ABSTRACT
Background: Primary breast tumors with neuroendocrine (NE) differentiation are a heterogeneous tumor group with diversity of biological behavior, with poorly defined prevalence and prognosis. Objective: To evaluate the chromogranin, synaptophysin, CD56, INSM1 markers expression prevalence and the association between NE differentiation and tumor molecular type. Material and methods: Observational, cross-sectional study which included 110 breast tissue samples with primary invasive carcinoma. Immunohistochemistry was performed for chromogranin, synaptophysin, CD56 and INMS1 markers. NE differentiation was considered with 10-90% positive cells, and NE tumor with > 90% positive cells. Results: 26.3% showed neuroendocrine differentiation. Out of these, 48.2% were luminal-A type, 24.1% luminal-B, 11.5% HER2neu, 17.2% triple-negative; 1.8% were NE tumors. Tumors were marker positive, and out of these to chromogranin in 24.5%, synaptophysin in 28.2%, CD56 in 2.7%, INSM1 in 16.4%. Synaptophysin was expressed in 17.3% luminal-A type, 6.4% luminal-B, 0.9% HER2neu, 3.6% triple-negative. NE differentiation showed association with synaptophysin expression (r = 0.586, p = 0.0001). Conclusion: The NE differentiation prevalence was 26.3% in primary invasive breast cancers, with luminal-A molecular type predominance.
Introducción: los tumores primarios de mama con diferenciación neuroendócrina (NEBC por sus siglas en inglés) son un grupo heterogéneo de tumores con diversidad de comportamiento biológico, con prevalencia y pronóstico poco definido. Objetivo: evaluar la prevalencia de la expresión los marcadores cromogranina, sinaptofisina, CD56, INSM1 y la asociación entre la diferenciación neuroendócrina y el tipo molecular del tumor. Material y métodos: estudio observacional, transversal que incluyó 110 muestras de tejido mamario con carcinoma invasor primario. Se realizó inmunohistoquímica para los marcadores cromogranina, sinaptofisina, CD56 y INMS1. La presencia 10-90% de células positivas se consideró diferenciación neuroendócrina y tumor neuroendócrino con > 90% de células positivas. Resultados: el 26.3% mostró diferenciación neuroendócrina. De estos, 48.2% fueron tipo luminal-A, 24.1% luminal-B, 11.5% HER2neu y 17.2% triple-negativo; 1.8% resultaron tumores neuroendócrinos. Los tumores presentaron marcadores positivos y de estos, 24.5% fueron a cromogranina, 28.2% a sinaptofisina, 2.7% a CD56 y 16.4% a INSM1. La sinaptofisina se expresó en 17.3% del tipo luminal-A, 6.4% luminal-B, 0.9% HER2neu, 3.6% triple-negativo. La diferenciación neuroendócrina mostró asociación con la expresión de sinaptofisina (r = 0.586, p = 0.0001). Conclusión: la prevalencia de la diferenciación neuroendócrina fue del 26.3% en los cánceres invasores primarios de mama, con predominio en el tipo molecular luminal-A.
Subject(s)
Biomarkers, Tumor , Synaptophysin , Humans , Female , Cross-Sectional Studies , Biomarkers, Tumor/metabolism , Middle Aged , Adult , Synaptophysin/metabolism , Aged , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , CD56 Antigen/metabolism , Immunohistochemistry , Repressor Proteins/metabolism , Chromogranins/metabolism , Receptor, ErbB-2/metabolism , Aged, 80 and overABSTRACT
Approximately 30%-40% of growth hormone-secreting pituitary adenomas (GHPAs) harbor somatic activating mutations in GNAS (α subunit of stimulatory G protein). Mutations in GNAS are associated with clinical features of smaller and less invasive tumors. However, the role of GNAS mutations in the invasiveness of GHPAs is unclear. GNAS mutations were detected in GHPAs using a standard polymerase chain reaction (PCR) sequencing procedure. The expression of mutation-associated maternally expressed gene 3 (MEG3) was evaluated with RT-qPCR. MEG3 was manipulated in GH3 cells using a lentiviral expression system. Cell invasion ability was measured using a Transwell assay, and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by immunofluorescence and western blotting. Finally, a tumor cell xenograft mouse model was used to verify the effect of MEG3 on tumor growth and invasiveness. The invasiveness of GHPAs was significantly decreased in mice with mutated GNAS compared with that in mice with wild-type GNAS. Consistently, the invasiveness of mutant GNAS-expressing GH3 cells decreased. MEG3 is uniquely expressed at high levels in GHPAs harboring mutated GNAS. Accordingly, MEG3 upregulation inhibited tumor cell invasion, and conversely, MEG3 downregulation increased tumor cell invasion. Mechanistically, GNAS mutations inhibit EMT in GHPAs. MEG3 in mutated GNAS cells prevented cell invasion through the inactivation of the Wnt/ß-catenin signaling pathway, which was further validated in vivo. Our data suggest that GNAS mutations may suppress cell invasion in GHPAs by regulating EMT through the activation of the MEG3/Wnt/ß-catenin signaling pathway.
