American Association for Cancer Research
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Supplementary Figure 5 from TMEM16A Induces MAPK and Contributes Directly to Tumorigenesis and Cancer Progression

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journal contribution
posted on 2023-03-30, 21:03 authored by Umamaheswar Duvvuri, Daniel J. Shiwarski, Dong Xiao, Carol Bertrand, Xin Huang, Robert S. Edinger, Jason R. Rock, Brian D. Harfe, Brian J. Henson, Karl Kunzelmann, Rainer Schreiber, Raja S. Seethala, Ann Marie Egloff, Xing Chen, Vivian W. Lui, Jennifer R. Grandis, Susanne M. Gollin

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).



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.

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