ANO1 Is Amplified, Overexpressed, and Associated with a Poor Prognosis in Breast Cancer.

In a search for potential oncogenes other than CCND1 within tumors exhibiting 11q13 amplification, we analyzed comparative genomic hybridization data of primary breast tumor samples. As expected, we found a significant gain in copy number in the 11q13 region. Genomic fine mapping revealed that the most frequently and highly amplified region spans ∼5 Mb (67–72 Mb) and contains ANO1 and 15 other protein-coding genes, including fibroblast growth factor 4 (FGF4), fibroblast growth factor 9 (FGF9), cyclin D1 (CCND1), Fas (TNFRSF6)-associated via death domain (FADD), and cortactin (CTTN) (3–5, 10, 27, 30, 31) (Fig. 1A). Interestingly, ANO1 was found repeatedly within the summit of amplification (i.e., in terms of copy number and frequency), suggesting that tumors with increased ANO1 copy number have a selective advantage (Fig. 1A, arrow). Similar results were obtained in HNSCC primary tumor samples, supporting the significance of ANO1 as a potential oncogenic driver in both cancer types (Fig. S1A).

Next, we assessed whether amplification of ANO1 correlates with overexpression and found that 11q13 amplification results in higher mRNA expression of ANO1 in breast and HNSCC tumors than in non–11q13-amplified tumors (Fig. 1B and Fig. S1B). As expected based on the close proximity, we found a significant correlation between the amplification of ANO1 and CCND1 in primary HNSCC and breast cancer tumor samples (Fig. S1C). However, we found no correlation between ANO1 and CCND1at the mRNA level (Fig. S1D) and only a weak correlation at the protein level (Fig. S1 E and F).

To determine whether there is an association between ANO1 amplification and clinical outcome in breast cancer patients, we analyzed a published dataset of copy number and overall survival in breast cancer patients (32) and found that amplification of ANO1 correlates with high grade disease and is a negative predictor for overall survival (Fig. 1C). In agreement with ANO1 being an important predictor for survival in breast cancer, we found a significant correlation between ANO1 expression levels and overall survival in breast cancer patients (Fig. S1H).

To examine whether the observed amplification and overexpression of ANO1 results in higher ANO1 protein levels, we stained primary breast tumors for ANO1. ANO1 staining was positive in 78% of breast tumors (Fig. 1D and Table S1). Staining of primary HNSCC and ESCC tumors for ANO1 revealed that 100% of primary HNSCC tumors and 90% of ESCC tumors are positive for ANO1 (Fig. S1G and Table S1). Thus, ANO1 is amplified and highly expressed in breast cancer and other tumors and associates with a poor prognosis.

ANO1 Is Critical for Cell Survival and Proliferation in 11q13-Amplified Breast Cancer, HNSCC, and ESCC Cells.

To find suitable models for testing the involvement of ANO1 in tumorigenesis, we analyzed a panel of established breast cancer cell lines for ANO1 amplification. A large subset of cell lines showed amplification of the same region identified in primary breast tumor samples (Fig. S1I and Table S2). Consistent with genomic amplification of ANO1, mRNA (Fig. S1J) and protein levels (Fig. S1M) were significantly higher in 11q13-amplified breast cancer cell lines than in non–11q13-amplified lines. Based on these results, we selected the ZR75-1, HCC1954, and MDA-MB-415 breast cancer cell lines for further experiments. Additionally, we profiled a set of established HNSCC and ESCC cell lines and selected FaDu (ESCC) and Te11 (HNSCC) as additional cell lines for our experiments (Fig. S1 K, L, and N).

Next, we tested whether ANO1 is important for the survival and proliferation of 11q13-amplified cancer cells by first constructing doxycycline (dox)-inducible lentiviral vectors expressing three independent shRNA constructs targeting ANO1 (shRNA_#1–#3) or a nontargeting shRNA (shRNA-NT). We then generated stable pools of ZR75-1, HCC1954, MDA-MB-415, Te11, and FaDu cells expressing these shRNAs. Induction of the expression of shRNA_#1–#3, but not shRNA-NT, led to pronounced knockdown of ANO1 in all five cell lines (Fig. S2 A and B). Knockdown of ANO1 resulted in decreased viability (Fig. 2A) and colony formation (Fig. 2B) and arrested the cells in G1 of the cell cycle (Fig. 2C). Moreover, we found increased expression of cleaved poly(ADP-ribose)polymerase and active caspase-3/9 and a reduction in B-cell lymphoma 2 (Bcl2), myeloid cell leukemia sequence 1 (Mcl-1), and survivin protein levels (Fig. 2D) upon knockdown of ANO1 in breast cancer cells, further identifying it as a protein with prosurvival activity. Similar results were obtained in Te11 and FaDu cells (Fig. S2 C–F). Thus, our results demonstrate that ANO1 expression is critical for cell survival and proliferation in 11q13-amplified breast cancer, HNSCC, and ESCC cells.

