RESUMEN
In oral squamous cell carcinoma (OSCC), involvement and activation of the Hedgehog pathway (HH) may be related to epithelial-mesenchymal transition and cell proliferation. The present study aimed to evaluate epithelial-mesenchymal transition and proliferative potential in OSCC cases demonstrating activation of the HH pathway. Twenty-three GLi-1-positive OSCC cases were submitted to immunohistochemical detection of Snail, Slug, N-cadherin, E-cadherin, ß-catenin, and MCM3 proteins. Clinical-pathologic immunoexpression data were obtained from the invasion front and tumor islets, and then compared. At the invasion front, OSCC cases presented positive Snail, Slug, and MCM3 expression in the nuclei of tumor cells. Loss of membrane and cytoplasmic expression of E-cadherin and ß-catenin was also observed. Positive N-cadherin expression was observed in 31.78% of the cases. GLi-1 immunoexpression was associated with loss of membrane E-cadherin (P<0.001), membrane ß-catenin (P<0.001), and cytoplasmic ß-catenin (P=0.02) expression. In the tumor islets, we observed nuclear expression of GLi-1, Snail, Slug, and MCM3. E-cadherin and ß-catenin showed positivity in tumor cell membranes. Statistically significant positive correlations between GLi-1 and Snail (P=0.05), E-cadherin (P=0.01), and cytoplasmic ß-catenin (P=0.04) were found. GLi-1 was associated with clinical staging, while membrane ß-catenin expression was related to the presence of metastasis in lymph nodes and to clinical staging. The HH pathway may be involved in regulating the expression of the mesenchymal phenotype. The loss of membrane E-cadherin and ß-catenin expression was observed at the tumor front region, whereas cell adhesion protein expression was detected in tumor islets regardless of MCM3.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Proliferación Celular , Transición Epitelial-Mesenquimal , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Proteína con Dedos de Zinc GLI1/biosíntesis , Femenino , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patologíaRESUMEN
Gli proteins are key transcription factors of the Hedgehog (HH) signaling pathway, which is associated with tumorigenesis and drug resistance. However, the role of the HH signaling pathway in epithelial ovarian cancer (EOC) remains unclear. Studies have demonstrated that in some tumors, homeobox protein NANOG (NANOG), a known stem cell marker, is a downstream effector of Gli. However, limited research has been conducted on the association between Gli and NANOG in EOC, particularly regarding their roles in the tumor stemness, such as tumor development, drug resistance and patient prognosis. Thus, the aim of the present study was to explore the aforementioned issues. In this study, Gli1, Gli2 and NANOG expression in EOC tissues was assessed using immunohistochemistry. Gene expression was also assessed using western blotting and reverse transcriptionquantitative PCR in SKOV3 cells treated with a Gli inhibitor and an HH agonist. Furthermore, cell proliferation, colonyforming ability and cisplatin sensitivity were assessed using Cell Counting Kit8 and colony formation assays. The results showed that both Gli1 and NANOG were associated with cisplatin resistance and EOC disease stage, while the nuclear expression of Gli2 was significantly associated with cisplatin resistance. Together, the expression of Gli and NANOG predicted poor patient prognosis. Targeting Gli with GANT61 impeded tumor proliferation, reversed cisplatin resistance and colony formation, and reduced NANOG expression. To conclude, Gli and NANOG may be effective indicators of platinum resistance and prognosis in EOC. Targeting Gli may reduce the stemness of ovarian cancer cell, which may be achieved via indirect targeting of NANOG.
Asunto(s)
Carcinoma Epitelial de Ovario/metabolismo , Proteína Homeótica Nanog/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Neoplasias Ováricas/metabolismo , Proteína con Dedos de Zinc GLI1/biosíntesis , Proteína con Dedos de Zinc GLI1/genética , Proteína Gli2 con Dedos de Zinc/biosíntesis , Proteína Gli2 con Dedos de Zinc/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/farmacología , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Proteína Homeótica Nanog/genética , Proteínas Nucleares/antagonistas & inhibidores , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Pronóstico , Piridinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Adulto Joven , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Proteína Gli2 con Dedos de Zinc/antagonistas & inhibidoresRESUMEN
Neural stem cells (NSCs) generate neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used two-photon microscopy and followed NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI family zinc finger 1 (Gli1) or achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny, we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they become exhausted. Using single-cell RNA sequencing, we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here identify long-term self-renewing NSCs that contribute to the generation of new neurons in the adult hippocampus.
