Alpha-tubulin is the loading control. Table 1 List of selected significantly differentially expressed genes. the mucosal epithelium of the upper Irbesartan (Avapro) aerodigestive tract and present high mortality rate. Lack of efficient targeted-therapies and biomarkers towards patients stratification are caveats in the disease treatment. Anoctamin 1 (gene, is a cyclin-dependent kinase inhibitor that regulates G1-S phase transition by binding to cyclin E-CDK2 . During early G1, p27Kip1 promotes the assembly and nuclear translocation of cyclin D1-Cdk complexes . Mitogenic signals promote the assembly of cyclin D-CDK4 complexes that sequester p27Kip1 restraining its activity and favor the timely activation of cyclin E-CDK2 complexes and progression of the cell cycle . Diverse mechanisms modulate p27Kip1 function at the level of its transcription, translation, post-translation, as well as subcellular localization . Oncogene-activated pathways are involved in p27Kip1 nuclear export or cytoplasmic retention in human malignancies of epithelial origin, facilitating aberrant functions of p27Kip1, such as enhanced cell motility and migration . In this study, we investigated the gene expression patterns involved in ANO1 functions and explored their molecular mechanisms in patient-derived HNSCC cell lines cultured in three-dimensional collagen matrix. We also studied the potential of ANO1 as a drug Irbesartan (Avapro) target in HNSCC cell lines with high endogenous ANO1 expression. Our data demonstrate that ANO1 expression facilitates the expression of MCL1 and regulates the expression, subcellular distribution and proteolytic degradation of p27Kip1, which may explain how ANO1 mediates cancer cell progression. 2. Results 2.1. ANO1 Expression Promotes Cell Proliferation and EMT of Primary Cancer Cells Derived from HNSCC To study the function and involvement of ANO1 in HNSCC progression, we utilized cell lines from primary tumors of patients established at the University of Turku (UT-SCC cell lines). We have previously analyzed a mutational panel, copy number and gene expression profiles of 45 HPV-negative UT-SCC cell lines . Among eight selected cell lines with diverse ANO1 mRNA expression (Figure S1A) , immunoblotting of ANO1 identified UT-SCC-8 and UT-SCC-14 as the cell lines with endogenously highest protein expression (ANO1HIGH, Figure 1A). We next verified the high level of amplification in 11q13 region, harboring ANO1 gene, by generating the copy number profiles of these cell lines (Figure 1B), based on array comparative genomic hybridization (aCGH) data . Open in a separate window Figure 1 Comparison of ANO1 amplification Irbesartan (Avapro) and expression in a panel of University of Turku-Squamous Cell Carcinoma (UT-SCC) cell lines, and the effect of ANO1 expression on epithelial-to-mesenchymal transition (EMT). (A) Western Blot illustrating the expression of ANO1 in a panel of UT-SCC cell lines with diverse endogenous ANO1 expression levels. (B) Array comparative genomic hybridization (aCGH) profiles of chromosome 11 for UT-SCC-8 and UT-SCC-14 cell lines illustrating the high copy number amplification of 11q13. (C) Western blot analysis of EMT markers E-cadherin Rabbit Polyclonal to HSF2 and N-cadherin, and stem cell marker BMI1 in shANO1 and shScramble cell lines. GAPDH and -tubulin are the loading controls. Because silencing of ANO1 in cancer cells with 11q13 amplification results in reduction of cell proliferation, migration, and tumor growth [10,13,14,17,19], we knocked down ANO1 using two different shRNA constructs in ANO1HIGH cell lines to study its role in HNSCC. In line with previous studies, ANO1 depletion in the ANO1HIGH UT-SCC cells showed inhibition of cell proliferation (Figure S1B,C). Because ANO1 expression is linked to epithelial-to-mesenchymal transition (EMT) [11,20], we next examined if expression of selected EMT markers was affected by ANO1 depletion in the ANO1HIGH UT-SCC cells. E-cadherin levels were increased (Figure 1C), suggesting that ANO1 depletion is sufficient to revert the phenotype to a less malignant one . N-cadherin expression, an EMT marker that correlates with malignant behavior in HNSCC , decreased in ANO1 shRNA UT-SCC-8 cells. Of interest, a regulator of stemness, BMI1, was downregulated upon ANO1 depletion (Figure 1C). BMI1, together with Twist, can induce repression of E-cadherin expression in HNSCC , thus providing a possible explanation for E-cadherin increase in the ANO1-knockdown cells. These observations are in an agreement with previous findings that ANO1 promotes cell proliferation and EMT in HNSCC. 2.2. ANO1 Knockdown Leads to Downregulation of Pro-Survival Protein MCL1 Conferring Apoptosis To elucidate in an unbiased fashion the role of ANO1 in the cancerous properties of the ANO1HIGH UT-SCC cell lines, we performed RNA sequencing from ANO1 silenced cell samples.