RESUMO
Kirsten rat sarcoma viral oncogene homologue (KRAS) is a frequent oncogenic driver of solid tumors, including non-small cell lung cancer (NSCLC). The treatment and outcomes of KRAS-mutant cancers have not been dramatically revolutionized by direct KRAS-targeted therapies because of the lack of deep binding pockets for specific small molecule inhibitors. Here, we demonstrated that the mRNA and protein levels of the class III histone deacetylase SIRT1 were upregulated by the KRASMut-Raf-MEK-c-Myc axis in KRASMut lung cancer cells and in lung tumors of a mouse model with spontaneous KrasG12D expression. KRASMut-induced SIRT1 bound to KRASMut and stably deacetylated KRASMut at lysine 104, which increased KRASMut activity. SIRT1 knockdown (K/D) or the SIRT1H363Y mutation increased KRASMut acetylation, which decreased KRASMut activity and sensitized tumors to the anticancer effects of cisplatin and erlotinib. Furthermore, in KrasG12D/+;Sirt1co/co mice, treatment with cisplatin and erlotinib robustly reduced the tumor burden and increased survival rates compared with those in spontaneous LSL-KrasG12D/+;Sirt1+/+ mice and mice in each single-drug treatment group. Then, we identified p300 as a KRASMut acetyltransferase that reinforced KRASMut lysine 104 acetylation and robustly decreased KRASMut activity. KRASMut lysine 104 acetylation by p300 and deacetylation by SIRT1 were confirmed by LCâMS/MS. Consistent with this finding, the SIRT1 inhibitor EX527 suppressed KRASMut activity, which synergistically abolished cell proliferation and colony formation, as well as the tumor burden in KRASMut mice, when combined with cisplatin or erlotinib. Our data reveal a novel pathway critical for the regulation of KRASMut lung cancer progression and provide important evidence for the potential application of SIRT1 inhibitors and p300 activators for the combination treatment of KRASMut lung cancer patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Camundongos , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Cisplatino/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Cloridrato de Erlotinib/uso terapêutico , Sirtuína 1/genética , Sirtuína 1/metabolismo , Lisina , Cromatografia Líquida , Espectrometria de Massas em Tandem , MutaçãoRESUMO
We previously found the SLC3A2-NRG1 (S-N) fusion gene in a lung adenocarcinoma specimen without known driver mutations and validated this in 59 invasive mucinous adenocarcinoma (IMA) samples. Interestingly, KRAS mutation coexisted (62.5%) in 10 out of 16 NRG1 fusions. In this study, we examined the role of mutant KRAS in regulating the S-N fusion protein in KRAS mutant (H358) and wild-type (Calu-3) cells. KRAS mutation-mediated increase in MEK1/2 and ERK1/2 activity enhanced disintegrin and metalloproteinase (ADAM)17 activity, which increased the shedding of NRG1 from the S-N fusion protein. The cleavage of NRG1 also increased the phosphorylation of ERBB2-ERBB3 heterocomplex receptors and their downstream signalling pathways, including PI3K/Akt/mTOR, even under activated KRAS mutation signalling. The concurrence of S-N fusion and KRAS mutation synergistically increased cell proliferation, colony formation, tumour growth, and the cells' resistance to EGFR kinase inhibitors more than KRAS mutation alone. Targeted inhibition of MEK1/2, and ADAM17 significantly induced apoptosis singly and when combined with each mutation singly or with chemotherapy in both the concurrent KRAS mutant and S-N fusion xenograft and lung orthotopic models. Taken together, this is the first study to report that KRAS mutation increased NRG1 cleavage from the S-N fusion protein through ADAM17, thereby enhancing the Ras/Raf/MEK/ERK and ERBB/PI3K/Akt/mTOR pathways. Moreover, the coexistence of KRAS mutant and S-N fusion in lung tumours renders them vulnerable to MEK1/2 and/or ADAM17 inhibitors, at least in part, due to their dependency on the strong positive loop between KRAS mutation and S-N fusion.
