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.

Extracellular glucose and dysfunctional insulin receptor signaling independently upregulate arterial smooth muscle TMEM16A expression.

Diabetes alters the function of ion channels responsible for regulating arterial smooth muscle membrane potential, resulting in vasoconstriction. Our prior research demonstrated an elevation of TMEM16A in diabetic arteries. Here, we explored the mechanisms involved in Transmembrane protein 16A (TMEM16A) gene expression. Our data indicate that a Snail-mediated repressor complex regulates arterial TMEM16A gene transcription. Snail expression was reduced in diabetic arteries while TMEM16A expression was upregulated. The TMEM16A promoter contained three canonical E-box sites. Electrophoretic mobility and super shift assays revealed that the -154 nt E-box was the binding site of the Snail repressor complex and binding of the repressor complex decreased in diabetic arteries. High glucose induced a biphasic contractile response in pressurized nondiabetic mouse hindlimb arteries incubated ex vivo. Hindlimb arteries incubated in high glucose also showed decreased phospho-protein kinase D1 and TMEM16A expression. In hindlimb arteries from nondiabetic mice, administration of a bolus dose of glucose activated protein kinase D1 signaling to induce Snail degradation. In both in vivo and ex vivo conditions, Snail expression exhibited an inverse relationship with the expression of protein kinase D1 and TMEM16A. In diabetic mouse arteries, phospho-protein kinase D1 increased while Akt2 and pGSK3ß levels declined. These results indicate that in nondiabetic mice, high glucose triggers a transient deactivation of the Snail repressor complex to increase arterial TMEM16A expression independently of insulin signaling. Conversely, insulin resistance activates GSK3ß signaling and enhances arterial TMEM16A channel expression. These data have uncovered the Snail-mediated regulation of arterial TMEM16A expression and its dysfunction during diabetes.NEW & NOTEWORTHY The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.

The role of anoctamin 1 in liver disease.

Liver diseases include all types of viral hepatitis, alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), cirrhosis, liver failure (LF) and hepatocellular carcinoma (HCC). Liver disease is now one of the leading causes of disease and death worldwide, which compels us to better understand the mechanisms involved in the development of liver diseases. Anoctamin 1 (ANO1), a calcium-activated chloride channel (CaCC), plays an important role in epithelial cell secretion, proliferation and migration. ANO1 plays a key role in transcriptional regulation as well as in many signalling pathways. It is involved in the genesis, development, progression and/or metastasis of several tumours and other diseases including liver diseases. This paper reviews the role and molecular mechanisms of ANO1 in the development of various liver diseases, aiming to provide a reference for further research on the role of ANO1 in liver diseases and to contribute to the improvement of therapeutic strategies for liver diseases by regulating ANO1.

The anoctamins: Structure and function.

When activated by increase in intracellular Ca2+, anoctamins (TMEM16 proteins) operate as phospholipid scramblases and as ion channels. Anoctamin 1 (ANO1) is the Ca2+-activated epithelial anion-selective channel that is coexpressed together with the abundant scramblase ANO6 and additional intracellular anoctamins. In salivary and pancreatic glands, ANO1 is tightly packed in the apical membrane and secretes Cl-. Epithelia of airways and gut use cystic fibrosis transmembrane conductance regulator (CFTR) as an apical Cl- exit pathway while ANO1 supports Cl- secretion mainly by facilitating activation of luminal CFTR and basolateral K+ channels. Under healthy conditions ANO1 modulates intracellular Ca2+ signals by tethering the endoplasmic reticulum, and except of glands its direct secretory contribution as Cl- channel might be small, compared to CFTR. In the kidneys ANO1 supports proximal tubular acid secretion and protein reabsorption and probably helps to excrete HCO3-in the collecting duct epithelium. However, under pathological conditions as in polycystic kidney disease, ANO1 is strongly upregulated and may cause enhanced proliferation and cyst growth. Under pathological condition, ANO1 and ANO6 are upregulated and operate as secretory channel/phospholipid scramblases, partly by supporting Ca2+-dependent processes. Much less is known about the role of other epithelial anoctamins whose potential functions are discussed in this review.

The physiological roles of anoctamin2/TMEM16B and anoctamin1/TMEM16A in chemical senses.

