional deletion of Lats2 severely enhanced YAP-induced tumorigenic phenotypes in a liver specific Sav1 knock-out mouse model while additional deletion of Lats1 mildly affected the phenotype. Only Sav1 and Lats2 double knock-down cells formed larger colonies in soft agar assay, thereby recapitulating accelerated tumorigenesis seen in vivo. Importantly, this negative feedback is evolutionarily conserved, as Drosophila Yorkie (YAP ortholog) induces transcription of Warts (LATS2 ortholog) with Scalloped (TEAD ortholog). Collectively, we demonstrated the existence and function of an evolutionarily conserved negative feedback mechanism in the Hippo pathway, as well as the functional difference between LATS1 and LATS2 in regulation of YAP.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Evolução Molecular , Retroalimentação Fisiológica , Via de Sinalização Hippo , Humanos , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Células Tumorais Cultivadas , Proteínas de Sinalização YAPRESUMO
The switch between stem/progenitor cell expansion and differentiation is critical for organ homeostasis. The mammalian Hippo pathway effector and oncoprotein YAP expands undifferentiated stem/progenitor cells in various tissues. However, the YAP-associated transcription factors and downstream targets underlying this stemness-promoting activity are poorly understood. Here we show that the SRF-IL6 axis is the critical mediator of YAP-induced stemness in mammary epithelial cells and breast cancer. Specifically, serum response factor (SRF)-mediated binding and recruitment of YAP to mammary stem cell (MaSC) signature-gene promoters induce numerous MaSC signature genes, among which the target interleukin (IL)-6 is critical for YAP-induced stemness. High SRF-YAP/TAZ expression is correlated with IL6-enriched MaSC/basal-like breast cancer (BLBC). Finally, we show that this high SRF expression enables YAP to more efficiently induce IL6 and stemness in BLBC compared with luminal-type breast cancer. Collectively, our results establish the importance of SRF-YAP-IL6 signalling in promoting MaSC-like properties in a BLBC-specific manner.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Células Epiteliais/metabolismo , Interleucina-6/metabolismo , Glândulas Mamárias Humanas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Imunofluorescência , Células HEK293 , Humanos , Imunoprecipitação , Técnicas In Vitro , Células MCF-7 , Glândulas Mamárias Humanas/citologia , Camundongos , Camundongos Nus , Análise em Microsséries , Transplante de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Análise Serial de Tecidos , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
[Purpose] This study compared the coordination patterns of the trunk and pelvis in the transverse plane between healthy subjects and patients with chronic low back pain during an anterior load carriage task at various walking speeds. [Subjects] Ten healthy subjects and 10 patients with chronic low back pain performed an anterior carriage task with a load of 10% body weight at walking speeds of 3.5, 4.5, or 5.5â km/h. [Methods] The trunk and pelvic kinematics were measured by using a motion analysis system. During the anterior carriage task, the continuous relative phase differed significantly between groups with respect to walking speed. [Results] The continuous relative phase was more anti-phase in the chronic low back pain group than the control group. The inter-group continuous relative phase pattern was affected by walking at 5.5â km/h. [Conclusion] Compared to controls, subjects with chronic low back pain are unable to establish an in-phase between the trunk and pelvis from walking at 3.5 to 5.5â km/h during an anterior carriage task.
RESUMO
Human pancreatic ductal adenocarcinoma (PDAC) is a cancer with a dismal prognosis. The efficacy of PDAC anticancer therapies is often short-lived; however, there is little information on how this disease entity so frequently gains resistance to treatment. We adopted the concept of cancer stem cells (CSCs) to explain the mechanism of resistance and evaluated the efficacy of a candidate anticancer drug to target these therapy-resistant CSCs. We identified a subpopulation of cells in PDAC with CSC features that were enriched for aldehyde dehydrogenase (ALDH), a marker expressed in certain stem/progenitor cells. These cells were also highly resistant to, and were further enriched by, treatment with gemcitabine. Similarly, surgical specimens from PDAC patients showed that those who had undergone preoperative chemo-radiation therapy more frequently displayed cancers with ALDH strongly positive subpopulations compared with untreated patients. Importantly, these ALDH-high cancer cells were sensitive to disulfiram, an ALDH inhibitor, when tested in vitro. Furthermore, in vivo xenograft studies showed that the effect of disulfiram was additive to that of low-dose gemcitabine when applied in combination. In conclusion, human PDAC-derived cells that express high levels of ALDH show CSC features and have a key role in the development of resistance to anticancer therapies. Disulfiram can be used to suppress this therapy-resistant subpopulation.