Assuntos
Proteína ADAM17/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Neuregulina-1/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Neuregulina-1/metabolismo , TransfecçãoRESUMO
Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.
Assuntos
Animais Geneticamente Modificados/genética , Técnicas de Transferência de Genes , Genes de Troca , Transgenes , Peixe-Zebra/genética , AnimaisRESUMO
Cell transplantation into immunodeficient recipients is a widely used approach to study stem cell and cancer biology; however, studying cell states post transplantation in vivo is inconvenient in mammals. Here, we generated a foxn1/Casper mutant zebrafish that is transparent and exhibits T cell deficiency. By employing the line for hematopoietic stem cell (HSC) transplantation (HSCT), we could achieve nonconditioned transplantation. Meanwhile, we found that fetal HSCs from 3 days post fertilization zebrafish embryos produce a better transplant outcome in foxn1/Casper mutants, compared with adult HSCs. In addition to HSCT, the foxn1/Casper mutant is feasible for allografts of myelodysplastic syndrome-like and muscle cells, as well as xenografts of medaka muscle cells. In summary, foxn1/Casper mutants permit the nonconditioned engraftment of multiple cell types and visualized characterization of transplanted cells in vivo.
Assuntos
Aloenxertos/transplante , Fatores de Transcrição Forkhead/genética , Xenoenxertos/transplante , Mutação/genética , Neoplasias/patologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Sequência de Bases , Células-Tronco Fetais/citologia , Fatores de Transcrição Forkhead/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Resultado do Tratamento , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
The endothelial cilium is a microtubule-based organelle responsible for blood flow-induced mechanosensation and signal transduction during angiogenesis. The precise function and mechanisms by which ciliary mechanosensation occurs, however, are poorly understood. Although posttranslational modifications (PTMs) of cytoplasmic tubulin are known to be important in angiogenesis, the specific roles of ciliary tubulin PTMs play remain unclear. Here, we report that loss of centrosomal protein 41 (CEP41) results in vascular impairment in human cell lines and zebrafish, implying a previously unknown pro-angiogenic role for CEP41. We show that proper control of tubulin glutamylation by CEP41 is necessary for cilia disassembly and that is involved in endothelial cell (EC) dynamics such as migration and tubulogenesis. We show that in ECs responding to shear stress or hypoxia, CEP41 activates Aurora kinase A (AURKA) and upregulates expression of VEGFA and VEGFR2 through ciliary tubulin glutamylation, as well as leads to the deciliation. We further show that in hypoxia-induced angiogenesis, CEP41 is responsible for the activation of HIF1α to trigger the AURKA-VEGF pathway. Overall, our results suggest the CEP41-HIF1α-AURKA-VEGF axis as a key molecular mechanism of angiogenesis and demonstrate how important ciliary tubulin glutamylation is in mechanosense-responded EC dynamics.
Assuntos
Aurora Quinase A , Tubulina (Proteína) , Animais , Aurora Quinase A/genética , Cílios , Humanos , Microtúbulos , Proteínas , Tubulina (Proteína)/genética , Peixe-Zebra/genéticaRESUMO
The host immune system plays a pivotal role in the emergence of tumor cells that are refractory to multiple clinical interventions including immunotherapy, chemotherapy, and radiotherapy. Here, we examined the molecular mechanisms by which the immune system triggers cross-resistance to these interventions. By examining the biological changes in murine and tumor cells subjected to sequential rounds of in vitro or in vivo immune selection via cognate cytotoxic T lymphocytes, we found that multimodality resistance arises through a core metabolic reprogramming pathway instigated by epigenetic loss of the ATP synthase subunit ATP5H, which leads to ROS accumulation and HIF-1α stabilization under normoxia. Furthermore, this pathway confers to tumor cells a stem-like and invasive phenotype. In vivo delivery of antioxidants reverses these phenotypic changes and resensitizes tumor cells to therapy. ATP5H loss in the tumor is strongly linked to failure of therapy, disease progression, and poor survival in patients with cancer. Collectively, our results reveal a mechanism underlying immune-driven multimodality resistance to cancer therapy and demonstrate that rational targeting of mitochondrial metabolic reprogramming in tumor cells may overcome this resistance. We believe these results hold important implications for the clinical management of cancer.
