DOG1 expression is common in human tumors: A tissue microarray study on more than 15,000 tissue samples.

DOG1 (Discovered on GIST1) is a voltage-gated calcium-activated chloride and bicarbonate channel that is highly expressed in interstitial cells of Cajal and in gastrointestinal stromal tumors (GIST) derived from Cajal cells. To systematically determine in what tumor entities and normal tissue types DOG1 may be further expressed, a tissue microarray (TMA) containing 15,965 samples from 121 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. DOG1 immunostaining was found in 67 tumor types including GIST (95.7%), esophageal squamous cell carcinoma (31.9%), pancreatic ductal adenocarcinoma (33.6%), adenocarcinoma of the Papilla Vateri (20%), squamous cell carcinoma of the vulva (15.8%) and the oral cavity (15.3%), mucinous ovarian cancer (15.3%), esophageal adenocarcinoma (12.5%), endometrioid endometrial cancer (12.1%), neuroendocrine carcinoma of the colon (11.1%) and diffuse gastric adenocarcinoma (11%). Low level-DOG1 immunostaining was seen in 17 additional tumor entities. DOG1 expression was unrelated to histopathological parameters of tumor aggressiveness and/or patient prognosis in cancers of the breast (n = 1002), urinary bladder (975), ovary (469), endometrium (173), stomach (233), and thyroid gland (512). High DOG1 expression was linked to estrogen receptor expression in breast cancer (p < 0.0001) and absence of HPV infection in squamous cell carcinomas (p = 0.0008). In conclusion, our data identify several tumor entities that can show DOG1 expression levels at similar levels as in GIST. Although DOG1 is tightly linked to a diagnosis of GIST in spindle cell tumors, the differential diagnosis is much broader in DOG1 positive epithelioid neoplasms.

(Mammary Analogue) Secretory Carcinoma of the Nasal Cavity: Report of a Rare Case with p63 and DOG1 Expression and Uncommon Exon 4-Exon 14 ETV6-NTRK3 Fusion Diagnosed with Next Generation Sequencing.

We present a 72 years old male with a left nasal cavity (mammary analogue) secretory carcinoma (SC) which exhibited classical morphological features on light microscopical examination, diffuse strong S100 and mammoglobin positivity on immunohistochemistry, and ETV6-NTRK3 gene fusion on next generation sequencing (NGS) analysis. Unusual features of this tumor are expression of p63 and DOG1 on immunohistochemistry and the atypical junction between Exon 4 of the ETV6 gene and Exon 14 of the NTRK3 gene.

Epigenetic Modification as a Regulatory Mechanism for Spatiotemporal Dynamics of ANO1 Expression in Salivary Glands.

Anoctamin1 (ANO1), a calcium activated chloride channel, is known to play a critical role in salivary secretion. In the salivary gland, ANO1 is expressed exclusively in the acinar cells, with no expression in the ductal cells. However, the mechanisms that determine this distinctive cell type-dependent expression pattern of ANO1 remain unknown. In this study, we discovered that the cell-dependent expression of ANO1 during salivary gland organogenesis is regulated by DNA methylation of ANO1 CpG islands. ANO1 CpG islands in e12 embryonic submandibular glands (eSMG) are highly methylated, but those in e14 eSMG or adult SMG are significantly unmethylated. The differential expression pattern of ANO1 in duct and acini is defined at e14. Artificial demethylation by treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR), induced the expression of ANO1 in both the ductal cell line Human Submandibular Gland (HSG) and in the duct cells of adult mouse SMG. During the trans-differentiation in Matrigel of duct-origin HSG cells into acinar-like phenotype, significant demethylation of ANO1 CpG islands is observed. This may be due to the reduced expression of DNA methyltransferase (DNMT) 3a and 3b. These results suggest that the differential expression of ANO1 in salivary glands during organogenesis and differentiation is mainly regulated by epigenetic demethylation of the ANO1 gene.

A mutual activation loop between the Ca2+-activated chloride channel TMEM16A and EGFR/STAT3 signaling promotes breast cancer tumorigenesis.