Assuntos
Aldeído Desidrogenase/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Dissulfiram/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Células-Tronco Neoplásicas/enzimologia , Neoplasias Pancreáticas/tratamento farmacológico , Aldeído Desidrogenase/antagonistas & inibidores , Análise de Variância , Animais , Western Blotting , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , GencitabinaRESUMO
Respiratory distress syndrome (RDS), which is induced by insufficient production of surfactant, is the leading cause of mortality in preterm babies. Although several transcription factors are known to be involved in surfactant protein expression, the molecular mechanisms and signaling pathways upstream of these transcription factors have remained elusive. Here, using mammalian Hippo kinases (Mst1/2, mammalian sterile 20-like kinase 1/2) conditional knockout mice, we demonstrate that Mst1/2 kinases are critical for orchestration of transcription factors involved in surfactant protein homeostasis and prevention of RDS. Mice lacking Mst1/2 in the respiratory epithelium exhibited perinatal mortality with respiratory failure and their lungs contained fewer type I pneumocytes and more immature type II pneumocytes lacking microvilli, lamellar bodies, and surfactant protein expression, pointing to peripheral lung immaturity and RDS. In contrast to previous findings of YAP (Yes-associated protein)-mediated canonical Hippo signaling in the liver and intestine, loss of Mst1/2 kinases induced the defects in pneumocyte differentiation independently of YAP hyperactivity. We instead found that Mst1/2 kinases stabilized and phosphorylated the transcription factor Foxa2 (forkhead box A2), which regulates pneumocyte maturation and surfactant protein expression. Taken together, our results suggest that the mammalian Hippo kinases play crucial roles in surfactant homeostasis and coordination of peripheral lung differentiation through regulation of Foxa2 rather than of YAP.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Células Epiteliais Alveolares/citologia , Animais , Apoptose , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cruzamentos Genéticos , Via de Sinalização Hippo , Homeostase , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Serina-Treonina Quinases/fisiologia , Serina-Treonina Quinase 3 , Transdução de Sinais , Fatores de TempoRESUMO
Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein-protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein-protein interactions as they occur in unpurified cell extracts. By analysing single Ras-Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein-protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein-protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein-protein interaction level.
Assuntos
Sistemas Computacionais , Imunoprecipitação/métodos , Neoplasias/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Cinética , Camundongos , Camundongos Nus , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
[Purpose] This study aimed to investigate the effects of Vojta therapy on spatiotemporal gait parameters in children with spastic diplegia. [Methods] The study population consisted of 3 children diagnosed with spastic diplegia. The subjects were treated with Vojta therapy for 8 weeks and followed up for 8 weeks after completion of the therapy. Vicon motion analysis was used to determine the subjects' spatiotemporal gait parameters. [Results] The following results were noted in the changes of each joint angle in the sagittal plane after Vojta therapy. Subject 1 remained in phase throughout the entire gait cycle and did not show any noticeable improvement, even demonstrating a negative range of motion when compared to the baseline. Subject 2 showed a normal anti-phase in heel strike, and the mid-stance, and swing phases. Subject 3 showed a normal anti-phase in heel strike and mid-stance, but the anti-phase during the swing phase was not significantly different from the baseline. For subjects 2 and 3, compared to the baseline, the range of motion of the hip and knee increased but the range of motion of the ankle decreased. [Conclusion] The findings of this study indicate that Vojta therapy can do a good role in improve the spatiotemporal gait parameters of children with spastic diplegia.