Assuntos
Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/deficiência , ATPases Mitocondriais Próton-Translocadoras/deficiência , Neoplasias/metabolismo , Neoplasias/terapia , Animais , Antioxidantes/administração & dosagem , Linhagem Celular Tumoral , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Translocases Mitocondriais de ADP e ATP/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Neoplasias/genética , Tolerância a Radiação , Evasão TumoralRESUMO
Wnt signaling controls critical developmental processes including tissue/body patterning. Here we report the identification of a novel regulator of Wnt signaling, OTTOGI (OTG), isolated from a large-scale expression screening of human cDNAs in zebrafish embryos. Overexpression of OTG in zebrafish embryos caused dorso-anteriorized phenotype, inhibited the expression of Wnt target genes, and prevented nuclear accumulation of ß-catenin. Conversely, knockdown of zebrafish otg using specific antisense morpholino promoted nuclear accumulation of ß-catenin and caused ventralization. However, OTG failed to rescue headless-like phenotype induced by inhibition of GSK-3ß activity, suggesting that OTG acts upstream of GSK-3ß. OTG bound specifically to Frizzled8 (Fz8) receptor and caused retention of Fz8 in the endoplasmic reticulum possibly by preventing N-linked glycosylation of Fz8. Taken together, our data indicate that OTG functions as a novel negative regulator of Wnt signaling during development by the modulation of cell surface expression of Fz receptor.
Assuntos
Membrana Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/metabolismo , Animais , DNA Complementar/genética , Desenvolvimento Embrionário/genética , Retículo Endoplasmático/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Glicosilação , Humanos , Fenótipo , Ligação Proteica , Transporte Proteico , Transcriptoma , Proteínas de Peixe-Zebra/genéticaRESUMO
Genetically engineered animal tumor models have traditionally been generated by the gain of single or multiple oncogenes or the loss of tumor suppressor genes; however, the development of live animal models has been difficult given that cancer phenotypes are generally induced by somatic mutation rather than by germline genetic inactivation. In this study, we developed somatically mutated tumor models using TALEN-mediated somatic gene inactivation of cdkn2a/b or rb1 tumor suppressor genes in zebrafish. One-cell stage injection of cdkn2a/b-TALEN mRNA resulted in malignant peripheral nerve sheath tumors with high frequency (about 39%) and early onset (about 35 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Injection of rb1-TALEN mRNA also led to the formation of brain tumors at high frequency (58%, 31 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Analysis of each tumor induced by somatic inactivation showed that the targeted genes had bi-allelic mutations. Tumors induced by rb1 somatic inactivation were characterized as medulloblastoma-like primitive neuroectodermal tumors based on incidence location, histopathological features, and immunohistochemical tests. In addition, 3' mRNA Quanti-Seq analysis showed differential activation of genes involved in cell cycle, DNA replication, and protein synthesis; especially, genes involved in neuronal development were up-regulated.