Subject(s)
Chromogranins , Epithelial-Mesenchymal Transition , GTP-Binding Protein alpha Subunits, Gs , Growth Hormone-Secreting Pituitary Adenoma , Mutation , Neoplasm Invasiveness , RNA, Long Noncoding , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Animals , Humans , Growth Hormone-Secreting Pituitary Adenoma/genetics , Growth Hormone-Secreting Pituitary Adenoma/pathology , Growth Hormone-Secreting Pituitary Adenoma/metabolism , Mice , Chromogranins/genetics , Chromogranins/metabolism , Epithelial-Mesenchymal Transition/genetics , RNA, Long Noncoding/genetics , Female , Male , Cell Line, Tumor , Adenoma/genetics , Adenoma/pathology , Adenoma/metabolism , Middle Aged , Adult , Cell Proliferation/genetics , Xenograft Model Antitumor Assays , Wnt Signaling Pathway/genetics , Gene Expression Regulation, NeoplasticABSTRACT
Keloids are pathological scar tissue resulting from skin trauma or spontaneous formation, often accompanied by itching and pain. Although GNAS antisense RNA 1 (GNAS-AS1) shows abnormal upregulation in keloids, the underlying molecular mechanism is unclear. The levels of genes and proteins in clinical tissues from patients with keloids and human keloid fibroblasts (HKFs) were measured using quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assay. The features of HKFs, including proliferation and migration, were evaluated using cell counting kit 8 and a wound healing assay. The colocalization of GNAS-AS1 and miR-196a-5p in HKFs was measured using fluorescence in situ hybridization. The relationships among GNAS-AS1, miR-196a-5p and C-X-C motif chemokine ligand 12 (CXCL12) in samples from patients with keloids were analysed by Pearson correlation analysis. Gene interactions were validated by chromatin immunoprecipitation and luciferase reporter assays. GNAS-AS1 and CXCL12 expression were upregulated and miR-196a-5p expression was downregulated in clinical tissues from patients with keloids. GNAS-AS1 knockdown inhibited proliferation, migration, and extracellular matrix (ECM) accumulation of HKFs, all of which were reversed by miR-196a-5p downregulation. Signal transducer and activator of transcription 3 (STAT3) induced GNAS-AS1 transcription through GNAS-AS1 promoter interaction, and niclosamide, a STAT3 inhibitor, decreased GNAS-AS1 expression. GNAS-AS1 positively regulated CXCL12 by sponging miR-196-5p. Furthermore, CXCL12 knockdown restrained STAT3 phosphorylation in HKFs. Our findings revealed a feedback loop of STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3 that promoted HKF proliferation, migration and ECM accumulation and affected keloid progression.
Subject(s)
Cell Proliferation , Chemokine CXCL12 , Fibroblasts , Keloid , MicroRNAs , RNA, Long Noncoding , STAT3 Transcription Factor , Keloid/metabolism , Keloid/genetics , Keloid/pathology , Humans , MicroRNAs/metabolism , MicroRNAs/genetics , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Chemokine CXCL12/metabolism , Chemokine CXCL12/genetics , Fibroblasts/metabolism , Cell Movement , Feedback, Physiological , Chromogranins/genetics , Chromogranins/metabolism , Male , Female , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Signal Transduction , Adult , Cells, Cultured , Up-RegulationABSTRACT
The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell-cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.
Subject(s)
Adrenal Gland Neoplasms , Pheochromocytoma , Humans , Adrenal Glands/metabolism , Catecholamines/metabolism , Chromogranins/metabolismABSTRACT
IMPLICATIONS: The study identifies an opportunity to discover a PKA-independent pathway downstream of oncogene GNAS for managing IPMN lesions and their progression to PDAC.
