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Kewei Wang - One of the best experts on this subject based on the ideXlab platform.
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discovery of 4 arylthiophene 3 carboxylic acid as inhibitor of ANO1 and its effect as analgesic agent
Acta Pharmaceutica Sinica B, 2020Co-Authors: Yuxi Wang, Kewei Wang, Jian Gao, Song Zhao, Yan Song, Han Huang, Guiwang Zhu, Peili Jiao, Guisen Zhang, Liangren ZhangAbstract:Abstract Anoctamin 1 (ANO1) is a kind of calcium-activated chloride channel involved in nerve depolarization. ANO1 inhibitors display significant analgesic activity by the local peripheral and intrathecal administration. In this study, several thiophenecarboxylic acid and benzoic acid derivatives were identified as novel ANO1 inhibitors through the shape-based virtual screening, among which the 4-arylthiophene-3-carboxylic acid analogues with the best ANO1 inhibitory activity were designed, synthesized and compound 42 (IC50 = 0.79 μmol/L) was finally obtained. Compound 42 selectively inhibited ANO1 without affecting ANO2 and intracellular Ca2+ concentration. Subsequently, the analgesic effect was investigated by intragastric administration in pain models. Compound 42 significantly attenuated allodynia which was induced by formalin and chronic constriction injury. Through homology modeling and molecular dynamics, the binding site was predicted to be located near the calcium-binding region between α6 and α8. Our study validates ANO1 inhibitors having a significant analgesic effect by intragastric administration and also provides selective molecular tools for ANO1-related research.
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The Ca2+-activated chloride channel ANO1/TMEM16A: An emerging therapeutic target for epithelium-originated diseases?
Acta Pharmaceutica Sinica B, 2020Co-Authors: Yani Liu, Zongtao Liu, Kewei WangAbstract:Abstract Anoctamin 1 (ANO1) or TMEM16A gene encodes a member of Ca2+ activated Cl– channels (CaCCs) that are critical for physiological functions, such as epithelial secretion, smooth muscle contraction and sensory signal transduction. The attraction and interest in ANO1/TMEM16A arise from a decade long investigations that abnormal expression or dysfunction of ANO1 is involved in many pathological phenotypes and diseases, including asthma, neuropathic pain, hypertension and cancer. However, the lack of specific modulators of ANO1 has impeded the efforts to validate ANO1 as a therapeutic target. This review focuses on the recent progress made in understanding of the pathophysiological functions of CaCC ANO1 and the current modulators used as pharmacological tools, hopefully illustrating a broad spectrum of ANO1 channelopathy and a path forward for this target validation.
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inhibition of ca2 activated chloride channel ANO1 suppresses ovarian cancer through inactivating pi3k akt signaling
International Journal of Cancer, 2019Co-Authors: Zongtao Liu, Sushan Zhang, Feng Hou, Congxiao Zhang, Jianjun Gao, Kewei WangAbstract:Most common ovarian cancers are epithelial carcinoma in which the etiology for carcinogenesis remains elusive. ANO1/TMEM16A, a member of Ca2+ -activated Cl- channels (CaCCs), has been demonstrated to promote epithelium-originated cancers and whether it plays a role in the pathogenesis of ovarian cancer is unknown. In our study we found that ANO1 proteins were overexpressed in human epithelial ovarian cancer cells and tissue samples. ANO1 protein upregulation was correlated with the clinical FIGO (International Federation of Gynecology and Obstetrics) staging and poor grade in ovarian cancer tissues. Interestingly, the upregulation of ANO1 gene expression was also detected in the peripheral blood mononuclear cells (PBMCs) from preoperative patients with ovarian tumors, and the down-regulation of ANO1 in the PBMCs from postoperative patients. Silencing of ANO1 inhibited proliferation and invasion of ovarian cancer cells. Mechanistically, ANO1 knockdown attenuated phosphorylation of PI3K/Akt, and inhibition of PI3K/Akt signaling by specific inhibitor LY294002 resulted in suppression of ovarian cancer cells growth promoted by ANO1 expression. Furthermore, intratumoral injection of ANO1 siRNA suppressed subcutaneous xenograft tumor growth in nude mice implanted with ovarian cancer SKOV3 cells. Taken together, our findings demonstrate that ANO1 overexpression is involved in the pathogenesis of human epithelial ovarian cancer. Inhibition of ANO1 upregulation or inactivating PI3K/Akt signaling may have therapeutic potential for epithelial ovarian cancer, and the detection of ANO1 expression level in PBMCs from patients may also serve as a biomarker for diagnosis and prognosis of epithelial ovarian cancers.