RESUMO
Rab escort protein 1 (REP1), a component of the Rab geranyl-geranyltransferase 2 complex, plays a role in Rab protein recruitment in proper vesicles during vesicle trafficking. In addition to having well-known tissue degenerative phenotypes in the REP1 mutant, REP1 is tightly associated with cancer development and contributes to cell growth and survival. However, the functional mechanism of REP1 in cancer progression is largely uninvestigated. Here, we show that REP1 plays a crucial role in regulating mammalian target of rapamycin (mTOR) signaling and its downstream pathways, as well as autophagy and macropinocytosis, which are essential for cancer cell survival during metabolic stresses including starvation. REP1 small interfering RNA (siRNA) treatment downregulates mTORC1 activity in growing media, but blocks autophagosome formation under nutrient-depleted conditions. In contrast to the mild decrease of lysosomal enzyme activity seen in REP1 depletion, in REP1 knockdown the subcellular localization of lysosomes is altered, and localization of REP1 itself is modulated by intracellular nutrient levels and mTOR activity. Furthermore, REP1 depletion increases macro pinocytosis which may be a feedback mechanism to compensate autophagy inhibition. Concomitant treatment with macropinocytosis inhibitor and REP1siRNAresults in more significant cell death than autophagy blockade with REP1 knockdown. Therefore, REP1-mediated autophagy and lysosomal degradation processes act as novel regulatory mechanisms to support cancer cell survival, which can be further investigated as a potential cancer-targeting pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Neoplasias Pancreáticas/metabolismo , Pinocitose , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Sobrevivência Celular , Regulação para Baixo , Células HeLa , Humanos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , CamundongosRESUMO
Ran-binding protein family member, RanBP9 has been reported in various basic cellular mechanisms and neuropathological conditions including schizophrenia. Previous studies have reported that RanBP9 is highly expressed in the mammalian brain and retina; however, the role of RanBP9 in retinal development is largely unknown. Here, we present the novel and regulatory roles of RanBP9 in retinal development of a vertebrate animal model, zebrafish. Zebrafish embryos exhibited abundant expression of ranbp9 in developing brain tissues as well as in the developing retina. Yeast two-hybrid screening demonstrated the interaction of RanBP9 with Mind bomb, a component of Notch signaling involved in both neurogenesis and neural disease autism. The interaction is further substantiated by co-localization studies in cultured cells. Knockdown of ranbp9 resulted in retinal dysplasia with defective proliferation of retinal cells, downregulation of neuronal differentiation marker huC, elevation of neural proliferation marker her4, and alteration of cell cycle marker p57kip2. Expression of the Müller glial cell marker glutamine synthase was also affected in knockdown morphants. Our results suggest that Mind bomb-binding partner RanBP9 plays a role during retinal cell development of zebrafish embryogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Retina/embriologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Células COS , Proliferação de Células , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Proteínas do Citoesqueleto/genética , Regulação para Baixo , Proteína Semelhante a ELAV 3/genética , Proteína Semelhante a ELAV 3/metabolismo , Células Ependimogliais/fisiologia , Técnicas de Silenciamento de Genes , Neurogênese/fisiologia , Proteínas Nucleares/genética , Retina/citologia , Retina/metabolismo , Displasia Retiniana/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Rab escort protein-1 (REP1) is linked to choroideremia (CHM), an X-linked degenerative disorder caused by mutations of the gene encoding REP1 (CHM). REP1 mutant zebrafish showed excessive cell death throughout the body, including the eyes, indicating that REP1 is critical for cell survival, a hallmark of cancer. In the present study, we found that REP1 is overexpressed in human tumor tissues from cervical, lung, and colorectal cancer patients, whereas it is expressed at relatively low levels in the normal tissue counterparts. REP1 expression was also elevated in A549 lung cancer cells and HT-29 colon cancer cells compared with BEAS-2B normal lung and CCD-18Co normal colon epithelial cells, respectively. Interestingly, short interfering RNA (siRNA)-mediated REP1 knockdown-induced growth inhibition of cancer cell lines via downregulation of EGFR and inactivation of STAT3, but had a negligible effect on normal cell lines. Moreover, overexpression of REP1 in BEAS-2B cells enhanced cell growth and anchorage-independent colony formation with little increase in EGFR level and STAT3 activation. Furthermore, REP1 knockdown effectively reduced tumor growth in a mouse xenograft model via EGFR downregulation and STAT3 inactivation in vivo. These data suggest that REP1 plays an oncogenic role, driving tumorigenicity via EGFR and STAT3 signaling, and is a potential therapeutic target to control cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Receptores ErbB/genética , Oncogenes/genética , Fator de Transcrição STAT3/genética , Células A549 , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Coroideremia/genética , Regulação para Baixo/genética , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação/genética , Transdução de Sinais/genéticaRESUMO
