RESUMEN
OBJECTIVE: Apart from the role of the retinoblastoma gene, the genomic events associated with poor outcomes in patients with ophthalmic tumors are poorly understood. METHODS: We retrospectively analyzed 48 patients with six types of ophthalmic tumors. We searched for high-frequency mutated genes and susceptibility genes in these patients using combined exome and transcriptome analysis. RESULTS: We identified four clearly causative genes (TP53, PTCH1, SMO, BAP1). Susceptibility gene analysis identified hotspot genes, including RUNX1, APC, IDH2, and BRCA2, and high-frequency gene analysis identified several genes, including TP53, TTN, and MUC16. Transcriptome analysis identified 5868 differentially expressed genes, of which TOP2A and ZWINT were upregulated in all samples, while CFD, ELANE, HBA1, and HBB were downregulated. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the phosphoinositide 3-kinase (PI3K)-Akt and Transcriptional misregulation in cancer signaling pathways may be involved in ophthalmic tumorigenesis. CONCLUSIONS: TP53 is clearly involved in ophthalmic tumorigenesis, especially in basal cell carcinoma, and the PI3K-Akt signaling pathway may be an essential pathway involved in ophthalmic tumorigenesis. RUNX1, SMO, TOP2A, and ZWINT are also highly likely to be involved in ophthalmic tumorigenesis, but further functional experiments are needed to verify the mechanisms of these genes in regulating tumorigenesis.
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Neoplasias del Ojo , Genómica , Mutación , Proteína p53 Supresora de Tumor , Humanos , Femenino , Masculino , Proteína p53 Supresora de Tumor/genética , Genómica/métodos , Neoplasias del Ojo/genética , Estudios Retrospectivos , Persona de Mediana Edad , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Receptor Patched-1/genética , Perfilación de la Expresión Génica , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas Supresoras de Tumor/genética , Anciano , Proteínas de Unión a Poli-ADP-Ribosa/genética , Adulto , Transducción de Señal/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Biomarcadores de Tumor/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ADN-Topoisomerasas de Tipo II , Ubiquitina Tiolesterasa , Subunidad alfa 2 del Factor de Unión al Sitio PrincipalRESUMEN
INTRODUCTION: Oral cancer poses a significant burden on public health in India, with higher incidence and mortality rates. Despite advancements in treatment modalities, prognosis remains poor due to factors such as localized recurrence and lymph node metastasis, potentially influenced by cancer stem cells. Among signaling pathways implicated in CSC regulation, the Hedgehog pathway plays a crucial role in oral squamous cell carcinoma (OSCC). MATERIAL & METHODS: 97 OSCC patients' tissue samples were collected and subjected to RNA isolation, cDNA synthesis and quantitative real-time PCR to analyze PTCH1 and SMO expression. Protein expression was assessed through immunohistochemistry. Clinicopathological parameters were correlated with gene and protein expression. Statistical analysis included Pearson chi-square tests, co-relation co-efficient tests, Kaplan-Meier survival analysis and ROC curve analysis. RESULTS: PTCH1 expression correlated with lymphatic permeation (p = 0.002) and tumor stage (p = 0.002), while SMO expression correlated with lymph node status (p = 0.034) and tumor stage (p = 0.021). PTCH1 gene expression correlated with lymph node status (p = 0.024). High PTCH1 gene expression was associated with shorter survival in tongue cancer patients. ROC curve analysis indicated diagnostic potential for PTCH1 and SMO gene and cytoplasmic SMO expression in distinguishing malignant tissues from adjacent normal tissues. CONCLUSION: PTCH1 and SMO play a crucial role in oral cancer progression, correlating with tumor stages and metastatic potential. Despite not directly influencing overall survival, PTCH1 expression at specific anatomical sites hints at its prognostic implications. PTCH1 and SMO exhibit diagnostic potential, suggesting their utility as molecular markers in oral cancer management and therapeutic strategies.
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Carcinoma de Células Escamosas , Neoplasias de la Boca , Receptor Patched-1 , Receptor Smoothened , Humanos , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/mortalidad , Neoplasias de la Boca/diagnóstico , Femenino , Masculino , Persona de Mediana Edad , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/diagnóstico , Adulto , Anciano , Regulación Neoplásica de la Expresión Génica , Pronóstico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Estimación de Kaplan-Meier , Curva ROC , Metástasis Linfática/genética , Estadificación de NeoplasiasRESUMEN
Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.
