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Supplemental packet contains supplemental figures for "TMEM16A/ANO1 overexpression inhibits apoptosis via down-regulation of Bim expression." It also contains uncropped blots corresponding to blots in the main and supplemental figures. Uncropped blots are labeled & grouped by figure and panel in which the cropped version of the blots are used.

PDF file - 196K, Table summarizing the antibodies used for immunoblotting and IHC.

PDF file - 202K, Knockdown of TMEM16A in T24 cancer cells decreases motility and migration.

PDF file - 133K, Table summarizing the data from Figure 6.

PDF file - 132K, TMEM16A expression influences cell size measured via flow cytometry.

Purpose: TMEM16A is a calcium-activated chloride channel that is amplified in a variety of cancers, including 30% of head and neck squamous cell carcinomas (HNSCCs), raising the possibility of an anti-apoptotic role in malignant cells. This study investigated this using a multimodal, translational investigation.Experimental Design: Combination of (i) in vitro HNSCC cell culture experiments assessing cell viability, apoptotic activation, and protein expression (ii) in vivo studies assessing similar outcomes, and (iii) molecular and staining analysis of human HNSCC samples.Results: TMEM16A expression was found to correlate with greater tumor size, increased Erk 1/2 activity, less Bim expression, and less apoptotic activity overall in human HNSCC. These findings were corroborated in subsequent in vitro and in vivo studies and expanded to include a cisplatin-resistant phenotype with TMEM16A overexpression. A cohort of 41 patients with laryngeal cancer demonstrated that cases that recurred after chemoradiation failure were associated with a greater TMEM16A overexpression rate than HNSCC that did not recur.Conclusions: Ultimately, this study implicates TMEM16A as a contributor to tumor progression by limiting apoptosis and as a potential biomarker of more aggressive disease. Clin Cancer Res; 23(23); 7324–32. ©2017 AACR.

PDF file - 3718K, TMEM16A shRNA resistant DNA can rescue the expression from the stable shRNA knockdown.

PDF file - 472K, Over expression of TMEM16A in T24 cancer cells decreases motility and invasion.

PDF file - 292K, Table of values for proteomics screen.

PDF file - 78K, TMEM16A expression levels in primary SCCHN does not correlate with the presence of nodal metastases.

PDF file - 267K, Proposed model describing the role of TMEM16A in tumor progression.

Purpose: Tumor metastasis is the leading cause of death in patients with cancer. However, the mechanisms that underlie metastatic progression remain unclear. We examined TMEM16A (ANO1) expression as a key factor shifting tumors between growth and metastasis.Experimental Design: We evaluated 26 pairs of primary and metastatic lymph node (LN) tissue from patients with squamous cell carcinoma of the head and neck (SCCHN) for differential expression of TMEM16A. In addition, we identified mechanisms by which TMEM16A expression influences tumor cell motility via proteomic screens of cell lines and in vivo mouse studies of metastasis.Results: Compared with primary tumors, TMEM16A expression decreases in metastatic LNs of patients with SCCHN. Stable reduction of TMEM16A expression enhances cell motility and increases metastases while decreasing tumor proliferation in an orthotopic mouse model. Evaluation of human tumor tissues suggests an epigenetic mechanism for decreasing TMEM16A expression through promoter methylation that correlated with a transition between an epithelial and a mesenchymal phenotype. These effects of TMEM16A expression on tumor cell size and epithelial-to-mesenchymal transition (EMT) required the amino acid residue serine 970 (S970); however, mutation of S970 to alanine does not disrupt the proliferative advantages of TMEM16A overexpression. Furthermore, S970 mediates the association of TMEM16A with Radixin, an actin-scaffolding protein implicated in EMT.Conclusions: Together, our results identify TMEM16A, an eight transmembrane domain Ca2+-activated Cl− channel, as a primary driver of the “Grow” or “Go” model for cancer progression, in which TMEM16A expression acts to balance tumor proliferation and metastasis via its promoter methylation. Clin Cancer Res; 20(17); 4673–88. ©2014 AACR.

PDF file - 3396K, Overexpression of TMEM16A S970A increase anchorage dependent and independent tumor cell proliferation.

