Abstract
Metastasis leads to poor prognosis in colorectal cancer patients, and there is a growing need for new therapeutic targets. TMEM16A (ANO1, DOG1 or TAOS2) has recently been identified as a calcium-activated chloride channel (CaCC) and is reported to be overexpressed in several malignancies; however, its expression and function in colorectal cancer (CRC) remains unclear. In this study, we found expression of TMEM16A mRNA and protein in high-metastatic-potential SW620, HCT116 and LS174T cells, but not in primary HCT8 and SW480 cells, using RT-PCR, western blotting and immunofluorescence labeling. Patch-clamp recordings detected CaCC currents regulated by intracellular Ca2+ and voltage in SW620 cells. Knockdown of TMEM16A by short hairpin RNAs (shRNA) resulted in the suppression of growth, migration and invasion of SW620 cells as detected by MTT, wound-healing and transwell assays. Mechanistically, TMEM16A depletion was accompanied by the dysregulation of phospho-MEK, phospho-ERK1/2 and cyclin D1 expression. Flow cytometry analysis showed that SW620 cells were inhibited from the G1 to S phase of the cell cycle in the TMEM16A shRNA group compared with the control group. In conclusion, our results indicate that TMEM16A CaCC is involved in growth, migration and invasion of metastatic CRC cells and provide evidence for TMEM16A as a potential drug target for treating metastatic colorectal carcinoma.
Highlights
Colorectal cancer (CRC) is the third most common malignancy worldwide [1, 2], and metastasis is a crucial factor for the poor prognosis of CRC patients [3]
Immunofluorescence labeling revealed that TMEM16A was highly expressed on the cellular membrane and plasma of SW620, HCT116 and LS174T cells (S1 Fig.), whereas TMEM16A was hardly observed in HCT8 and SW480 cells (Fig. 2)
Previous studies showed that TMEM16A is frequently over-expressed in several malignancies, including SCCHN, esophageal cancer and gastrointestinal stromal tumors (GIST) [28, 36, 40]
Summary
Colorectal cancer (CRC) is the third most common malignancy worldwide [1, 2], and metastasis is a crucial factor for the poor prognosis of CRC patients [3]. Alterations of multiple gene, such as activation of oncogenes and inactivation of tumor suppressor genes, are involved in the progression of normal colonic epithelium into adenoma and into malignant adenocarcinoma [4, 5] there is limited information about the molecular changes that confer to the colorectal cancer metastasis [6, 7]. The analysis of the differential expression of genes located in this region led to the identification of TMEM16A, which is called ANO1 (anoctamin-1), DOG1 (discovered on gastrointestinal stromal tumors protein 1), ORAOV2 (oral cancer overexpressed 2) and TAOS2 (tumor-amplified and overexpressed sequence 2) [9, 10, 11, 12, 13]. TMEM16A is composed of 26 exons and contains eight transmembrane segaments with the N- and C-termini faced the cytoplasm and a reentrant loop located between TM5 and TM6 possibly forming the pore region [14]
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