Inhibition of TMEM16A Expression Suppresses Growth and Invasion in Human Colorectal Cancer Cells

<div><p>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.</p></div

Effect of T16Ainh-A01 on growth of SW480 and SW620 cells.

<p>Cells were treated with different concentrations of T16Ainh-A01 for 24 h and cell viability was measured by MTT assay. Data are expressed as Mean ± SD (n = 3).</p

TMEM16A knockdown induced a decrease in proliferation of SW620 cells.

<p>A, The growth of SW620 cells was inhibited by TMEM16A shRNA #1 and #2 compared to the control group (Mean ± SD; n = 3; * P<0.05). B, Representative immunoblots confirming knockdown of TMEM16A. Ctrl, control.</p

Expression of TMEM16A in CRC cell lines, HCT8, SW480, SW620, HCT116 and LS174T cells.

<p>A, RT-PCR reveals TMEM16A mRNA expression in SW620, HCT116 and LS174T cells, but not in HCT8 and SW480 cells. B, TMEM16A protein expression in HCT8 and SW480, SW620, HCT116 and LS174T cells was detected by Western blotting. Null FRT cells acts as negative control. FRT cells transfected with human TMEM16A serve as positive control.</p

Scratch wound assay of TMEM16A shRNA on SW620 cells.

<p>A, Migration of SW620 cells was assessed by a wound-healing assay in the presence of TMEM16A shRNA #1 and shRNA #2, compared with control shRNA. Representative images of wound closure were taken at 0 h, 24 h, 48 h and 72 h after wounding under 100× magnification. B, Bar graphs of panel A are shown. Values are the means ± SD; n = 3; ** P<0.01. Ctrl, control.</p

TMEM16A knockdown decreased activation of the MEK-ERK1/2 pathway and expression of cyclin D1.

<p>Western blot results show inhibition of TMEM16A led to a decrease in phospho-MEK, phospho-ERK1/2 and cyclin D1. Ctrl, control.</p

Immunofluorescence labeling of TMEM16A in CRC cell lines.

<p>Cells were grown on coverslips and stained with anti-TMEM16A antibodies. Left column, anti-TMEM16A immunofluorescence (Cy3, red). Middle column, DAPI staining to visualize nuclei. Right column, merged images are composites of anti-TMEM16A immunofluorescence and DAPI staining (200×).</p

Inhibition of TMEM16A decreased SW620 cell migration and invasion.

<p>A, Migration (without Matrigel) and invasion (with Matrigel) of SW620 cells are significantly suppressed by knockdown of TMEM16A compared with the control group through transwell penetration assays. Non-migrated cells were scraped with a cotton swab. Cells that penetrated the transwell filters were stained with Coomassie blue. Representative images are shown. B and C, Bar graphs of panel A are shown. Values are the means ± SD; n = 3; ** P<0.01. Ctrl, control.</p

Flow cytometric analysis of the cell cycle distribution in SW620 cells.

<p>A, B and C, Flow cytometric analysis of the cell cycle distribution of SW620 cells transfected with control shRNA,TMEM16A shRNA #1 and #2 respectively for 72 h. D, Bar graphs showing the relative percentage of cells in the indicated phases of the cell cycle after knockdown of TMEM16A. Data are expressed as the mean ± SD; n = 3; * P<0.05.</p

Knockdown of TMEM16A in SW620 cells.

<p>A, Western blot analysis of TMEM16A protein expression in SW620 cells that were transfected with shRNA #1 and #2. B, The bar graph summarizes the relative expression level of TMEM16A protein from panel A. Expression of TEMEM16A protein was normalized with the expression level of β-actin (n = 3). C, Representative immunofluence images of SW620 cells are shown after TMEM16A is knockdown by shRNA #1 and shRNA #2, compared with control shRNA (200×). D, The bar graph summarizes the peak currents recorded at 100 mV voltage in SW620 cells after TMEM16A was knocked down, compared with control shRNA. **p<0.01. All data are shown as mean ± SD. E, Whole-cell currents were recorded from SW620 cells transfected with control shRNA, shRNA #1 and #2 at a holding potential of 0 mV followed by pulsing voltages between ±100 mV in steps of 20 mV.</p