Asunto(s)
Hipocampo/crecimiento & desarrollo , Células-Madre Neurales/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linaje de la Célula , Femenino , Perfilación de la Expresión Génica , Hipocampo/citología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Microscopía Intravital , Masculino , Metalotioneína 3 , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación Multifotónica , Regeneración Nerviosa , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Análisis de la Célula Individual , Proteína con Dedos de Zinc GLI1/biosíntesis , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Increased expression of GLI1, the main Hedgehog signalling pathway effector, is related to unfavourable prognosis and progressive disease of certain breast cancer subtypes. We used conditional transgenic mice induced to overexpress GLI1 in the mammary epithelium either alone or in combination with deletion of one Trp53 allele to address the role of elevated GLI1 expression in breast tumour initiation and progression. Induced GLI1 expression facilitates mammary gland tumour formation and this was further increased upon heterozygous deletion of Trp53. The GLI1-induced primary tumours were of different murine molecular subtypes, including Normal-likeEx , Class8Ex , Claudin-LowEx and Erbb2-likeEx . The gene expression profiles of some of the tumours correlated well with the PAM50 subtypes for human breast cancer. Whole-exome sequencing revealed somatic mutation profiles with only little overlap between the primary tumours. Orthotopically serially transplanted GLI1-induced tumours maintained the main morphological characteristics of the primary tumours for ≥10 generations. Independent of Trp53 status and molecular subtype, the serially transplanted GLI1-induced tumours were able to grow both in the absence of transgenic GLI1 expression and in the presence of the GLI1 inhibitor GANT61. These data suggest that elevated GLI1 expression has a determinant role in tumour initiation; however, additional genetic events are required for tumour progression.
Asunto(s)
Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Proteína con Dedos de Zinc GLI1/genética , Animales , Femenino , Expresión Génica , Heterogeneidad Genética , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Trasplante de Neoplasias , Proteína con Dedos de Zinc GLI1/biosíntesisRESUMEN
BACKGROUND: Gastric Cancer (GC) is a frequently common malignancy. Recent studies have reported Rab1A as an activator of mTORC1, and the mTOR1 pathway is involved in regulating Gli1 expression in several cancers. Only a few studies have been performed to explore the relationship between Rab1A and p-S6K/Gli1in GC. METHODS: Immunohistochemistry (IHC) was performed to explore the association of Rab1A/p-S6K/Gli1 expression and prognosis in 117 GC tissue samples and adjacent normal tissues. RESULTS: Our results indicated that Rab1A/p-S6K/Gli1 was significantly overexpressed in GC tissues. High expression of Rab1A was closely related to the tumor size and the depth of tumor invasion. In addition, Rab1A expression was closely related with p-S6K/Gli1 expression in GC, and high level of Rab1A/p-S6K/Gli1 caused worse prognosis of GC patients. The univariate and multivariate analysis indicated that the expression of Rab1A was an independent prognostic factor. Moreover, both high Rab1A and p-S6K expression led to a worse prognosis when compared to a single positive expression as well as both high Rab1A/Gli1 expression also led to a worse prognosis than the single positive expression of Rab1A/Gli1. Strikingly, the overexpression of p-S6K also led to a worse prognosis in Rab1A positive patients, as did Gli1. CONCLUSION: Our results indicate that Rab1A/mTOR/S6K/Gli1 axis played a crucial role in GC, which may provide a novel field on targeted therapy of GC, especially for mTORC1-targeted therapy-resistant cancers.