ANO1 Is Sufficient to Promote Cell Proliferation in the Absence of 11q13 Amplification.

Having shown that ANO1 is amplified and overexpressed in breast cancer, we asked whether overexpression of ANO1 in non–11q13-amplified cells would be sufficient to promote cell viability. We generated pools of the immortalized but nontransformed breast epithelial cells MCF10A expressing ANO1, CCND1, a control vector (β-glucuronidase; GUS), or GFP (Fig. 2E). Overexpression of ANO1 significantly increased cell viability. Furthermore, we found that expression of ANO1 in MCF10A cells increased the levels of the antiapoptotic proteins Bcl2 and Mcl-1, suggesting that ANO1 has a prosurvival and antiapoptotic function. Despite its established role in inducing cell-cycle progression, cyclin D1 alone did not increase cell viability or enhance the effect of ANO1 (Fig. 2F). Thus, ANO1 is sufficient to promote cell viability in the absence of 11q13 amplification.

Chloride Channel Activity of ANO1 Is Required for Its Prosurvival Properties.

To determine the effect of ANO1 knockdown on chloride flux, we used a manual patch-clamp assay or the planar patch (QPatch) platform and found that ANO1 knockdown led to a significant reduction in calcium-dependent chloride currents (Fig. S3 A–D). Therefore, as expected, genetic ablation of ANO1 results in a significant loss of chloride channel activity.

We next used calcium-activated chloride channel inhibitor (CaCCinh)-A01, a low molecular weight inhibitor of ANO1 activity (25), to test whether the chloride channel activity of ANO1 is required for proliferation. Treatment of ZR75-1, HCC1954, and MDA-MB-415 cells with CaCCinh-A01 reduced cell viability (Fig. 3A) and inhibited colony formation (Fig. 3B). Similar results were obtained for FaDu and Te11 cells (Fig. S3 E and F). The sensitivity to CaCCinh-A01 correlated with both ANO1 amplification and expression levels (Table 1 and Fig. S1), suggesting that ANO1 biochemical activity is required for the promotion of cell viability in cell lines overexpressing ANO1.

The residues R621 and K668 of ANO1 map to a highly conserved region between transmembrane domains TM5 and TM6. Mutation of these residues to glutamate reduced permeability for anions while promoting cation permeability (19). In contrast to the effect of wild-type ANO1, overexpression of similar levels of the R621 or K668 mutants in MCF10A cells had no effect on cell viability (Fig. 3C and Fig. S3G). These results confirm that biochemical activity of ANO1 is required for its effects on cell viability. Treatment with CaCCinh-A01 decreased cell viability in MCF10A-ANO1 wild-type cells in a concentration-dependent manner with an IC₅₀ similar to its effect on ANO1-amplified breast cancer cell lines (IC₅₀ ∼8 µM) described above. At the same time, CaCCinh-A01 had no effect on the viability of MCF10A cells overexpressing ANO1 mutants, confirming the selectivity of the inhibitor (Fig. 3D). These data suggest that ANO1 overexpression in non–11q13-amplified cells establishes an addiction to ANO1 biochemical activity, which sensitizes the cells to the inhibition of ANO1.

ANO1 Is Required for HNSCC, ESCC, and Breast Cancer Tumor Growth and Maintenance in Vivo.

The effects of ANO1 on the growth and maintenance of 11q13-amplified cancers in vivo are unknown. To test the effects of ANO1 knockdown after overt tumor development, pools of two breast cancer cell lines (ZR75-1 and HCC1954) expressing shRNA-NT or shRNA targeting ANO1 were injected into immunodeficient mice. The growth of tumors arising from shRNA-NT cells with or without dox and from cells with ANO1 shRNA but without dox was similar. In contrast, dox treatment resulting in ANO1 knockdown significantly impaired the growth of xenografts expressing ANO1 shRNA (Fig. 4 A and B). Again, similar results were obtained in HNSCC (Te11) and ESCC (FaDu) cell lines (Fig. 4 C and D). These results show that ANO1 is critical for tumor growth and maintenance in 11q13-amplified breast cancer, HNSCC, and ESCC cells.

ANO1 Regulates EGFR- and Calcium-Dependent Signaling Pathways That Promote Cancer Cell Viability.

To define the biochemical mechanisms underlying the effects of ANO1 in breast cancer, we performed an antibody array on ZR75-1 and HCC1954 breast tumor lysates after ANO1 knockdown. We found that EGFR phosphorylation was strongly inhibited after ANO1 knockdown in ZR75-1 and HCC1954 breast tumor lysates (Fig. S4A). Subsequent analysis by immunoblotting validated these findings in ZR75-1 and HCC1954 breast tumor lysates and cell lysates (Fig. 5 A and B). In addition, the phosphorylation of ERK1/2, AKT, and v-src sarcoma viral oncogene homolog (SRC) was dramatically decreased (Fig. 5 A and B and Fig. S4B). Consistently, knockdown of ANO1 in HNSCC (Te11) and ESCC (FaDu) cells resulted in a similar inhibition of EGFR, ERK1/2, AKT, and SRC phosphorylation, supporting the hypothesis that the inhibition of EGFR signaling is a general rather than breast cancer-specific consequence of ANO1 knockdown (Fig. 5C and Fig. S4C). Furthermore, treatment of ZR75-1 and HCC1954 cells with CaCCinh-A01 decreased EGFR phosphorylation, suggesting that the chloride channel activity of ANO1 is important for its role in regulating EGFR activation (Fig. 5D). Together, these results suggest that ANO1 inhibition reduces EGFR signaling, contributing to a reduction in AKT, ERK1/2, and SRC phosphorylation and a decrease in cell viability.

To test whether the decrease in EGFR activation after knockdown of ANO1 is caused by a reduction in autocrine EGFR-ligand secretion, we measured the levels of EGF and TGF-α in the supernatant of HCC1954 and ZR75-1 cells after knockdown of ANO1. The levels of secreted EGF and TGF-α were reduced upon ANO1 knockdown (Fig. S4 E and F). These results suggest that knockdown of ANO1 reduces EGFR signaling by decreasing autocrine EGFR-ligand secretion in breast cancer cells. Next, we asked whether the reconstitution of EGFR signaling is sufficient to rescue the effect of ANO1 inhibition on cell viability. Treatment of ZR75-1 and HCC1954 cells with 20 ng/mL EGF was sufficient to reverse the inhibitory effect of CaCCinh-A01 on EGFR phosphorylation (Fig. 5D). Notably, although 20 ng/mL of EGF restored EGFR phosphorylation, the inhibitory effect of CaCCinh-A01 on cell viability was reversed only partially, suggesting that additional mechanisms are involved in the effect of ANO1 on cell viability (Fig. 5E). Given that the chloride channel activity of ANO1 is important for its oncogenic function, we hypothesized that chloride-driven activation of calcium-dependent pathways also contributes to the effects of ANO1 on cell viability. Intracellular calcium can activate the calcium/calmodulin-dependent protein kinase kinase (CAMKK) and lead to the phosphorylation and activation of CaMKII (33). To test this hypothesis, we first assessed the phosphorylation of CaMKII and found it to be dramatically reduced by ANO1 inhibition (Fig. 5 D and F). Next, we treated the cells with carbachol, a cholinergic agonist that leads to intracellular calcium release via activation of the acetylcholine receptor. Similar to EGF, carbachol restored CaMKII phosphorylation but only partially rescued the inhibitory effect of CaCCinh-A01 on cell viability (Fig. 5 E and F). Notably, combined EGF and carbachol treatments completely rescued the inhibitory effect of CaCCinh-A01 application on cell viability. Taken together, these results indicate that ANO1 regulates cell viability by modulation of both EGFR and CaMKII signaling.

Finally, we examined whether overexpression of ANO1 enhances the phosphorylation of EGFR and CAMKII. Immunoblotting revealed an increase in EGFR, SRC, and CAMKII phosphorylation in lysates of MCF10A cells overexpressing ANO1 compared with control cells (Fig. 5G and Fig. S4D). Notably, MCF10A cells overexpressing ANO1 and ANO1-amplified HNSCC, ESCC, and breast cancer cells were more sensitive to SRC inhibition (Table S3). Consistent with the observed reduction of EGFR-ligand secretion after knockdown of ANO1, overexpression of ANO1 in MCF10A cells led to increased secretion of EGF and TGF-α (Fig. S4G). Treatment of MCF10A-ANO1 cells with either the EGFR inhibitor AEE788 or with the CAMKII inhibitor KN93 reduced EGFR and CAMKII phosphorylation, respectively, to the levels of the parental cells (Fig. 5 G and H) but only partially reversed ANO1 promotion of cell viability (Fig. 5I). Consistently, ANO1-amplified HNSCC and ESCC cell lines were more sensitive than nonamplified lines to several EGFR inhibitors (Table S4). Notably, the combination of the EGFR and CAMKII inhibitors completely abrogated the effect of ANO1 overexpression on cell viability (Fig. 5I). Last, we analyzed the expression of ANO1 and the phosphorylation of EGFR and CAMKII in lysates of primary human breast tumors. Consistent with our results in human breast cancer cell lines, expression of ANO1 correlated with the phosphorylation of EGFR and CAMKII in primary human breast tumor samples (Fig. S4H). In summary, these findings suggest that ANO1 promotes oncogenesis in ANO1-amplified and -overexpressed cancers by activating EGFR- and calcium-dependent signaling pathways.

Cell Culture.

Te11, Te1, Te9, and KYSE70 cells were maintained in Roswell Park Memorial Institute medium (RPMI); KYSE150 and KYSE450 cells were maintained in 45% (vol/vol) F-12/45% (vol/vol) RPMI; and FaDu cells were maintained in Eagle's Minimum Essential Medium supplemented with 10% (vol/vol) FBS at 37 °C, 5% CO₂. ZR75-1, HCC1954, and MDA-MB-415 (MDA-415) cells were propagated in RPMI with 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin (PenStrep) at 37 °C, 5% CO₂. For experiments, the serum concentration was reduced to 0.5% (vol/vol) to avoid masking the effects of growth factors present under full-serum conditions. MCF10A cells were obtained from J. Brugge (Harvard Medical School, Boston, MA) and propagated in DMEM/F-12 medium (Invitrogen) supplemented with 5% (vol/vol) horse serum (HyClone), 20 ng/mL human recombinant EGF (Peprotech), 0.5 µg/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin (Sigma), 10 µg/mL insulin (Sigma), 100 IU/mL penicillin, and 100 µg/mL streptomycin. Te1, Te9, and Te11 cells were purchased form the RIKEN cell bank; KYSE70, KYSE150, and KYSE450 cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (the German cell bank), and all other cell lines were obtained from the American Type Culture Collection.

Compounds.

CaCCinh-A01 (Specs), KN93 (Tocris, Sigma Aldrich) and AEE788, gefitinib, erlotinib, BIBX1382, lapatinib, saracatinib, dasatinib, and bosutinib (Tocris) were dissolved in DMSO to a final concentration of 10 mM. Carbamoylcholine chloride (carbachol; Sigma Aldrich) was dissolved in ethanol to a final concentration of 10 mM. Cells were treated with the indicated concentrations of inhibitors or matching volumes of DMSO and/or ethanol.

Lentivirus Preparation and Generation of Stable Cell Lines.

For lentiviral production, HEK 293T cells were cotransfected using Fugene (Promega) with pLKO1-tet on ANO1 shRNA or pLKO1-tet on nonsilencing shRNA and packaging plasmid mix (Sigma).The culture medium was replaced with fresh medium after 16 h, and supernatants were collected 48 h and 72 h after transfection. For generation of stable lines, 0.8 × 10⁶ cells per well were infected with lentiviruses in the presence of 8 μg/mL Polybrene (Applied Bioanalytical) at a multiplicity of infection of 20. Cells stably expressing the shRNA were selected with 1.5–2 µg/mL puromycin. MCF10A cells were transduced with the lentiviral internal ribosome entry site (IRES)-GFP vector (pLKO-IRES-GFP) expressing human ANO1 (ac-splice-form) cDNA and/or the lentiviral pSD69-human phosphoglycerate kinase 1 (hPGK)-Puro-GUS vector human CCND1 cDNA or the empty vector. Cells stably expressing pSD69-hPGK-Puro-GUS were selected with 1.5 µg/mL puromycin, and GFP-expressing cells were selected using one to three rounds of cell sorting by FACS.

Copy Number Analysis.

Genomic DNA was isolated using Qiagen’s DNeasy Kit according to the manufacturer’s instructions. RT-PCR analysis was performed as described using a probe for ANO1: Hs04399219_cn, CCND1: Hs69455941_cn, and RNAseP (4401631; Invitrogen). Copy number was calculated using Copy Caller v1.0 freeware (Applied Biosystems), normalized to RNAseP and expressed relative to normal human tissue (human placenta, female D3035; Sigma).

Bioinformatics Analysis.

Clinical annotation, copy number (SNP 6.0; Affymetrix), and expression (Illumina HiSeq) calls for breast cancer and HNSCC tissue samples were obtained from ref. 32 or The Cancer Genome Atlas (https://tcga-data.nci.nih.gov/tcga/) using the Data Matrix functionality and cgdsr package, available from cBIO Cancer Genomics Portal (ww.cbioportal.org/). Breast cancer and HNSCC cell line data came from the Cancer Cell Line Encyclopedia effort, a collaboration among Novartis Institutes for Biomedical Research, the Broad Institute, and the Genomics Institute of the Novartis Foundation (57). All data were formatted, filtered, and analyzed in R. Plots were created using the ggplot2 package and ref. 58.

Cell-Viability and Colony-Formation Assays.

For measurement of cell viability, 3 × 10³ cells per well were seeded into a 96-well plate, adhered overnight, and treated with the indicated concentrations of inhibitor or solvent for 72 h. Cell viability was assessed using the Cell Proliferation Reagent WST-1 (Roche) or Cell Titer Glo (Promega). Colony-formation assays were performed by seeding 1,000 cells per well in six-well plates (for breast cancer cell lines) or 1,000 Te11 or FaDu cells per well in 24-well plates. Cells were allowed to adhere overnight before treatment with CaCCinh-A01 or DMSO as indicated. Colonies were stained after 10–18 d with 0.2% (wt/vol) crystal violet in PBS/4% (vol/vol) formalin. Colony area was quantified using the Odyssey scanner and software (LI-COR Biosciences).

Cell-Cycle Analysis.

Cells were detached using trypsin-EDTA, resuspended in growth medium, and counted. Then 1 × 10⁶ cells and supernatant were washed in PBS, fixed in 70% (vol/vol) ethanol for 60 min at 4 °C, washed twice, and resuspended in PI buffer (PBS supplemented with 50 μg/mL propidium iodide, 10 μg/mL RNase A, 0.1% (wt/vol) sodium citrate, and 0.1% (vol/vol) Triton X-100). At least 2 × 10⁴ cells per sample were analyzed with a FACScan flow cytometer (Becton Dickinson).

Conventional Patch-Clamp and QPatch Recordings.

Whole-cell currents were measured using conventional and planar (QPatch; Sophion Bioscience) patch-clamp electrophysiology (59). For conventional patch-clamp recordings, cells were plated onto glass coverslips. All measurements were performed at room temperature (21–23 °C) 24 h after plating. Currents were recorded using an Axopatch 200B patch-clamp amplifier, low-pass filtered at 2 kHz, and subsequently digitized at 10 kHz with a Digidata 1322A and pClamp 9.0 data acquisition software (Molecular Devices). For QPatch recordings, cells were harvested in serum-free medium containing trypsin inhibitor. QPatch was operated in accordance with the manufacturer’s specification. Gigaseal, whole-cell configuration, and series resistance compensation were established using QPatch software (Sophion Biosciences). Cells were measured 72 h after dox addition. The voltage protocol used for QPatch recordings combined a voltage step with a voltage ramp protocol from a holding potential of −70 mV. The protocol was repeated every 30 s. The same buffers were used for both conventional and QPatch recordings. The extracellular recording solution contained (in mM): NaCl (156), Hepes (10), glucose (10), CaCl2 (5), at pH 7.4 adjusted with NaOH. The intracellular solution contained (in mM): N-methyl-D-glucamine (130), EGTA (20), Hepes (10), MgCl2 (1), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (10), Mg ATP (2), CaCl2, at pH 7.2 adjusted with HCl; osmolarity adjusted to 320 with sucrose. The pipette solution contained either 338 nM or 1 µM free Ca²⁺.

