Anoctamin-1 Expression Research Articles

Effects of protein diet on expression of Anoctamin1 of Cajal cell in the nervous plexus of the stomach in male mice.

To determine the association between a protein-rich diet and the expression of anoctamin 1 in Interstitial cells of Cajal within the muscle layer of the stomach wall in male mice. The experimental study was conducted at the Anatomy Department of the College of Medicine, Al- Nahrain University, Iraq, from November 2020 to April 2021, and comprised male adult healthy male mice. They were divided randomly into two equal groups. Group A was fed a high protein diet, while control group B was fed a standard pellet diet. The tissue samples were harvested at day 30 post-surgery. The stomach samplings were placed in 10% neutral formalin for 24 hours to obtain paraffin sections for routine histological and immunohistochemical staining. The protein expression in stomach smooth muscle of each group was detected by immunohistochemical staining. Data was analysed using SPSS 24. Of the 20 mice, 10(50%) were in each of the two groups. Group A exhibited significant weight-gain compared to group B (p≤ 0.05). There was significant elevation in muscle wall thickness in group A, compared to group B (p≤0.05). Tunica muscularis of the stomach in group A significantly thickened compared to group B (p≤0.05). The expression of anoctamin 1 was significantly more intense in group A compared to group B (p≤0.01). Unbalanced food had a significant impact on the stomach, affecting the thickness of the muscularis layer and the expression of anoctamin 1 in cajal cells in the muscularis externa.

Promoter hypermethylation as a novel regulator of ANO1 expression and function in prostate cancer bone metastasis

Despite growing evidence implicating the calcium-activated chloride channel anoctamin1 (ANO1) in cancer metastasis, its direct impact on the metastatic potential of prostate cancer and the possible significance of epigenetic alteration in this process are not fully understood. Here, we show that ANO1 is minimally expressed in LNCap and DU145 prostate cancer cell lines with low metastatic potential but overexpressed in high metastatic PC3 prostate cancer cell line. The treatment of LNCap and DU145 cells with DNMT inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) potentiates ANO1 expression, suggesting that DNA methylation is one of the mechanisms controlling ANO1 expression. Consistent with this notion, hypermethylation was detected at the CpG island of ANO1 promoter region in LNCap and DU145 cells, and 5-Aza-CdR treatment resulted in a drastic demethylation at promoter CpG methylation sites. Upon 5-Aza-CdR treatment, metastatic indexes, such as cell motility, invasion, and metastasis-related gene expression, were significantly altered in LNCap and DU145 cells. These 5-Aza-CdR-induced metastatic hallmarks were, however, almost completely ablated by stable knockdown of ANO1. These in vitro discoveries were further supported by our in vivo observation that ANO1 expression in xenograft mouse models enhances the metastatic dissemination of prostate cancer cells into tibial bone and the development of osteolytic lesions. Collectively, our results help elucidate the critical role of ANO1 expression in prostate cancer bone metastases, which is epigenetically modulated by promoter CpG methylation.

Airway basal cell‑derived exosomes suppress epithelial‑mesenchymal transition of lung cells by inhibiting the expression of ANO1.

Disruption of the epithelial-mesenchymal transition (EMT) of activated lung cells is an important strategy to inhibit the progression of idiopathic pulmonary fibrosis (IPF). The present study investigated the role of exosomes derived from airway basal cells on EMT of lung cells and elucidate the underlying mechanism. Exosomes were characterized by nanoparticle tracking analysis, transmission electron microscopy imaging and markers detection. The role of exosome on the EMT of lung epithelial cells and lung fibroblasts induced by TGF-β1 was detected. RNA sequencing screened dysregulated genes in exosome-treated group, followed by the bioinformatical analysis. One of the candidates, anoctamin-1 (ANO1), was selected for further gain-and-loss phenotype assays. A bleomycin-induced pulmonary fibrosis model was used to evaluate the treatment effect of exosomes. Exosomes were round-like and positively expressed CD63 and tumor susceptibility gene 101 protein. Treatment with exosomes inhibited the EMT of lung cells activated by TGF-β1. 4158 dysregulated genes were identified in exosome-treated group under the threshold of |log2 fold-change| value >1 and they were involved in the metabolism of various molecules, as well as motility-related biological processes. A key gene, ANO1, was verified by reverse transcription-quantitative PCR, whose overexpression induced the EMT of lung cells. By contrast, ANO1 knockdown reversed the EMT induced by TGF-β1. In vivo assay indicated that exosome treatment ameliorated pulmonary fibrosis and inhibited the upregulation of ANO1 induced by bleomycin. In conclusion, airway basal cell-derived exosomes suppressed the EMT of lung cells via the downregulation of ANO1. These exosomes represent a potential therapeutic option for patients with IPF.