Subject(s)
Cell Proliferation , Chromogranins , Cyclic AMP-Dependent Protein Kinases , GTP-Binding Protein alpha Subunits, Gs , Organoids , Pancreatic Neoplasms , Humans , GTP-Binding Protein alpha Subunits, Gs/metabolism , GTP-Binding Protein alpha Subunits, Gs/genetics , Organoids/metabolism , Organoids/pathology , Chromogranins/genetics , Chromogranins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Pancreatic Ducts/metabolism , Pancreatic Ducts/pathology , Pancreatic Ducts/cytology , Acinar Cells/metabolism , Acinar Cells/pathologyABSTRACT
Approximately 5% of colorectal cancers (CRCs) have a gain-of-function mutation in the GNAS gene, which leads to the activation of cAMP-dependent signaling pathways and associates with poor prognosis. We investigated the effect of an activating GNAS mutation in CRC cell lines on gene expression and cell proliferation in vitro, and tumor growth in vivo. GNAS-mutated (GNASmt) HCT116 cells showed stimulated synthesis of cAMP as compared to parental (Par) cells. The most upregulated gene in the GNASmt cells was cAMP-hydrolyzing phosphodiesterase 4D (PDE4D) as detected by RNA sequencing. To further validate our finding, we analyzed PDE4D expression in a set of human CRC tumors (n = 35) and demonstrated overexpression in GNAS mutant CRC tumors as compared to GNAS wild-type tumors. The GNASmt HCT116 cells proliferated more slowly than the Par cells. PDE4 inhibitor Ro 20-1724 and PDE4D subtype selective inhibitor GEBR-7b further suppressed the proliferation of GNASmt cells without an effect on Par cells. The growth inhibitory effect of these inhibitors was also seen in the intrinsically GNAS-mutated SK-CO-1 CRC cell line having high levels of cAMP synthesis and PDE4D expression. In vivo, GNASmt HCT116 cells formed smaller tumors than the Par cells in nude mice. In conclusion, our findings demonstrate that GNAS mutation results in the growth suppression of CRC cells. Moreover, the GNAS mutation-induced overexpression of PDE4D provides a potential avenue to impede the proliferation of CRC cells through the use of PDE4 inhibitors.
Subject(s)
Chromogranins , Colorectal Neoplasms , Cyclic Nucleotide Phosphodiesterases, Type 4 , GTP-Binding Protein alpha Subunits, Gs , Animals , Humans , Mice , Chromogranins/genetics , Chromogranins/metabolism , Colorectal Neoplasms/genetics , Cyclic Nucleotide Phosphodiesterases, Type 4/genetics , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , HCT116 Cells , Mice, Nude , Mutation , Phosphodiesterase 4 Inhibitors/pharmacologyABSTRACT
The chromogranin-secretogranin secretory proteins-granins-are acidic proteins localized in granules of endocrine cells and neurons. The chromogranin family includes chromogranins A (CgA) and B, as well as secretogranin II (once called chromogranin C). Members of this family undergo catalytic proteolysis to produce active peptides. The CgA-derived peptides vasostatin-1 and vasostatin-2, in particular, appear to protect against atherosclerosis, suppressing the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, as well as exerting vasodilatory effects by enhancing nitric oxide bioavailability. Vasostatin-1 also suppresses vasoconstriction and abnormal angiogenesis. Vasostatin-1 and vasostatin-2 may be novel therapeutic targets for atherosclerosis and coronary heart disease, also protecting the myocardium against ischaemic damage.
Subject(s)
Atherosclerosis , Calreticulin , Chromogranins , Peptide Fragments , Humans , Chromogranins/chemistry , Chromogranins/metabolism , Angiogenesis , Proteins/metabolism , PeptidesABSTRACT
Several human disorders are caused by genetic or epigenetic changes involving the GNAS locus on chromosome 20q13.3 that encodes the alpha-subunit of the stimulatory G protein (Gsα) and several splice variants thereof. Thus, pseudohypoparathyroidism type Ia (PHP1A) is caused by heterozygous inactivating mutations involving the maternal GNAS exons 1-13 resulting in characteristic abnormalities referred to as Albright's hereditary osteodystrophy (AHO) that are associated with resistance to several agonist ligands, particularly to parathyroid hormone (PTH), thereby leading to hypocalcemia and hyperphosphatemia. GNAS mutations involving the paternal Gsα exons also cause most of these AHO features, but without evidence for hormonal resistance, hence the term pseudopseudohypoparathyroidism (PPHP). Autosomal dominant pseudohypoparathyroidism type Ib (PHP1B) due to maternal GNAS or STX16 mutations (deletions, duplications, insertions, and inversions) is associated with epigenetic changes at one or several differentially methylated regions (DMRs) within GNAS. Unlike the inactivating Gsα mutations that cause PHP1A and PPHP, hormonal resistance is caused in all PHP1B variants by impaired Gsα expression due to loss of methylation at GNAS exon A/B, which can be associated in some familial cases with epigenetic changes at the other maternal GNAS DMRs. The genetic defect(s) responsible for sporadic PHP1B, the most frequent variant of this disorder, remain(s) unknown for the majority of patients. However, characteristic epigenetic GNAS changes can be readily detected that include a gain of methylation at the neuroendocrine secretory protein (NESP) DMR. Multiple genetic or epigenetic GNAS abnormalities can thus impair Gsα function or expression, consequently leading to inadequate cAMP-dependent signaling events downstream of various Gsα-coupled receptors.
