tmem16a induces mapk and contributes directly …...whitney rank-sum test was used to test the...

Molecular and Cellular Pathobiology

TMEM16A Induces MAPK and Contributes Directly toTumorigenesis and Cancer Progression

Umamaheswar Duvvuri1,7, Daniel J. Shiwarski1, Dong Xiao1, Carol Bertrand2, Xin Huang6, Robert S. Edinger5,JasonR.Rock10, BrianD.Harfe11, Brian J.Henson8,9, Karl Kunzelmann12, Rainer Schreiber12, RajaS. Seethala3,Ann Marie Egloff1, Xing Chen1, Vivian W. Lui1, Jennifer R. Grandis1,4, and Susanne M. Gollin1,3,8,9

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 notbeen previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16Aoverexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreasedoverall 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 andin vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by anincrease in extracellular signal–regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologicinhibition 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 ofTMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation in vitro. Together, our findings provide amechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially noveltherapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevantfor treatment of human cancers, including SCCHN. Cancer Res; 72(13); 3270–81. �2012 AACR.

IntroductionAmplification of the chromosomal band 11q13 is frequently

seen in malignancies arising from breast, bladder, head andneck (SCCHN), and esophagus (1). Detailed molecular analysisof the 11q13 region led to identification of TMEM16A (alsoknown as TAOS2, DOG1, and ANO1; refs. 2–4). This genebelongs to the TMEM16 family, characterized by the presence

of 8 transmembrane domains and a highly conserved domainof unknown function (DUF590).

Recently, TMEM16A was shown to be a calcium-activatedchloride channel (CaCC; refs. 5–7). The biologic importance ofTmem16a is underscored by the lethal phenotype in the knock-out mouse because of abnormal tracheal development (8–10).TMEM16Acontributes tomany important physiologic functions,including the control of epithelial fluid transport, saliva produc-tion, and gastrointestinal tract motility (10–14). The third extra-cellular loop of TMEM16A (between transmembrane regions5 and 6) has been suggested to be the putative pore-formingdomain (6). Using mutagenesis approaches, the lysine residue atamino acid 610 (in the putative pore region) was found to becritical for its channel function (15). Specifically, mutation of thisresidue from lysine to alanine (TMEM16A-K610A) was shown tobe hypomorphic, with greatly reduced chloride conductance.

CaCCs can be proapoptotic and suppress tumor formationin breast epithelia (16). However, there are exceptions, theputative CaCC, bestrophin (BEST1) is upregulated in coloncancer cells, and can promote proliferation (17). In addition,several reports have shown that TMEM16A is overexpressed inmany tumor types including esophageal cancers, gastrointes-tinal stromal tumors, and SCCHN (18–20). These seeminglydiscordant findings prompted us to investigate the role ofTMEM16A in epithelial malignancies.

We initially determined the effects of TMEM16A on tumorproliferation (in vitro and in vivo) through gain- and loss-of-function experiments. Our results show that TMEM16A

Authors' Affiliations: Departments of 1Otolaryngology, 2Cell Biology andPhysiology, 3Pathology, 4Pharmacology andChemical Biology, and 5Renaland Electrolyte Division, University of Pittsburgh Medical Center; 6Depart-ment of Obstetrics, Gynecology, and Reproductive Sciences, University ofPittsburgh School of Medicine and Magee-Women's Research Institute,University of Pittsburgh Medical Center; 7Veterans Affairs PittsburghHealth System; 8University of Pittsburgh Cancer Institute; 9Human Genet-ics, University of Pittsburgh Graduate School of Public Health, Pittsburgh,Pennsylvania; 10Department of Cell Biology, Duke University MedicalCenter, Durham, North Carolina; 11Department of Molecular Genetics andMicrobiology and the Genetics Institute, University of Florida, Gainesville,Florida; and 12Department of Physiology, University of Regensburg,Regensburg, Germany

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for J.R. Rock: Department of Anatomy, University ofCalifornia San Francisco.

Corresponding Author: Umamaheswar Duvvuri, University of Pittsburgh,200 Lothrop Street, W948 Biomedical Science Tower, Pittsburgh, PA15213. Phone: 412-647-0954; Fax: 412-647-2080; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-0475-T

�2012 American Association for Cancer Research.

