PDF file - 97K, TMEM16A manipulation via shRNA mediated knock-down or overexpression leads to a measurable change in chloride conductance. Quantitative PCR demonstrates a significant decrease (~75%) in TMEM16A levels after shRNA treatment (A; mean � SEM; n=3, ***P<0.001). T24 cells stably overexpressing TMEM16A demonstrate elevated gene expression by qPCR (B; mean � SEM; n=3, ***P<0.001). MQAE assays were used to demonstrate chloride fluxes in T24 cells treated with shRNA or cDNA expressing TMEM16A (C, D & E; mean � SEM; n=3, ***P<0.001). TMEM16A knock-down leads to a ~50% decrease in chloride-flux associated fluorescence. A comparison of the chloride fluxes for the TMEM16A overexpressing and shRNA knock-down cells to control demonstrates the relative differences in chloride channel activity over time (E). Evaluation of the TMEM16A-K610A mutant shows reduced channel activity when transfected into HEK-293T cells (mean � SEM; *P<0.05). HEK-293T cells were transfected with the indicated plasmids. Chloride conductance was evaluated by patch clamping. The TMEM16A-K610A mutant demonstrates ~20% of the activity of wild-type protein, suggesting that it functions as a hypomorphic version of the protein (F).
ARTICLE ABSTRACT
Frequent 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.
