American Association for Cancer Research
00085472can120475t-sup-f3_97k.pdf (98 kB)

Supplementary Figure 3 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 - 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).



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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