RESUMO
The IL6-GP130-STAT3 pathway facilitates lung cancer progression and resistance to tyrosine kinase inhibitors. Although glycosylation alters the stability of GP130, its effect on the ligand IL6 remains unclear. We herein find that N-glycosylated IL6, especially at Asn73, primarily stimulates JAK-STAT3 signaling and prolongs STAT3 phosphorylation, whereas N-glycosylation-defective IL6 (deNG-IL6) induces shortened STAT3 activation and alters the downstream signaling preference for the SRC-YAP-SOX2 axis. This signaling shift induces epithelial-mesenchymal transition (EMT) and migration in vitro and metastasis in vivo, which are suppressed by targeted inhibitors and shRNAs against SRC, YAP, and SOX2. Osimertinib-resistant lung cancer cells secrete a large amount of deNG-IL6 through reduced N-glycosyltransferase gene expression, leading to clear SRC-YAP activation. deNG-IL6 contributes to drug resistance, as confirmed by in silico analysis of cellular and clinical transcriptomes and signal expression in patient specimens. Therefore, the N-glycosylation status of IL6 not only affects cell behaviors but also shows promise in monitoring the dynamics of lung cancer evolution.
Assuntos
Acrilamidas , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Interleucina-6 , Neoplasias Pulmonares , Inibidores de Proteínas Quinases , Fator de Transcrição STAT3 , Humanos , Glicosilação , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Interleucina-6/metabolismo , Interleucina-6/genética , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Linhagem Celular Tumoral , Acrilamidas/farmacologia , Camundongos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Compostos de Anilina/farmacologia , Receptor gp130 de Citocina/metabolismo , Receptor gp130 de Citocina/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Fosforilação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Quinases da Família src/metabolismo , Quinases da Família src/genética , Camundongos Nus , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Metástase Neoplásica , Regulação Neoplásica da Expressão Gênica , Feminino , Indóis , PirimidinasRESUMO
Pluripotent mouse embryonic stem cells (ESCs) can differentiate to all germ layers and serve as an in vitro model of embryonic development. To better understand the differentiation paths traversed by ESCs committing to different lineages, we track individual differentiating ESCs by timelapse imaging followed by multiplexed high-dimensional Imaging Mass Cytometry (IMC) protein quantification. This links continuous live single-cell molecular NANOG and cellular dynamics quantification over 5-6 generations to protein expression of 37 different molecular regulators in the same single cells at the observation endpoints. Using this unique data set including kinship history and live lineage marker detection, we show that NANOG downregulation occurs generations prior to, but is not sufficient for neuroectoderm marker Sox1 upregulation. We identify a developmental cell type co-expressing both the canonical Sox1 neuroectoderm and FoxA2 endoderm markers in vitro and confirm the presence of such a population in the post-implantation embryo. RNASeq reveals cells co-expressing SOX1 and FOXA2 to have a unique cell state characterized by expression of both endoderm as well as neuroectoderm genes suggesting lineage potential towards both germ layers.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Fator 3-beta Nuclear de Hepatócito , Células-Tronco Embrionárias Murinas , Fatores de Transcrição SOXB1 , Animais , Camundongos , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Rastreamento de Células/métodos , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Linhagem da Célula , Endoderma/metabolismo , Endoderma/citologia , Análise de Célula Única/métodos , Desenvolvimento Embrionário/genética , Placa Neural/metabolismo , Placa Neural/embriologia , Placa Neural/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologiaRESUMO
Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene, Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the three Klf4 enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site in Klf4 enhancer E2 rescued enhancer function and Klf4 expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities of Klf4 enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.
Assuntos
Elementos Facilitadores Genéticos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Fator 3 de Transcrição de Octâmero , Fatores de Transcrição SOXB1 , Animais , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Camundongos , Sítios de Ligação , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
The median eminence (ME), located at the base of the hypothalamus, is an essential centre of information exchange between the brain and the pituitary. We and others previously showed that mutations and duplications affecting the transcription factor SOX3/Sox3 result in hypopituitarism, and this is likely of hypothalamic origin. We demonstrate here that the absence of Sox3 predominantly affects the ME with phenotypes that first occur in juvenile animals, despite the embryonic onset of SOX3 expression. In the pituitary, reduction in hormone levels correlates with a lack of endocrine cell maturation. In parallel, ME NG2-glia renewal and oligodendrocytic differentiation potential are affected. We further show that low-dose aspirin treatment, which is known to affect NG2-glia, or changes in gut microbiota, rescue both proliferative defects and hypopituitarism in Sox3 mutants. Our study highlights a central role of NG2-glia for ME function during a transitional period of post-natal development and indicates their sensitivity to extrinsic signals.