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inhibition of ANO1 tmem16a induces apoptosis in human prostate carcinoma cells by activating tnf α signaling
Cell Death and Disease, 2018Co-Authors: Yan Song, Kewei Wang, Lizhao Guan, Jian Gao, Jianjun Gao, Xiaoling ChenAbstract:Overexpression of the Ca2+-activated chloride channel ANO1/TMEM16A is implicated in tumorigenesis, and inhibition of ANO1 overexpression suppresses xenograft tumor growth and invasiveness. However, the underlying molecular mechanism for ANO1 inhibition in suppression of tumorigenesis remains unknown. Here, we show that silencing or inhibition of endogenous ANO1 inhibits cell growth, induces apoptosis and upregulates TNF-α expression in prostate cancer PC-3 cells. Enhancement of TNF-α signaling by ANO1 knockdown leads to upregulation of phosphorylated Fas-associated protein with death domain and caspase activation. Furthermore, silencing of ANO1 inhibits growth of PC-3 xenograft tumors in nude mice and induces apoptosis in tumors via upregulation of TNF-α signaling. Taken together, our findings provide mechanistic insight into promoting apoptosis in prostate cancer cells by ANO1 inhibition through upregulation of TNF-α signaling.
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inhibition of calcium activated chloride channel ANO1 suppresses proliferation and induces apoptosis of epithelium originated cancer cells
Oncotarget, 2016Co-Authors: Lizhao Guan, Kewei Wang, Jian Gao, Yan Song, Jianjun GaoAbstract:// Lizhao Guan 1 , Yan Song 1 , Jian Gao 1 , Jianjun Gao 2 , KeWei Wang 1, 2 1 Department of Molecular and Cellular Pharmacology, State Key Laboratory of Natural and Biomimetic Drugs, Peking University School of Pharmaceutical Sciences, Beijing 100191, China 2 Department of Pharmacology, Qingdao University School of Pharmacy, Qingdao 266021, China Correspondence to: KeWei Wang, email: wangkw@bjmu.edu.cn , wangkw@qdu.edu.cn Keywords: ANO1, proliferation, apoptosis, migration, cancer, CaCC inh -A01, T16A inh -A01 Received: March 02, 2016 Accepted: October 01, 2016 Published: October 08, 2016 ABSTRACT ANO1, a calcium-activated chloride channel, has been reported to be amplified or overexpressed in tissues of several cancers. However, reports on its roles in tumor progression obtained from cancer cell lines are inconsistent, suggesting that the role of ANO1 in tumorigenesis is likely dependent on either its expression level or cell-type expressing ANO1. To investigate the biological roles of ANO1 in different tumor cells, we, in this study, selected several cancer cell lines and a normal HaCaT cell line with high expression levels of ANO1, and examined the function of ANO1 in these cells using approaches of lentiviral knockdown and pharmacological inhibition. We found that ANO1 knockdown significantly inhibited cell proliferation and induced cell apoptosis in either tumor cell lines or normal HaCaT cell line. Moreover, silencing ANO1 arrested cancer cells at G1 phase of cell cycle. Treatment with ANO1 inhibitor CaCC inh -A01 reduced cell viability in a dose-dependent manner. Furthermore, both ANO1 inhibitors CaCC inh -A01 and T16A inh -A01 significantly suppressed cell migration. Our findings show that ANO1 overexpression promotes cancer cell proliferation and migration; and genetic or pharmacological inhibition of ANO1 induces apoptosis and cell cycle arrest at G1 phase in different types of epithelium-originated cancer cells.
Poole, Alastair W. - One of the best experts on this subject based on the ideXlab platform.