The Ca2+-activated chloride channel TMEM16A (anoctamin 1) is overexpressed in breast cancer. It remains unclear how TMEM16A overexpression plays a role in carcinogenesis in breast cancer. In this study, we found that high TMEM16A expression in combination with high EGFR or STAT3 expression was significantly associated with shorter overall survival in ER-positive breast cancer patients without tamoxifen treatment, and longer overall survival in patients with tamoxifen treatment. EGFR/STAT3 signaling activation by EGF promoted TMEM16A expression, and TMEM16A overexpression activated EGFR/STAT3 signaling in breast cancer cells. Both in vitro and in animal studies showed that TMEM16A overexpression promoted, and TMEM16A knockdown inhibited breast cancer cell proliferation and tumor growth. In addition, TMEM16A overexpression-induced cell proliferation was blocked by EGFR/STAT3 inhibitors, and TMEM16A knockdown reduced EGF-induced proliferation and tumorigenesis in breast cancer. Furthermore, inhibition of TMEM16A channel function effectively reduced breast cancer cell proliferation, especially in combination with EGFR inhibitors. Our findings identify a mutual activation loop between TMEM16A and EGFR/STAT3 signaling, which is important for breast cancer proliferation and growth. TMEM16A inhibition may represent a novel therapy for EGFR-expressing breast cancer.

Direct repression of anoctamin 1 (ANO1) gene transcription by Gli proteins.

The Ca2+-activated Cl- channel, anoctamin 1 (Ano1, also known as transmembrane protein 16A) contributes to intestinal pacemaking, fluid secretion, cellular excitability, and tissue development. The human ANO1 promoter contains binding sites for the glioma-associated oncogene (Gli) proteins. We investigated regulation of ANO1 transcription by Gli. ANO1 promoter activity was determined using a luciferase reporter system. Binding and functional effects of Glis on ANO1 transcription and expression were demonstrated by chromatin immunoprecipitation, small interfering RNA knockdown, PCR, immunolabeling, and recordings of Ca2+-activated Cl- currents in human embryonic kidney 293 (HEK293) cells. Results from previous genome-wide association studies were used to test ANO1 promoter polymorphisms for association with disease. Gli1 and Gli2 bound to the promoter and repressed ANO1 transcription. Repression depended on Gli binding to a site close to the ANO1 transcriptional start site. Mutation of this site prevented Gli binding and transcriptional repression. Knockdown of Gli expression and inhibition of Gli activity increased expression of ANO1 RNA and Ca2+-activated Cl- currents in HEK293 cells. A single-nucleotide polymorphism prevented Gli binding and showed association with irritable bowel syndrome. We conclude that Gli1 and Gli2 repress ANO1 by a novel mechanism that is independent of Gli cleavage and that has a role in gastrointestinal function.-Mazzone, A., Gibbons, S. J., Eisenman, S. T., Strege, P. R., Zheng, T., D'Amato, M., Ordog, T., Fernandez-Zapico, M. E., Farrugia, G. Direct repression of anoctamin 1 (ANO1) gene transcription by Gli proteins.

Characterisation of DOG-1 Expression in Salivary Gland Tumours and Comparison with Myoepithelial Markers.

DOG1 is an established diagnostic marker for gastrointestinal stromal tumours (GIST), but has been reported in salivary gland tumours (SGT) as an acinar and intercalated duct marker. However, its specificity and distribution is not well established. The aim of this study was to evaluate the diagnostic utility of DOG-1 expression in SGT in addition to comparing it with myoepithelial markers. Normal salivary tissue and SGT (n = 184) were examined for expression of DOG1 and a range of myoepithelial markers. SGT included: acinic cell carcinoma (ACC, n = 15), secretory carcinoma (SC, n = 9), pleomorphic adenoma (PA, n = 49), carcinoma ex-PA (Ca ex-PA, n = 11), adenoid cystic carcinoma (AdCC, n = 20), polymorphous adenocarcinoma (PAC, n = 6), myoepithelioma (n = 6), myoepithelial carcinoma (MC, n = 2), basal cell adenoma (BCA, n = 14), canalicular adenoma (CA, n = 19), mucoepidermoid carcinoma (MEC, n = 11), oncocytoma (n = 2), adenocarcinoma NOS (AdNOS, n = 4), basal cell adenocarcinoma (BCAC, n = 2), salivary duct carcinoma (SDC, n = 3) and papillary cystadenocarcinoma (PCAC, n = 1). Normal acini and ACC (14/15) showed strong luminal DOG1 staining; SC were largely negative with only focal expression in 3/9 cases. Luminal staining was seen in PA (14/49), PAC (4/6), Ca ex-PA (4/11) and AdCC (6/20). 8/11 MEC showed luminal and/or mucous cell staining. No staining was seen in myoepithelioma, MC, CA, adNOS and BCAC. BCA showed strong staining of myoepithelial cells in some cases (5/14). Variable myoepithelial DOG1 staining was seen in PA, Ca ex PA, BCA, SDC and PCAC which was not as consistent as myoepithelial markers such as calponin, p63 and αSMA. Absence of DOG1 can differentiate ACC from SC, but staining is variable in PA, PLGA and Ca ex-PA. Myoepithelial staining in some tumours but not in normal gland suggests a wider distribution in SGT than originally envisaged.