Immunoblotting.

Cells were lysed with RIPA buffer [50 mM Tris⋅HCl (pH 8), 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, 0.1% (vol/vol) SDS] supplemented with 1× protease inhibitor mixture (Complete Mini; Roche), 0.2 mM sodium orthovanadate, 20 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride. Lysates from xenografts and human primary breast tumors were prepared by lysing kryo-homogenized tumor powder in RIPA buffer. Whole-cell lysates were subjected to SDS/PAGE and immunoblotting. Membranes were incubated with antibodies as indicated and exposed to secondary HRP-/IRDye-coupled antibodies (LI-COR Biosciences). The following primary antibodies were used: ANO1 (SP31; Abcam), Tubulin (Sigma), ERK2 (Santa Cruz), phoshpo-CAMKII (pCAMKII) (T286; Abcam) and phospho-EGFR (pEGFR) (Y1086; Epitomics); all other primary antibodies were from Cell Signaling Technology.

PathScan and Receptor Tyrosine Kinase Arrays.

Cell lysates were analyzed using the PathScan receptor tyrosine kinase (RTK) Signaling Antibody Array Kit (Cell Signaling Technology) and the Human Phospho-RTK Array Kit (R&D Systems) according to the manufacturers’ instructions. Signals were normalized to the respective positive controls on the array.

EGF and TGF-α ELISA.

Human EGF and TGF-α in the supernatant were measured by ELISA (Invitrogen and R&D Systems, respectively) according to the manufacturers’ instructions. Cells were cultured for 24 h in starving conditions, and values were normalized to total cell number at the end of the experiment.

Animal Experiments.

All work involving laboratory animals was carried out in strict accordance with the Swiss and US federal, state, local and institutional guidelines governing the use of laboratory animals in research and were approved by the Swiss Cantonal Veterinary Office of Basel or the American Association for Laboratory Animal Science and the Novartis Institutes for BioMedical Research Institutional Animal Care and Use Committee (IACUC). For HNSCC xenograft assays, 7-wk-old female outbred athymic nu/nu mice (Taconic) were inoculated s.c. in the dorsal axillary region with 2 × 10⁶ FaDu_shRNA or 5 × 10⁶ Te-11_shRNA cells plus 25% (vol/vol) Matrigel. Breast cancer xenograft assays were carried out with 7- to 9-wk-old SCID/beige and SCID/NOD mice (Jackson Labs). For orthotopic engraftment of the ZR75-1 and HCC1954 cell lines, 2 × 10⁶ cells were suspended in a 100-μL mixture of Basement Membrane Matrix Phenol Red-free (BD Biosciences) and PBS 1:1 and were injected into mouse mammary gland 4 or between mammary glands 2 and 3. For growth of the ZR75-1 cell line, mice were switched to estrogen-containing drinking water 2 d before injection. When the average tumor volume reached 100 mm³, some animals were switched to food containing dox at 400 ppm. Tumor volume and animal weight were determined every 3 or 4 d.

Immunohistochemistry.

Surgically resected tissue samples were procured under Institutional Review Board approval by Maine Medical Center Tissue Bank, Maine Medical Center Pathology Department, Portland, ME. Primary breast tumors were obtained with the appropriate informed consent. Human tissue microarray for esophageal squamous cell carcinoma (ESC961; Pantomics), for head-and-neck tumors (HN802; BioMax), normal tissues (FDA955; BioMax), and sections of archival formalin-fixed, paraffin-embedded human tumor specimen were deparaffinized and immunostained with a 1:50 dilution of anti-ANO1 antibody (SP31; Abcam) using the Ventana Discovery XT system. UltraMap HRP-conjugated secondary antibody was used, followed by 3,3'-diaminobenzidine and hematoxylin counterstaining. Staining was assessed by a pathologist, and the intensity of staining was scored as negative (0), mild (1+), moderate (2+), and strong (3+).

Statistical Analysis.

All data are expressed as means ± SEM. Statistical analyses were performed in GraphPad Prism using the Student t test or ANOVA with Tukey’s post test as appropriate. The Mann–Whitney test was used to calculate the significance of the difference in copy number in Fig. 1C.