Low dose cadmium exposure regulates miR-381–ANO1 interaction in airway epithelial cells

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death worldwide. Cigarette smoke which has approximately 2–3 µg of Cadmium (Cd) per cigarette contributes to the environmental exposure and development and severity of COPD. With the lack of a cadmium elimination mechanism in humans, the contribution of cadmium induced stress to lung epithelial cells remains unclear. Studies on cadmium responsive miRNAs suggest regulation of target genes with an emphasis on the critical role of miRNA–mRNA interaction for cellular homeostasis. Mir-381, the target miRNA in this study is negatively regulated by cadmium in airway epithelial cells. miR-381 is reported to also regulate ANO1 (Anoctamin 1) expression negatively and in this study low dose cadmium exposure to airway epithelial cells was observed to upregulate ANO1 mRNA expression via mir-381 inhibition. ANO1 which is a Ca2+-activated chloride channel has multiple effects on cellular functions such as proliferation, mucus hypersecretion and fibroblast differentiation in inflamed airways in chronic respiratory diseases. In vitro studies with cadmium at a high concentration range of 100–500 µM is reported to activate chloride channel, ANO1. The secretory epithelial cells are regulated by chloride channels like CFTR, ANO1 and SLC26A9. We examined “ever” smokers with COPD (n = 13) lung tissue sections compared to “never” smoker without COPD (n = 9). We found that “ever” smokers with COPD had higher ANO1 expression. Using mir-381 mimic to inhibit ANO1, we demonstrate here that ANO1 expression is significantly (p < 0.001) downregulated in COPD derived airway epithelial cells exposed to cadmium. Exposure to environmental cadmium contributes significantly to ANO1 expression.

#3679 ANOCTAMIN 1 INHIBITION SUPPRESSES CYSTOGENESIS BY ENHANCING CILIOGENESIS AND THE CILIARY DOSAGE OF POLYCYSTINS

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy which is characterized by abnormal tubular epithelial proliferation and fluid secretion. Anoctamin 1 (ANO1) is a calcium-dependent chloride channel, which has recently been shown to be involved in ADPKD progression. However, how ANO1 contributes to ADPKD is largely unexplored. Method 1. Based on mRNA gene chips, the expression of ANO1 abnormally increased in human ADPKD kidneys. 2. Immunohistochemical staining showed ANO1 location. 3. Real-time PCR and Western blot showed mRNAs and protein of ANO1 in ADPKD patients and animal model-PKD1RC/RC mice. 4. Confocal immunofluorescence showed ANO1 and PC2 location in the cilia. 5. 3D gel culture to verify the function of ANO1 in cysts formation. 6. ANO1 inhibitors and siRNA to discover STAT3 and ERK pathway in ADPKD cell by WB. Results We found that ANO1 is consistently upregulated inhuman and mouse PKD kidneys. Intriguingly, ANO1 located in a vesicle-like patten at the ciliary base but not on the ciliary surface. ANO1 deficiency promotes both ciliogenesis and the ciliary dosage of polycystin-2 in human Pkd1-/- cells, and reduced cyst formation in 3D culture models. Moreover, inhibition of ANO1 abolished the activation of STAT3 and ERK pathways in PKD cells. Conclusion Our data indicate ANO1 may aggravate ADPKD by regulating the cilia length and PC2 expression. Moreover, ANO1 could support cyst growth by activating STAT3 and ERK pathways. Thus ANO1 is a negative regulator for both cilia formation and cilia trafficking of polycystins and provide mechanistic insights regarding the therapeutic potential of ANO1 pathway in ADPKD treatment.