Assuntos
Aspirina , Microbioma Gastrointestinal , Hipopituitarismo , Eminência Mediana , Neuroglia , Animais , Hipopituitarismo/genética , Aspirina/farmacologia , Camundongos , Microbioma Gastrointestinal/genética , Eminência Mediana/metabolismo , Neuroglia/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular , Hipófise/metabolismo , Camundongos Knockout , MasculinoRESUMO
Metastasis is the leading cause of cancerrelated death in osteosarcoma (OS). OS stem cells (OSCs) and anoikis resistance are considered to be essential for tumor metastasis formation. However, the underlying mechanisms involved in the maintenance of a stemcell phenotype and anoikis resistance in OS are mostly unknown. Foslike antigen 1 (FOSL1) is important in maintaining a stemlike phenotype in various cancers; however, its role in OSCs and anoikis resistance remains unclear. In the present study, the dynamic expression patterns of FOSL1 were investigated during the acquisition of cancer stemlike properties using RNA sequencing, PCR, western blotting and immunofluorescence. Flow cytometry, tumorsphere formation, clone formation assays, anoikis assays, western blotting and in vivo xenograft and metastasis models were used to further investigate the responses of the stemcell phenotype and anoikis resistance to FOSL1 overexpression or silencing in OS cell lines. The underlying molecular mechanisms were evaluated, focusing on whether SOX2 is crucially involved in FOSL1mediated stemness and anoikis in OS. FOSL1 expression was observed to be upregulated in OSCs and promoted tumorsphere formation, clone formation and tumorigenesis in OS cells. FOSL1 expression correlated positively with the expression of stemnessrelated factors (SOX2, NANOG, CD117 and Stro1). Moreover, FOSL1 facilitated OS cell anoikis resistance and promoted metastases by regulating the expression of apoptosis related proteins BCL2 and BAX. Mechanistically, FOSL1 upregulated SOX2 expression by interacting with the SOX2 promoter and activating its transcription. The results also showed that SOX2 is critical for FOSL1mediated stemlike properties and anoikis resistance. The current findings indicated that FOSL1 is an important regulator that promotes a stem celllike phenotype and anoikis resistance to facilitate tumorigenesis and metastasis in OS by regulating the transcription of SOX2. Thus, FOSL1 might represent an attractive target for therapeutic interventions in OS.
Assuntos
Anoikis , Carcinogênese , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas , Osteossarcoma , Proteínas Proto-Oncogênicas c-fos , Fatores de Transcrição SOXB1 , Osteossarcoma/patologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Anoikis/genética , Animais , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Camundongos , Carcinogênese/genética , Carcinogênese/patologia , Metástase Neoplásica , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Camundongos Nus , Masculino , Feminino , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Increased cancer stem cell (CSC) content and SOX2 overexpression are common features in the development of resistance to therapy in hormone-dependent breast cancer, which remains an important clinical challenge. SOX2 has potential as biomarker of resistance to treatment and as therapeutic target, but targeting transcription factors is also challenging. Here, we examine the potential inhibitory effect of different polyoxometalate (POM) derivatives on SOX2 transcription factor in tamoxifen-resistant breast cancer cells. METHODS: Various POM derivatives were synthesised and characterised by infrared spectra, powder X-ray diffraction pattern and nuclear magnetic resonance spectroscopy. Estrogen receptor (ER) positive breast cancer cells, and their counterparts, which have developed resistance to the hormone therapy tamoxifen, were treated with POMs and their consequences assessed by gel retardation and chromatin immunoprecipitation to determine SOX2 binding to DNA. Effects on proliferation, migration, invasion and tumorigenicity were monitored and quantified using microscopy, clone formation, transwell, wound healing assays, flow cytometry and in vivo chick chorioallantoic membrane (CAM) models. Generation of lentiviral stable gene silencing and gene knock-out using CRISPR-Cas9 genome editing were applied to validate the inhibitory effects of the selected POM. Cancer stem cell subpopulations were quantified by mammosphere formation assays, ALDEFLUOR activity and CD44/CD24 stainings. Flow cytometry and western blotting were used to measure reactive oxygen species (ROS) and apoptosis. RESULTS: POMs blocked in vitro binding activity of endogenous SOX2. [P2W18O62]6- (PW) Wells-Dawson-type anion was the most effective at inhibiting proliferation in various cell line models of tamoxifen resistance. 10 µM PW also reduced cancer cell migration and invasion, as well as SNAI2 expression levels. Treatment of tamoxifen-resistant cells with PW impaired tumour formation by reducing CSC content, in a SOX2-dependent manner, which led to stem cell depletion in vivo. Mechanistically, PW induced formation of reactive oxygen species (ROS) and inhibited Bcl-2, leading to the death of tamoxifen-resistant cells. PW-treated tamoxifen-resistant cells showed restored sensitivity to tamoxifen. CONCLUSIONS: Together, these observations highlight the potential use of PW as a SOX2 inhibitor and the therapeutic relevance of targeting SOX2 to treat tamoxifen-resistant breast cancer.
Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição SOXB1 , Tamoxifeno , Compostos de Tungstênio , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Tamoxifeno/farmacologia , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Compostos de Tungstênio/farmacologia , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , AnimaisRESUMO
Cancer stem cells (CSC) play an important role in carcinogenesis and are acknowledged to be responsible for chemoresistance in cholangiocarcinoma (CCA). Studying CCA CSC has been challenging, due to lack of consensus CSC markers, and to their plastic nature. Since dual expression of the core pluripotent factors SOX2/OCT4 has been shown to correlate with poor outcome in CCA patients, we selected the SOX2/OCT4 activating short half-life GFP-based live reporter (SORE6-dsCopGFP) to study CSC dynamics at the single-cell level. Transduction of five human CCA cell lines resulted in the expression of 1.8-13.1% GFP-positive (SORE6POS) cells. By live imaging, we found that SORE6POS CCA cells possess self-renewal capacity and that they can be induced to differentiate. Significantly, the SORE6POS cells were highly tumorigenic, both in vitro and in vivo, thus implicating the characteristics of primary CSCs. When we then analyzed for selected CSC-related markers, we found that the majority of both CD133+/CD44+, and CD133+/LGR5+ CCA cells were SORE6POS cells. Exposing transduced cells to standard CCA chemotherapy revealed higher growth rate inhibition at 50% (GR50s) for SORE6POS cells compared to GFP-negative (SORE6NEG) ones indicating that these CSC-like cells were more resistant to the treatment. Moreover, the chemotherapy induced SORE6POS from SORE6NEG cells, while retaining the existing SORE6POS population. Finally, treatment of transduced cells with CDK4/6 inhibitors in vitro for 3 days resulted in a lowered CSC number in the culture. Thus, applying a live reporter system allowed us to elucidate the stem cell diversity and drug-induced plasticity of CCA CSCs. These findings have clear implications for future management of such patients.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Células-Tronco Neoplásicas , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Humanos , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Genes Reporter , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Análise de Célula Única/métodos , Animais , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Antineoplásicos/farmacologia , Plasticidade Celular/efeitos dos fármacos , Resistencia a Medicamentos AntineoplásicosRESUMO
During the aging process, elastin is degraded and the level of elastin-derived peptides (EDPs) successively increases. The main peptide released from elastin during its degradation is a peptide with the VGVAPG sequence. To date, several papers have described that EDPs or elastin-like peptides (ELPs) affect human mesenchymal stem cells (hMSCs) derived from different tissues. Unfortunately, despite the described effect of EDPs or ELPs on the hMSC differentiation process, the mechanism of action of these peptides has not been elucidated. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG and VVGPGA peptides on the hMSC stemness marker and elucidation of the mechanism of action of these peptides. Our data show that both studied peptides (VGVAPG and VVGPGA) act with the involvement of ERK1/2 and c-SRC kinases. However, their mechanism of activation is probably different in hMSCs derived from adipose tissue. Both studied peptides increase the KI67 protein level in hMSCs, but this is not accompanied with cell proliferation. Moreover, the changes in the NANOG and c-MYC protein expression and in the SOX2 and POU5F1 mRNA expression suggest that EDPs reduced the hMSC stemness properties and could initiate cell differentiation. The initiation of differentiation was evidenced by changes in the expression of AhR and PPARγ protein as well as specific genes (ACTB, TUBB3) and proteins (ß-actin, RhoA) involved in cytoskeleton remodeling. Our data suggest that the presence of EDPs in tissue can initiate hMSC differentiation into more tissue-specific cells.