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Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage
'FASEB', 2016Co-Authors: Mattheij, Nadine J. A., Braun Attila, Castoldi Elisabetta, Pircher Joachim, Baaten, Constance C. F. M. J., Wülling Manuela, Kuijpers, Marijke J. E., Köhler Ralf, Van Kruchten Roger, Poole, Alastair W.Abstract:Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, ANO1, or Ca2+-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in ANO1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P 0.05) with reduced PS exposure (−65 to 90%); 2) lowered Ca2+-dependent swelling (−80%) and membrane blebbing (−90%); 3) reduced calpain-dependent protein cleavage (−60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.—Mattheij, N. J. A., Braun, A., van Kruchten, R., Castoldi, E., Pircher, J., Baaten, C. C. F. M. J., Wülling, M., Kuijpers, M. J. E., Köhler, R., Poole, A. W., Schreiber, R., Vortkamp, A., Collins, P. W., Nieswandt, B., Kunzelmann, K., Cosemans, J. M. E. M., Heemskerk, J. W. M. Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage
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Survival protein anoctamin‐6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage
'FASEB', 2016Co-Authors: Mattheij, Nadine J. A., Braun Attila, Kruchten Roger, Castoldi Elisabetta, Pircher Joachim, Baaten, Constance C. F. M. J., Wülling Manuela, Kuijpers, Marijke J. E., Köhler Ralf, Poole, Alastair W.Abstract:Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano) 6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, ANO1, or Ca2+-dependent K(Ca)3.1Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in ANO1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6min (control 6.4min, P 0.05) with reduced PS exposure (265 to 90%); 2) lowered Ca2+-dependent swelling (280%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome
Criss H Hartzell - One of the best experts on this subject based on the ideXlab platform.
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anoctamins tmem16 proteins chloride channels flirting with lipids and extracellular vesicles
Annual Review of Physiology, 2017Co-Authors: Jarred M Whitlock, Criss H HartzellAbstract:Anoctamin (ANO)/TMEM16 proteins exhibit diverse functions in cells throughout the body and are implicated in several human diseases. Although the founding members ANO1 (TMEM16A) and ANO2 (TMEM16B) are Ca2+-activated Cl- channels, most ANO paralogs are Ca2+-dependent phospholipid scramblases that serve as channels facilitating the movement (scrambling) of phospholipids between leaflets of the membrane bilayer. Phospholipid scrambling significantly alters the physical properties of the membrane and its landscape and has vast downstream signaling consequences. In particular, phosphatidylserine exposed on the external leaflet of the plasma membrane functions as a ligand for receptors vital for cell-cell communication. A major consequence of Ca2+-dependent scrambling is the release of extracellular vesicles that function as intercellular messengers by delivering signaling proteins and noncoding RNAs to alter target cell function. We discuss the physiological implications of Ca2+-dependent phospholipid scrambling, the extracellular vesicles associated with this activity, and the roles of ANOs in these processes.
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hypoxia augments the calcium activated chloride current carried by anoctamin 1 in cardiac vascular endothelial cells of neonatal mice
British Journal of Pharmacology, 2014Co-Authors: Jie Lou, Criss H Hartzell, Binlin Song, Yuanfeng Gong, Qiushi Wang, K E, Dayue Darrel Duan, Dan Zhao, Zhiren ZhangAbstract:Background and Purpose The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (ANO1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of ANO1 in mouse cardiac vascular endothelial cells (CVECs). Experimental Approach Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether ANO1 was expressed and functioned as CaCC in CVECs. Key Results ANO1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca2+ and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl(Ca) detected in CVECs was significantly inhibited by T16Ainh-A01, an ANO1 inhibitor, and a pore-targeting, specific anti-ANO1 antibody, and was markedly decreased in ANO1 gene knockdown CVECs. The density of ICl(Ca) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl(Ca) was inhibited by T16Ainh-A01 or anti-ANO1 antibody. Hypoxia also increased the current density of ICl(Ca) in ANO1 gene knockdown CVECs. Conclusions and Implications ANO1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances ANO1-mediated ICl(Ca) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca2+. ANO1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, ANO1 might be a potential therapeutic target to prevent ischaemic damage.
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activation of the ANO1 tmem16a chloride channel by calcium is not mediated by calmodulin
The Journal of General Physiology, 2014Co-Authors: Jinqiu Zhu, Yuanyuan Cui, Criss H HartzellAbstract:The Ca2+-activated Cl channel anoctamin-1 (ANO1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. ANO1 is activated by increases in intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to ANO1 or whether phosphorylation or additional Ca2+-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of ANO1 by Ca2+ for the following reasons. (a) Exogenous CaM has no effect on ANO1 currents in inside-out excised patches. (b) Overexpression of Ca2+-insensitive mutants of CaM have no effect on ANO1 currents, whereas they eliminate the current mediated by the small-conductance Ca2+-activated K+ (SK2) channel. (c) ANO1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, ANO1 binds very weakly to CaM in pull-down assays. (d) ANO1 is activated in excised patches by low concentrations of Ba2+, which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca2+ because the current can be repeatedly activated in excised patches in the absence of ATP or other high-energy compounds. Although ANO1 is blocked by the CaM inhibitor trifluoperazine (TFP), we propose that TFP inhibits the channel in a CaM-independent manner because TFP does not inhibit ANO1 when applied to the cytoplasmic side of excised patches. These experiments lead us to conclude that CaM is not required for activation of ANO1 by Ca2+. Although CaM is not required for channel opening by Ca2+, work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel.