Inhibition of STAT6/Anoctamin-1 Activation Suppresses Proliferation and Invasion of Gastric Cancer Cells.

BACKGROUND: Gastric carcinoma is the most popular cancer worldwide. Anoctamin-1 is a calcium-activated channel and highly expressed in various tumors. A previous study indicated that suppressed Anoctamin-1 expression decreased cancer cell proliferation or migration. As a signal transduction and transcription activator, STAT6 is a novel agonist for Anoctamin-1 promoter. However, its role in tumor cell proliferation or migration remains unclear. Therefore, this study aimed to suppress STAT6 and Anoctamin-1 protein expression in gastric cancer cells to test the inhibitory effects on gastric cancer cell migration or invasion. MATERIALS AND METHODS: MTT colorimetry was used to test cell proliferation. Western blot was used to measure STAT6 and Anoctamin-1 expression before and after small interfering RNA (siRNA) treatment. A scratch assay was performed to measure cell migration, followed by Transwell chamber assay analysis of cell invasion. RESULTS: After STAT6 siRNA interference, the expression of STAT6 and Anoctamin-1 was significantly decreased in the gastric carcinoma cell line. Anoctamin-1 siRNA interference only decreased its protein expression, but not STAT6 protein expression. Interference of STAT6 or Anoctamin-1 reduced their protein expression and inhibited proliferation, migration, or invasion of gastric cancer cells. CONCLUSIONS: Inhibition of STAT6/Anoctamin-1 activation decreased proliferation, migration, or invasion of gastric cancer cells, suggesting that the STAT6/Anoctamin-1 pathway might be a novel target for treating gastric cancer.

DNA methylation regulates TMEM16A/ANO1 expression through multiple CpG islands in head and neck squamous cell carcinoma.

ANO1 is a calcium-activated chloride channel that is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and other cancers. While ANO1 expression negatively correlates with survival in several cancers, its epigenetic regulation is poorly understood. We analyzed HNSCC samples from TCGA and a separate dataset of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) samples to identify differentially methylated regions. E6 and E7 transfected normal oral keratinocytes (NOK) were used to induce hypermethylation of the ANO1 promoter. We found three CpG islands that correlated with ANO1 expression, including two positively correlated with expression. Using two HNSCC datasets with differential expression of ANO1, we showed hypermethylation of positively correlated CpG islands potentiates ANO1 expression. E7 but not E6 transfection of NOK cells led to hypermethylation of a positively correlated CpG island without a change in ANO1 expression. ANO1 promoter methylation was also correlated with patient survival. Our results are the first to show the contribution of positively correlated CpG's for regulating gene expression in HNSCC. Hypermethylation of the ANO1 promoter was strongly correlated with but not sufficient to increase ANO1 expression, suggesting methylation of positively correlated CpG's likely serves as an adjunct to other mechanisms of ANO1 activation.

Ca2+-activated Cl- current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization.

The role of Ca2+-activated Cl- current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

Sox10 and DOG1 Expression in Primary Adnexal Tumors of the Skin.