Assuntos
Diferenciação Celular , Elastina , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/metabolismo , Elastina/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/citologia , Antígeno Ki-67/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Peptídeos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Células Cultivadas , Oligopeptídeos/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Proliferação de Células , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismoRESUMO
Sensorineural hearing loss can be caused by lesions to the inner ear during development. Understanding the events and signaling pathways that drive inner ear formation is crucial for determining the possible causes of congenital hearing loss. We have analyzed the innervation and expression of SOX2, JAGGED1, ß-catenin (CTNNB1), and vitamin D receptor (VDR) in the inner ears of human conceptuses aged 5 to 10 weeks after fertilization (W) using immunohistochemistry. The prosensory domains of the human inner ear displayed SOX2 and JAGGED1 expression throughout the analyzed period, with SOX2 expression being more extensive in all the analyzed timepoints. Innervation of vestibular prosensory domains was present at 6 W and extensive at 10 W, while nerve fibers reached the base of the cochlear prosensory domain at 7-8 W. CTNNB1 and VDR expression was mostly membranous and present during all analyzed timepoints in the inner ear, being the strongest in the non-sensory epithelium. Their expression was stronger in the vestibular region compared to the cochlear duct. CTNNB1 and VDR expression displayed opposite expression trends during the analyzed period, but additional studies are needed to elucidate whether they interact during inner ear development.
Assuntos
Orelha Interna , Proteína Jagged-1 , Receptores de Calcitriol , Fatores de Transcrição SOXB1 , beta Catenina , Humanos , beta Catenina/metabolismo , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , Orelha Interna/metabolismo , Orelha Interna/inervação , Orelha Interna/embriologia , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Regulação da Expressão Gênica no Desenvolvimento , FemininoRESUMO
Mammalian gene expression is controlled by transcription factors (TFs) that engage sequence motifs in a chromatinized genome, where nucleosomes can restrict DNA access. Yet, how nucleosomes affect individual TFs remains unclear. Here, we measure the ability of over one hundred TF motifs to recruit TFs in a defined chromosomal locus in mouse embryonic stem cells. This identifies a set sufficient to enable the binding of TFs with diverse tissue specificities, functions, and DNA-binding domains. These chromatin-competent factors are further classified when challenged to engage motifs within a highly phased nucleosome. The pluripotency factors OCT4-SOX2 preferentially engage non-nucleosomal and entry-exit motifs, but not nucleosome-internal sites, a preference that also guides binding genome wide. By contrast, factors such as BANP, REST, or CTCF engage throughout, causing nucleosomal displacement. This supports that TFs vary widely in their sensitivity to nucleosomes and that genome access is TF specific and influenced by nucleosome position in the cell.
Assuntos
Células-Tronco Embrionárias Murinas , Nucleossomos , Fatores de Transcrição , Nucleossomos/metabolismo , Nucleossomos/genética , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco Embrionárias Murinas/metabolismo , Sítios de Ligação , Ligação Proteica , Genoma/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Cromatina/metabolismo , Cromatina/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Montagem e Desmontagem da CromatinaRESUMO
The inflammatory tumor microenvironment (TME) is a key driver for tumor-promoting processes. Tumor-associated macrophages are one of the main immune cell types in the TME and their increased density is related to poor prognosis in prostate cancer. Here, we investigated the influence of pro-inflammatory (M1) and immunosuppressive (M2) macrophages on prostate cancer lineage plasticity. Our findings reveal that M1 macrophage secreted factors upregulate genes related to stemness while downregulating genes associated with androgen response in prostate cancer cells. The expression of cancer stem cell (CSC) plasticity markers NANOG, KLF4, SOX2, OCT4, and CD44 was stimulated by the secreted factors from M1 macrophages. Moreover, AR and its target gene PSA were observed to be suppressed in LNCaP cells treated with secreted factors from M1 macrophages. Inhibition of NFκB signaling using the IKK16 inhibitor resulted in downregulation of NANOG, SOX2, and CD44 and CSC plasticity. Our study highlights that the secreted factors from M1 macrophages drive prostate cancer cell plasticity by upregulating the expression of CSC plasticity markers through NFκB signaling pathway.