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Cilios , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Pulmón , Transducción de Señal , Animales , Ratones , Tipificación del Cuerpo/genética , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Pulmón/embriología , Pulmón/metabolismo , Morfogénesis/genética , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli2 con Dedos de Zinc/genéticaRESUMEN
The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we show that Spinal Cord Organoids (SCOs) can be applied to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. We validate this system by measuring the effects of mutating the HH receptor PTCH1 and the impact of HH agonists and antagonists on NPC specification. SCOs represent an accessible and reliable in-vitro tool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation.
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Proteínas Hedgehog , Organoides , Transducción de Señal , Médula Espinal , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Animales , Organoides/metabolismo , Organoides/citología , Médula Espinal/metabolismo , Médula Espinal/citología , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Receptor Patched-1/metabolismo , Receptor Patched-1/genéticaRESUMEN
The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.
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Proteínas Hedgehog , Transducción de Señal , Papilas Gustativas , Lengua , Animales , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Lengua/metabolismo , Lengua/crecimiento & desarrollo , Ratones , Papilas Gustativas/metabolismo , Papilas Gustativas/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Homeostasis , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli2 con Dedos de Zinc/genética , Proteína Gli3 con Dedos de Zinc/metabolismo , Proteína Gli3 con Dedos de Zinc/genética , Proteínas del Tejido Nervioso , Proteínas de Ciclo Celular , Proteínas Ligadas a GPIRESUMEN
The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 µg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.
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Enterotoxinas , Proteínas Hedgehog , Transducción de Señal , Linfocitos T , Timo , Animales , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Femenino , Embarazo , Ratas , Timo/metabolismo , Timo/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Efectos Tardíos de la Exposición Prenatal/inmunología , Diferenciación Celular/efectos de los fármacos , Ratas Sprague-Dawley , MasculinoRESUMEN
BACKGROUND: Sonic Hedgehog (SHH) is a fundamental signaling pathway that controls tissue reconstruction, stem cell biology, and differentiation and has a role in gut tissue homeostasis and development. Dysregulation of SHH leads to the development of HCC. METHODS, AND RESULTS: The present study was conducted to compare the effects of mesenchymal stem cells (MSCs) and curcumin on SHH molecular targets in an experimental model of HCC in rats. One hundred rats were divided equally into the following groups: control group, HCC group, HCC group received MSCs, HCC group received curcumin, and HCC group received MSCs and curcumin. Histopathological examinations were performed, and gene expression of SHH signaling target genes (SHH, PTCH1, SMOH, and GLI1) was assessed by real-time PCR in rat liver tissue. Results showed that SHH target genes were significantly upregulated in HCC-untreated rat groups and in MSC-treated groups, with no significant difference between them. Administration of curcumin with or without combined administration of MSCs led to a significant down-regulation of SHH target genes, with no significant differences between both groups. As regards the histopathological examination of liver tissues, both curcumin and MSCs, either through separate use or their combined use, led to a significant restoration of normal liver pathology. CONCLUSIONS: In conclusion, SHH signaling is upregulated in the HCC experimental model. MSCs do not inhibit the upregulated SHH target genes in HCC. Curcumin use with or without MSCs administration led to a significant down-regulation of SHH signaling in HCC and a significant restoration of normal liver pathology.
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Carcinoma Hepatocelular , Curcumina , Proteínas Hedgehog , Neoplasias Hepáticas , Células Madre Mesenquimatosas , Transducción de Señal , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Animales , Curcumina/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Transducción de Señal/efectos de los fármacos , Ratas , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Trasplante de Células Madre Mesenquimatosas/métodos , Masculino , Modelos Animales de Enfermedad , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Hígado/efectos de los fármacosRESUMEN
Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.
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Neuronas Dopaminérgicas , Proteínas Hedgehog , Células Madre Pluripotentes Inducidas , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad de Parkinson , Receptor Patched-1 , Proteína con Dedos de Zinc GLI1 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Neuronas Dopaminérgicas/metabolismo , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , AMP Cíclico/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Mutación/genética , Calcio/metabolismo , Diferenciación Celular/genética , Transducción de Señal/genéticaRESUMEN
Tumors of the head and neck, more specifically the squamous cell carcinoma, often show upregulation of the Hedgehog signaling pathway. However, almost nothing is known about its role in the sinonasal adenocarcinoma, either in intestinal or non-intestinal subtypes. In this work, we have analyzed immunohistochemical staining of six Hedgehog pathway proteins, sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched1 (PTCH1), Gli family zinc finger 1 (GLI1), Gli family zinc finger 2 (GLI2), and Gli family zinc finger 3 (GLI3), on 21 samples of sinonasal adenocarcinoma and compared them with six colon adenocarcinoma and three salivary gland tumors, as well as with matching healthy tissue, where available. We have detected GLI2 and PTCH1 in the majority of samples and also GLI1 in a subset of samples, while GLI3 and the ligands SHH and IHH were generally not detected. PTCH1 pattern of staining shows an interesting pattern, where healthy samples are mostly positive in the stromal compartment, while the signal shifts to the tumor compartment in tumors. This, taken together with a stronger signal of GLI2 in tumors compared to non-tumor tissues, suggests that the Hedgehog pathway is indeed activated in sinonasal adenocarcinoma. As Hedgehog pathway inhibitors are being tested in combination with other therapies for head and neck squamous cell carcinoma, this could provide a therapeutic option for patients with sinonasal adenocarcinoma as well.