Chemical senses allow animals to detect and discriminate a vast array of molecules. The olfactory system is responsible of the detection of small volatile molecules, while water dissolved molecules are detected by taste buds in the oral cavity. Moreover, many animals respond to signaling molecules such as pheromones and other semiochemicals through the vomeronasal organ. The peripheral organs dedicated to chemical detection convert chemical signals into perceivable information through the employment of diverse receptor types and the activation of multiple ion channels. Two ion channels, TMEM16B, also known as anoctamin2 (ANO2) and TMEM16A, or anoctamin1 (ANO1), encoding for Ca2+-activated Cl¯ channels, have been recently described playing critical roles in various cell types. This review aims to discuss the main properties of TMEM16A and TMEM16B-mediated currents and their physiological roles in chemical senses. In olfactory sensory neurons, TMEM16B contributes to amplify the odorant response, to modulate firing, response kinetics and adaptation. TMEM16A and TMEM16B shape the pattern of action potentials in vomeronasal sensory neurons increasing the interspike interval. In type I taste bud cells, TMEM16A is activated during paracrine signaling mediated by ATP. This review aims to shed light on the regulation of diverse signaling mechanisms and neuronal excitability mediated by Ca-activated Cl¯ channels, hinting at potential new roles for TMEM16A and TMEM16B in the chemical senses.

Effect of Calcium Supplementation and TMEM16A Inhibition on Endoplasmic Reticulum Stress Induced by Dental Fluorosis in Mice.

BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.

High ANO1 expression is a prognostic factor and correlated with an immunosuppressive tumor microenvironment in pancreatic cancer.

Background: Aminooctylamine (ANO1) plays an oncogenic role in various cancers. However. its role in pancreatic cancer (PC) has rarely been studied. This study investigated the prognostic value of ANO1 and its correlation with the tumor microenvironment (TME) in PC. Methods: Consecutive patients with PC (n = 119) were enrolled. The expression of ANO1 in cancer cells, the expression of fibroblast activation protein (FAP) and alpha smooth muscle actin in cancer-associated fibroblasts (CAFs), and the numbers of CD8- and FOXP3-positive tumor-infiltrating lymphocytes (TILs) were evaluated using immunohistochemistry. The prognostic value of ANO1 and its correlation with CAF subgroups and TILs were analyzed. The possible mechanism of ANO1 in the TME of PC was predicted using the the Cancer Genome Atlas (TCGA) dataset. Results: The expression of AN01 was correlated with overall survival (OS) and disease-free survival. Multi-factor analysis showed that high ANO1 expression was an independent adverse prognostic factor for OS (hazard ratio, 4.137; P = 0.001). ANO1 expression was positively correlated with the expression of FAP in CAFs (P < 0.001) and negatively correlated with the number of CD8-positive TILs (P = 0.005), which was also validated by bioinformatics analysis in the TCGA dataset. Moreover, bioinformatic analysis of the TCGA dataset revealed that ANO1 may induce an immunosuppressive tumor microenvironment in pancreatic cancer in a paracrine manner. Conclusion: ANO1 is a prognostic factor in patients with PC after radical resection. ANO1 may induce an immunosuppressive tumor microenvironment in PC in a paracrine manner, suggesting that ANO1 may be a novel therapeutic target.

p90RSK pathway inhibition synergizes with cisplatin in TMEM16A overexpressing head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) constitutes one of the most common types of human cancers and often metastasizes to lymph nodes. Platinum-based chemotherapeutic drugs are commonly used for treatment of a wide range of cancers, including HNSCC. Its mode of action relies on its ability to impede DNA repair mechanisms, inducing apoptosis in cancer cells. However, due to acquired resistance and toxic side-effects, researchers have been focusing on developing novel combinational therapeutic strategies to overcome cisplatin resistance. In the current study, we identified p90RSK, an ERK1/2 downstream target, as a key mediator and a targetable signaling node against cisplatin resistance. Our results strongly support the role of p90RSK in cisplatin resistance and identify the combination of p90RSK inhibitor, BI-D1870, with cisplatin as a novel therapeutic strategy to overcome cisplatin resistance. In addition, we have identified TMEM16A expression as a potential upstream regulator of p90RSK through the ERK pathway and a biomarker of response to p90RSK targeted therapy in the context of cisplatin resistance.

