PDF file - 69K, TMEM16A knock-down alters progression through cell cycle. T24 cells were transfected with TMEM16A siRNA or non-targeting control. These cells were then subjected to cell cycle analysis using FACS with propidium iodide staining. Transient knock-down of TMEM16A by siRNA blocks cell cycle progression from G1 to S phase (E; mean � SEM; n=3, **P<0.01, ***P<0.001). Caspase 3/7 activation was measured as a surrogate for apoptosis in T24 control and TMEM16A overexpressing cells. TMEM16A overexpression led to a reduction in caspase3/7 activity (B). Control and TMEM16A overexpressing cells were treated with AZD6244 (a specific ERK1/2 inhibitor), leading to an abrogation of TMEM16A-induced cell growth (C). Genetic knock-down of ERK1/2 with siRNA also abolished TMEM16A-induced growth (D).
ARTICLE ABSTRACTFrequent gene amplification of the receptor-activated calcium-dependent chloride channel TMEM16A (TAOS2 or ANO1) has been reported in several malignancies. However, its involvement in human tumorigenesis has not been previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16A overexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreased overall survival in patients with SCCHN. TMEM16A overexpression significantly promoted anchorage-independent growth in vitro, and loss of TMEM16A resulted in inhibition of tumor growth both in vitro and in vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by an increase in extracellular signal–regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologic inhibition of MEK/ERK and genetic inactivation of ERK1/2 (using siRNA and dominant-negative constructs) abrogated the growth effect of TMEM16A, indicating a role for mitogen-activated protein kinase (MAPK) activation in TMEM16A-mediated proliferation. In addition, a developmental small-molecule inhibitor of TMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation in vitro. Together, our findings provide a mechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially novel therapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevant for treatment of human cancers, including SCCHN. Cancer Res; 72(13); 3270–81. ©2012 AACR.