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Published OnlineFirst May 7, 2012; DOI: 10.1158/0008-5472.CAN-12-0475-T

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induces potent and specific stimulation of the extracellularsignal–regulated kinase (ERK)1/2 (as determined by phospho-ERK1/2), and contributes to the growth of cancer cell lines.These studies provide, to the best of our knowledge, the firstmechanistic description of the role of TMEM16A in humanmalignancies and suggest that this protein may play an impor-tant role in facilitating tumor growth.

Materials and MethodsTMEM16A antibody and immunoblottingA rabbit polyclonal serum was obtained by immunizing

rabbits with an epitope (CARVLEKSLKKESRNKEKR) fromexon 14 of TMEM16A. This epitope was chosen on the basisof a BLAST search that defined a unique 21 amino-acidsequence. For immunoblotting, equal amounts of protein wereseparated on SDS-PAGE and transferred to nitrocellulosemembranes. The membranes were then probed with anti-TMEM16A serum, phospho-ERK1/2, pERK1/2, p-ERK5, ERK5,B-Raf, C-Raf, phospho-AKT Ser 472, AKT (Cell Signaling), andCyclin D1 M-20 (Santa Cruz). b-Tubulin or actin was used as aloading control. Ras activation kit was used according toinstructions (Millipore, EMD). All immunoblots were scannedat 600 dpi and postprocessed using Photoshop software (Ado-be Systems). Anymanipulation was applied to the entire imageto preserve image integrity.

Cell cultureHEK-293T cells were obtained from American Type Culture

Collection (ATCC; Manassas, VA). UM-SCC1 and T24 cellswere obtained from the University of Michigan (a gift fromDr Thomas Carey). EPC1 cells were a gift fromDrH. Nakagawa.All cell lines were genotyped to establish identity within 6months of experimentation. Stable overexpressing clones weremade using DNA transfection or retroviral infection. Cells wereselected after transduction and viable cells were pooled. Indi-vidual clones were identified by the method of limiting dilu-tions. Each clone was kept for 10 passages, after which an earlypassage sample was thawed and used.

Plasmid/siRNA transfections, retrovirus generation,shRNA transductionPlasmid transfections were conducted using Fugene (DNA)

or Lipofectamine 2000 (siRNA) according to the manufac-turer's instructions. TMEM16A and TMEM16A-K610A mutantwere subcloned into pBabe-puro vectors. Retroviruses weregenerated by transfecting PhoenixAmpho cells with theseplasmids. Lentiviral shRNA and retroviral particles were usedto transduce cells. Antibiotic selection was carried out. Alltransduction experiments were repeated at least 3 separatetimes to ensure reproducibility. GFP-tagged dominant nega-tive ERK2 construct was previously described (21). ERK1/2siRNA was obtained from Cell Signaling.

Whole-cell patch clampingWhole-cell patch clamping was conducted as previously

described (22). All pipette solutions were previously described(22). All experiments used a standard protocol that alternated acurrent–voltage (I/V) step measurement. The I/V measure-

ment stepped the holding potential from�100 toþ100 mV in20 mV steps. All patch clamp results were normalized by thecell capacitance recorded at the start of the experiment.

MQAE fluorescence assaysMQAE chloride efflux assays were conducted on cells plated

onto optical Petri dishes (Matek) precoated with poly-L-lysineas previously described (23). MQAE was introduced into thecells using a hypotonic shock followed by recovery for 10minutes before the start of the experiment. The MQAE-loadedcells were then mounted on the stage of an IX-81 Olympusmicroscope and perfused to 37�C. MQAE fluorescence inten-sity was captured every 15 seconds at the 445-nm wavelengthin response to excitation at 340 nm.

The magnitude of fluorescence in each cell in the field wasquantified froma circular region of interest (ROI) drawnwithinthe cell, and the time course offluorescence changewas plottedas the average � SEM of all ROIs in the field (typically 10–50cells, "n") for a single coverslip. The rate of change in fluores-cence upon the switch from high chloride to low chloride wasdetermined for each ROI. All ROIs exhibiting a positive, linearrate of change (R2 � 0.75) from at least 3 separate, identicalexperiments (coverslips, "N") were pooled, and statistical sig-nificance was assessed for all ROIs using Student's t test.