Asunto(s)
Proteínas Quinasas S6 Ribosómicas/biosíntesis , Neoplasias Gástricas/diagnóstico , Proteína con Dedos de Zinc GLI1/biosíntesis , Proteínas de Unión al GTP rab1/biosíntesis , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Quinasas S6 Ribosómicas/análisis , Proteínas Quinasas S6 Ribosómicas/metabolismo , Neoplasias Gástricas/metabolismo , Proteína con Dedos de Zinc GLI1/análisis , Proteína con Dedos de Zinc GLI1/metabolismo , Proteínas de Unión al GTP rab1/análisis , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
OBJECTIVE: Patched (PTCH1) is an important receptor protein in the Hedgehog pathway and plays a tumor suppressor role in a variety of tumors. This study was to detect the expression of PTCH1 in ovarian cancer (OC) tissues to analyze the relationship between expression of PTCH1 and prognosis, and to explore its role in regulating OC cell proliferation and apoptosis. MATERIALS AND METHODS: OC tissues and normal ovarian tissues were collected to detect PTCH1 expression. SKOV3, A2780, Caov3, and IOSE80 cells were cultured in vitro to test PTCH1 expression. pIRES2-Scramble and pIRES2-PTCH1 were transfected into SKOV3 and A2780 cells, respectively. PTCH1 and Gli1 expressions were detected by western blot. Cell apoptosis was determined by flow cytometry. Cell proliferation was assessed by EdU staining. RESULTS: PTCH1 expression was significantly decreased in OC tissue compared with normal ovarian tissue and was associated with tumor size, TNM stage, and pathological grade (p < 0.05). The prognosis of patients with low PTCH1 expression was obviously worse than that of patients with high PTCH1 expression. The expression of PTCH1 in OC SKOV3, A2780, and Caov3 cells was markedly lower than that in normal ovarian epithelial IOSE80 cells. Transfection of pIRES2-PTCH1 apparently upregulated PTCH1 level, inhibited GLI1 expression and cell proliferation, and promoted apoptosis of SKOV3 and A2780 cells. CONCLUSION: PTCH1 level in OC was abnormally decreased and related to prognosis. Overexpression of PTCH1 inhibited GLI1 expression, attenuated OC cell proliferation, and induced apoptosis, suggesting that manipulation of PTCH1 expression might be a novel approach for the treatment of OC.
Asunto(s)
Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Receptor Patched-1/biosíntesis , Adulto , Anciano , Apoptosis/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/genética , Receptor Patched-1/genética , Pronóstico , Transfección , Proteína con Dedos de Zinc GLI1/biosíntesisRESUMEN
Tumor microenvironment provides a specialized niche in which a population of stem-like cells is enriched and contributes to cancer progression. Moreover, cancer stem cell (CSC) phenotype has been associated with epithelial-mesenchymal transition (EMT). Here we investigated the effect of tumor microenvironment on the phenotypic characteristics of head and neck cancer cells and expression of CSC markers using a three-dimensional (3D), spheroid, culture system of CAL33 cell line from human tongue squamous cell carcinoma. CAL33 cells derived from 2D monolayer cultures were grown in spheroid cultures containing serum-free medium (epidermal growth factor [EGF], fibroblast growth factor [FGF], and insulin). Adherent CAL33 cells from spheroids or standard control cultures were grown in the presence/absence of serum in combination with hypoxia/normoxia. Markers of EMT, CSC, and hypoxia were analyzed either by Western blotting, immunofluorescence, or reverse transcription quantitative PCR. Spheroid cultures showed hypoxic microenvironment (high carbonic anhydrase IX [CAIX] expression), mesenchymal-like characteristics (reduced E-cadherin and increased vimentin and N-cadherin expression, presence of larger colonies comprised of larger, spread cells with lower density), and increased expression of the CSC marker glioma-associated oncogene homolog 1 (Gli1). These effects were recapitulated in serum-free adherent CAL33 cells maintained for prolonged periods in hypoxia (1% O2) but, in contrast, were completely abolished by the presence of serum. Overall, we found that a combination of hypoxia, EGF and FGF was essential to induce the EMT in adherent CAL33 cell cultures. The addition of serum rapidly reverts the EMT of cells, affects CSC phenotype and, thus, prevents the detection of such cells in tumor cell lines.
Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Proteína con Dedos de Zinc GLI1/biosíntesis , Anhidrasa Carbónica IX/biosíntesis , Anhidrasa Carbónica IX/genética , Adhesión Celular/efectos de los fármacos , Hipoxia de la Célula , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Factores de Crecimiento de Fibroblastos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias de la Lengua/genética , Microambiente Tumoral , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Hedgehog (Hh) signaling is involved in the progression of hepatocellular carcinoma (HCC), while its detailed mechanisms are not well illustrated. Our present study revealed that the expression of Gli1, while not Gli2 or Gli3, is significantly increased in HCC cell lines and 20/28 (71.4%) HCC tissues as compared with their corresponding controls. Over expression of Gli1 can promote the migration, invasion and epithelial-mesenchymal transition (EMT) of HCC cells. Gli1 can increase the expression of Twist, while not other EMT transcription factors such as Snail, ZEB1 or Slug. Gli1 increases the transcription of Twist while it has no significant effect on the protein or mRNA stability. Chromatin immunoprecipitation-polymerase chain reaction confirms that Gli1 can directly bind to the promoter of Twist, in which the third binding site is essential for Gli1 induced transcription. Collectively, our data suggest that upregulation of Twist is involved in Gli1 induced migration and invasion of HCC cells.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Movimiento Celular , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Regulación hacia Arriba , Proteína con Dedos de Zinc GLI1/metabolismo , Células Cultivadas , Cromatina/química , Transición Epitelial-Mesenquimal , Humanos , Inmunoprecipitación , Neoplasias Hepáticas/patología , Invasividad Neoplásica , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína con Dedos de Zinc GLI1/biosíntesis , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Hedgehog/metabolismo , Glicoproteínas de Membrana/biosíntesis , MicroARNs/genética , Desarrollo de Músculos/fisiología , Músculo Esquelético/embriología , Proteínas del Tejido Nervioso/biosíntesis , Receptores Patched/biosíntesis , Proteína Gli3 con Dedos de Zinc/biosíntesis , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Embrión de Pollo , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Cultivo Primario de Células , Proteína con Dedos de Zinc GLI1/biosíntesisRESUMEN
BACKGROUND This study assessed the prognostic value of GLI1 in gastric cancer and analyzed the possible GLI1-related signaling network in chemosensitivity. MATERIAL AND METHODS Bioinformatic data mining was performed by using data in the TCGA-Stomach Cancer (TCGA-STAD) and the Kaplan-Meier plotter. GLI1 co-expressed genes in TCGA-STAD were subjected to KEGG pathway analysis. The genes enriched in the KEGG pathways were further subjected to Protein-Protein Interaction (PPI) analysis. RESULTS In TCGA-STAD, high GLI1 gene/exon expression was associated with significantly worse survival (p=0.016 and 0.0023 respectively). In the Kaplan-Meier plotter, high GLI1 expression was associated with unfavorable overall survival (OS) (HR: 1.68, 95%CI: 1.42-2, p<0.0001) and first progression-free survival (FPS) (HR: 1.72, 95%CI: 1.4-2.11, p<0.0001). In TCGA-STAD, 600 GLI1 co-expressed genes were identified (absolute Pearson's r ≥0.5). The most significant pathways were pathways in cancer (p=230.0E-12) and the Hedgehog signaling pathway (p=6.9E-9). PI3K-AKT pathway (p=17.0E-9) has the largest proportion of gene enrichment. Some GLI1 co-expressed genes in the PI3K-AKT pathway are central nodes in the PPI network and also play important roles in chemosensitivity of gastric cancer. Nevertheless, the mechanisms underlying their co-expression are still largely unexplored. CONCLUSIONS High GLI1 expression is associated with unfavorable OS and FPS in patients with gastric cancer. As a member of the Hedgehog signaling pathway, GLI1 co-expressed genes are also largely enriched in PI3K/AKT pathway in gastric cancer, which is closely related to chemoresistance. The underlying mechanisms are still largely unexplored and need further study.
Asunto(s)
Neoplasias Gástricas/genética , Proteína con Dedos de Zinc GLI1/genética , Adulto , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Biología Computacional , Supervivencia sin Enfermedad , Femenino , Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Pronóstico , Mapas de Interacción de Proteínas , Transducción de Señal , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Proteína con Dedos de Zinc GLI1/biosíntesis , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Epithelia-mesenchymal transition (EMT) is critical for invasion and metastasis of esophageal carcinoma. Gli1, a transcriptional factor in Hedgehog pathway, is correlated with EMT, invasion and metastasis of tumors. However, its role in esophageal cancer is still unknown. Bioinformatics analysis revealed relationship between microRNA (miR)-361 and 3'-UTR of Gli1 gene. This study thus investigated the role of miR-361 and Gli1 in invasion and metastasis of esophageal cancer. Both tumor and adjacent tissues were collected from 58 esophageal cancer patients to test the expressions of miR-361 and Gli1, the relationship of which was confirmed by dual-luciferase reporter gene assay. Cultured esophageal cancer cells EC9706 were transfected with mimic NC, miR-361 mimic, si-NC, si-Gli1, miR-361 mimics+si-Glil, pQC or pQC-FU-Gli1. Transwell and colony formation assays were performed for cell invasion and attachment-independent growth. Expressions of Gli1, Snail, E-cadherin and N-cadherin proteins were revealed by Western blotting. The expression of Gli1 was significantly elevated in esophageal cancer tissues, along with lower miR-361 expression which was correlated with TNM stage. MiR-361 inhibited the expression of Gli1 via targeting on 3'-UTR of Gli1 gene. The transfection of miR-361 mimics and/or si-Gli1 significantly suppressed the growth of malignant cells. The over-expression of miR-361 and/or silencing of Gli1 decreased intracellular expression of Gli1, Snail and N-cadherin, and increased E-cadherin expression to suppress EMT and invasion of tumor cells while the opposite effects were obtained by over-expression of Gli1. Abnormal elevation of Gli1 and decrease of miR-361 were found in esophageal cancer tissues. MiR-361 weakened invasion of cancer cells and impeded EMT process via the inhibition of Gli1.