Subject(s)
Chromogranins , Pseudohypoparathyroidism , Humans , Chromogranins/genetics , Chromogranins/metabolism , Pseudohypoparathyroidism/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Epigenesis, Genetic , DNA MethylationABSTRACT
BACKGROUND: Additional studies may be needed when evaluating parathyroid fine needle aspirates (FNA) due to cytomorphologic similarities. The liquid-based cytology (LBC) is an effective cytology method which provides additional slides for ancillary techniques. We aimed to examine the immunocytochemical (ICC) GATA 3, TTF-1, which chromogranin and PTH expressions and to determine their diagnostic importance in parathyroid FNAs which were prepared via LBC technique. METHODS: In total, 45 parathyroid FNA cases were examined in our laboratory for 12-month period. Thirty five cases without cell blocks were included in the study. Ten cases were excluded from study (five cases were nondiagnostic, five cases had cell blocks). For main LBC cytology slides SurePath pap test kit was used. For ICC study 4 PAP-stained LBC slides were obtained from each case's residual material. The slides were then stained with GATA 3, TTF-1, PTH and chromogranin. RESULTS: Of 35 cases, 8 were male and 27 were female. The mean age of the cases was 52.25 ± 11.44 (range 27-72). The mean diameter of the lesions was 18.54 ± 7.66 mm (range 6-32 mm).The most used antibody was TTF-1 (in all 35 cases). The positivity rates were 84.8%, 83.9% and 81.8% for GATA 3, PTH and chromogranin, respectively. TTF-1 was completely negative in 100% of cases. All patients had high PTH washout values (29 patients, PTH washout value was >5000 pg/mL). In 30 cases, histopathologic follow-up was available. The cyto-histological correlation was 100%. Based on the PTH washout values for cases without resection, the diagnostic accuracy of FNA was determined as 100% in detecting the parathyroid origin. Among the antibodies, the most specific and sensitive antibody was TTF-1 (100%). The sensitivity values of the antibodies indicating parathyroid origin for GATA3, PTH and chromogranin were 86.67%, 83.33% and 81.82%, respectively. CONCLUSION: The addition of ICC studies applied to LBC slides is also very helpful in diagnosis, especially in FNAs with limited material such as parathyroid. GATA3 is a more reliable ICC marker determining parathyroid origin. In differentiation from thyroid origin, the addition of TTF-1 to the ICC panel increases the diagnostic accuracy to 96.67%. Although PTH and chromogranin have lower sensitivity, they are still reliable markers to detect parathyroid origin.
Subject(s)
Chromogranins , Parathyroid Hormone , Humans , Male , Female , Chromogranins/metabolism , Biopsy, Fine-Needle/methods , Parathyroid Hormone/metabolism , Parathyroid Glands/pathology , Thyroid Gland/pathologyABSTRACT
PURPOSE OF REVIEW: Pseudohypoparathyroidism (PHP) is a disorder caused by mutations and/or epigenetic changes at the complex GNAS locus. It is characterized by hypocalcemia, hyperphosphatemia, and an elevated parathyroid hormone concentration secondary to the resistance of target tissues to the biological actions of parathyroid hormone. PHP is divided into several subtypes with different yet overlapping phenotypes. Research on the bone status in patients with PHP is sparse and has yielded inconsistent results. This review was performed to summarize the current knowledge on the bone phenotypes and possible mechanisms of PHP. RECENT FINDINGS: Patients with PHP exhibit highly variable bone phenotypes and increased concentrations of bone turnover markers. Long-standing elevation of the parathyroid hormone concentration may lead to hyperparathyroid bone diseases, including rickets and osteitis fibrosa. Compared with normal controls, patients with PHP may exhibit similar, increased, or decreased bone mineral density. Higher bone mineral density has been found in patients with PHP type 1A than in normal controls, whereas decreased bone mass, osteosclerosis, and osteitis fibrosa cystica have been reported in patients with PHP type 1B, indicating more variable bone phenotypes in PHP type 1B. Bone tissues show partial sensitivity to parathyroid hormone in patients with PHP, leading to heterogeneous reactions to parathyroid hormone in different individuals and even in different regions of bone tissues in the same individual. Regions rich in cancellous bone are more sensitive and show more obvious improvement after therapy. Active vitamin D and calcium can significantly improve abnormal bone metabolism in patients with PHP.
Subject(s)
Bone Diseases , Pseudohypoparathyroidism , Humans , Bone and Bones/metabolism , Pseudohypoparathyroidism/genetics , Pseudohypoparathyroidism/complications , Pseudohypoparathyroidism/metabolism , Parathyroid Hormone/metabolism , Bone Diseases/complications , Phenotype , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Chromogranins/genetics , Chromogranins/metabolismABSTRACT
The granin neuropeptide family is composed of acidic secretory signaling molecules that act throughout the nervous system to help modulate synaptic signaling and neural activity. Granin neuropeptides have been shown to be dysregulated in different forms of dementia, including Alzheimer's disease (AD). Recent studies have suggested that the granin neuropeptides and their protease-cleaved bioactive peptides (proteoforms) may act as both powerful drivers of gene expression and as a biomarker of synaptic health in AD. The complexity of granin proteoforms in human cerebrospinal fluid (CSF) and brain tissue has not been directly addressed. We developed a reliable nontryptic mass spectrometry assay to comprehensively map and quantify endogenous neuropeptide proteoforms in the brain and CSF of individuals diagnosed with mild cognitive impairment and dementia due to AD compared to healthy controls, individuals with preserved cognition despite AD pathology ("Resilient"), and those with impaired cognition but no AD or other discernible pathology ("Frail"). We drew associations between neuropeptide proteoforms, cognitive status, and AD pathology values. Decreased levels of VGF proteoforms were observed in CSF and brain tissue from individuals with AD compared to controls, while select proteoforms from chromogranin A showed the opposite effect. To address mechanisms of neuropeptide proteoform regulation, we showed that the proteases Calpain-1 and Cathepsin S can cleave chromogranin A, secretogranin-1, and VGF into proteoforms found in both the brain and CSF. We were unable to demonstrate differences in protease abundance in protein extracts from matched brains, suggesting that regulation may occur at the level of transcription.
Subject(s)
Alzheimer Disease , Neuropeptides , Humans , Alzheimer Disease/pathology , Chromogranins/metabolism , Chromogranin A/metabolism , Peptide Fragments/metabolism , Neuropeptides/metabolism , Brain/metabolism , Biomarkers , Peptide Hydrolases/metabolism , Amyloid beta-Peptides/metabolismABSTRACT
Pregnancy is a state of physiological and hormonal changes. One of the endocrine factors involved in these processes is chromogranin A, an acidic protein produced, among others, by the placenta. Although it has been previously linked to pregnancy, no existing articles have ever managed to clarify the role of this protein regarding this subject. Therefore, the aim of the present study is to gather knowledge of chromogranin A's function with reference to gestation and parturition, clarify elusive information, and, most importantly, to formulate hypotheses for the future studies to verify.
