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: Chemotherapy failure derived from drug resistance is the most important reason causing the recurrence in colorectal cancer patients. Therefore, it is necessary to shed light on the mechanism of chemotherapy resistance in colorectal cancer patients. : We looked into the contribution of and ANO1 to chemoresistance in the human colorectal carcinoma cell lines. We silence and overexpress in HCT116 and HT29 cells with lentivirus and siRNA knockdown technique in the absence or presence of , oxaliplatin or 5-fluorouracil (5-FU). ANO1, p-pg, cleaved PARP, cleaved caspase-3, and EGFR expression was measured by Western blot. Cell apoptosis was measured by flow cytometry. : We found that promoted ANO1 expression on colon cancer cells. Moreover, ANO1 prevent colon cancer apoptosis from oxaliplatin and 5-FU. Additionally, knockdown expression could block protective effects and increase the apoptosis effects induced by oxaliplatin and 5-FU. Therefore, might be biologically involved in the development of colon cancer chemoresistance via ANO1 pathway. : Taken together, our findings provide a valuable insight into clinical management and therapy, which may ameliorate colorectal cancer patient outcomes.

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‐β 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. This work highlights ANO1 as a promising target for gastrointestinal (GI) cancers’ precision treatment. ANO1 is highly amplified or expressed in GI cancers. Through inhibiting cancer cell ferroptosis in a PI3K‐Akt‐dependent manner, ANO1 stimulates the production and secretion of TGF‐β by cancer cells, subsequently strengthens cancer‐associated fibroblast recruitment, and cripples CD8+ T cell‐mediated anti‐tumor immune responses, generating resistance to immunotherapy.

Anoctamin 1 (ANO1) has been observed to be overexpressed in gastrointestinal and pulmonary epithelial cells, as well as in a number of cancers. Although Ano1 is involved in the prognosis of colorectal cancer (CRC), its mechanism of action in metastatic CRC has not been fully elucidated. The expression of Ano1 was assessed in samples obtained from The Cancer Genome Atlas (TCGA) database. Then, we used Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Gene set enrichment analysis (GSEA), Gene set variation analysis (GSVA), and Weighted Correlation Network Analysis (WGCNA) to determine the functions of Ano1. Additionally, random survival forest, Cox multivariate analysis, Kaplan Meier analysis, and ROC were used to determine the predictive value of Ano1 on clinical outcomes in CRC patients. Finally, HE staining, immunohistochemical (IHC) analysis and qRT-PCR were used to explore the expression of the Ano1 gene in CRC tissue. The expression level of Ano1 in CRC was significantly elevated, and the prognosis was poor. The modules with a higher proportion of upregulated genes tended to be positively correlated with Ano1-high. KNG1, GNG4, F2, POSTN, THBS2, SPP1 and FGA were identified as hub proteins of the PPI network. The heatmap showed that the expression level of the Ano1-high group was significantly negatively correlated with immune infiltrate. The overexpression of the Ano1 gene in CRC tissue samples was also confirmed by HE staining, immunohistochemical (IHC) analysis and qRT-PCR. High expression of Ano1 is closely related to a poor prognosis in patients with colorectal cancer. Ano1 may participate in the metastasis and progression, as well as the immune regulation of CRC. In summary, Ano1 can act as a potential prognostic biomarker and a novel target for CRC therapy.

Some thermosensitive transient receptor potential (TRP) channels form a protein complex with anoctamin 1 (ANO1, also called TMEM16A). TRP channels have high calcium permeability, and the calcium entering cells through TRP channel activation activates ANO1, a calcium-activated chloride channel, involved in many physiological and pathological conditions. The physiological significance of TRP channels is often mediated by their ability to activate ANO1, which controls chloride flux across the plasma membrane. This review summarizes the latest understanding on the interactions between ANO1 and thermosensitive TRP channels, including TRPV1, TRPV3, and TRPV4, which are involved in pain sensitization in primary sensory neurons, proliferation and migration of human keratinocytes, and fluid secretion such as sweat, respectively. [Display omitted]

Before anoctamins (TMEM16 proteins) were identified as a family of Ca2+-activated chloride channels and phospholipid scramblases, the founding member anoctamin 1 (ANO1, TMEM16A) was known as DOG1, a marker protein for gastrointestinal stromal tumors (GIST). Meanwhile, ANO1 has been examined in more detail, and the role of ANO1 in cell proliferation and the development of different types of malignomas is now well established. While ANO5, ANO7, and ANO9 may also be relevant for growth of cancers, evidence has been provided for a role of ANO6 (TMEM16F) in regulated cell death. The cellular mechanisms by which anoctamins control cell proliferation and cell death, respectively, are just emerging; however, the pronounced effects of anoctamins on intracellular Ca2+ levels are likely to play a significant role. Recent results suggest that some anoctamins control membrane exocytosis by setting Ca2+i levels near the plasma membrane, and/or by controlling the intracellular Cl− concentration. Exocytosis and increased membrane trafficking induced by ANO1 and ANO6 may enhance membrane expression of other chloride channels, such as CFTR and volume activated chloride channels (VRAC). Notably, ANO6-induced phospholipid scrambling with exposure of phosphatidylserine is pivotal for the sheddase function of disintegrin and metalloproteinase (ADAM). This may support cell death and tumorigenic activity of IL-6 by inducing IL-6 trans-signaling. The reported anticancer effects of the anthelminthic drug niclosamide are probably related to the potent inhibitory effect on ANO1, apart from inducing cell cycle arrest through the Let-7d/CDC34 axis. On the contrary, pronounced activation of ANO6 due to a large increase in intracellular calcium, activation of phospholipase A2 or lipid peroxidation, can lead to ferroptotic death of cancer cells. It therefore appears reasonable to search for both inhibitors and potent activators of TMEM16 in order to interfere with cancer growth and metastasis.

Volume-regulated anion channel (VRAC) is ubiquitously expressed and plays a pivotal role in vertebrate cell volume regulation. A heterologous complex of leucine-rich repeat containing 8A (LRRC8A) and LRRC8B-E constitutes the VRAC, which is involved in various processes such as cell proliferation, migration, differentiation, intercellular communication, and apoptosis. However, the lack of a potent and selective inhibitor of VRAC limits VRAC-related physiological and pathophysiological studies, and most previous VRAC inhibitors strongly blocked the calcium-activated chloride channel, anoctamin 1 (ANO1). In the present study, we performed a cell-based screening for the identification of potent and selective VRAC inhibitors. Screening of 55,000 drug-like small-molecules and subsequent chemical modification revealed 3,3′-((2-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (VI-116), a novel potent inhibitor of VRAC. VI-116 fully inhibited VRAC-mediated I− quenching with an IC50 of 1.27 ± 0.18 μM in LN215 cells and potently blocked endogenous VRAC activity in PC3, HT29 and HeLa cells in a dose-dependent manner. Notably, VI-116 had no effect on intracellular calcium signaling up to 10 μM, which completely inhibited VRAC, and showed high selectivity for VRAC compared to ANO1 and ANO2. However, DCPIB, a VRAC inhibitor, significantly affected ATP-induced increases in intracellular calcium levels and Eact-induced ANO1 activation. In addition, VI-116 showed minimal effect on hERG K+ channel activity up to 10 μM. These results indicate that VI-116 is a potent and selective VRAC inhibitor and a useful research tool for pharmacological dissection of VRAC.

Calcium-activated chloride channels (CaCCs) are widespread chloride channels which rely on calcium activation to perform their functions. In 2008, TMEM16A (also known as anoctamin1, ANO1) was identified as the molecular basis of the CaCCs, which provided the possibility to study the physiological function of CaCCs. TMEM16A is widely expressed in various cells and controls basic physiological functions, including neuronal and cardiac excitability, nerve transduction, smooth muscle contraction, epithelial Cl− secretion and fertilization. However, the abnormal function of TMEM16A may cause a variety of diseases, including asthma, gastrointestinal motility disorder and various cancers. Therefore, TMEM16A is a putative drug target for many diseases, and it is important to determine specific and efficient modulators of TMEM16A channel. In recent years, we and others have screened several natural modulators of TMEM16A against cancers and gastrointestinal motility dysfunction. This article reviews the screening methods, efficacy of TMEM16A modulators and pharmacological effects of TMEM16A modulators on different diseases.Graphic Abstact

Calcium-activated chloride channels (CaCCs) are essential for epithelial secretion, neuronal transmission, and smooth muscle function. Among the Anoctamin family, Anoctamin 1 (ANO1) and Anoctamin 2 (ANO2) are classical CaCCs proteins. ANO1 has been identified as a potential therapeutic target due to its involvement in diseases such as cancer and cystic fibrosis. However, current high-throughput screening (HTS) systems face limitations in achieving subtype-specific detection and optimizing screening strategies. Stable HTS cell models expressing ANO1 or ANO2 were constructed via lentiviral transduction in Fischer mouse thyroid (FRT) cells. The models were validated using flow cytometry, Reverse Transcription Polymerase Chain Reaction(RT-PCR), and YFP-H148Q/I152L-based iodide fluorescence quenching assays. Patch-clamp electrophysiology was employed to characterize ANO1 and ANO2 current properties. Although these electrophysiological features have been previously reported, their application in HTS workflows had not been systematically evaluated. ANO1 displayed notable current rundown under sustained stimulation with high Ca 2+ or agonist concentrations, whereas ANO2 maintained stable currents under identical conditions. Based on these findings, an optimized screening strategy was developed, incorporating agonist concentration gradients and the timing of inhibitor application. This approach improved the specificity and reliability of modulator detection. A robust and functionally validated cell-based HTS platform for CaCCs modulator discovery was established. By integrating the electrophysiological characteristics of ANO1 into the screening design, the optimized strategy enhances the accuracy of identifying selective ANO1 modulators. This work provides a methodological basis for future mechanism-driven screening of CaCCs-targeted compounds.

Transient receptor potential vanilloid 4 (TRPV4) is a widely expressed cation channel that plays an important role in many physiological and pathological processes. However, most TRPV4 drugs carry a risk of side effects. Moreover, existing screening methods are not suitable for the high-throughput screening (HTS) of drugs. In this study, a cell model and HTS method for targeting TRPV4 channel drugs were established based on a calcium-activated chloride channel protein 1 Anoctamin 1 (ANO1) and a double mutant (YFP-H148Q/I152L) of the yellow fluorescent protein (YFP). Patch-clamp experiments and fluorescence quenching kinetic experiments were used to verify that the model could sensitively detect changes in intracellular Ca2+ concentration. The functionality of the TRPV4 cell model was examined through temperature variations and different concentrations of TRPV4 modulators, and the performance of the model in HTS was also evaluated. The model was able to sensitively detect changes in the intracellular Ca2+ concentration and also excelled at screening TRPV4 drugs, and the model was more suitable for HTS. We successfully constructed a drug cell screening model targeting the TRPV4 channel, which provides a tool to study the pathophysiological functions of TRPV4 in vitro.

The Piezo1 channel, a mechanosensitive non-selective cation channel, plays a critical role in mediating calcium (Ca 2+ ) influx in response to mechanical stimuli, which is vital for numerous physiological functions. However, the specificity and potency of current Piezo1 modulators are limited, and the existing screening methodologies are not sufficiently designed for high-throughput screening (HTS). To address these challenges, we developed a customized cellular model aimed at enhancing HTS efficiency for identifying potent and selective Piezo1 modulators. We utilized a cellular model that incorporates anoctamin-1 (ANO1), a calcium-activated chloride channel, along with a green fluorescent protein mutant with ultra-high halide sensitivity (YFP-H148Q/I152L) to monitor intracellular ion concentrations through fluorescence. This model can accurately detect changes in intracellular iodine ion (I ⁻ ) concentration and rapidly and sensitively screen drugs targeting the Piezo1 channel, demonstrating exceptional performance in high-throughput screening (HTS). Our development of a pharmacological cell screening model specifically designed for Piezo1 provides a valuable tool for investigating Piezo1 modulators and their pathophysiological roles in vitro.