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ANO1 activation does not require calmodulin
Biophysical Journal, 2013Co-Authors: Jinqiu Zhu, Yuanyuan Cui, Criss H HartzellAbstract:The Ca2+-activated Cl channel anoctamin-1 (ANO1, also called TMEM16A), a member of the Anoctamin superfamily, plays a variety of important physiological roles including epithelial fluid secretion. ANO1 is activated and gated by intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to ANO1 or whether an additional Ca2+-binding subunit such as calmodulin (CaM) is required. Here, we report that CaM is not a necessary component for ANO1 activation for the following reasons: 1) ANO1 activation by photolysis of caged Ca2+ is very rapid (<1ms), suggesting that Ca2+ binds directly to the channel. 2) Exogenous CaM has no effect on the currents generated by either the ac or abc isoforms of ANO1 in inside-out excised patches. 3) Over-expression of functionally defective CaM has no effect on ANO1 (abc isoform) currents while it eliminates the small conductance Ca-activated K (SK2) current. 4) CaM does not physically interact with ANO1 as determined by co-immunoprecipitation. 5) ANO1 is activated in excised patches by Ba, which is a very poor CaM activator. We also re-examined the effect of the CaM inhibitor trifluoperazine (TFP). TFP blocks ANO1 in a voltage-dependent manner, suggesting that TFP blocks the current by lodging in the ANO1 pore. These results show CaM is not required for ANO1 activation and supports the hypothesis that Ca binds directly to ANO1.
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anoctamin 1 tmem16a ca2 activated chloride channel stoichiometrically interacts with an ezrin radixin moesin network
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Patricia Perezcornejo, Charity Duran, Criss H Hartzell, Avanti Gokhale, Qinghuan Xiao, Victor FaundezAbstract:The newly discovered Ca2+-activated Cl− channel (CaCC), Anoctamin 1 (ANO1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of ANO1 in HEK cells or Xenopus oocytes is sufficient to generate Ca2+-activated Cl− currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the ANO1 channel. These studies provide a comprehensive footprint of putative ANO1 regulatory networks. We find that ANO1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. ANO1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and ANO1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies ANO1 current without affecting its surface expression level. Another network associated with ANO1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca2+-binding proteins, kinases, and lipid-interacting proteins are enriched in the ANO1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating ANO1 function and trafficking to polarized domains of the plasma membrane.
Anke Bill - One of the best experts on this subject based on the ideXlab platform.
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The Mechanistic Role of the Calcium-Activated Chloride Channel ANO1 in Tumor Growth and Signaling.
Advances in experimental medicine and biology, 2017Co-Authors: Anke Bill, Larry Alex GaitherAbstract:Multiple studies have described the high expression and amplification of Anoctamin 1 (ANO1) in various cancers, including, but not limited to breast cancer, head and neck cancer, gastrointestinal stromal tumors and glioblastoma. ANO1 has been demonstrated to be critical for tumor growth in breast and head and neck cancers through its regulation of EGFR signaling and pathway modulators like MAPK and protein kinase B. However, the discovery of ANO1 as a calcium activated chloride channel came as a surprise to the field and has given rise to many questions. How does a chloride channel promote oncogenesis? Is the chloride channel function of ANO1 important for its role in cancer? Does ANO1 exhibits chloride-independent functions in cancer cells? This review summarizes the current understanding of ANO1’s function in cancer, provides a synopsis of the findings addressing the open questions in the field and gives an outlook on the promising future of ANO1 as a potential therapeutic target for the treatment of various cancers.