Subject(s)
Chromogranins , Endocrine System , Pregnancy , Female , Humans , Chromogranin A/metabolism , Chromogranins/metabolism , Endocrine System/metabolism , Parturition , Placenta/metabolism , Peptide Fragments/metabolismABSTRACT
Genetic defects of GNAS, the imprinted gene encoding the stimulatory G protein α-subunit, are responsible for multiple diseases. Abnormal GNAS imprinting causes pseudohypoparathyroidism type 1B (PHP1B), a prototype of mammalian end-organ hormone resistance. Hypomethylation at the maternally methylated GNAS A/B region is the only shared defect in patients with PHP1B. In autosomal dominant (AD) PHP1B kindreds, A/B hypomethylation is associated with maternal microdeletions at either the GNAS NESP55 differentially methylated region or the STX16 gene located approximately 170 kb upstream. Functional evidence is meager regarding the causality of these microdeletions. Moreover, the mechanisms linking A/B methylation and the putative imprinting control regions (ICRs) NESP-ICR and STX16-ICR remain unknown. Here, we generated a human embryonic stem cell model of AD-PHP1B by introducing ICR deletions using CRISPR/Cas9. With this model, we showed that the NESP-ICR is required for methylation and transcriptional silencing of A/B on the maternal allele. We also found that the SXT16-ICR is a long-range enhancer of NESP55 transcription, which originates from the maternal NESP-ICR. Furthermore, we demonstrated that the STX16-ICR is an embryonic stage-specific enhancer enabled by the direct binding of pluripotency factors. Our findings uncover an essential GNAS imprinting control mechanism and advance the molecular understanding of PHP1B pathogenesis.
Subject(s)
Chromogranins , Pseudohypoparathyroidism , Animals , Humans , Darbepoetin alfa/genetics , Darbepoetin alfa/metabolism , Chromogranins/genetics , Chromogranins/metabolism , Pseudohypoparathyroidism/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , DNA Methylation , Genomic Imprinting , Mammals/metabolismABSTRACT
BACKGROUND: Basal cell carcinoma (BCC) is the most common cutaneous malignancy and may show various differentiations. The possible pluripotent stem cell lineage of BCCs, whose origins are controversial today, is thought to be the main reason for the different morphologies. The aim of the study is to evaluate the expression of some neuroendocrine and smooth muscle markers of differentiation in BCCs and investigate the relationship between histopathologic subtypes and recurrence. METHODS: A total of 128 cases diagnosed as BCC in our center were included. Immunohistochemical studies of CD56, synaptophysin, chromogranin-A, smooth muscle actin (SMA), desmin, caldesmon, and Ki67 were applied. RESULTS: CD56, chromogranin-A, and synaptophysin immunoreactivity were detected in 77.3%, 13.3%, and 0.8% of the cases, respectively. 78.1% showed SMA positivity while no tumor expressed desmin or caldesmon. A correlation between histopathologic recurrence risk groups and CD56 expression was found (p < 0.05). CONCLUSIONS: CD56 and SMA immunoreactivity is present in the majority of BCCs. However, the available findings do not support neuroendocrine or smooth muscle differentiation. CD56 antigen can be used for prognostic purposes in detecting high recurrence risk tumors. After the investigation of the expression rates of these two antigens in different cutaneous tumors, it may be appropriate to use them for diagnostic purposes in BCCs.
Subject(s)
Carcinoma, Basal Cell , Skin Neoplasms , Humans , Synaptophysin/metabolism , Biomarkers, Tumor/metabolism , Actins/metabolism , Chromogranins/metabolism , Desmin/metabolism , Carcinoma, Basal Cell/pathology , Skin Neoplasms/pathology , CD56 Antigen , Muscle, Smooth/pathology , Calmodulin-Binding Proteins , Cell DifferentiationABSTRACT
Keloid is a common dermis tumor, occurring repeatedly, affecting the quality of patients' life. Long non-coding RNAs (lncRNAs) have crucial regulatory capacities in skin scarring formation and subsequent scar carcinogenesis. The intention of this study was to investigate the mechanism and function of GNAS antisense-1 (GNAS-AS1) in keloids. Clinical samples were collected to evaluate the expression of GNAS-AS1, RUNX2, and miR-188-5p by qRT-PCR. The proliferation, migration, and invasion of HKF cells were detected by CCK-8, wound healing, and Transwell assays. The expression levels of mRNA and protein were examined through qRT-PCR and Western blot assay. Luciferase reporter assay was used to identify the binding relationship among GNAS-AS1, miR-188-5p, and Runt-related transcription factor 2 (RUNX2). GNAS-AS1 and RUNX2 expressions were remarkably enhanced, and miR-188-5p expression was decreased in keloid clinical tissues and HKF cells. GNAS-AS1 overexpression promoted cells proliferation, migration, and invasion, while GNAS-AS1 knockdown had the opposite trend. Furthermore, overexpression of GNAS-AS1 reversed the inhibitory effect of 5-FU on cell proliferation, migration, and invasion. MiR-188-5p inhibition or RUNX2 overexpression could enhance the proliferation, migration, and invasion of HKF cells. GNAS-AS1 targeted miR-188-5p to regulate RUNX2 expression. In addition, the inhibition effects of GNAS-AS1 knockdown on HKF cells could be reversed by inhibition of miR-188-5p or overexpression of RUNX2, while RUNX2 overexpression eliminated the suppressive efficaciousness of miR-188-5p mimics on HKF cells growth. GNAS-AS1 knockdown could regulate the miR-188-5p/RUNX2 signaling axis to inhibit the growth and migration in keloid cells. It is suggested that GNAS-AS1 may become a new target for the prevention and treatment of keloid.