PDF file - 87K

PDF file - 109K, TMEM16A expression can be rescued by expression of cDNA that is resistant to the effects of anti-TMEM16A shRNA. Quantitative RT-PCR was used to demonstrate that the knock-down of endogenous TMEM16A induced by shRNA can be rescued by the expression of DNA engineered to be shRNA-resistant, labeled TMEM16A DNA* (mean � SEM; n=4, *P

PDF file - 72K, Patient survival is not impacted by TMEM16A amplification status. Disease-specific and overall survival of the cohort of SCCHN patients was not significantly correlated with TMEM16A amplification (A, B).

PDF file - 407K, TMEM16A overexpression promotes cell proliferation in HEK-293T cells. TMEM16A overexpression induced cellular proliferation (*P

PDF file - 109K, TMEM16A inhibition with T16A-inh01 (A01) alone or in combination with the MEK/ERK inhibitor UO126 leads to decreased tumor cell viability in vitro. UM-SCC1 and T24 cells were treated with increasing concentrations of A01. A dose dependent reduction in cell viability was observed (A, B *P

PDF file - 101K, Cyclin D1 expression is reduced in the proliferating tissues derived from Tmem16a -/- mice when compared to wild-type littermates. Tissues harvested from Tmem16a -/- and wild-type mice were subjected to immunohistochemical staining for cyclin-D1. The proliferating mesoderm demonstrated intense nuclear staining of cyclin-D1 in wild-type mice, but not in knock-out littermates (A). Levels of cyclinD1 mRNA are correlated with TMEM16A expression in tissues derived from TMEM16A (-/-), (+/-) and (+/+) mice (B). TMEM16A overexpression did not induce activation of ERK5, another MAPK that can contribute to proliferation (C).

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

PDF file - 139K, Anti-TMEM16A antibody recognizes endogeneous TMEM16A in immunohistochemistry. A rabbit polyclonal antibody was raised against a human TMEM16A epitope (as described in the Materials and Methods). This antibody was validated in immunohistochemistry using tissues obtained from Tmem16a -/- mice and wild-type littermates. Gastrointestinal stromal tumor (a tumor type that is known to overexpress TMEM16A) is used a positive control. The in-house antibody was compared against a commercial rabbit monoclonal DOG1 antibody (Thermo Fisher Scientific, Rockford, IL) which is a validated antibody that is used by our clinical Pathology laboratory for IHC (A). Surface biotinylation was used to demonstrate that TMEM16A is present on the cell membrane of T24 cells, and migrates at the expected molecular weight (~115kDa). Murine Tmem16a migrates at a higher molecular weight (~150 kDa), in accordance with previously published data (B).

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.

TMEM16A, a calcium-activated chloride channel, was recently isolated from oral cavity cancer cells. TMEM16A is amplified in head and neck cancers and overexpressed in gastrointestinal stromal tumors (GIST's); however, the role that TMEM16A plays in promoting tumor growth remains unclear. The goal of this research was to establish the biologic function of TMEM16A in the context of epithelial malignancies. We hypothesized that TMEM16A contributes to tumor cell survival and may be a useful prognostic factor for patients with squamous cell carcinoma of the head and neck (SCCHN). Knockdown and overexpressing SCCHN and T24 cell lines were transduced with lentiviral particles encoding RNAi and engineered to stably overexpress TMEM16A. For functional verification, electrophysiologic assays were performed to establish that the altered TMEM16A expression was modulating the chloride current. To elucidate a mechanism of TMEM16A's proliferative effects, immunoblotting and pharmacologic inhibition of potential downstream targets were performed. Our results demonstrate that the levels of TMEM16A expression modulates proliferation in vitro and in vivo, and that stable overexpression of TMEM16A increases resistance to cisplatin therapy as compared to cancer cell lines expressing endogenous TMEM16A. Specificity of these phenotypic results were confirmed using genetic rescue experiments. Next, we identified that the MAPK/Cyclin-D1 axis was a critical signaling pathway in TMEM16A-induced proliferation, and that pMAPK/MAPK and Cyclin-D1 levels paralleled expression of TMEM16A. Finally, using pharmacologic agents niflumic acid or NPPB to inhibit the TMEM16A chloride channel activity in combination with a MAPK inhibitor (U0126) demonstrated additive effects significantly decreasing cancer cell proliferation in vitro. In summary, our results provide novel insights into the biologic function of TMEM16A and its role in tumor cell proliferation. This research has enhanced our understanding about the pathogenesis of SCCHN tumors, and can potentially be translated to many other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-220. doi:10.1158/1538-7445.AM2011-LB-220