Enhanced Expression of ANO1 in Head and Neck Squamous Cell Carcinoma Causes Cell Migration and Correlates with Poor Prognosis

Abstract

Head and neck squamous cell carcinoma (HNSCC) has the potential for early metastasis and is associated with poor survival. Ano1 (Dog1) is an established and sensitive marker for the diagnosis of gastrointestinal stromal tumors (GIST) and has recently been identified as a Ca2+ activated Cl− channel. Although the ANO1 gene is located on the 11q13 locus, a region which is known to be amplified in different types of human carcinomas, a detailed analysis of Ano1 amplification and expression in HNSCC has not been performed. It is thus still unclear how Ano1 contributes to malignancy in HNSCC. We analyzed genomic amplification of the 11q13 locus and Ano1 together with Ano1-protein expression in a large collection of HNSCC samples. We detected a highly significant correlation between amplification and expression of Ano1 and showed that HNSCC patients with Ano1 protein expression have a poor overall survival. We further analyzed the expression of the Ano1 protein in more than 4′000 human samples from 80 different tumor types and 76 normal tissue types and detected that besides HNSCC and GISTs, Ano1 was rarely expressed in other tumor samples or healthy human tissues. In HNSCC cell lines, expression of Ano1 caused Ca2+ activated Cl− currents, which induced cell motility and cell migration in wound healing and in real time migration assays, respectively. In contrast, knockdown of Ano1 did not affect intracellular Ca2+ signaling and surprisingly did not reduce cell proliferation in BHY cells. Further, expression and activity of Ano1 strongly correlated with the ability of HNSCC cells to regulate their volume. Thus, poor survival in HNSCC patients is correlated with the presence of Ano1. Our results further suggest that Ano1 facilitates regulation of the cell volume and causes cell migration, which both can contribute to metastatic progression in HNSCC.

Role of anoctamins in cancer and apoptosis

“…scale), which express much higher levels of Ano1, when compared with HT 29 cells (cf. western blot, figure 3a) [27]. In a previous study, we did not detect inhibition of proliferation of BHY cells by siRNA rstb.royalsocietypublishing.org Phil.…”

“…Surprisingly, downregulation of Ano1 contributes to cerebrovascular remodelling by promoting basilar smooth muscle cell proliferation, which is through inhibition of expression of cyclin D1 and cyclin E [26]. We performed additional experiments and compared proliferation of two HNSCC (CAL33, BHY) and one colonic epithelial cell line (HT 29 ) using an online impedance-based xCELLigence proliferation assay system [27] (figure 1a). Proliferation was clearly higher in the HNSCC cell lines (cf.…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…[132][133][134] The coding sequence for ANO1 is located on amplicon 11q13, which contains the coding sequence for several proteins related to cell cycle, proliferation and apoptosis, and which is often amplified in cancers with poor prognosis. 118,[135][136][137][138] ANO1 is upregulated in several cancer tissues including head and neck squamous cell carcinoma, prostate, breast, pancreatic cancer and gastrointestinal stromal tumor cells. 120,[139][140][141][142] Figure 14A and 14B show overexpression of ANO1 at the mRNA level and the protein level, respectively, in PDAC cells compared to normal human pancreatic ductal epithelium cells (HPDE).…”

“…147 Other studies, however, show that cell proliferation in certain cell types is unaffected by ANO1, e.g. ANO1 overexpression or knockdown do not affect proliferation in head and neck squamous cell carcinoma (HNSCC) 137,139 and ANO1 knockdown does not affect proliferation in pancreatic ductal adenocarcinoma cells. 120 ANO1 isoforms are shown to produce different channel characteristics (voltage and Ca 2C dependency).…”

“…120 ANO1 knockdown resulted in a decreased migratory rate. Although many studies have shown the importance of ANO1 in migration 137,139,141,145,146,156 there are also studies demonstrating that ANO1 might act indirectly. Thus, Ubby et al overexpressed ANO1 in HEK293 cells and measured large ANO1-like currents, but no effect on migration was observed.…”