Subject(s)
Keloid , MicroRNAs , RNA, Long Noncoding , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Keloid/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Cell Movement/genetics , Gene Expression Regulation, Neoplastic , Chromogranins/genetics , Chromogranins/metabolism , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolismABSTRACT
BACKGROUND/AIM: Cholangioblastic variant of intrahepatic cholangiocarcinoma (CVICC) is an exceedingly rare primary biliary tract tumor and typically occurs in young patients with a median age of 24.5-year-old. It can mimic metastatic well-differentiated neuroendocrine tumors in the liver with its similar histologic and immunophenotypic features. CASE REPORT: We hereby report a CVICC in a 68-year-old female patient with distinctive biphasic cytologic features. The patient was diagnosed and treated as a metastatic well differentiated neuroendocrine tumor. The recurrent liver tumor was resected and the tumor cells were strongly positive for Inhibin A and cytokeratin 19 (CK19), focally and weakly positive for synaptophysin and chromogranin, and negative for Insulinoma associated protein 1 (INSM1). Ribonucleic acid (RNA) sequencing showed that the tumor bared a characteristic Nipped-B-like protein (NIPBL)-Nucleus accumbens-associated protein 1 (NACC1) gene fusion. CONCLUSION: To the best of our knowledge, this is the first documented case in an elder patient of this entity with NIPPL-NACC1 gene fusion. Acknowledgment of the biphasic cytology, screening with Inhibin A in suspicious cases, and coupled with a molecular study may facilitate accurate classification of this aggressive tumor and lead to proper clinical management.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Liver Neoplasms , Female , Humans , Aged , Young Adult , Adult , Synaptophysin/metabolism , Keratin-19/metabolism , Chromogranins/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cholangiocarcinoma/genetics , Cholangiocarcinoma/surgery , Cholangiocarcinoma/diagnosis , Liver Neoplasms/pathology , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/surgery , Bile Duct Neoplasms/metabolism , Bile Ducts, Intrahepatic/pathology , RNA/metabolism , Repressor Proteins/metabolism , Cell Cycle Proteins/metabolism , Neoplasm Proteins/metabolismABSTRACT
Chromogranin A (CgA) is a 439 amino acid protein secreted by neuroendocrine cells. Proteolytic processing of CgA results in the production of different bioactive peptides. These peptides have been associated with inflammatory bowel disease, diabetes, and cancer. One of the chromogranin A-derived peptides is â¼52 amino acid long Pancreastatin (PST: human (h)CgA250-301, murine (m)CgA263-314). PST is a glycogenolytic peptide that inhibits glucose-induced insulin secretion from pancreatic islet ß-cells. In addition to this metabolic role, evidence is emerging that PST also has inflammatory properties. This review will discuss the immunomodulatory properties of PST and its possible mechanisms of action and regulation. Moreover, this review will discuss the potential translation to humans and how PST may be an interesting therapeutic target for treating inflammatory diseases.
Subject(s)
Chromogranins , Pancreatic Hormones , Humans , Animals , Mice , Chromogranin A/pharmacology , Pancreatic Hormones/metabolism , Chromogranins/metabolism , Peptides , Amino AcidsABSTRACT
Insulin is synthesized by pancreatic ß-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus and deliver insulin to the bloodstream. The mechanism of how proinsulin and its processing enzymes are sorted and targeted from the trans-Golgi network (TGN) to SGs remains mysterious. No cargo receptor for proinsulin has been identified. Here, we show that chromogranin (CG) proteins undergo liquid-liquid phase separation (LLPS) at a mildly acidic pH in the lumen of the TGN, and recruit clients like proinsulin to the condensates. Client selectivity is sequence-independent but based on the concentration of the client molecules in the TGN. We propose that the TGN provides the milieu for converting CGs into a "cargo sponge" leading to partitioning of client molecules, thus facilitating receptor-independent client sorting. These findings provide a new receptor-independent sorting model in ß-cells and many other cell types and therefore represent an innovation in the field of membrane trafficking.