Cell viability assay and drug treatmentsFor proliferation and viability analysis, cells were plated

in 96-well optical plates at 5� 103 cells/well. The plates weretreated the following day, as indicated. One to 3 days aftertreatment, the CellTiter-Glo Assay (Promega) was usedaccording to the manufacturer's directions.

Soft agar assay, anchorage-independent viabilitySoft agar assays were conducted as previously described

(24). Briefly, 5� 104 cells suspended in 0.7% agar solution wereplated in a 35-mm dish on top of 1.4% agar. Colonies werecounted 3weeks after plating. Colonies with a diameter greaterthan 100 mm were counted using crystal violet. Anchorage-independent viability was determined by plating cells (5� 103)in poly-HEMA coated plates as previously described (25). Cellviability was assessed 3 days after plating.

In vivo growthAll animal studies were conducted under approval from the

University of Pittsburgh and were conducted in accordancewith established guidelines. Nude mice were injected on eachflank and subsequent tumor volumes were measured when apalpable tumor was noticed. Measurements of length andwidth were recorded and used to determine the volume ofeach tumor. At the conclusion of the experiment, tumors wereharvested and processed for further evaluation.

Primary tissue samples, tissue arrayPaired normal and tumor tissueswere collected after obtain-

ing informed consent and approval from our InstitutionalReview Board. Normal adjacent mucosa is defined as histo-logically benign appearing mucosa (as judged by an experi-enced pathologist) acquired from the margins of the tumor

Role of TMEM16A in Tumor Progression

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resection. Tissue arrays containing replicate cores werecreated from patients who underwent curative surgery forSCCHN at our institution. Staining was conducted withanti-TMEM16A antisera and scored using a semiquantitativesystem (H-score) and the results correlated with survival.H-score was defined as the relative intensity, scored on a 0 to 3scale, multiplied by the percentage of positively stained cells.The H-scores for the population were analyzed to determinethemedian score. High and low expressors were categorized ashaving H-scores above or below the median.

Oncomine analysisData were abstracted from the Oncomine database, and

used to assess the relative expression of TMEM16A in tumorsversus normal adjacent mucosa. We specifically evaluated theexpression the RefSeq version of TMEM16A: NM_018043.5. Afold-change of at least 3 was used as a cut-off value.

FISH studiesFISH studies were carried out on the tissuemicroarray using

a probe for the centromere of chromosome 11 (CEP11) labeledwith SpectrumGreen (AbbottMolecular) and a probe preparedfrom a BAC clone (RP11-805J14; CHRI) and labeled by nicktranslation with SpectrumOrange. Slide processing and scor-ing were described previously (26).

Knockout mouse experimentsTmem16a knockout mice were generated as previously

described (8, 9). Tissues were obtained by dissecting the oralcavity mucosa from mice after genotyping. Tissues were snapfrozen and subsequently used for immunochemistry, RT-PCR,and immunoblotting as described earlier.

Quantitative reverse transcription-PCRThe reverse transcriptions were carried out with prede-

signed TaqMan primer and probe pairs as described earlier(2). Reverse transcriptase (RT) controls were carried out foreach RNA input each time. Quantitative PCR (qPCR) wasconducted for TMEM16A and GAPDH was used as an endog-enous control. The primer and probe sequences, conditions,and concentration have been described previously (4).

Statistical analysisAll data are reported as mean � SEM unless stated other-

wise. Cell viability and tumor xenograft measurements wereanalyzed with a 2-tailed Student's t test. A paired t test wasused to test for differences in TMEM16A expression betweenmatched tumor and benign mucosa. A two-sided Mann–Whitney rank-sum test was used to test the association ofoverexpression of TMEM16A with 11q13 amplification andTMEM16A expression on IHC. Kaplan–Meier and log ranktests of equality of survivors were used to evaluate differencesin overall survival or disease-specific survival by high versuslow TMEM16A IHC scores, as defined by the median value.Multivariable Cox proportional hazards models were usedto evaluate TMEM16A tumor protein levels as a predictorof overall survival after adjusting for age, sex, and diseasestage. The assumption of proportional hazards was tested by

evaluation of Schoenfeld residuals. P < 0.05 was considered asstatistically significant.