Assuntos
Receptores de Hialuronatos , Fator 4 Semelhante a Kruppel , Macrófagos , NF-kappa B , Proteína Homeobox Nanog , Células-Tronco Neoplásicas , Neoplasias da Próstata , Fatores de Transcrição SOXB1 , Transdução de Sinais , Masculino , Humanos , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 4 Semelhante a Kruppel/metabolismo , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Macrófagos/metabolismo , Regulação para Cima , Microambiente Tumoral/imunologia , Plasticidade Celular/genética , Regulação Neoplásica da Expressão Gênica , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologia , Animais , CamundongosRESUMO
Osteogenesis is tightly coupled with angiogenesis spatiotemporally. Previous studies have demonstrated that type H blood vessel formed by endothelial cells with high expression of CD31 and Emcn (CD31hi Emcnhi ECs) play a crucial role in bone regeneration. The mechanism of the molecular communication around CD31hi Emcnhi ECs and bone mesenchymal stem cells (BMSCs) in the osteogenic microenvironment is unclear. This study indicates that exosomes from bone mesenchymal stem cells with 7 days osteogenic differentiation (7D-BMSCs-exo) may promote CD31hi Emcnhi ECs angiogenesis, which was verified by tube formation assay, qRT-PCR, Western blot, immunofluorescence staining and µCT assays etc. in vitro and in vivo. Furthermore, by exosomal miRNA microarray and WGCNA assays, we identified downregulated miR-150-5p as the most relative hub gene coupling osteogenic differentiation and type H blood vessel angiogenesis. With bioinformatics assays, dual luciferase reporter experiments, qRT-PCR and Western blot assays, SOX2(SRY-Box Transcription Factor 2) was confirmed as a novel downstream target gene of miR-150-5p in exosomes, which might be a pivotal mechanism regulating CD31hi Emcnhi ECs formation. Additionally, JC-1 immunofluorescence staining, Western blot and seahorse assay results showed that the overexpression of SOX2 could shift metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis to enhance the CD31hi Emcnhi ECs formation. The PI3k/Akt signaling pathway might play a key role in this process. In summary, BMSCs in osteogenic differentiation might secrete exosomes with low miR-150-5p expression to induce type H blood vessel formation by mediating SOX2 overexpression in ECs. These findings might reveal a molecular mechanism of osteogenesis coupled with type H blood vessel angiogenesis in the osteogenic microenvironment and provide a new therapeutic target or cell-free remedy for osteogenesis impaired diseases.