Transcriptome and Proteome Profiling of Primary Human Gastric Interstitial Cells of Cajal Predicts Pacemaker Networks

Interstitial cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells required for normal GI motility. Dysfunctions in ICC have been reported in patients with GI motility disorders, such as gastroparesis, who exhibit debilitating symptoms and greatly reduced quality of life. While the proteins, calcium-activated chloride channel anoctamin-1 (ANO1) and the receptor tyrosine kinase (KIT), are known to be expressed by human ICC, relatively little is known about the broad molecular circuitry underpinning human ICC functions. The present study therefore investigates the transcriptome and proteome of ANO1-expressing, KITlow/CD45-/CD11B- ICC obtained from primary human gastric tissue. Excess human gastric tissue resections were obtained from sleeve gastrectomy patients. ICC were purified using fluorescence-activated cell sorting (FACSorting). Then, ICC were characterized by using immunofluorescence, real-time polymerase chain reaction, RNA-sequencing and mass spectrometry. Compared to unsorted cells, real-time polymerase chain reaction showed the KITlow/CD45-/CD11B- ICC had: a 9-fold (P < 0.05) increase in ANO1 expression; unchanged KIT expression; and reduced expression for genes associated with hematopoietic cells (CD68, > 10-fold, P < 0.001) and smooth muscle cells (DES, > 4-fold, P < 0.05). RNA-sequencing and gene ontology analyses of the KITlow/CD45-/CD11B- cells revealed a transcriptional profile consistent with ICC function. Similarly, mass spectrometry analyses of the KITlow/CD45-/CD11B- cells presented a proteomic profile consistent with ICC activities. STRING-based protein interaction analyses using the RNA-sequencing and proteomic datasets predicted protein networks consistent with ICC-associated pacemaker activity and ion transport. These new and complementary datasets provide a valuable molecular framework for further understanding how ICC pacemaker activity regulates smooth muscle contraction in both normal GI tissue and GI motility disorders.

ANO1 reprograms cholesterol metabolism and the tumor microenvironment to promote cancer metastasis.

Tumor metastasis is a complex process. A better understanding of the mechanistic underpinnings of the metastatic cascade could uncover approaches to better prevent and target metastases. Here, we integrated transcriptomic profiling in primary esophageal squamous cell carcinoma (ESCC) and matched lymph node metastatic tissues and genome-wide CRISPR/Cas9 functional screening to identify the key drivers of cancer metastasis. Anoctamin 1 (ANO1) expression was found to be elevated in primary tumors and further increased in metastatic tissues. High expression of ANO1 was associated with poor patient survival, and ANO1 overexpression promoted cancer invasion and metastasis. Mechanistically, ANO1 inactivated the LXR pathway and increased intracellular cholesterol accumulation by interacting with JUN to repress the transcription of cholesterol hydroxylase, CYP27A1. In a paracrine manner, interleukin 1β (IL-1β) secreted from ANO1-expressing ESCC cancer cells activated NFκB signaling in fibroblasts to stimulate production of the chemokine CCL1, which in turn enhanced invasion of ESCC cancer cells. Furthermore, molecular docking and in silico screening of 1,500,000 small molecules identified K786-4469 as a lead compound that targeted ANO1 and suppressed cancer metastasis in vitro and in vivo. Overall, these findings uncover a non-canonical function of ANO1 in regulating cholesterol metabolism and fibroblast function, revealing ANO1 as a potential prognostic biomarker and therapeutic target for metastatic ESCC.

High level of ANO1 promotes pancreatic cancer growth in concert with oncogenic KRAS.

Anoctamin-1 (ANO1) was identified as an unfavorable prognostic marker in pancreatic cancer. However, the exact implication of ANO1 in pancreatic cancer is still poorly understood. Here we investigated the effect of ANO1 in pancreatic cancer progression under the context of oncogenic KRAS, aiming at finding a new therapeutic target. Knockdown and overexpression of oncogenic KRAS as well as ANO1 in PDAC cell lines were performed by lentivirus infection. Cell proliferation and migration assay, RNA seq analysis were performed in PDAC cells bearing different status of ANO1 and KRAS. In vivo mice model was used to investigate the xenograft tumor growth with different status of KRAS and ANO1. Our results showed that ANO1 expression level is elevated in poorly differentiated cancer cells. Overexpression of ANO1 in PDAC cancer cells was found to promote cancer cell proliferation in vitro and in vivo, which synergized with the introduction of oncogenic KRAS. Consistently, knockdown of ANO1 expression was found to suppress cancer growth in vitro and in vivo. RNA seq analysis revealed that the observed synergistic cancer-promoting effect from ANO1 and oncogenic KRAS is likely due to concurrent activating key genes involved in lipid metabolism including HMGCS1. The outcome from our study suggests that ANO1 plays an important role in promoting pancreatic cancer development, especially at the presence of oncogenic KRAS. Considering the prevalence of KRAS mutation in pancreatic cancer patients, suppression ANO1 may represent a potential effective therapeutic measure in pancreatic cancer treatment.