Asunto(s)
Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , MicroARNs/biosíntesis , Proteína con Dedos de Zinc GLI1/biosíntesis , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Neoplasias Esofágicas/genética , Femenino , Células HEK293 , Humanos , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Sonic hedgehog (Shh) regulates a wave of oligodendrocyte production for extensive myelination during postnatal development. During this postnatal period of oligodendrogenesis, we fate-labeled cells exhibiting active Shh signaling to examine their contribution to the regenerative response during remyelination. Bitransgenic mouse lines were generated for induced genetic fate-labeling of cells actively transcribing Shh or Gli1. Gli1 transcription is an effective readout for canonical Shh signaling. ShhCreERT2 mice and Gli1CreERT2 mice were crossed to either R26tdTomato mice to label cells with red fluorescence, or, R26IAP mice to label membranes with alkaline phosphatase. When tamoxifen (TMX) was given on postnatal days 6-9 (P6-9), Shh ligand synthesis was prevalent in neurons of ShhCreERT2; R26tdTomato mice and ShhCreERT2;R26IAP mice. In Gli1CreERT2 crosses, TMX from P6-9 detected Gli1 transcription in cells that populated the corpus callosum (CC) during postnatal myelination. Delaying TMX to P14-17, after the peak of oligodendrogenesis, significantly reduced labeling of Shh synthesizing neurons and Gli1 expressing cells in the CC. Importantly, Gli1CreERT2;R26tdTomato mice given TMX from P6-9 showed Gli1 fate-labeled cells in the adult (P56) CC, including cycling progenitor cells identified by EdU incorporation and NG2 immunolabeling. Furthermore, after cuprizone demyelination of the adult CC, Gli1 fate-labeled cells incorporated EdU and were immunolabeled by NG2 early during remyelination while forming myelin-like membranes after longer periods for remyelination to progress. These studies reveal a postnatal cell population with transient Shh signaling that contributes to oligodendrogenesis during CC myelination, and gives rise to cells that continue to proliferate in adulthood and contribute to CC remyelination.
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Linaje de la Célula/genética , Proteínas Hedgehog/genética , Vaina de Mielina/patología , Oligodendroglía/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Quelantes , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Cuprizona , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/prevención & control , Femenino , Masculino , Ratones , Vaina de Mielina/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Proteína con Dedos de Zinc GLI1/biosíntesis , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Hedgehog signaling pathway has been implicated in the pathology of ovarian cancer, and Survivin (BIRC5) has been suggested as a novel target of this pathway. Herein we investigated the role of Hedgehog signaling pathway and Survivin in ovarian carcinoma and borderline tumor samples. We aimed to determine possible ways of pathway modulation on primary ovarian cancer cells and an established cell line. RNA was extracted from fresh tumors and control tissues and gene expression was examined using qRT-PCR. Pathway activity in cell lines was examined after treatment with cyclopamine, SHH protein, GANT-61 or lithium chloride using qRT-PCR, western blot and confocal microscopy. The difference between control tissue, borderline tumors and carcinomas can be seen in GLI1 and SUFU gene expression, which is significantly higher in borderline tumors compared to carcinomas. SUFU also shows lower expression levels in higher FIGO stages relative to lower stages. BIRC5 is expressed in all tumors and in healthy ovarian tissues compared to our control tissue, healthy fallopian tube samples. Primary cells developed from ovarian carcinoma tissue respond to cyclopamine treatment with a short-term decrease in cell proliferation, downregulation of Hedgehog pathway genes, including BIRC5, and changes in protein dynamics. Stimulation with SHH protein results in increased cell migration, while GLI1 transfection or PTCH1 silencing demonstrate pathway upregulation. The pathway activity can be modulated by LiCl at the GSK3ß-SUFU-GLI level, suggesting at least partial non-canonical activation. Downregulation of the pathway with GANT-61 has proved to be more effective than cyclopamine. GLI inhibitors may be a superior treatment option in ovarian cancer compared to SMO inhibitors.
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Carcinoma/metabolismo , Proteínas Hedgehog/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Carcinoma/genética , Carcinoma/patología , Carcinoma Epitelial de Ovario , Estudios de Casos y Controles , Femenino , Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Estadificación de Neoplasias , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Survivin , Proteína con Dedos de Zinc GLI1/biosíntesis , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Activation of the Hedgehog (Hh) pathway effector GLI1 is linked to tumorigenesis and invasiveness in a number of cancers, with targeting of GLI1 by small molecule antagonists shown to be effective. We profiled a collection of GLI antagonists possessing distinct mechanisms of action for efficacy in phenotypic models of inflammatory and non-inflammatory breast cancer (IBC and non-IBC) that we showed expressed varying levels of Hh pathway mediators. Compounds GANT61, HPI-1, and JK184 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in TN-IBC (SUM149) and TNBC (MDA-MB-231 and SUM159) cell lines. In addition, GANT61 and JK184 significantly down-regulated GLI1 targets that regulate cell cycle (cyclin D and E) and apoptosis (Bcl2). GANT61 reduced SUM149 spheroid growth and emboli formation, and in orthotopic SUM149 tumor models significantly decreased tumor growth. We successfully utilized phenotypic profiling to identify a subset of GLI1 antagonists that were prioritized for testing in in vivo models. Our results indicated that GLI1 activation in TN-IBC as in TNBC, plays a vital role in promoting cell proliferation, motility, tumor growth, and formation of tumor emboli.
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Compuestos Heterocíclicos con 2 Anillos/farmacología , Neoplasias Inflamatorias de la Mama/tratamiento farmacológico , Piridinas/farmacología , Pirimidinas/farmacología , Tiazoles/farmacología , Proteína con Dedos de Zinc GLI1/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Neoplasias Inflamatorias de la Mama/metabolismo , Neoplasias Inflamatorias de la Mama/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nonmelanoma skin cancer is the most common cancer in humans, comprising mainly basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). BCC proliferation is highly dependent on the Hedgehog signaling pathway. We aimed to investigate a panel of anticancer drugs with known activity against skin cancer for their therapeutic potential in localized, enhanced topical treatment of SCC and BCC. Cytotoxicity profiles for vismodegib, 5-fluorouracil (5-FU), methotrexate (MTX), cisplatin, bleomycin, and vorinostat were established in terms of half maximal inhibitory concentration values in a panel of immortalized keratinocytes (HaCaT), BCC (UWBCC1 and BCC77015), and SCC (A431 and SCC25) cell lines. The impact of treatment on the regulation of Hedgehog pathway target genes (GLI1 and PTCH1), measured by real-time PCR, was compared between UWBCC1 and HaCaT. Varying cell line sensitivity profiles to the examined anticancer drugs were observed. Generally, 24-h drug exposure was sufficient to reduce cell viability. We found that 5-FU, MTX, and cisplatin significantly downregulated the expression of two genes controlled by the Hedgehog pathway (≤25-, 2.9-, and 12.5-fold, respectively, for GLI1 in UWBCC1 cells at 48 h, P<0.0001). The gene regulation showed clear concentration dependence and correlated with cytotoxicity for both 5-FU and MTX. We find a potential for the use of anticancer drugs in localized and enhanced topical treatment of nonmelanoma skin cancer. Of importance in the clinical setting, 24-h drug exposure may be sufficient for significant cytotoxicity for vismodegib, 5-FU, cisplatin, and bleomycin. MTX, 5-FU, and cisplatin may offer particular promise through combined cytotoxicity and downregulation of Hedgehog pathway genes GLI1 and PTCH1.