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ANO1 tmem16a interacts with egfr and correlates with sensitivity to egfr targeting therapy in head and neck cancer
Oncotarget, 2015Co-Authors: Anke Bill, Abraham Gutierrez, Sucheta Kulkarni, Carolyn Kemp, Debora Bonenfant, Hans Voshol, Umamaheswar Duvvuri, Alex L GaitherAbstract:// Anke Bill 1 , Abraham Gutierrez 1 , Sucheta Kulkarni 2 , Carolyn Kemp 2 , Debora Bonenfant 3 , Hans Voshol 3 , Umamaheswar Duvvuri 2, 4, * , L. Alex Gaither 1, * 1 Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA 2 University of Pittsburgh, Medical Center, Department of Otolaryngology, Pittsburgh, PA 15213, USA 3 Novartis Institutes for Biomedical Research, Basel, CH-4002, Switzerland 4 VA Pittsburgh HealthCare System, Pittsburgh, PA 15213, USA * These authors have contributed equally to this work Correspondence to: L. Alex Gaither, e-mail: alex.gaither@novartis.com Umamaheswar Duvvuri, e-mail: duvvuriu@upmc.edu Keywords: epidermal growth factor receptor (EGFR), EGFR-targeted therapy, biomarker, calcium-activated chloride channel, protein-protein interaction Received: December 01, 2014 Accepted: February 07, 2015 Published: March 16, 2015 ABSTRACT The epidermal growth factor receptor (EGFR) contributes to the pathogenesis of head&neck squamous cell carcinoma (HNSCC). However, only a subset of HNSCC patients benefit from anti-EGFR targeted therapy. By performing an unbiased proteomics screen, we found that the calcium-activated chloride channel ANO1 interacts with EGFR and facilitates EGFR-signaling in HNSCC. Using structural mutants of EGFR and ANO1 we identified the trans/juxtamembrane domain of EGFR to be critical for the interaction with ANO1. Our results show that ANO1 and EGFR form a functional complex that jointly regulates HNSCC cell proliferation. Expression of ANO1 affected EGFR stability, while EGFR-signaling elevated ANO1 protein levels, establishing a functional and regulatory link between ANO1 and EGFR. Co-inhibition of EGFR and ANO1 had an additive effect on HNSCC cell proliferation, suggesting that co-targeting of ANO1 and EGFR could enhance the clinical potential of EGFR-targeted therapy in HNSCC and might circumvent the development of resistance to single agent therapy. HNSCC cell lines with amplification and high expression of ANO1 showed enhanced sensitivity to Gefitinib, suggesting ANO1 overexpression as a predictive marker for the response to EGFR-targeting agents in HNSCC therapy. Taken together, our results introduce ANO1 as a promising target and/or biomarker for EGFR-directed therapy in HNSCC.
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variomics screen identifies the re entrant loop of the calcium activated chloride channel ANO1 that facilitates channel activation
Journal of Biological Chemistry, 2015Co-Authors: Anke Bill, Oana M Popa, Michiel T Van Diepen, Abraham Gutierrez, Sarah Lilley, Maria Velkova, Kathryn Acheson, Hedaythul Choudhury, Nicole A Renaud, Douglas S AuldAbstract:The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.
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small molecule facilitated degradation of ANO1 protein a new targeting approach for anticancer therapeutics
Journal of Biological Chemistry, 2014Co-Authors: Anke Bill, Christopher Rothwell, Michelle Lynn Hall, Jason Borawski, Catherine Hodgson, Jeremy L Jenkins, Philippe Piechon, Oana Popa, Pamela Tranter, Scott TriaAbstract:ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCCinh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCCinh-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCCinh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCCinh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCCinh-A01-resistant cell pools was not affected by CaCCinh-A01 as compared with the parental cells. Consistently, CaCCinh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCCinh-A01, indicating that CaCCinh-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers.