Inhibition of ANO1 by Cis- and Trans-Resveratrol and Their Anticancer Activity in Human Prostate Cancer PC-3 Cells.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is involved in the proliferation, migration, and invasion of various cancer cells including head and neck squamous cell carcinoma, lung cancer, and prostate cancer. Inhibition of ANO1 activity or downregulation of ANO1 expression in these cancer cells is known to exhibit anticancer effects. Resveratrol, a natural polyphenol abundant in wines, grapes, berries, soybeans, and peanuts, shows a wide variety of biological effects including anti-inflammatory, antioxidant, and anticancer activities. In this study, we investigated the effects of two stereoisomers of resveratrol on ANO1 activity and found that cis- and trans-resveratrol inhibited ANO1 activity with different potencies. Cis- and trans-resveratrol inhibited ANO1 channel activity with IC50 values of 10.6 and 102 μM, respectively, and had no significant effect on intracellular calcium signaling at 10 and 100 μM, respectively. In addition, cis-resveratrol downregulated mRNA and protein expression levels of ANO1 more potently than trans-resveratrol in PC-3 prostate cancer cells. Cis- and trans-resveratrol significantly reduced cell proliferation and cell migration in an ANO1-dependent manner, and both resveratrol isomers strongly increased caspase-3 activity, PARP cleavage, and apoptotic sub-G1 phase ratio in PC-3 cells. These results revealed that cis-resveratrol is a potent inhibitor of ANO1 and exhibits ANO1-dependent anticancer activity against human metastatic prostate cancer PC-3 cells.

Characterization of Neurochemical Signature Alterations in the Enteric Nervous System in Autoimmune Encephalomyelitis

To date, it has remained unclear whether gastrointestinal symptoms, which are frequently observed in patients with multiple sclerosis (MS), are accompanied by pathology of the enteric nervous system (ENS). Here, the neurotransmitter signature of ENS neurons and morphological alterations of interstitial cells of Cajal (ICCs) were studied in patients with MS and mice with experimental autoimmune encephalomyelitis (EAE), which is an animal model of MS. Immunohistochemical analysis was performed on colonic whole mounts from mice with EAE and on paraffin-embedded sections of intestinal tissue from patients with MS. Antibodies against neurotransmitters or their enzymes (including vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS), and choline acetyltransferase (ChAT)) were used in conjunction with pan-neuronal markers. In addition, the presence of anoctamin 1 (ANO1)-expressing ICCs was studied. ENS changes were observed in the myenteric plexus, but they were absent in the submucosal plexus of both EAE mice and patients with MS. There was a significant decrease in the percentage of ChAT-positive neurons in EAE mice as opposed to a trend toward an increase in patients with MS. Moreover, while ANO1 expression was decreased in EAE mice, patients with MS displayed a significant increase. Although additional studies are necessary to accomplish an in-depth characterization of ENS alterations in MS, our results imply that such alterations exist and may reveal novel insights into the pathophysiology of MS.

The Novel lncRNA ENST00000530525 Affects ANO1, Contributing to Blood-Brain Barrier Injury in Cultured hCMEC/D3 Cells Under OGD/R Conditions.