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Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptor Patched-1/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Proteína con Dedos de Zinc GLI1/genética , Anilidas/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Fluorouracilo/farmacología , Proteínas Hedgehog/metabolismo , Humanos , Metotrexato/farmacología , Receptor Patched-1/biosíntesis , Piridinas/farmacología , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Proteína con Dedos de Zinc GLI1/biosíntesisRESUMEN
In damaged kidneys, increased extracellular matrix (ECM) and tissue stiffness stimulate kidney fibrosis through incompletely characterized molecular mechanisms. The transcriptional coactivators yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz) function as mechanosensors in cancer cells and have been implicated in the regulation of myofibroblasts in the kidney. We hypothesized that the development of kidney fibrosis depends on Yap-induced activation and proliferation of kidney fibroblasts. In mice, Yap expression increased in renal fibroblasts after unilateral ureteral obstruction (UUO), in association with worsening of interstitial fibrosis. In cultured fibroblasts, inhibition of Yap/Taz signaling blocked TGF-ß1-induced fibroblast-to-myofibroblast transformation and ECM production, whereas constitutive activation of Yap promoted fibroblast transformation and ECM production even in the absence of TGF-ß1. Moreover, in the absence of TGF-ß1, fibroblasts seeded on a stiffened ECM transformed into myofibroblasts in a process dependent on the activation of Yap. In mice with UUO, the Yap inhibitor verteporfin reduced interstitial fibrosis. Furthermore, Gli1+ cell-specific knockout of Yap/Taz in mice suppressed UUO-induced ECM deposition, myofibroblast accumulation, and interstitial fibrosis. In a UUO-release model, induction of Gli1+ cell-specific Yap/Taz knockout partially reversed the development of interstitial fibrosis. Thus, in the kidney, Yap is a tissue mechanosensor that can be activated by ECM and transforms fibroblasts into myofibroblasts; the interaction of Yap/Taz and ECM forms a feed-forward loop resulting in kidney fibrosis. Identifying mechanisms that interrupt this profibrotic cycle could lead to the development of anti-fibrosis therapy.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Riñón/patología , Miofibroblastos , Fosfoproteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Matriz Extracelular , Fibroblastos/fisiología , Fibrosis/etiología , Eliminación de Gen , Masculino , Ratones , Miofibroblastos/metabolismo , Fosfoproteínas/genética , Transactivadores , Obstrucción Ureteral/patología , Proteínas Señalizadoras YAP , Proteína con Dedos de Zinc GLI1/biosíntesisRESUMEN
Previous studies report aberrant activation of the hedgehog signaling pathway in the progression of various cancers. This study aimed to investigate the expressions of smoothened and downstream glioma-associated oncogene homology-1 in gastric cancer and the underlying molecular mechanisms. Here, we first detected the expression in 58 cases of primary gastric cancer tissue and matched normal tissue specimens by western blot analysis and quantitative reverse transcription polymerase chain reaction. Cell proliferation and cycle were assayed in gastric cancer cells after blocking the hedgehog pathway by lentiviral-short hairpin RNA knockdown. In vitro inhibition of hedgehog pathway resulted in decreased cell proliferation and migration. Our studies demonstrate an important role for smoothened and glioma-associated oncogene homology-1 in gastric cancer and suggest inhibition of hedgehog pathway as a novel and potent strategy to treat gastric cancer patients.
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Receptor Smoothened/genética , Neoplasias Gástricas/genética , Proteína con Dedos de Zinc GLI1/genética , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Hedgehog/genética , Humanos , Lentivirus/genética , Masculino , Transducción de Señal , Receptor Smoothened/biosíntesis , Neoplasias Gástricas/patología , Proteína con Dedos de Zinc GLI1/biosíntesisRESUMEN
Complement components have been implicated in a wide variety of functions including neurogenesis, proliferation, cell migration, differentiation, cancer, and more recently early development and regeneration. Following our initial observations indicating that C3a/C3aR signaling induces chick retina regeneration, we analyzed its role in chick eye morphogenesis. During eye development, the optic vesicle (OV) invaginates to generate a bilayer optic cup (OC) that gives rise to the retinal pigmented epithelium (RPE) and neural retina. We show by immunofluorescence staining that C3 and the receptor for C3a (the cleaved and active form of C3), C3aR, are present in chick embryos during eye morphogenesis in the OV and OC. Interestingly, C3aR is mainly localized in the nuclear compartment at the OC stage. Loss of function studies at the OV stage using morpholinos or a blocking antibody targeting the C3aR (anti-C3aR Ab), causes eye defects such as microphthalmia and defects in the ventral portion of the eye that result in coloboma. Such defects were not observed when C3aR was disrupted at the OC stage. Histological analysis demonstrated that microphthalmic eyes were unable to generate a normal optic stalk or a closed OC. The dorsal/ventral patterning defects were accompanied by an expansion of the ventral markers Pax2, cVax and retinoic acid synthesizing enzyme raldh-3 (aldh1a3) domains, an absence of the dorsal expression of Tbx5 and raldh-1 (aldh1a1) and a re-specification of the ventral RPE to neuroepithelium. In addition, the eyes showed overall decreased expression of Gli1 and a change in distribution of nuclear ß-catenin, suggesting that Shh and Wnt pathways have been affected. Finally, we observed prominent cell death along with a decrease in proliferating cells, indicating that both processes contribute to the microphthalmic phenotype. Together our results show that C3aR is necessary for the proper morphogenesis of the OC. This is the first report implicating C3aR in eye development, revealing an unsuspected hitherto regulator for proper chick eye morphogenesis.