Predicting Mutational Effects on Ca2+-Activated Chloride Conduction of TMEM16A Based on a Simulation Study.

The dysfunction and defects of ion channels are associated with many human diseases, especially for loss-of-function mutations in ion channels such as cystic fibrosis transmembrane conductance regulator mutations in cystic fibrosis. Understanding ion channels is of great current importance for both medical and fundamental purposes. Such an understanding should include the ability to predict mutational effects and describe functional and mechanistic effects. In this work, we introduce an approach to predict mutational effects based on kinetic information (including reaction barriers and transition state locations) obtained by studying the working mechanism of target proteins. Specifically, we take the Ca2+-activated chloride channel TMEM16A as an example and utilize the computational biology model to predict the mutational effects of key residues. Encouragingly, we verified our predictions through electrophysiological experiments, demonstrating a 94% prediction accuracy regarding mutational directions. The mutational strength assessed by Pearson's correlation coefficient is -0.80 between our calculations and the experimental results. These findings suggest that the proposed methodology is reliable and can provide valuable guidance for revealing functional mechanisms and identifying key residues of the TMEM16A channel. The proposed approach can be extended to a broad scope of biophysical systems.

Identification of determinants of lipid and ion transport in TMEM16/anoctamin proteins through a Bayesian statistical analysis.

The TMEM16/Anoctamin protein family (TMEM16x) is composed of members with different functions; some members form Ca2+-activated chloride channels, while others are lipid scramblases or combine the two functions. TMEM16x proteins are typically activated in response to agonist-induced rises of intracellular Ca2+; thus, they couple Ca2+-signalling with cell electrical activity or plasmalemmal lipid homeostasis. The structural domains underlying these functions are not fully defined. We used a Naïve Bayes classifier to gain insights into these domains. The method enabled identification of regions involved in either ion or lipid transport, and suggested domains for possible pharmacological exploitation. The method allowed the prediction of the transport property of any given TMEM16x. We envisage this strategy could be exploited to illuminate the structure-function relationship of any protein family composed of members playing different molecular roles.

A Pharmacological Investigation of the TMEM16A Currents in Murine Skeletal Myogenic Precursor Cells.

TMEM16A is a Ca2+-activated Cl- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

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.

Anoctamin-1 is induced by TGF-ß and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

Niclosamide, but not ivermectin, inhibits anoctamin 1 and 6 and attenuates inflammation of the respiratory tract.

Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.

Mechanistic basis of ligand efficacy in the calcium-activated chloride channel TMEM16A.

Agonist binding in ligand-gated ion channels is coupled to structural rearrangements around the binding site, followed by the opening of the channel pore. In this process, agonist efficacy describes the equilibrium between open and closed conformations in a fully ligand-bound state. Calcium-activated chloride channels in the TMEM16 family are important sensors of intracellular calcium signals and are targets for pharmacological modulators, yet a mechanistic understanding of agonist efficacy has remained elusive. Using a combination of cryo-electron microscopy, electrophysiology, and autocorrelation analysis, we now show that agonist efficacy in the ligand-gated channel TMEM16A is dictated by the conformation of the pore-lining helix α6 around the Ca2+ -binding site. The closure of the binding site, which involves the formation of a π-helix below a hinge region in α6, appears to be coupled to the opening of the inner pore gate, thereby governing the channel's open probability and conductance. Our results provide a mechanism for agonist binding and efficacy and a structural basis for the design of potentiators and partial agonists in the TMEM16 family.

LXR Signaling-Mediated Cholesterol Metabolism Reprogramming Regulates Cancer Cell Metastasis.

Metastasis is a key contributor to mortality in patients with cancer. While many regulators of metastasis have been identified, critical targets to prevent and inhibit metastatic tumor growth remain elusive. A recent study in this issue of Cancer Research by Deng and colleagues compared gene expression signatures between primary esophageal squamous cell carcinoma tumors and metastatic tumors and combined the analysis with genes induced in metastatic cancer cell lines, which identified anoctamin 1 (ANO1) as a key driver of metastasis. ANO1 caused cholesterol accumulation by inhibiting LXR signaling and decreased cholesterol hydroxylation by downregulating the expression of cholesterol hydroxylase CYP27A1. ANO1 also regulated tumor cell-fibroblast cross-talk that contributed to inflammatory cytokine signaling (IL1ß) and metastasis. Through in silico analysis, the study identified a novel small-molecule inhibitor of ANO1 that decreased tumor burden at a metastatic site. These studies provide novel insights into the role of ANO1 in cellular cholesterol metabolism and associated signaling in mediating metastasis. See related article by Deng et al., p. 1851.