Asunto(s)
Adenocarcinoma , Proteínas Hedgehog , Inmunohistoquímica , Transducción de Señal , Proteína Gli2 con Dedos de Zinc , Humanos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Masculino , Femenino , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli2 con Dedos de Zinc/genética , Persona de Mediana Edad , Proyectos Piloto , Anciano , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Proteína Gli3 con Dedos de Zinc/metabolismo , Proteína Gli3 con Dedos de Zinc/genética , Neoplasias de los Senos Paranasales/metabolismo , Neoplasias de los Senos Paranasales/patología , Adulto , Regulación Neoplásica de la Expresión Génica , Proteínas del Tejido Nervioso , Proteínas NuclearesRESUMEN
Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, GLI1, and PTCH1 expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the GLI1 and PTCH1 gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.
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Diferenciación Celular , Proteínas Hedgehog , Neuroblastoma , Transducción de Señal , Tretinoina , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Humanos , Diferenciación Celular/efectos de los fármacos , Tretinoina/farmacología , Transducción de Señal/efectos de los fármacos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuroblastoma/genética , Línea Celular Tumoral , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones , Animales , Proteínas Supresoras de TumorRESUMEN
Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.
Asunto(s)
Cerebelo , Cilios , Proteínas Hedgehog , Proteínas del Tejido Nervioso , Receptor Patched-1 , Transducción de Señal , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Cilios/metabolismo , Animales , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Cerebelo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Endocitosis , Diferenciación Celular , Proliferación Celular , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Ratones NoqueadosRESUMEN
PURPOSE: The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth. METHODS: Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting. RESULTS: Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth. CONCLUSION: Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Receptores ErbB , Proteínas Hedgehog , Neoplasias Pulmonares , Estabilidad Proteica , Transducción de Señal , Humanos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ligandos , Animales , Línea Celular Tumoral , Transducción de Señal/genética , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Ratones Desnudos , Ratones , Regulación Neoplásica de la Expresión Génica , Femenino , Proliferación Celular/genética , Ubiquitinación , MasculinoRESUMEN
BACKGROUND AND PURPOSE: Medulloblastoma (MB) is a common primary brain cancer in children. Proton therapy in pediatric MB is intensively studied and widely adopted. Compared to photon, proton radiations offer potential for reduced toxicity due to the characteristic Bragg Peak at the end of their path in tissue. The aim of this study was to compare the effects of irradiation with the same dose of protons or photons in Patched1 heterozygous knockout mice, a murine model predisposed to cancer and non-cancer radiogenic pathologies, including MB and lens opacity. MATERIALS AND METHODS: TOP-IMPLART is a pulsed linear proton accelerator for proton therapy applications. We compared the long-term health effects of 3 Gy of protons or photons in neonatal mice exposed at postnatal day 2, during a peculiarly susceptible developmental phase of the cerebellum, lens, and hippocampus, to genotoxic stress. RESULTS: Experimental testing of the 5 mm Spread-Out Bragg Peak (SOBP) proton beam, through evaluation of apoptotic response, confirmed that both cerebellum and hippocampus were within the SOBP irradiation field. While no differences in MB induction were observed after irradiation with protons or photons, lens opacity examination confirmed sparing of the lens after proton exposure. Marked differences in expression of neurogenesis-related genes and in neuroinflammation, but not in hippocampal neurogenesis, were observed after irradiation of wild-type mice with both radiation types. CONCLUSION: In-vivo experiments with radiosensitive mouse models improve our mechanistic understanding of the dependence of brain damage on radiation quality, thus having important implications in translational research.
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Animales Recién Nacidos , Apoptosis , Hipocampo , Fotones , Terapia de Protones , Animales , Ratones , Apoptosis/efectos de la radiación , Terapia de Protones/efectos adversos , Hipocampo/efectos de la radiación , Meduloblastoma/radioterapia , Meduloblastoma/patología , Carcinogénesis/efectos de la radiación , Ratones Noqueados , Neoplasias Cerebelosas/radioterapia , Neoplasias Cerebelosas/patología , Encéfalo/efectos de la radiación , Receptor Patched-1/genética , Modelos Animales de Enfermedad , Protones/efectos adversosRESUMEN
A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.
