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      Small Molecule-facilitated Degradation of ANO1 Protein : A NEW TARGETING APPROACH FOR ANTICANCER THERAPEUTICS

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          Abstract

          Background: The calcium-activated chloride channel ANO1 is highly expressed in cancer.

          Results: Inhibition of ANO1 activity alone is not sufficient to inhibit cancer cell proliferation, suggesting a novel function of ANO1 protein in cancer.

          Conclusion: The ANO1 inhibitor CaCC inh-A01 inhibits cancer cell proliferation by facilitating degradation of ANO1.

          Significance: Our results may provide a new targeting approach for antitumor therapy in ANO1-amplified cancers.

          Abstract

          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 CaCC inh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCC inh-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 CaCC inh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCC inh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCC inh-A01-resistant cell pools was not affected by CaCC inh-A01 as compared with the parental cells. Consistently, CaCC inh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCC inh-A01, indicating that CaCC inh-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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          TMEM16A confers receptor-activated calcium-dependent chloride conductance.

          Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.
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            TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity.

            Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.
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              Expression cloning of TMEM16A as a calcium-activated chloride channel subunit.

              Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                18 April 2014
                5 March 2014
                5 March 2014
                : 289
                : 16
                : 11029-11041
                Affiliations
                From the []Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139,
                the [§ ]Novartis Institutes for Biomedical Research, Horsham, West Sussex, RH12 5AB, United Kingdom, and
                the []Novartis Institutes for Biomedical Research, Basel CH-4002, Switzerland
                Author notes
                [2 ] To whom correspondence should be addressed: Novartis Institutes for Biomedical Research, 500 Technology Square, Cambridge, MA 02139. Tel.: 617-871-7209; Fax: 617-871-5783; E-mail: alex.gaither@ 123456novartis.com .
                [1]

                Present address: Schrödinger, Inc., Cambridge, MA 02139.

                Article
                M114.549188
                10.1074/jbc.M114.549188
                4036244
                24599954
                f3f9ed8e-ccbf-4f59-a2c2-e233fda7b1be
                © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Unported License applies to Author Choice Articles

                History
                : 10 January 2014
                : 24 February 2014
                Categories
                Cell Biology

                Biochemistry
                anticancer drug,cancer biology,cancer therapy,chloride channels,computational biology,er-associated degradation,ion channels,molecular modeling,protein degradation,small molecules

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