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abstract lb 205 the calcium activated chloride channel ANO1 promotes breast cancer progression by activating egfr and camk signaling
Cancer Research, 2013Co-Authors: Adrian Britschgi, Anke Bill, Heike Brinkhaus, Christopher Rothwell, Ieuan Clay, Stephan Duss, Michael Rebhan, Pichai Raman, Chantale T Guy, Kristie WetzelAbstract:Genomic alterations are the underlining cause of many human cancers: Amplified and overexpressed genes can drive neoplastic transformation and become essential survival factors for cancer cells. Thus, they represent promising targets for anti-cancer therapies and their identification and validation is of paramount importance. In a search for novel survival factors contributing to breast cancer oncogenesis, we performed genomic fine mapping of the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human neoplasia, in a large dataset of breast cancer patients. Remarkably, we found ANO1, a calcium-activated chloride channel, to be located within the summit of the most frequently and highly amplified genomic region. Furthermore, amplification of ANO1 correlates with its overexpression and elevated protein levels and is a negative predictor for overall survival in breast cancer patients. Next, we examined how ANO1 contributes to cancer cell survival and proliferation. We found that inhibition of ANO1 expression or function reduced cancer cell viability and colony formation, and triggered apoptosis in 11q13 amplified breast cancer, HNSCC and ESCC. Furthermore, expression of ANO1 in non-transformed mammary cells increased cell viability and established a novel addiction to ANO1 biochemical activity. We next applied inducible shRNAs against ANO1 in vivo to assess the effect of ANO1 inhibition on the maintenance of established tumors. We found in four different 11q13-amplified tumor models that knockdown of ANO1 reduced tumor growth, indicating an important role for ANO1 not only in oncogenesis, but also in tumor maintenance of 11q13 amplified cancers. To explore the mechanism by which inhibition of ANO1 contributes to cancer cell viability and tumor growth, we performed antibody arrays to measure the activity of major oncogenic signaling pathways after knockdown of ANO1. Upon depletion of ANO1, activation of EGFR and several survival signaling pathways (AKT-, SRC- and ERK1/2 pathways) were reduced in different models of human cancer. Subsequent experiments showed that ANO1 modulates both EGFR- and Ca2+/calmodulin-dependent protein kinase (CAMK) signaling in breast cancer and HNSCC cells. Consistently, activation of EGFR- and CAMK correlated with the expression of ANO1 in human primary breast tumor samples. Lastly, we found that only the simultaneous stimulation of EGFR- and CAMK-signaling pathways rescued the effect of the ANO1-inhibition. In summary, our study establishes ANO1 as a key tumor-promoting factor in 11q13-amplified breast and other malignancies. Our results highlight the importance of chloride channels in cancer and provide the first detailed mechanistic insight into the activity of ANO1 in tumorigenesis. Most importantly, our findings open up new opportunities for therapeutic intervention in several prevalent cancers. Citation Format: Adrian Britschgi, Anke Bill, Heike Brinkhaus, Christopher Rothwell, Ieuan Clay, Stephan Duss, Michael Rebhan, Pichai Raman, Chantale Guy, Kristie Wetzel, Elizabeth George, M. Oana Popa, Sarah Lilley, Hedaythul Choudhury, Martin Gosling, Louis Wang, Stephanie Fitzgerald, Jason Borawski, Jonathan Baffoe, Mark Labow, L. Alex Gaither, Mohamed Bentires-Alj. The calcium activated chloride channel ANO1 promotes breast cancer progression by activating EGFR- and CAMK-signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-205. doi:10.1158/1538-7445.AM2013-LB-205
Mattheij, Nadine J. A. - One of the best experts on this subject based on the ideXlab platform.
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Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage
'FASEB', 2016Co-Authors: Mattheij, Nadine J. A., Braun Attila, Castoldi Elisabetta, Pircher Joachim, Baaten, Constance C. F. M. J., Wülling Manuela, Kuijpers, Marijke J. E., Köhler Ralf, Van Kruchten Roger, Poole, Alastair W.Abstract:Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, ANO1, or Ca2+-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in ANO1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P 0.05) with reduced PS exposure (−65 to 90%); 2) lowered Ca2+-dependent swelling (−80%) and membrane blebbing (−90%); 3) reduced calpain-dependent protein cleavage (−60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.—Mattheij, N. J. A., Braun, A., van Kruchten, R., Castoldi, E., Pircher, J., Baaten, C. C. F. M. J., Wülling, M., Kuijpers, M. J. E., Köhler, R., Poole, A. W., Schreiber, R., Vortkamp, A., Collins, P. W., Nieswandt, B., Kunzelmann, K., Cosemans, J. M. E. M., Heemskerk, J. W. M. Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage
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Survival protein anoctamin‐6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage
'FASEB', 2016Co-Authors: Mattheij, Nadine J. A., Braun Attila, Kruchten Roger, Castoldi Elisabetta, Pircher Joachim, Baaten, Constance C. F. M. J., Wülling Manuela, Kuijpers, Marijke J. E., Köhler Ralf, Poole, Alastair W.Abstract:Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano) 6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, ANO1, or Ca2+-dependent K(Ca)3.1Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in ANO1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6min (control 6.4min, P 0.05) with reduced PS exposure (265 to 90%); 2) lowered Ca2+-dependent swelling (280%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome