ANO1 Knockout Cell Lines

Investigate the role of anoctamin 1 (ANO1), a calcium-activated chloride channel, in cancer cell proliferation, migration, and apoptosis using ANO1 gene knockout (KO) cell lines, and evaluate its potential as a therapeutic target for prostate and oral squamous cell carcinoma.

1. ANO1 in Cancer Progression

ANO1 (TMEM16A) is overexpressed in various cancers, including prostate, oral squamous cell carcinoma (OSCC), and breast cancer, where it promotes tumor growth, metastasis, and invasion. Pharmacological inhibition of ANO1 has shown anticancer effects, but the precise mechanism remains unclear .

2. Therapeutic Need

Current treatments for advanced prostate and OSCC have limited efficacy, highlighting the need for novel targets. ANO1’s role in cancer cell survival and migration makes it a promising candidate for therapeutic intervention .

3. Technical Gap

Constitutive ANO1 KO cell lines are essential for understanding ANO1’s cell-autonomous functions in cancer, but such models have not been systematically characterized for functional studies.

1. Generation of ANO1 KO Cell LinesMethod: CRISPR/Cas9 technology was used to disrupt ANO1 in PC-3 (prostate cancer) and CAL-27 (OSCC) cells. Lentiviral vectors expressing ANO1-specific sgRNA were used, followed by puromycin selection to establish stable KO clones 17.Validation: Western blotting and RT-qPCR confirmed complete loss of ANO1 protein and mRNA expression in KO cells. Electrophysiological assays showed abolished calcium-activated chloride currents, verifying functional KO 36.2. Functional CharacterizationProliferation Inhibition: MTS assays showed reduced viability in ANO1 KO PC-3 and CAL-27 cells compared to wild-type (WT) cells, with \(IC_{50}\) values for Ani-D2 (a novel ANO1 inhibitor) increasing from 7.29 µM in WT to >30 µM in KO cells 78.Migration Suppression: Wound healing assays revealed 68–88% reduced migration in ANO1 KO PC-3 cells, with similar results in CAL-27 cells, indicating ANO1-dependent regulation of cell motility 1112.Apoptosis Induction: ANO1 KO cells showed increased caspase-3 activity and PARP cleavage, with 2.3-fold more apoptotic cells than WT cells, confirming ANO1’s role in suppressing apoptosis 1415.3. Mechanistic InsightsRNA Sequencing: Transcriptomic analysis of ANO1 KO PC-3 cells identified downregulation of MYC, HIF1A, and ERBB2 signaling pathways, and upregulation of IFIT2, NDRG1, and other immune response genes 1316.Target Specificity: ANO1 KO had minimal effect on CFTR chloride channel activity and intracellular calcium signaling, confirming ANO1-specific effects 910.

1. Mechanistic Insights

ANO1 promotes cancer progression through:

Cell Survival: Suppression of caspase-3 activation and PARP cleavage, maintaining cell viability .

Migration: Regulation of cytoskeletal remodeling and cell motility, as seen in wound healing assays .

Signaling Pathways: Activation of MYC and HIF1A pathways, critical for tumor growth and angiogenesis .

2. Translational Significance

Biomarker Potential: High ANO1 expression correlates with poor prognosis in prostate and oral cancer patients, making ANO1 a valuable prognostic biomarker. ANO1 KO cell lines enable validation of ANO1 as a predictive marker for therapeutic response .

Drug Discovery: ANO1 KO cell lines serve as robust models for screening ANO1 inhibitors, such as natural products from Mallotus apelta (e.g., Ani-D2), which reduce ANO1 protein levels without affecting mRNA expression, indicating post-translational regulation .

3. Product Utility

ANO1 KO cell lines offer unique value for:

Mechanistic Research: Dissecting ANO1’s role in calcium signaling, chloride channel activity, and cross-talk with EGFR/STAT3 pathways .

Preclinical Modeling: Recapitulating ANO1-dependent tumor progression in vitro, including cell migration and invasion, to evaluate candidate therapeutics .

Combination Therapy Testing: Assessing synergistic effects of ANO1 inhibitors with existing anticancer agents, such as cisplatin or immune checkpoint blockers .