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Diferenciación Celular , Cóclea , Receptor Patched-1 , Estría Vascular , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Animales , Ratones , Estría Vascular/metabolismo , Estría Vascular/citología , Cóclea/metabolismo , Cóclea/embriología , Cóclea/citología , Transducción de Señal , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli2 con Dedos de Zinc/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genéticaRESUMEN
Adult medulloblastoma (MB) is a rare disease affecting 0.6 persons per million adults over 19 years of age. The SHH-activated/TP53-wild type is the most common subtype, accounting for 60% of adult MBs, being characterized by mutations in PTCH1, SMO, or the TERT promoter. Several small studies demonstrate objective but short-lived responses to SMO inhibitors such as vismodegib or sonidegib. Like other oncogene-addicted solid tumors, detection of the corresponding drivers through liquid biopsy could aid in the molecular diagnosis and monitoring of the disease through less invasive procedures. However, most studies have only evaluated cerebrospinal fluid as the ctDNA reservoir, and very limited evidence exists on the role of liquid biopsy in plasma in patients with primary central nervous system tumors, including MB. We present the case of a 26-year-old patient with a recurrent MB, in which next-generation sequencing (FoundationOne CDx) revealed a mutation in PTCH1, allowing the patient to be treated with vismodegib in second line, resulting in a durable benefit lasting for 1 year. Using an in-house digital PCR probe, the PTCH1 mutation could be tracked in ctDNA during treatment with first-line chemotherapy and while on treatment with vismodegib, demonstrating a precise correlation with the radiological and clinical behavior of the disease.
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Anilidas , ADN Tumoral Circulante , Meduloblastoma , Mutación , Receptor Patched-1 , Piridinas , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Meduloblastoma/sangre , Meduloblastoma/patología , Piridinas/uso terapéutico , Receptor Patched-1/genética , Adulto , Anilidas/uso terapéutico , ADN Tumoral Circulante/genética , ADN Tumoral Circulante/sangre , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Neoplasias Cerebelosas/sangre , Masculino , FemeninoRESUMEN
Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.
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Neoplasias Cerebelosas , Células Madre Pluripotentes Inducidas , Meduloblastoma , Humanos , Ratones , Animales , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patología , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Proteínas Hedgehog/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Transformación Celular Neoplásica , Carcinogénesis/genética , Organoides/metabolismo , Receptores PatchedRESUMEN
Immune checkpoint inhibitors (ICIs) have become prominent therapies for gastrointestinal cancer (GC). However, it is urgent to screen patients who can benefit from ICIs. Protein patched homolog 1 (PTCH1) is a frequently altered gene in GC. We attempt to explore the association between PTCH1 mutation and immunotherapy efficacy. The Memorial Sloan Kettering Cancer Center (MSKCC) cohort (nâ =â 236) with GC (esophageal, gastric and colorectal cancers) patients receiving ICIs was used for discovery and the Peking University Cancer Hospital (PUCH) GC cohort (nâ =â 92) was used for validation. Overall survival (OS) and tumor mutational burden (TMB) of the PTCH1 mutant-type (PTCH1-MUT) and PTCH1 wild-type (PTCH1-WT) groups were compared. Furthermore, GC data were collected from The Cancer Genome Atlas to assess the potential mechanisms. In the MSKCC cohort, PTCH1-MUT group showed significantly better OS (Pâ =â 0.017) and higher TMB. Multivariate analysis showed that PTCH1 mutation was associated with better OS. In the PUCH cohort, PTCH1-MUT group showed significantly longer OS (Pâ =â 0.036) and progression-free survival, and higher durable clinical benefit and TMB. Immune cell infiltration analysis revealed that PTCH1-MUT group had significantly higher distributions of CD8 T cells, CD4 T cells, NK cells, mast cells and M1 cells. The PTCH1-MUT group showed significantly higher expression of most immune-related genes. Gene set enrichment analysis showed that the PTCH1-MUT group had enriched INF-γ response, INF-α response, glycolysis and reactive oxygen species pathway gene sets. PTCH1 mutation may represent a potential biomarker for predicting ICIs response in GC. Nevertheless, prospective cohort studies should be performed to further validate our results.
