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.
Highlights
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
ANO1 has been described under multiple names [discovered on gastrointestinal stromal tumors protein 1 (DOG1), oral cancer overexpressed 2 (ORAOV2), tumor-amplified and overexpressed sequence 2 (TAOS2), and tumor-amplified and overexpressed sequence 2 (TMEM16A)] derived from its upregulation in different cancer types, including gastrointestinal stromal tumor, squamous cell carcinoma of the upper aerodigestive tract and the esophagus (ESCC), and pancreatic cancer [3,4,5, 17, 18]
In the present study we provide evidence that ANO1 is amplified, overexpressed, and contributes to breast cancer, head-and-neck squamous cell carcinoma (HNSCC), and ESCC tumorigenesis by activating EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII), subsequently inducing activation of AKT and mitogen-activated protein kinase 1 (MAPK) signaling
Summary
ANO1 Is Amplified, Overexpressed, and Associated with a Poor Prognosis in Breast Cancer. 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. 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). 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. Data represent the mean of three independent experiments. n.a., not amplified
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