Rab escort protein 1 (REP1) is a component of Rab geranyl-geranyl transferase 2 complex. Mutations in REP1 cause a disease called choroideremia (CHM), which is an X-linked eye disease. Although it is postulated that REP1 has functions in cell survival or death of various tissues in addition to the eye, how REP1 functions in normal and cancer cells remains to be elucidated. Here, we demonstrated that REP1 is required for the survival of intestinal cells in addition to eyes or a variety of cells in zebrafish, and also has important roles in tumorigenesis. Notably, REP1 is highly expressed in colon cancer tissues and cell lines, and silencing of REP1 sensitizes colon cancer cells to serum starvation- and 5-FU-induced apoptosis. In an effort to elucidate the molecular mechanisms underlying REP1-mediated cell survival under those stress conditions, we identified FOXO3 as a binding partner of REP1 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that REP1 blocked the nuclear trans-localization of FOXO3 through physically interacting with FOXO3, thereby suppressing FOXO3-mediated apoptosis. Importantly, the inhibition of REP1 combined with 5-FU treatment could lead to significant retarded tumor growth in a xenograft tumor model of human cancer cells. Thus, our results suggest that REP1 could be a new therapeutic target in combination treatment for colon cancer patients.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Neoplasias do Colo/genética , Proteína Forkhead Box O3/genética , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Coroideremia/genética , Coroideremia/patologia , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Fluoruracila/administração & dosagem , Proteína Forkhead Box O3/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mutação , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/genéticaRESUMO
Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system. As a model for oculocutaneous albinism (OCA) and color vision impairment, tyrosinase mutants demonstrated diminished color sensation, indicated mainly by hypopigmentation of the retinal pigment epithelium (RPE). Due to its relative simplicity and ease, color preference screening using zebrafish larvae is suitable for high-throughput screening applications. This system may potentially be applied to the analysis of drug effects on larval behavior or the detection of sensory deficits in neurological disorder models, such as autism-related disorders, using mutant larvae generated by the CRISPR/Cas9 technique.
Assuntos
Análise do Comportamento Aplicada/métodos , Comportamento Animal/fisiologia , Cor , Peixe-Zebra/fisiologia , Animais , Feminino , MasculinoRESUMO
Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.
RESUMO
Galanin is a multifunctional neuropeptide that is implicated in the modulation of physiological processes, including nociception, cognition, feeding behavior, neuronal growth, and reproduction. The physiological effects of galanin are mediated through its interaction with three different G protein-coupled receptors, i.e., GALR1, GALR2, and GALR3. Unlike mammals, zebrafish have four different receptors for galanin, diversified from GALR1 (GAL1a and GALR1b) and GALR2 (GALR2a and GALR2b). Despite the importance of galanin in the central nervous system (CNS), no information has been reported regarding GalR2 in zebrafish CNS. In this study, we found that galr2a is expressed at low levels in restricted areas of the brain; however, galr2b was widely expressed in CNS including olfactory bulb, midbrain tegmentum, preoptic region, dorsal thalamus, posterior tuberculum, postoptic commissure, hindbrain, and spinal cord. To further analyze the distribution of GALR2b neurons and their interaction with GAL, we generated Tg(galr2b:egfp) zebrafish, which express enhanced green fluorescent protein (EGFP) under the control of a galr2b promoter. Investigation of the CNS of transgenic reporter zebrafish revealed that galr2b:EGFP(+) neurons are distributed and interact with galanin-immunoreactive (galanin-IR) cells in various regions of the brain and spinal cord. We found that in some regions of the brain and spinal cord, galanin-IR nerve cells were not observed near galr2b:EGFP neurons, suggesting that GALR2b may have the potential to interact with other ligands instead of galanin in these regions.
Assuntos
Encéfalo/metabolismo , Galanina/metabolismo , Neurônios/metabolismo , Receptor Tipo 2 de Galanina/análise , Receptor Tipo 2 de Galanina/metabolismo , Medula Espinal/metabolismo , Animais , Feminino , Proteínas de Peixes/análise , Masculino , Peixe-ZebraRESUMO
The importance of PPARγ (peroxisome proliferator-activated receptor γ) in gastric cancer (GC) is unclear. We investigated the role of PPARγ in GC cell lines and an animal model, and its prognostic significance of PPARγ in GC patients. We controlled PPARγ and galectin-9 expression by using siRNAs and lentiviral constructs. Interaction between PPARγ and galectin-9 was evaluated using luciferase and chromatin immunoprecipitation assays. PPARγ expression in GCs was determined by immunohistochemical staining of tissue microarrays and survival analysis was done. Overexpression of PPARγ was accompanied by increased galectin-9. Enhanced PPARγ or galectin-9 expression increased E-cadherin expression; decreased expression of N-cadherin, fibronectin, snail, twist and slug and reduced cell invasion and migration. PPARγ bound to the galectin-9 promoter region. Galectin-9 activity increased in PPARγ-overexpressing cells but decreased in PPARγ siRNA-treated cells. In a zebrafish xenograft model, the number of migrated cancer cells and number of fish with AGS cells in the tail vein were reduced in PPARγ-overexpressing GC cells. PPARγ was expressed in 462 of the 688 patients (69.2%) with GC. In 306 patients with intestinal-type GC, those with PPARγ-positive tumors had lower overall and cancer-specific mortalities than those with PPARγ-negative tumors. PPARγ expression was an independent prognostic factor for overall and GC-specific mortality in patients with intestinal-type GC (adjusted hazard ratio, 0.42; 95% CI, 0.22-0.81). PPARγ inhibits cell invasion, migration and epithelial-mesenchymal transition through upregulation of galectin-9 in vitro and in vivo.
Assuntos
Galectinas/genética , PPAR gama/fisiologia , Neoplasias Gástricas/metabolismo , Animais , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Galectinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Invasividade Neoplásica , Transplante de Neoplasias , PPAR gama/agonistas , Prognóstico , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Ligação Proteica , Rosiglitazona , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Tiazolidinedionas/farmacologia , Regulação para Cima , Peixe-ZebraRESUMO
A subset of ventral spinal cord precursors, known as pMN precursor cells, initially generate motor neurons and then oligodendrocyte progenitor cells (OPCs), which migrate and differentiate as myelinating oligodendrocytes in the developing neural tube. The switch between motor neuron and oligodendrocyte production by the pMN neural precursors is an important step in building a functional nervous system. However, the precise mechanism that orchestrates the sequential generation of motor neurons and oligodendrocytes within the common population of pMN precursors is still unclear. The current study demonstrates that Indian Hedgehog b (Ihhb), previously known as Echidna Hedgehog, begins to be expressed in the floor plate cells of the ventral spinal cord at the time of OPC specification in zebrafish embryos. Ihhb loss-of-function analysis revealed that Ihhb function is required for OPC specification from pMN precursors by negatively regulating the proliferation of neural precursors. Finally, results showed that Sonic Hedgehog (Shh) could not replace Ihhb function in OPC specification, suggesting that Ihhb and Shh play separate roles in OPC specification. Altogether, data from the present study suggested a novel mechanism, mediated by Ihhb, for the sequential generation of motor neurons and oligodendrocytes from pMN precursors in the ventral spinal cord of zebrafish embryos.