Assuntos
Diferenciação Celular , Células Endoteliais , Exossomos , Células-Tronco Mesenquimais , MicroRNAs , Neovascularização Fisiológica , Osteogênese , MicroRNAs/genética , MicroRNAs/metabolismo , Exossomos/metabolismo , Osteogênese/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/genética , Neovascularização Fisiológica/genética , Animais , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Camundongos , Humanos , Células Cultivadas , Transdução de Sinais , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Reprogramação Metabólica , AngiogêneseRESUMO
How the dorsal-ventral axis of the vertebrate jaw, particularly the position of tooth initiation site, is established remains a critical and unresolved question. Tooth development starts with the formation of the dental lamina, a localized thickened strip within the maxillary and mandibular epithelium. To identify transcriptional regulatory networks (TRN) controlling the specification of dental lamina from the naïve mandibular epithelium, we utilized Laser Microdissection coupled low-input RNA-seq (LMD-RNA-seq) to profile gene expression of different domains of the mandibular epithelium along the dorsal-ventral axis. We comprehensively identified transcription factors (TFs) and signaling pathways that are differentially expressed along mandibular epithelial domains (including the dental lamina). Specifically, we found that the TFs Sox2 and Tfap2 (Tfap2a/Tfap2b) formed complimentary expression domains along the dorsal-ventral axis of the mandibular epithelium. Interestingly, both classic and novel dental lamina specific TFs-such as Pitx2, Ascl5 and Zfp536-were found to localize near the Sox2:Tfap2a/Tfap2b interface. To explore the functional significance of these domain specific TFs, we next examined loss-of-function mouse models of these domain specific TFs, including the dental lamina specific TF, Pitx2, and the ventral surface ectoderm specific TFs Tfap2a and Tfap2b. We found that disruption of domain specific TFs leads to an upregulation and expansion of the alternative domain's TRN. The importance of this cross-repression is evident by the ectopic expansion of Pitx2 and Sox2 positive dental lamina structure in Tfap2a/Tfap2b ectodermal double knockouts and the emergence of an ectopic tooth in the ventral surface ectoderm. Finally, we uncovered an unappreciated interface of mesenchymal SHH and WNT signaling pathways, at the site of tooth initiation, that were established by the epithelial domain specific TFs including Pitx2 and Tfap2a/Tfap2b. These results uncover a previously unknown molecular mechanism involving cross-repression of domain specific TFs including Pitx2 and Tfap2a/Tfap2b in patterning the dorsal-ventral axis of the mouse mandible, specifically the regulation of tooth initiation site.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox PITX2 , Proteínas de Homeodomínio , Mandíbula , Fatores de Transcrição SOXB1 , Fator de Transcrição AP-2 , Fatores de Transcrição , Animais , Camundongos , Linhagem da Célula/genética , Epitélio/metabolismo , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mandíbula/metabolismo , Odontogênese/genética , Transdução de Sinais , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Dente/metabolismo , Dente/crescimento & desenvolvimento , Dente/embriologia , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Parkinson's disease is a progressive neurodegenerative disease that affects the motor and non-motor circuits of the brain. Currently, there are no promising therapeutic measures for Parkinson's disease, and most strategies designed to alleviate the Parkinson's disease are palliative. The dearth of therapeutic interventions in Parkinson's disease has driven attention in the search for targets that may augment dopamine secretion, promote differentiation towards dopaminergic neuronal lineage, or aid in neuroprotection from neuronal stress and inflammation, and prevent Parkinson's disease associated motor impairment and behavioural chaos. The study first reports that Rev-erbα plays an important role in regulating the differentiation of undifferentiated neuronal cells towards dopaminergic neurons through abating Sox2 expression in human SH-SY5Y cells. Rev-erbα directly binds to the human Sox2 promoter region and represses their expression to promote differentiation towards dopaminergic neurons. We have reported a novel mechanism of Rev-erbα which effectively abrogates 1-methyl-4-phenylpyridinium induced cytotoxicity, inflammation, and oxidative stress, exerted a beneficial effect on transmembrane potential, and suppressed apoptosis in the neuronal in vitro model of Parkinson's disease. Rev-erbα ligand SR9011 was observed to ease the disease severity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced mouse model of Parkinson's disease. Rev-erbα alleviates the locomotor behavioural impairment, prevents cognitive decline and promotes motor coordination in mice. Administration of Rev-erbα ligand also helps in replenishing the dopaminergic neurons and abrogating the neurotoxin mediated toxicity in an in vitro and in vivo Parkinson's disease model. We conclude that Rev-erbα emerges as a moonlighting nuclear receptor that could be targeted in the treatment and alleviation of Parkinson disease.
Assuntos
Neurônios Dopaminérgicos , Neurogênese , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares , Fatores de Transcrição SOXB1 , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Animais , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Humanos , Camundongos , Neurogênese/efeitos dos fármacos , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Diferenciação Celular , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/genética , Estresse Oxidativo/efeitos dos fármacos , ApoptoseRESUMO
The gene encoding the NUAK family kinase 1 (NUAK1) is frequently amplified and its expression is upregulated, activating oncogenic signaling in various cancers. However, little is known about its role in gastric cancer (GC). We investigate the mechanistic links among NUAK1, Hedgehog signaling, and tumorigenesis in GC. NUAK1 overexpression is validated in local and public GC cohorts. Patient-derived xenograft and transgenic mouse models demonstrate that NUAK1 depletion or inhibition dramatically ameliorates gastric tumorigenesis. NUAK1 upregulates GLI1 expression by activating STAT5-mediated transcription and stabilizing GLI1 protein. NUAK1 depletion or inhibition impairs cancer cell expansion, tumor formation, and chemotherapy resistance in in vitro and in vivo models. Clinicopathological analysis confirms that upregulated NUAK1 expression correlates with poor prognosis and chemotherapy resistance in human GC. Our findings demonstrate that the signaling axis NUAK1/STAT5/GLI1 promotes cancer cell expansion and tumorigenesis and indicate that NUAK1 is an attractive therapeutic target and prognostic factor in GC.
Assuntos
Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição SOXB1 , Fator de Transcrição STAT5 , Transdução de Sinais , Neoplasias Gástricas , Proteína GLI1 em Dedos de Zinco , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/tratamento farmacológico , Humanos , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT5/genética , Animais , Linhagem Celular Tumoral , Camundongos , Proliferação de Células/efeitos dos fármacos , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Masculino , Feminino , Carcinogênese/patologia , Carcinogênese/genéticaRESUMO
Objective To evaluate the value of SOX1 and PAX1 gene methylation detection in the secondary triage of high-grade cervical lesions.Methods Exfoliated cervical cells were collected from 122 patients tested positive for human papilloma virus (HPV) and subjected to thin-prep cytologic test (TCT) and SOX1/PAX1 gene methylation tests.Results The HPV test combined with TCT showed the sensitivity of 95.24% and the specificity of 23.75% for detecting cervical intraepithelial neoplasia (CIN) grade 2 and above (CIN2+).After the addition of the SOX1/PAX1 gene methylation detection in secondary triage,the sensitivity for detecting CIN2+ was 83.33%,which had no statistically significant difference from the sensitivity of TCT combined with HPV test (P=0.078).However,the specificity reached 77.50%,which was significantly higher than that of HPV test combined with TCT (P<0.001).The SOX1/PAX1 gene methylation level in the CIN2+ group was higher than those in the normal cervical tissue and the CIN1 group(P<0.001).The cut-off values of SOX1 and PAX1 gene methylation for CIN2+ detection were -11.81 and -11.98,respectively.Conclusion Adding the detection of SOX1/PAX1 gene methylation in secondary triage significantly improves the efficiency and accuracy of CIN2+ detection.
Assuntos
Metilação de DNA , Fatores de Transcrição Box Pareados , Fatores de Transcrição SOXB1 , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Humanos , Feminino , Fatores de Transcrição Box Pareados/genética , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Fatores de Transcrição SOXB1/genética , Adulto , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
Lung squamous cell carcinoma (LSCC) is a deadly cancer in the world. Histone demethylase Jmjd2c is a key epigenetic regulator in various tumors, while the molecular mechanism underlying Jmjd2c regulatory in LSCC is still unclear. We used the aldehyde dehydrogenasebright (ALDHbri+) subtype as a research model for cancer stem cells (CSCs) in LSCC and detected the sphere formation ability and the proportion of ALDHbri+ CSCs with Jmjd2c interference and caffeic acid (CA) treatment. Additionally, we carried out bioinformatic analysis on the expression file of Jmjd2c RNAi mice and performed western blotting, qRT-PCR, Co-IP and GST pull-down assays to confirm the bioinformatic findings. Moreover, we generated Jmjd2c-silenced and Jmjd2c-SOX2-silenced ALDHbri+ tumor-bearing BALB/c nude mice to detect the effects on tumor progression. The results showed that Jmjd2c downregulation inhibited the sphere formation and the proportion of ALDHbri+ CSCs. The SOX2 decreased expression significantly in Jmjd2c RNAi mice, and they were positively co-expressed according to the bioinformatic analysis. In addition, SOX2 expression decreased in Jmjd2c shRNA ALDHbri+ CSCs, Jmjd2c and SOX2 proteins interacted with each other. Furthermore, Jmjd2c interference revealed significant blocking effect, and Jmjd2c-SOX2 interference contributed even stronger inhibition on ALDHbri+ tumor progression. The Jmjd2c and SOX2 levels were closely related to the development and prognosis of LSCC patients. This study indicated that Jmjd2c played key roles on maintaining ALDHbri+ CSC activity in LSCC by interacting with transcription factor SOX2. Jmjd2c might be a novel molecule for therapeutic targets and biomarkers in the diagnosis and clinical treatment of lung cancer.