ANO1-Mediated Inhibition of Cancer Ferroptosis Confers Immunotherapeutic Resistance through Recruiting Cancer-Associated Fibroblasts.

The application of immunotherapy in gastrointestinal (GI) cancers remains challenging because of the limited response rate and emerging therapeutic resistance. Combining clinical cohorts, multi-omics study, and functional/molecular experiments, it is found that ANO1 amplification or high-expression predicts poor outcomes and resistance to immunotherapy for GI cancer patients. Knocking-down or inhibiting ANO1 suppresses the growth/metastasis/invasion of multiple GI cancer cell lines, cell-derived xenograft, and patient-derived xenograft models. ANO1 contributes to an immune-suppressive tumor microenvironment and induces acquired resistance to anti-PD-1 immunotherapy, while ANO1 knockdown or inhibition enhances immunotherapeutic effectiveness and overcomes resistance to immunotherapy. Mechanistically, through inhibiting cancer ferroptosis in a PI3K-Akt signaling-dependent manner, ANO1 enhances tumor progression and facilitates cancer-associated fibroblast recruitment by promoting TGF-ß release, thus crippling CD8+ T cell-mediated anti-tumor immunity and generating resistance to immunotherapy. This work highlights ANO1's role in mediating tumor immune microenvironment remodeling and immunotherapeutic resistance, and introduces ANO1 as a promising target for GI cancers' precision treatment.

Identification of a druggable pocket of the calcium-activated chloride channel TMEM16A in its open state.

The calcium-activated chloride channel TMEM16A is a potential drug target to treat hypertension, secretory diarrhea, and several cancers. However, all reported TMEM16A structures are either closed or desensitized, and direct inhibition of the open state by drug molecules lacks a reliable structural basis. Therefore, revealing the druggable pocket of TMEM16A exposed in the open state is important for understanding protein-ligand interactions and facilitating rational drug design. Here, we reconstructed the calcium-activated open conformation of TMEM16A using an enhanced sampling algorithm and segmental modeling. Furthermore, we identified an open-state druggable pocket and screened a potent TMEM16A inhibitor, etoposide, which is a derivative of a traditional herbal monomer. Molecular simulations and site-directed mutagenesis showed that etoposide binds to the open state of TMEM16A, thereby blocking the ion conductance pore of the channel. Finally, we demonstrated that etoposide can target TMEM16A to inhibit the proliferation of prostate cancer PC-3 cells. Together, these findings provide a deep understanding of the TMEM16A open state at an atomic level and identify pockets for the design of novel inhibitors with broad applications in chloride channel biology, biophysics, and medicinal chemistry.

Mechanical stimulation controls osteoclast function through the regulation of Ca2+-activated Cl- channel Anoctamin 1.

Mechanical force loading is essential for maintaining bone homeostasis, and unloading exposure can lead to bone loss. Osteoclasts are the only bone resorbing cells and play a crucial role in bone remodeling. The molecular mechanisms underlying mechanical stimulation-induced changes in osteoclast function remain to be fully elucidated. Our previous research found Ca2+-activated Cl- channel Anoctamin 1 (Ano1) was an essential regulator for osteoclast function. Here, we report that Ano1 mediates osteoclast responses to mechanical stimulation. In vitro, osteoclast activities are obviously affected by mechanical stress, which is accompanied by the changes of Ano1 levels, intracellular Cl- concentration and Ca2+ downstream signaling. Ano1 knockout or calcium binding mutants blunts the response of osteoclast to mechanical stimulation. In vivo, Ano1 knockout in osteoclast blunts loading induced osteoclast inhibition and unloading induced bone loss and. These results demonstrate that Ano1 plays an important role in mechanical stimulation induced osteoclast activity changes.