Subject(s)
Cytoplasmic Granules , Golgi Apparatus , Insulin-Secreting Cells , Proinsulin , Secretory Vesicles , Chromogranins/metabolism , Cytoplasmic Granules/metabolism , Golgi Apparatus/metabolism , Humans , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Proinsulin/metabolism , Secretory Vesicles/metabolismABSTRACT
Pseudohypoparathyroidism type Ib (PHP1B) is characterized predominantly by resistance to parathyroid hormone (PTH) leading to hypocalcemia and hyperphosphatemia. These laboratory abnormalities are caused by maternal loss-of-methylation (LOM) at GNAS exon A/B, which reduces in cis expression of the stimulatory G protein α-subunit (Gsα). Paternal Gsα expression in proximal renal tubules is silenced through unknown mechanisms, hence LOM at exon A/B reduces further Gsα protein in this kidney portion, leading to PTH resistance. In a previously reported PHP1B family, affected members showed variable LOM at exon A/B, yet no genetic defect was found by whole-genome sequencing despite linkage to GNAS. Using targeted long-read sequencing (T-LRS), we discovered an approximately 2800-bp maternally inherited retrotransposon insertion nearly 1200 bp downstream of exon XL not found in public databases or in 13,675 DNA samples analyzed by short-read whole-genome sequencing. T-LRS data furthermore confirmed normal methylation at exons XL, AS, and NESP and showed that LOM comprising exon A/B is broader than previously thought. The retrotransposon most likely causes the observed epigenetic defect by impairing function of a maternally derived NESP transcript, consistent with findings in mice lacking full-length NESP mRNA and in PHP1B patients with deletion of exon NESP and adjacent intronic sequences. In addition to demonstrating that T-LRS is an effective strategy for identifying a small disease-causing variant that abolishes or severely reduces exon A/B methylation, our data demonstrate that this sequencing technology has major advantages for simultaneously identifying structural defects and altered methylation. © 2022 American Society for Bone and Mineral Research (ASBMR).
Subject(s)
Chromogranins , Pseudohypoparathyroidism , Animals , Chromogranins/genetics , Chromogranins/metabolism , DNA Methylation/genetics , Darbepoetin alfa/genetics , Darbepoetin alfa/metabolism , Exons/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Mice , Pseudohypoparathyroidism/genetics , Retroelements , PseudohypoparathyroidismABSTRACT
Loss of methylation (LOM) at GNAS-A/B:TSS-differentially methylated regions (DMRs) in the GNAS locus is observed in pseudohypoparathyroidism type 1B (PHP1B). Many PHP1B cases are sporadic, but autosomal dominant-PHP1B has a deletion involving NESP55 expressed from the maternal allele or STX16 located upstream of the GNAS locus on the maternal allele. We report the possible first familial PHP1B cases with retrotransposon insertion in the GNAS locus on the maternal allele. To our knowledge, they are the possible first cases with imprinting disorders caused by retrotransposon insertion. The two sibling cases experienced tetany and/or cramps from school age and had hypocalcemia and an increased serum intact parathyroid hormone (PTH) level together with overweight, round face, and normal intellectual levels. Methylation analysis for DMRs in the GNAS locus showed only LOM of the GNAS-A/B:TSS-DMR. Copy number abnormalities at STX16 and the GNAS locus were not detected by array comparative genomic hybridization. Whole-genome sequencing and Sanger sequencing revealed an approximately 1000-bp SVA retrotransposon insertion upstream of the first exon of A/B on the GNAS locus in these siblings. Whole-genome methylome analysis by Enzymatic Methyl-Seq in the siblings showed normal methylation status in the region surrounding the insertion site and mild LOM of the GNAS-A/B:TSS-DMR. We conducted transcriptome analysis using mRNA from skin fibroblasts and induced pluripotent stem cells (iPSCs) derived from the siblings and detected no aberrant NESP55 transcripts. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis in skin fibroblasts showed increased A/B expression in the patients and no NESP55 expression, even in a control. qRT-PCR analysis in iPSCs showed decreased NESP55 expression with normal methylation status of the GNAS-NESP:TSS-DMR in the patients. The retrotransposon insertion in the siblings likely caused decreased NESP55 expression that could lead to increased A/B expression via LOM of the GNAS-A/B:TSS-DMR, subsequent reduced Gsα expression, and finally, PHP1B development. © 2022 American Society for Bone and Mineral Research (ASBMR).