ResultsTMEM16A overexpression correlates with decreasedsurvival in SCCHN

To examine the protein expression profile, rabbit polyclonalantibody to TMEM16A, was generated and used for immuno-blotting and IHC (Supplementary Fig. S1a and S1b). Asexpected, Tmem16a derived from murine tissues migrates ata higher molecular weight (�150 kDa) than human TMEM16A(�115 kDa). This is consistent with previously published data(6). We further validated this antibody's specificity in IHC byevaluating tissues obtained fromwild-type and knockoutmice.

TMEM16A protein is overexpressed by 5-fold in tumorswhen compared with paired adjacent normal mucosa (Fig.1A and B; P < 0.001). Overexpression was noted in 14/17 (83%)samples. Next, we confirmed that TMEM16A mRNA is over-expressed by 5-fold in a separate cohort of primary tumors andpaired adjacent normal mucosa (Fig. 1C; P < 0.05).

TMEM16A is endogenously expressed in benign secretorytissues such as salivary gland and breast tissue (14). Wetherefore wanted to confirm that breast malignancies alsooverexpress TMEM16A. We used the Oncomine database todetermine the expression of TMEM16A in normal and malig-nant tissues from a variety of tumor types. We found thatalthough TMEM16Amay be expressed at a high level in normalbreast tissue, its expression is even higher in neoplastic breasttissue (Fig. 1D). This suggests that although endogenousTMEM16A expression may be high in some normal tissues,malignant cells derived from these tissues further upregulateTMEM16A. This finding implicates TMEM16A as a potentialtarget gene in malignant transformation.

We then determined the association between TMEM16Aoverexpression and clinical outcome of patients with SCCHN.TMEM16A expression was detected in approximately 85% ofSCCHN tumors. Kaplan–Meier survival analysis showed thatpatients with high-level tumor expression of TMEM16A haddecreased overall survival (P¼ 0.04, log rank test; Fig. 1E). HighTMEM16A tumor levels tended to be associated withdecreased overall survival in a univariable Cox proportionalhazards model (HR ¼ 3.04; 95% CI, 0.97–9.46) and in amultivariable Cox proportional hazards model adjusting forage, sex, and nodal stage (HR ¼ 2.8; 95% CI, 0.86–9.16).

However, Kaplan–Meier analysis showed no significantcorrelation between overall or disease-free survival andTMEM16A gene amplification (Supplementary Fig. S2a andS2b). Although patients whose tumors harbored TMEM16Aamplification had a median survival of 50.7 months, whereasthose not amplified for TMEM16A did not reach a mediansurvival, suggesting that theremay be a trend toward improvedsurvival in patients without gene amplification. Gene ampli-fication was not strongly correlated with protein expression.

TMEM16A promotes tumor growth and proliferationNext, we investigated the effects of TMEM16Amanipulation

(through gain-of-function and loss-of-function) on the

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proliferation of cancer cells (Supplementary Fig. S3a and S3b).To evaluate the role of TMEM16A independently of 11q13amplification, we chose to use the UM-SCC1 SCCHN cell line(that harbors 11q13 amplification) and the T24 bladder cancercell line (that does not contain the 11q13 amplicon). LentiviralshRNA was used to "knock-down" TMEM16A in both cell lines.We identified 2 independent shRNA sequences that caused asignificant reduction (�80%) in TMEM16A protein expression.TMEM16A knockdown led to a measurable change in whole-cell chloride conductance (�50%), as assayed by MQAE fluo-rescence assays (Supplementary Fig. S3c–S3e; ref. 27). Forcedoverexpression of a mutant version of TMEM16A (TMEM16A-K610A) led to significantly smaller chloride conductance (Sup-plementary Fig. S3f). For further experiments, we used one ofthe sequences (shRNA#5).To determine the consequences of TMEM16A knockdown