Ischemic stroke (IS) is a major neurological disease with high fatality and residual disability burdens. Long noncoding RNAs (lncRNAs) have been found to play an important role in IS. However, the roles and significance of most lncRNAs in IS are still unknown. This study was performed to identify differentially expressed (DE) lncRNAs using a lncRNA microarray in whole blood samples of patients suffering from acute cerebral ischemia. Bioinformatics analyses, including GO, KEGG pathway enrichment analysis, and proximity to putative stroke risk location analysis were performed. The novel lncRNA, ENST00000530525, significantly decreased after IS. Furthermore, we evaluated lncRNA ENST00000530525 expression in cultured hCMEC/D3 cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions using fluorescent in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (RT–qPCR) analysis. To investigate the function of lncRNA ENST00000530525, its over-expression (OE) and negative control (NC) plasmids were transfected into hCMEC/D3 cells, and cell viability was detected by a cell counting kit-8 (CCK-8) assay after OGD/R. LncRNA ENST00000530525 and ANO1 expression were investigated using RT–qPCR and immunofluorescence. For blood–brain barrier (BBB) permeability, FITC-dextran transendothelial permeability assay and tight junction (TJ) protein immunofluorescence assays were performed. There were 3352 DE lncRNAs in the blood samples of acute IS patients. The validation results were consistent with the gene chip data. The GO and KEGG results showed that these lncRNAs were mainly related to oxygen and glucose metabolism, leukocyte transendothelial migration, mitophagy and cellular senescence. Among these, lncRNA ENST00000530525 was the most highly downregulated lncRNA and it was mapped within the IS-associated gene anoctamin-1 (ANO1). We further found that lncRNA ENST00000530525 was downregulated in hCMEC/D3 cells under 4 h OGD and 20 h reoxygenation (OGD4/R20) conditions. Upregulating lncRNA ENST00000530525 by plasmid transfection decreased cell viability while increasing ANO1 expression and it contributed to BBB injury in hCMEC/D3 cells after OGD4/R20. The lncRNA ENST00000530525 might play deleterious roles in post-stroke pathogenesis. These results show that some DE lncRNAs in humans participate through characteristic roles in post-stroke pathogenesis; thus, the roles and significance of some novel lncRNAs in IS warrant further study.

Downregulation of ANO1 activity in PC‐9 cells modulated by a new novel candidate TMEMinh001

Relevant to multiple studies, calcium activated chloride channel called Anoctamin1 (ANO1) is identified as an excellent biomarker for tumor growth reported in different cancer cells such as prostate, breast, and lung cancer. Growing evidence suggests, ANO1 promotes tumor growth via regulating EGFR signaling and modulating MAPK and protein kinase B pathway. In non‐small cell lung cancer (NSCLC), the ANO1 expression level is high, leading to high cancer‐related mortality. To date, several natural and synthetic compounds have shown their modulatory effect on ANO1 activity but are out of the limelight due to selectivity. Our present study is carried out based on finding a selective and potent modulator of ANO1 activity by in silico and in vitro assay. According to the hypothesis, inhibitors of ANO1 are screened out, where an inhibitor reduced ANO1 expression without modulating intracellular Ca+ level and cystic fibrosis transmembrane conductance regulator (CFTR) channel activity. The molecular docking study revealed negative high binding free energy than known inhibitor Ani9 and other candidate compounds. Furthermore, our potential compound suppressed cell viability and migration process in ANO1 expressing PC9 cells, while not in H1975 cells not expressing ANO1. Besides, it initiated the apoptosis process by up‐regulating caspase‐3 activity. Thus, the above results provided significant evidence of the anticancer effect of the final compound against ANO1, reducing its protein level in NSCLC.