Primary skin adnexal tumors can be challenging to classify and must be discerned from cutaneous adenocarcinoma metastases from various sites. We evaluated expression of Sox10 and DOG1 in normal cutaneous adnexa and in 194 primary skin adnexal tumors, and compared their performance in discriminating primary skin adnexal tumors from cutaneous metastatic adenocarcinomas with that of p40 and p63. In normal skin adnexa, we noted Sox10 expression in both the secretory and myoepithelial cells in eccrine glands, but only in myoepithelial cells in apocrine glands. DOG1 demonstrated canalicular expression in eccrine glands, and weak expression in myoepithelial cells of apocrine glands, germinative cells of sebaceous glands, and outer root sheath of follicular infundibulum. Sox10 was expressed in 100% of cylindromas and spiradenomas, and in variable frequency in other benign and malignant tumors of sweat glands. DOG1 was positive in most cylindromas (87.5%), in only 10.5% of spiradenomas, and was variably expressed in other benign and malignant tumors of sweat glands. All syringomas (n = 20) were negative for Sox10 and DOG1. One out of the 33 follicular neoplasms was positive for Sox10 and DOG1 (3%). All sebaceous neoplasms were negative for Sox10, and 28.1% of them were positive for DOG1. Sox10 was specific (91.9%) but not sensitive (28.4%) for primary skin origin, and was far less accurate (38.5%) than p63 or p40 (95.5% accuracy). Combining Sox10 with p63 or p40 showed only very minimal gain in accuracy (96%). DOG1 expression in tumors showed low sensitivity and specificity for skin adnexal origin.

Expression of anoctamin 1 is associated with advanced tumor stage in patients with non-small cell lung cancer and predicts recurrence after surgery.

PURPOSE: Anoctamin 1 (ANO1), a recently identified calcium-activated chloride channel, has been found to have a critical role in tumorigenesis and tumor progression in several types of cancer. However, its role in non-small cell lung cancer (NSCLC) remains to be elucidated. In this study, we evaluated the utility of ANO1 as a prognostic marker. PATIENTS AND METHODS: ANO1 expression was detected in tumor tissues and paraneoplastic tissues of I-IV stage NSCLC patients who received surgical treatment by using immunohistochemical and quantitative RT-PCR analyses. Epidermal growth factor receptor (EGFR) was investigated using immunohistochemistry. Then the TNM stage of the tumor samples was assessed and patients were followed up for developing recurrence. RESULTS: ANO1 expression was significantly increased in NSCLC tumor tissues compared to the paraneoplastic tissues at both RNA and protein level. In addition, ANO1 overexpression was correlated with the high expression of EGFR and led to an advanced tumor stage. And also high ANO1 expression was significantly correlated with high recurrence rate at 1-year follow-up. CONCLUSIONS: ANO1 overexpression associated with the high expression of EGFR can be a predictive marker of recurrence after surgery in NSCLC patients.

ANO1 protein as a potential biomarker for esophageal cancer prognosis and precancerous lesion development prediction.

OBJECTIVES: Anoctamin 1 (ANO1) has been found to be overexpressed in esophageal squamous cell carcinoma (ESCC) in our previous study. Herein we showed the clinical relevance of ANO1 alterations with ESCC and esophageal precancerous lesion progression. RESULTS: ANO1 was detected in 38.1% (109/286) and 25.4% (77/303) of tumors in the two cohorts, but in none of morphologically normal operative margin tissues. ANO1 expression was significantly associated with a shorter overall survival (OS), especially in patients with moderately differentiated and stage IIA tumors. In 499 iodine-unstained biopsies from the endoscopic screening cohort in 2005-2007, all the 72 pathologically normal epithelial mucosa presented negative immunostaining, whereas ANO1 expression was observed in 3/11 tumors and 5/231 intraepithelial lesions. 7/8 ANO1-positive cases had developed unfavorable outcomes revealed by endoscopic follow-up in 2012. Analysis of another independent cohort of 148 intraepithelial lesions further confirmed the correlation between ANO1 expression and progression of precancerous lesions. 3/4 intraepithelial lesions with ANO1 expression had developed ESCC within 4-9 years after the initial endoscopic examination. METHODS: Immunohistochemistry (IHC) was performed to examine ANO1 expression in surgical ESCC specimens and two independent cohorts of esophageal biopsies from endoscopic screening in high-incidence area of ESCC in northern China. Association between ANO1 expression, clinico-pathologic parameters, and the impact on overall survival was analyzed. CONCLUSIONS: Positive ANO1 is a promising biomarker to predict the unfavorable outcome for ESCC patients. More importantly, it can predict disease progression of precancerous lesions.