ANO1 amplification and expression in HNSCC with a high propensity for future distant metastasis and its functions in HNSCC cell lines

Abstract

Background:Head and neck squamous cell carcinoma (HNSCC) is associated with poor survival. To identify prognostic and diagnostic markers and therapeutic targets, we studied ANO1, a recently identified calcium-activated chloride channel (CaCC).Methods:High-resolution genomic and transcriptomic microarray analysis and functional studies using HNSCC cell line and CaCC inhibitors.Results:Amplification and overexpression of genes within the 11q13 amplicon are associated with the propensity for future distance metastasis of HPV-negative HNSCC. ANO1 was selected for functional studies based on high correlations, cell surface expression and CaCC activity. ANO1 overexpression in cells that express low endogenous levels stimulates cell movement, whereas downregulation in cells with high endogenous levels has the opposite effect. ANO1 overexpression also stimulates attachment, spreading, detachment and invasion, which could account for its effects on migration. CaCC inhibitors decrease movement, suggesting that channel activity is required for the effects of ANO1. In contrast, ANO1 overexpression does not affect cell proliferation.Interpretation:ANO1 amplification and expression could be markers for distant metastasis in HNSCC. ANO1 overexpression affects cell properties linked to metastasis. Inhibitors of CaCCs could be used to inhibit the tumourigenic properties of ANO1, whereas activators developed to increase CaCC activity could have adverse effects.

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

“…149 There are several reports highlighting the importance of ANO1 in cancer cell metastasis, i.e., Shi and coworkers demonstrated a correlation between ANO1 overexpression and lymph node metastasis of ESCC and advanced clinical stage of cancers 157 and Ayoub and collaborators found that increased ANO1 expression correlates with increased risk of developing metastases in oral and head and neck squamous cell carcinomas. 139 In contrast, Shiwarshi and coworkers find that reduced ANO1 expression slows proliferation and allow metastatic progression and they suggest that ANO1 expression is a switch between growth and metastasis. 143 …”

“…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.…”

“…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). Finally, the functional expression of ANO1 was assessed using the whole-cell patch-clamp technique (steady-state I-V relationship) and cells transfected with scrambled siRNA or siRNA targeting ANO1.…”

Role of anoctamins in cancer and apoptosis

“…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.…”