Epigenetic regulation of Anoctamin1 in pulmonary arterial hypertension

Introduction/HypothesisPulmonary arterial hypertension (PAH) is associated with substantial pulmonary vascular remodeling wherein endothelial cells (ECs) within the lumen hyper‐proliferate leading to occlusive plexiform lesions. We previously reported that Anoctamin‐1 (ANO1) expression is increased in lung ECs from patients with PAH and is associated with increased EC proliferation. However, the mechanism of ANO1 expression regulation in PAH remains unclear. DNA methylation has been recognized as an important mechanism in gene regulation. In this study, we test the hypothesis that ANO1 expression in PAH is epigenetically mediated via DNA methylation.Methods/ResultsDNA was extracted from lung ECs of patients with idiopathic PAH (n=7) and controls (n=6), followed by Bisulfite Next‐Generation Sequencing (EpigenDx, MA) to determine DNA methylation profile in human ANO1 gene (Ensembl Gene ID: ENSG00000131620). We used 19 assays to cover 97 CpG sites in the human ANO1 gene, including regions of 5’‐upstream, 5’‐UTR, and different introns. Our data show that the methylation of 29 of 97 (30%) CpG sites in human ANO1 gene is significantly altered in lung ECs from PAH patients than that from control subjects. Among the significantly altered 29 CpG sites, the methylation at 22 CpG sites (76%) is significantly decreased. To determine whether decreased DNA methylation changes human ANO1 gene expression, we then exposed normal human pulmonary artery ECs to RG108, which is a DNA methyltransferase inhibitor that broadly reduces DNA methylation. Our data show that cells treated with RG108 for 72 hours have a significant 2‐fold increase in ANO‐1 mRNA expression in comparison to control cells, suggesting that decreased DNA methylation is sufficient to upregulate human ANO1 expression. We are developing CRISPR‐based approaches to target these identified CpG sites to determine the specific contribution of their methylation in regulation of human ANO1 gene.ConclusionsOur results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH.

Hypertriglyceridemia Impairs Urethral Spontaneous Tone Through Down-regulation of ANO1 in Mouse Urethral Smooth Muscle Cells

ObjectiveTo investigate the effect of hypertriglyceridemia (HTG) on the Anoctamin 1 (ANO1)-centered signal on urinary incontinence. MethodsFemale mice were fed with high fat diet to set up HTG mice model. ANO1 expression in urethrae were detected by Flow Cytometry and Western Blotting. The habit of micturition was recorded through urine spot test. Urethral Smooth Muscle Myography was used to record the urethral spontaneous tone (STT) and contraction. Patch-clamp Recording confirmed the currents of ANO1 in urethral smooth muscle cells. ResultsHTG mice showed almost abolished STT and significantly decreased contraction induced by EACT. Meanwhile, a change in urination habits was detected in HTG mice which suggested that mice could not hold the urethral pressure. Furthermore, ANO1 expression in single urethral smooth muscle cells from HTG mice decreased almost 30% than that found in cells from normal group. Moreover, EACT (an N-aroylaminothiazole, ANO1 activator) induced decreased intracellular calcium ([Ca2+]i) and currents in single urethral smooth muscle cells from HTG mice. ConclusionThese results suggested that hypertriglyceridemia decreased the STT on urethral motility by down-regulating the expression of ANO1 in urethral smooth muscles. These findings provide a new perspective on the mechanism of urinary incontinence which is common in HTG patients.

Cinobufagin Exerts Anticancer Activity in Oral Squamous Cell Carcinoma Cells through Downregulation of ANO1.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including oral squamous cell carcinoma (OSCC). OSCC is a highly aggressive cancer and the most common oral malignancy. ANO1 has been proposed as a potential candidate for targeted anticancer therapy. In this study, we performed a cell-based screening to identify novel regulators leading to the downregulation of ANO1, and discovered cinobufagin, which downregulated ANO1 expression in oral squamous cell carcinoma CAL-27 cells. ANO1 protein levels were significantly reduced by cinobufagin in a dose-dependent manner with an IC50 value of ~26 nM. Unlike previous ANO1 inhibitors, short-term (≤10 min) exposure to cinobufagin did not alter ANO1 chloride channel activity and ANO1-dependent intestinal smooth muscle contraction, whereas long-term (24 h) exposure to cinobufagin significantly reduced phosphorylation of STAT3 and mRNA expression of ANO1 in CAL-27 cells. Notably, cinobufagin inhibited cell proliferation of CAL-27 cells expressing high levels of ANO1 more potently than that of ANO1 knockout CAL-27 cells. In addition, cinobufagin significantly reduced cell migration and induced caspase-3 activation and PARP cleavage in CAL-27 cells. These results suggest that downregulation of ANO1 by cinobufagin is a potential mechanism for the anticancer effect of cinobufagin in OSCC.

Anoctamin1 Induces Hyperproliferation of HaCaT Keratinocytes and Triggers Imiquimod-Induced Psoriasis-Like Skin Injury in Mice.

