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. 2013 Mar 12;110(11):E1026-34.
doi: 10.1073/pnas.1217072110. Epub 2013 Feb 19.

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

Adrian Britschgi  1 , Anke Bill, Heike Brinkhaus, Christopher Rothwell, Ieuan Clay, Stephan Duss, Michael Rebhan, Pichai Raman, Chantale T Guy, Kristie Wetzel, Elizabeth George, M Oana Popa, Sarah Lilley, Hedaythul Choudhury, Martin Gosling, Louis Wang, Stephanie Fitzgerald, Jason Borawski, Jonathan Baffoe, Mark Labow, L Alex Gaither, Mohamed Bentires-Alj
Affiliations

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

Adrian Britschgi et al. Proc Natl Acad Sci U S A. .

Abstract

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.

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Conflict of interest statement

Conflict of interest statement: A. Bill, C.R., I.C., M.R., P.R., C.T.G., K.W., E.G., M.O.P., S.L., H.C., M.G., L.W., S.F., J. Borawski, J. Baffoe, M.L. and L.A.G. are employees of Novartis Institutes for Biomedical Research.

Figures

Fig. 1.
Fig. 1.
ANO1 is amplified and highly expressed in breast cancer and predicts survival. (A) Copy number (CN) variation (chromosome 11) in breast cancer tissue is shown by genomic location. Histograms depict the aggregation statistic for each chromosomal region (n = 819). Dashed lines highlight the 11q13 area shown at right as a zoom-in view. The arrow indicates the genomic location of ANO1. Data source: The Cancer Gene Atlas. (B) Box plots of ANO1 mRNA levels in non–11q13-amplified and 11q13-amplified breast tumor tissues. Normalized gene expression values (z-scores) were plotted (n = 469). Data source: The Cancer Gene Atlas. (C) Average copy number variation (the gray region depicts 95% confidence interval) in 11q13 is shown by genomic location for patients who survived the 7-y observation period (solid line) or died during the study (dashed line). The significance of the differences in copy number at the ANO1 promoter (vertical dashed line) is given above each plot. Data source: ref. . (D) Representative images of ANO1 expression in breast carcinoma. Note that myoepithelial cells in normal breast tissue stain positive for ANO1. (Scale bars: 5 µm.)
Fig. 2.
Fig. 2.
Knockdown of ANO1 decreases breast cancer cell viability and colony-formation capacity. (A and B) Bar graphs showing relative viability (A) and colony formation (B) of the indicated cancer cell lines after dox-induced knockdown of ANO1. Data were normalized to the respective non–dox-treated samples. Data are expressed as mean ± SEM, n = 5; *P < 0.05; **P < 0.01; ***P < 0.001, here and in the following panels. (C) Bar graphs showing the relative percentage of cells in the indicated phases of the cell cycle after dox-induced knockdown of ANO1. Data were normalized to the percentage of non–dox-treated cells in the respective cell-cycle phase. Data are expressed as mean ± SEM; n = 4. (D) Knockdown of ANO1 induces apoptosis. Immunoblots of lysates from the indicated cell lines after knockdown of ANO1. ERK2 was used as loading control. (E) Immunoblots of lysates from MCF10A cells stably transfected with ANO1 and/or CCND1. MCF10A cells expressing GFP and/or GUS were used as controls. (F) ANO1 increases MCF10A viability. Bar graphs showing relative viability of MCF10A cells stably transfected as in E. Data were normalized to the respective control vector cell lines. Data are expressed as mean ± SEM; n = 5.
Fig. 3.
Fig. 3.
Inhibition of ANO1 function decreases breast cancer cell viability and colony formation. (A and B) Bar graphs showing relative viability (A) or colony formation (B) of breast cancer cell lines after inhibition of ANO1 with CaCCinh-A01. Data were normalized to the respective DMSO-treated samples. Data are expressed as mean ± SEM; n = 5. (C) Bar graphs showing relative viability of MCF10A cells stably transfected with wild-type ANO1, the pore-mutants ANO1-R621E, or ANO1-K668E, respectively. Data were normalized to the GFP control vector cell line. Data are expressed as mean ± SEM; n = 5. (D) Bar graphs showing relative viability of MCF10A modified as in C after treatment with the indicated concentrations of CaCCinh-A01. Data were normalized within the same cell line to the DMSO-treated cells. Data are expressed as means ± SEM; n = 5. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4.
Fig. 4.
Inhibition of ANO1 reduces tumor growth and maintenance. (A and B) Growth curves of breast cancer tumors with or without knockdown of ANO1. Cells expressing shRNA-NT or shRNA_3 were orthotopically injected into SCID/beige mice, and dox treatment was started when tumors were palpable. Data are expressed as mean ± SEM; n = 5–8; P < 0.001. (C and D) Growth curves of HNSCC and ESCC tumors with or without knockdown of ANO1. nu/nu mice were s.c. injected with cells expressing shRNA-NT or shRNA_2 and were fed with dox-containing food when average tumor volume reached 100 mm3. Data are expressed as mean ± SEM; n = 8; P < 0.001.
Fig. 5.
Fig. 5.
ANO1 induces proliferation by activating both EGFR- and calcium-dependent signaling mechanisms. (A) Immunoblots of lysates from breast cancer tumors at the study end point as described in Fig. 4. (B) Immunoblots of lysates from breast cancer cell lines as indicated. (C) Immunoblots of lysates from Te11 (HNSCC) and FaDu (ESCC) cells as indicated. (D) Immunoblots of lysates from breast cancer cells treated for 6 h with 10 µM CaCCinh-A01 and/or 20 ng/mL EGF as indicated. (E) Bar graphs showing relative viability of breast cancer cell lines after treatment with 10 µM CaCCinh-A01 and/or 20 ng/mL EGF or 10 µM carbachol as indicated. Data were normalized to the respective vehicle-treated samples. Data are expressed as mean ± SEM; n = 5; ***P < 0.001; ns, not significant. (F) Immunoblots of lysates from breast cancer cells treated for 6 h with 10 µM CaCCinh-A01 and/or 10 µM carbachol as indicated. (G) Immunoblots of lysates from MCF10A cells stably expressing WT-ANO1 or the GFP vector control and treated for 6 h with DMSO or 100 nM AEE788 as indicated. (H) Immunoblots of lysates from MCF10A cells stably expressing wt-ANO1 or the GFP-empty vector control and treated for 6 h with DMSO or 5 µM KN93 as indicated. (I) Bar graphs showing relative viability of MCF10A cells expressing wild-type ANO1 or GFP-empty vector control after treatment with DMSO or 100 nM AEE788 and/or 5 µM KN93 as indicated. Data were normalized to the values of vector control cells and are expressed as mean ± SEM; n = 5; **P < 0.01; ***P < 0.001; ns, not significant.
Fig. P1.
Fig. P1.
Effects of ANO1 in breast cancer oncogenesis. Proposed model for the effect of the calcium-activated chloride channel ANO1 in oncogenesis. 11q13-amplified breast cancers depend on the promotion of cell viability and tumor maintenance by ANO1. Knockdown or inhibition of ANO1 leads to reduced activation of EGFR- and CAMK-dependent pathways and a subsequent inhibition of AKT and MAPK signaling. This inhibition results in decreased cell viability, induction of apoptosis, and inhibition of tumor growth and maintenance in several in vitro and in vivo models.
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