Assuntos
Diferenciação Celular/fisiologia , Proteínas Hedgehog/metabolismo , Neurogênese/fisiologia , Oligodendroglia/citologia , Células-Tronco/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Separação Celular , Imuno-Histoquímica , Hibridização In Situ , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Oligodendroglia/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Células-Tronco/metabolismoRESUMO
The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.
Assuntos
Antineoplásicos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Receptores ErbB/metabolismo , Modelos Animais , Neoplasias/tratamento farmacológico , Animais , Butadienos/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Humanos , Mutação , Nitrilas/farmacologia , Fenótipo , Estrutura Terciária de Proteína , Quinazolinas/farmacologia , Transgenes , Tirfostinas/farmacologiaRESUMO
Previous studies have shown that Notch signaling not only regulates the number of early differentiating neurons, but also maintains proliferating neural precursors in the neural tube. Although it is well known that Notch signaling is closely related to the differentiation of adult neural stem cells, none of transgenic zebrafish provides a tool to figure out the relationship between Notch signaling and the differentiation of neural precursors. The goal of this study was to characterize Her4-positive cells by comparing the expression of a fluorescent Her4 reporter in Tg[her4-dRFP] animals with a GFAP reporter in Tg[gfap-GFP] adult zebrafish. BrdU incorporation indicated that dRFP-positive cells were proliferating and a double labeling assay revealed that a significant fraction of the Her4-dRFP positive population was also GFAP-GFP positive. Our observations suggest that a reporter line with Notch-dependent gene expression can provide a tool to examine proliferating neural precursors and/or neuronal/glial precursors in the development of the adult nervous system to examine the model in which Notch signaling maintains proliferating neural precursors in the neural tube.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Neurais/metabolismo , Colículos Superiores/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Encéfalo/citologia , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Confocal , Células-Tronco Neurais/fisiologia , Receptor Notch3 , Receptores Notch/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Serrate-JaggedRESUMO
INTRODUCTION: C-C chemokine receptor type 7 (CCR7) plays an important role in chemotactic and metastatic responses in various cancers, including breast cancer. In the present study, the authors demonstrated that microRNA (miRNA) let-7a downregulates CCR7 expression and directly influences the migration and invasion of breast cancer cells. METHODS: The expression of CCR7, its ligand CCL21, and let-7a was detected in breast cancer cell lines and in breast cancer patient tissues. Synthetic let-7a and an inhibitor of let-7a were transfected into MDA-MB-231 and MCF-7 breast cancer cells, respectively, and cell proliferation, cell migration, and invasion assays were performed. To confirm the fact that 3'UTR of CCR7 is a direct target of let-7a, a luciferase assay for the reporter gene expressing the let-7a binding sites of CCR7 3'UTR was used. An in vivo invasion animal model system using transparent zebrafish embryos was also established to determine the let-7a effect on breast cancer cell invasion. RESULTS: First, a higher expression of both CCR7 and CCL21 in malignant tissues than in their normal counterparts from breast cancer patients was observed. In addition, a reverse correlation in the expression of CCR7 and let-7a in breast cancer cell lines and breast cancer patient tissues was detected. Synthetic let-7a decreased breast cancer cell proliferation, migration, and invasion, as well as CCR7 protein expression in MDA-MB-231 cells. The let-7a inhibitor reversed the let-7a effects on the MCF-7 cells. The 3'UTR of CCR7 was confirmed as a direct target of let-7a by using the luciferase assay for the reporter gene expressing let-7a CCR7 3'UTR binding sites. Notably, when analyzing in vivo invasion, MDA-MB 231 cells after synthetic let-7a transfection were unable to invade the vessels in zebrafish embryos. CONCLUSIONS: The results from the present study suggest that targeting of CCL21-CCR7 signaling is a valid approach for breast cancer therapy and that let-7a directly binds to the 3'UTR of CCR7 and blocks its protein expression, thereby suppressing migration and invasion of human breast cancer cells. Furthermore, the present study underscores the therapeutic potential of let-7a as an antitumor and antimetastatic manager in breast cancer patients.