Psoriasis, a long-lasting and multifactorial skin disease, is related to comorbidities such as metabolic disease, depression, and psoriatic arthritis. Psoriasis occurs due to a variety of factors including keratinocyte hyperproliferation, inflammation, and abnormal differentiation. Proinflammatory cytokines upregulated by increased activation of keratinocytes and immune cells in the skin trigger progression of psoriasis. This study aimed to investigate the effects of anoctamin1 (ANO1) on psoriasis development in vitro and in vivo. We analyzed the proliferation of HaCaT keratinocytes and ANO1-related ERK and AKT signaling pathways after ANO1 inhibitor (T16Ainh-A01 and Ani9) treatment and knock-down of ANO1. Furthermore, after applying imiquimod (IMQ) cream or coapplying IMQ cream and T16Ainh-A01 on mouse ears, we not only observed psoriatic symptoms, including ear thickening, but also quantified the effects of treatment on ERK and AKT signaling-involved proteins and proinflammatory cytokines. Inhibition of ANO1 attenuated the proliferation of HaCaT cells and induced reduction of pERK1/2. Coapplication of IMQ and T16Ainh-A01 on ears of mice reduced not only symptoms of IMQ-induced psoriasis such as thickening and erythema, but also expression of ANO1 and pERK1/2 compared to that of application of IMQ alone. In addition, the expression levels of IL-17A, IL-17F, IL-22, IL-23, IL-6, IL-1β, and TNF-α increased after applying IMQ and were significantly reduced by coapplying IMQ and T16Ainh-A01. These results aid in understanding the underlying mechanisms of ANO1 in epidermal layer keratinocyte hyperproliferation and suggest the potential of ANO1 as a target to treat psoriasis.

ANO1 Expression Orchestrates p27Kip1/MCL1-Mediated Signaling in Head and Neck Squamous Cell Carcinoma.

Simple SummaryOur aim was to elucidate the molecular mechanisms of how ANO1 contributes to oncogenic processes in squamous cell carcinoma of the head and neck (HNSCC). We explored transcriptional programs influenced by ANO1 knockdown in patient-derived UT-SCC cell lines with 11q13 amplification and ANO1 overexpression. ANO1 depletion led to downregulation of broad pro-survival BCL2 family protein members, including MCL1, and simultaneously induced upregulation of the cell cycle inhibitor p27Kip1 and its redistribution from the cytoplasm into the nucleus in the studied HNSCC cells. Gene set enrichment analysis highlighted pathways associated with perturbed cell cycle and apoptosis in the ANO1-depleted samples. Silencing of ANO1 and application of an ANO1-targeting small-molecule inhibitor led to ANO1 degradation and reduction of cell viability. These findings suggest that ANO1 has drug target potential that deserves further evaluation in preclinical in vivo models.Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that derive from the mucosal epithelium of the upper aerodigestive tract and present high mortality rate. Lack of efficient targeted-therapies and biomarkers towards patients’ stratification are caveats in the disease treatment. Anoctamin 1 (ANO1) gene is amplified in 30% of HNSCC cases. Evidence suggests involvement of ANO1 in proliferation, migration, and evasion of apoptosis; however, the exact mechanisms remain elusive. Aim of this study was to unravel the ANO1-dependent transcriptional programs and expand the existing knowledge of ANO1 contribution to oncogenesis and drug response in HNSCC. We cultured two HNSCC cell lines established from primary tumors harboring amplification and high expression of ANO1 in three-dimensional collagen. Differential expression analysis of ANO1-depleted HNSCC cells demonstrated downregulation of MCL1 and simultaneous upregulation of p27Kip1 expression. Suppressing ANO1 expression led to redistribution of p27Kip1 from the cytoplasm to the nucleus and associated with a cell cycle arrested phenotype. ANO1 silencing or pharmacological inhibition resulted in reduction of cell viability and ANO1 protein levels, as well as suppression of pro-survival BCL2 family proteins. Collectively, these data provide insights of ANO1 involvement in HNSCC carcinogenesis and support the rationale that ANO1 is an actionable drug target.

Prognostic significance of ANO1 expression in cancers.