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Tipificación del Cuerpo/fisiología , Complemento C3a/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores de Complemento/metabolismo , Epitelio Pigmentado de la Retina/embriología , Aldehído Deshidrogenasa/metabolismo , Animales , Apoptosis/fisiología , Proliferación Celular/fisiología , Embrión de Pollo , Proteínas Hedgehog/metabolismo , Microftalmía/embriología , Morfogénesis/fisiología , Factor de Transcripción PAX2/metabolismo , Receptores de Complemento/genética , Retinal-Deshidrogenasa/metabolismo , Proteínas de Dominio T Box/metabolismo , Vía de Señalización Wnt/fisiología , Proteína con Dedos de Zinc GLI1/biosíntesis , beta Catenina/metabolismoRESUMEN
AIMS: Activation of hepatic stellate cells (HSCs) plays a pivotal role at the center of the fibrogenic progression in nonalcoholic steatohepatitis (NASH). However, it is poorly understood that how various molecules interact within HSCs during the progression of NASH to fibrosis. The aim of the present study is to delineate how inflammasome molecules, hedgehog signaling and autophagy provoke HSC activation using palmitic acid (PA) as a major insult. MAIN METHODS: Inflammasome activation, hedgehog signaling activity and autophagy in PA-exposed HSCs were determined to investigate their role in activation of human and rodent HSC lines or primary HSCs. KEY FINDINGS: PA treatment elicited HSC activation reflected by increased mRNA levels of transforming growth factor-ß1, connective tissue growth factor, tissue inhibitor of metalloproteinase-1 and procollagen type I (α1). In addition, expression levels of NOD-like receptor protein 3 (NLRP3) and hedgehog signaling transcription factor Gli-1 were increased in PA-exposed HSCs. It's evident that PA treatment resulted in increased production of light chain 3-II and autophagosomes, as well as enhanced autophagy flux reflected by transduction of an adeno-associated viral vector. Whereas, reduced autophagy, which is often seen in the late stage of NASH, provoked inflammasome activation. Moreover, suppressing the Hh signaling pathway by LDE225 blocked production of light chain 3-II and autophagy flux. SIGNIFICANCE: Saturated fatty acids, such as PA, stimulate HSC activation through inflammasomes and hedgehog signaling. Meanwhile, compromised autophagy may facilitate HSC activation, implicating valuable candidates for pharmacologic intervention against the progression of fibrogenesis in NASH.
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Proteínas Hedgehog/metabolismo , Células Estrelladas Hepáticas/metabolismo , Inflamasomas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Ácido Palmítico/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Células Estrelladas Hepáticas/patología , Humanos , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/biosíntesis , Enfermedad del Hígado Graso no Alcohólico/patología , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta1/biosíntesis , Proteína con Dedos de Zinc GLI1/biosíntesisRESUMEN
PURPOSE: Keratin 17 (KRT17) has been suggested as a potential diagnostic marker of squamous cell carcinoma including oral squamous cell carcinoma (OSCC). The current study was conducted to clarify the function of KRT17 and its expression mechanism in OSCC. METHODS: Immunohistochemical analyses were carried out to examine the expression of KRT17, GLI family zinc finger (GLI)-1, GLI-2, or cleaved caspase-3 in OSCCs. The expression of KRT17, GLI-1, or GLI-2 was investigated among OSCC cell lines, and the effects of loss-of-function of KRT17 or GLI, using siRNA or inhibitor, on the cell growth of the OSCC cell line HSC-2 particularly with respect to apoptosis were examined. RESULTS: Immunohistochemical analyses of tissue specimens obtained from 78 OSCC patients revealed that KRT17 was not observed in non-tumor regions but was strongly expressed at high frequencies in tumor regions. Knockdown of KRT17 increased the number of cleaved caspase-3-positive cells, leading to the reduction of cell number. Loss-of-function of GLI-1 or GLI-2 also increased the cell numbers of apoptotic cells positive for staining of Annexin-V and propidium iodide (PI) and the terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling (TUNEL) method, and induced DNA fragmentation. This inhibitory effect on cell growth was partially rescued by exogenous KRT17 expression. In the KRT17-positive regions in OSCCs, GLI-1 or GLI-2 was frequently detected, and the number of cells with cleaved caspase-3 positive was decreased. CONCLUSIONS: KRT17 promotes tumor cell growth, at least partially, through its anti-apoptotic effect as a result of the KRT17 overexpression by GLIs in OSCC.