Assuntos
Carcinoma de Células Escamosas , Histona Desmetilases com o Domínio Jumonji , Neoplasias Pulmonares , Células-Tronco Neoplásicas , Fatores de Transcrição SOXB1 , Animais , Feminino , Humanos , Masculino , Camundongos , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genéticaRESUMO
Salivary gland squamous cell carcinomas (SG-SCCs) constitute a rare type of head and neck cancer which is linked to poor prognosis. Due to their low frequency, the molecular mechanisms responsible for their aggressiveness are poorly understood. In this work we studied the role of the phosphatase DUSP1, a negative regulator of MAPK activity, in controlling SG-SCC progression. We generated DUSP1 KO clones in A253 human cells. These clones showed a reduced ability to grow in 2D, self-renew in ECM matrices and to form tumors in immunodeficient mice. This was caused by an overactivation of the stress and apoptosis kinase JNK1/2 in DUSP1-/+ clones. Interestingly, RNAseq analysis revealed that the expression of SOX2, a well-known self-renewal gene was decreased at the mRNA and protein levels in DUSP1-/+ cells. Unexpectedly, CRISPR-KO of SOX2 did not recapitulate DUSP1-/+ phenotype, and SOX2-null cells had an enhanced ability to self-renew and to form tumors in mice. Gene expression analysis demonstrated that SOX2-null cells have a decreased squamous differentiation profile -losing TP63 expression- and an increased migratory phenotype, with an enhanced epithelial to mesenchymal transition signature. In summary, our data indicates that DUSP1 and SOX2 have opposite functions in SG-SCC, being DUSP1 necessary for tumor growth and SOX2 dispensable showing a tumor suppressor function. Our data suggest that the combined expression of SOX2 and DUSP1 could be a useful biomarker to predict progression in patients with SG-SCCs.
Assuntos
Carcinoma de Células Escamosas , Progressão da Doença , Fosfatase 1 de Especificidade Dupla , Fatores de Transcrição SOXB1 , Neoplasias das Glândulas Salivares , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatase 1 de Especificidade Dupla/genética , Humanos , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Animais , Camundongos , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Neoplasias das Glândulas Salivares/metabolismo , Linhagem Celular Tumoral , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genéticaRESUMO
SRY (Sex Determining Region) box 2 (SOX2) is an essential transcription factor that plays crucial roles in activating genes involved in pre- and post-embryonic development, adult tissue homeostasis, and lineage specifications. SOX2 maintains the self-renewal property of stem cells and is involved in the generation of induced pluripotency stem cells. SOX2 protein contains a particular high-mobility group domain that enables SOX2 to achieve the capacity to participate in a broad variety of functions. The information about the involvement of SOX2 with gene regulatory elements, signaling networks, and microRNA is gradually emerging, and the higher expression of SOX2 is functionally relevant to various cancer types. SOX2 facilitates the oncogenic phenotype via cellular proliferation and enhancement of invasive tumor properties. Evidence are accumulating in favor of three dimensional (higher order) folding of chromatin and epigenetic control of the SOX2 gene by chromatin modifications, which implies that the expression level of SOX2 can be modulated by epigenetic regulatory mechanisms, specifically, via DNA methylation and histone H3 modification. In view of this, and to focus further insights into the roles SOX2 plays in physiological functions, involvement of SOX2 during development, precisely, the advances of our knowledge in pre- and post-embryonic development, and interactions of SOX2 in this scenario with various signaling pathways in tumor development and cancer progression, its potential as a therapeutic target against many cancers are summarized and discussed in this article.
Assuntos
Desenvolvimento Embrionário , Neoplasias , Fatores de Transcrição SOXB1 , Transdução de Sinais , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Humanos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Desenvolvimento Embrionário/genética , Animais , Progressão da Doença , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão GênicaRESUMO
SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.