Background:Anoctamin-1 (ANO1) plays a pivotal role in cancer progression. A meta-analysis was conducted to assess the potential prognostic role of ANO1 in cancers.Methods:A total of 1760 patients from 7 eligible studies were included into the analysis. Pooled hazard ratios or odds ratios were extracted and calculated with a random-effects model, and analyses of heterogeneity bias were conducted.Results:Our results showed that over expression of ANO1 was significantly correlated with poor overall survival in all cancers (HR = 1.52; 95% CI: 1.19–1.92; P = .0006). Subgroup analysis indicated that there was a significant association between over expression of ANO1 and poor prognosis breast cancer (HR = 3.24; 95% CI: 1.74–6.04), head and neck squamous cell carcinoma (HR = 1.14; 95% CI: 1.00–1.30), esophageal squamous cell carcinoma (HR = 1.93; 95% CI: 1.07–3.50), gastric cancer (HR = 1.62; 95% CI: 1.12–2.34) and colorectal cancer (HR = 1.38; 95% CI: 1.03–1.85). In addition, over expression of ANO1 was not associated with TNM stage, histological grade, lymph node metastasis, tumor size, age and gender. However, ANO1 was significantly associated with human epidermal growth factor receptor 2, but not associated with progesterone receptor or estrogen receptor in breast cancer.Conclusions:Our results indicate that ANO1 can be a predictive factor for prognosis of cancer.

Opposing roles of E3 ligases TRIM23 and TRIM21 in regulation of ion channel ANO1 protein levels

Anoctamin-1 (ANO1) (TMEM16A) is a calcium-activated chloride channel that plays critical roles in diverse physiological processes, such as sensory transduction and epithelial secretion. ANO1 levels have been shown to be altered under physiological and pathological conditions, although the molecular mechanisms that control ANO1 protein levels remain unclear. The ubiquitin–proteasome system is known to regulate the levels of numerous ion channels, but little information is available regarding whether and how ubiquitination regulates levels of ANO1. Here, we showed that two E3 ligases, TRIM23 and TRIM21, physically interact with the C terminus of ANO1. In vitro and in vivo assays demonstrated that whereas TRIM23 ubiquitinated ANO1 leading to its stabilization, TRIM21 ubiquitinated ANO1 and induced its degradation. Notably, ANO1 regulation by TRIM23 and TRIM21 is involved in chemical-induced pain sensation, salivary secretion, and heart-rate control in mice, and TRIM23 also mediates ANO1 upregulation induced by epidermal growth factor treatment. Our results suggest that these two antagonistic E3 ligases act together to control ANO1 expression and function. Our findings reveal a previously unrecognized mechanism for regulating ANO1 protein levels and identify a potential molecular link between ANO1 regulation, epidermal growth factor, and other signaling pathways.

LncRNA GIHCG Promotes the Development of Esophageal Cancer by Modulating miR-29b-3p/ANO1 Axis.

BackgroundEsophageal cancer is one of the most frequent cancers with a higher mortality worldwide. Although many long non-coding RNAs (LncRNAs) are reported to play important roles in the progression of esophageal cancer, the function of lncRNA GIHCG in esophageal cancer remains unclear.MethodsThe expression of GIHCG in esophageal cancer tissues and cancer cell lines was detected by qRT-PCR. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay, EdU staining assay and colony formation assay. Cell invasion and migration were measured by transwell assay. Cell apoptosis was detected by a flow cytometer. Luciferase reporter assay and RIP assay were used to determine the interaction between GIHCG and miR-29b-3p, and their subsequent regulation of anoctamin 1 (ANO1). The expression of ANO1 in esophageal cancer tissues and cell lines was detected by Western blot. The effect of GIHCG/miR-29b-3p in tumor formation was assessed by the xenograft nude mice model in vivo.ResultsGIHCG was significantly upregulated in esophageal cancer tissues and relevant cancer cell lines. Downregulation of GIHCG significantly inhibited the growth, colony formation, invasion, migration and induced apoptosis of esophageal cancer cells in vitro. Bioinformatic analysis and RIP assay determined that GIHCG was a sponge of miR-29b-3p, and ANO1 was a direct target of miR-29b-3p. Moreover, functional experiments showed that GIHCG upregulated ANO1 expression by directly sponging miR-29b-3p. Furthermore, in vivo experiment revealed that knockdown of GIHCG significantly inhibited tumor growth in nude mice.ConclusionOur study revealed that lncRNA GIHCG promoted the progression of esophageal cancer by targeting the miR-29b-3p/ANO1 axis, suggesting that GIHCG might be a novel therapeutic target for esophageal cancer.