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Biomarcadores de Tumor , Neoplasias Gastrointestinales , Inhibidores de Puntos de Control Inmunológico , Mutación , Receptor Patched-1 , Humanos , Receptor Patched-1/genética , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Biomarcadores de Tumor/genética , Femenino , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/patología , Masculino , Persona de Mediana Edad , Anciano , Pronóstico , AdultoRESUMEN
The exploration of molecular genetic mechanisms that underlie carcinogenesis, hereditary factors of various oncological diseases, including basal cell carcinoma, the most common type of skin cancer is especially actual and significant for target strategies of public health. The diagnosis of basal cell carcinoma is based on complex clinical, radiologic and genetic examination data. The further research in the field of somatic or hereditary mutations in genes associated with basal cell carcinoma, including Patched 1 (PTCH1), Patched 2 (PTCH2), Smoothed (SMO) continue to be topical. The strategies of primary prevention of basal cell carcinoma, discussions of complex issues of decision-making concerning treatment at primary health care level, training courses and development of guidelines for general practitioners and interdisciplinary recommendations for effective early diagnosis and comprehensive care of basal cell carcinoma are to be suggested.
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Carcinoma Basocelular , Neoplasias Cutáneas , Humanos , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Carcinoma Basocelular/diagnóstico , Carcinoma Basocelular/genética , Carcinoma Basocelular/prevención & control , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/prevención & control , Biología MolecularRESUMEN
Patched homolog 1 (PTCH1) has been proven to facilitate cell proliferation and self-renewal in esophageal cancer (EC). The present study intended to exploit the influence of PTCH1 on EC cells and the potential mechanisms. PTCH1 and methyltransferase-like 3 (METTL3) expression were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in EC cell lines. Following the loss- and gain-of-function assays, cell proliferation was examined by cell counting kit (CCK)-8 and clone formation assays, invasion and migration by Transwell and scratch assays, and the sphere-forming ability of stem cells by cell sphere-forming assay. The expression of stemness genes NANOG homeobox protein (NANOG), octamer-binding transcription factor 4 (Oct4), and sex-determining region Y-box 2 (SOX2) was detected by Western blot. Methylated RNA immunoprecipitation (Me-RIP) assay was performed to test N6-methyladenosine (m6A) modification levels of PTCH1 mRNA, RIP and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) assays to assess the binding of METTL3 to PTCH1, and actinomycin D treatment to examine PTCH1 mRNA stability. A xenograft tumor model in nude mice was established for further in vivo verification. PTCH1 and METTL3 expression was high in EC cells. Knockdown of METTL3 reduced m6A level and stability of PTCH1 mRNA. Knockdown of PTCH1 or METTL3 declined invasion, proliferation, migration, and NANOG, Oct4, and SOX2 levels in EC cells, and reduced sphere-forming abilities of EC stem cells. Overexpression of PTCH1 abolished the suppressive effect of METTL3 knockdown on EC cells in vitro. METTL3 knockdown repressed tumor growth in nude mice, which was negated by further overexpressing PTCH1. METTL3 facilitated growth and stemness of EC cells via upregulation of PTCH1 expression by enhancing PTCH1 m6A modification.NEW & NOTEWORTHY PTCH1 has been proved to facilitate cell proliferation and self-renewal in esophageal cancer. We studied the upstream regulation mechanism of PTCH1 by METTL3 through m6A modification. Our results provide a new target and theoretical basis for the treatment of esophageal cancer.
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Neoplasias Esofágicas , Metiltransferasas , Ratones , Animales , Humanos , Metilación , Metiltransferasas/genética , Ratones Desnudos , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Células Madre/metabolismo , Neoplasias Esofágicas/genética , ARN Mensajero/genéticaRESUMEN
Two male patients with bifid rib-basal cell nevus-jaw cyst syndrome (BCNS) were admitted to Department of Stomatology, the First Affiliated Hospital of Bengbu Medical College due to radiological findings of multiple low density shadows in the jaw. Clinical and imaging findings showed thoracic malformation, calcification of the tentorium cerebellum and falx cerebrum as well as widening of the orbital distance. Whole exon high-throughput sequencing was performed in two patients and their family members. The heterozygous mutations of c.C2541C>A(p.Y847X) and c.C1501C>T(p.Q501X) in PTCH1 gene were detected in both patients. Diagnosis of BCNS was confirmed. The heterozygous mutations of PTCH1 gene locus were also found in the mothers of the two probands. Proband 1 showed clinical manifestations of low intelligence, and heterozygous mutations of c.C2141T(p.P714L) and c.G3343A(p.V1115I) were detected in FANCD2 gene. Proband 2 had normal intelligence and no FANCD2 mutation. The fenestration decompression and curettage of jaw cyst were performed in both patients. Regular follow-up showed good bone growth at the original lesion, and no recurrence has been observed so far.