RESUMO
The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in "normal" tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Nitrilas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Quinolinas/farmacologia , Proteínas ras/genética , Animais , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Skeletal muscles of vertebrates are typically composed of slow- and fast-twitch fibers that differ in their morphology, gene expression profiles, contraction speeds, metabolic properties and patterns of innervation. During myogenesis, how muscle precursors are induced to mature into distinct slow- or fast-twitch fiber-types is inadequately understood. We have previously shown that within the somites of the zebrafish embryo, the activity of the zinc finger and SET domain-containing transcriptional regulator Blimp1 is essential for the specification of slow muscle fibers. Here, we have investigated the mechanism by which Blimp1 programs myoblasts to adopt the slow-twitch fiber fate. In slow myoblasts, expression of the Blimp1 protein is transient, and precedes the expression of slow muscle-specific differentiation genes. We demonstrate that the competence of somitic myoblasts to commit to the slow lineage in response to Blimp1 changes as a function of developmental time. Furthermore, we provide evidence that mammalian Blimp1 can recapitulate the slow myogenic program in zebrafish, suggesting that zebrafish Blimp1 can recognize the same consensus DNA sequence that is bound by the mammalian protein. Finally, we show that zebrafish Blimp1 can repress the expression of fast muscle-specific myosin light chain, mylz2, through direct binding near the promoter of this gene, indicating that an important function of the transcriptional activity of Blimp1 in slow muscle development is the suppression of fast muscle-specific gene expression. Taken together, these findings provide new insights into the molecular basis of vertebrate muscle fiber-type specification, and underscore Blimp1 as the central determinant of this process.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Muscular/genética , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Sítios de Ligação , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Diferenciação Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Mioblastos Esqueléticos/metabolismo , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Proteínas Nucleares/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo , Regiões Promotoras Genéticas , Transcrição Gênica , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Mouse double minute (Mdm) genes span an evolutionary timeframe from the ancient eukaryotic placozoa Trichoplax adhaerens to Homo sapiens, implying a significant and possibly conserved cellular role throughout history. Maintenance of DNA integrity and response to DNA damage involve many key regulatory pathways, including precise control over the tumour suppressor protein p53. In most vertebrates, degradation of p53 through proteasomal targeting is primarily mediated by heterodimers of Mdm2 and the Mdm2-related protein Mdm4 (also known as MdmX). Both Mdm2 and Mdm4 have p53-binding regions, acidic domains, zinc fingers, and C-terminal RING domains that are conserved throughout evolution. Vertebrates typically have both Mdm2 and Mdm4 genes, while analyses of sequenced genomes of invertebrate species have identified single Mdm genes, suggesting that a duplication event occurred prior to emergence of jawless vertebrates about 550-440 million years ago. The functional relationship between Mdm and p53 in T. adhaerens, an organism that has existed for 1 billion years, implies that these two proteins have evolved together to maintain a conserved and regulated function.