on in vivo tumor growth, shRNA-treated UM-SCC1 cells were

inoculated subcutaneously into nude mice. TMEM16A shRNAled to a significant decrease in xenograft growth (Fig. 2A andB).Furthermore, we found that TMEM16A shRNA-treated tumorsexhibited decreased Ki-67 staining (Fig. 2C and D). Severalreports have showed that themitogen-activated protein kinase(MAPK)/ERK signaling pathway enhances the growth of epi-thelial cancer cells, in particular, bladder cancer, and SCCHN(28). We noted that pERK staining was less prominent intumors derived from TMEM16A shRNA-treated cells versuscontrol shRNA tumors (Fig. 2D).

Next, we exogenously overexpressed TMEM16A in severalcell lines that do not harbor endogenous gene amplification. InT24 cells, TMEM16A was trafficked to the cell surface (Sup-plementary Fig. S1b), led to increased chloride fluxes, andresulted in enhanced xenograft growth (Fig. 3A–C). Tumorxenografts confirmed overexpression of TMEM16A andshowed increased pERK1/2 and Ki-67 staining (Fig. 3D).

Figure 1. TMEM16A isoverexpressed in SCCHN andcorrelates with decreased survival.TMEM16A expression is upregulatedin 17 paired tumor (T) versus pairedadjacent normal mucosa (N) asdetected by immunoblotting (A).There is a 5-fold increase ofTMEM16A expression in tumorsrelative to normal adjacent mucosa(B; mean � SEM; n ¼ 17; ��, P <0.001). A 5-fold increase in tumor:normal TMEM16A mRNA wasobserved in a separate cohort(C; n ¼ 15; �, P < 0.05). TMEM16Aexpression data were abstractedfrom Oncomine and analyzed forrelative overexpression (D). HighTMEM16A expression wasassociated with decreased overallpatient survival (E; n ¼ 34, P < 0.05).Representative images of TMEM16Astaining are shown.






TMEM16A overexpression led to increased in vitro prolifer-ation of HEK-293T cells, and increased anchorage-indepen-dent viability in immortalized keratinocytes (SupplementaryFig. S4a and S4b). TMEM16A knockdown in 2 other cell lines(Cal33 and PCI-15B) suppressed cell growth in vitro (Supple-mentary Fig. S4c). Furthermore, TMEM16A cooperated withconstitutively active H-RAS to induce focus formation inimmortalizedMEFs (Supplementary Fig. S4d). Similarly, forcedoverexpression of TMEM16A conferred increased colonogenicsurvival in immortalized keratinocytes and tumor cell lines(Supplementary Fig. S4e). Taken together, these data suggestthat TMEM16A overexpression can facilitate oncogenic trans-formation through cooperation with potent oncogenes.

We next determined the effect of TMEM16A knockdown onin vitro growth. Treatment with TMEM16A shRNA led to asignificant retardation in proliferation when compared withcontrol shRNA (Fig. 4A andB).TMEM16A shRNA abrogated theincrease in both anchorage-dependent and -independent cellproliferation noted in overexpressing cells (Fig. 4C and D).TMEM16A knockdown induced an accumulation of cells inG0/G1 and a concomitant decrease in the S/G2 phase, suggest-

ing a block in cell-cycle progression (Supplementary Fig. S5a).Interestingly, TMEM16A overexpression impaired caspase-3/7activation, suggesting that TMEM16A may in fact impairapoptotic cell death, along with promoting cell growth (Sup-plementary Fig. S5b).

TMEM16A induces phosphorylation of ERK1/2Because we observed differential expression of pERK1/2 in

tumor xenografts, we examinedwhether TMEM16A expressioninfluenced ERK1/2 activation. TMEM16A overexpression wasassociated with an increase in phosphorylated ERK1/2 (�2-fold) and cyclin D1 (�5-fold; Fig. 5A). TMEM16A siRNA led to adecrease in phospho-ERK1/2 and cyclin D1 (Fig. 5B). We notedthat AKT and phospho-AKT levels were not significantlyinfluenced by TMEM16A overexpression, suggesting specificityto the ERK1/2 pathway (Fig. 5B). Similarly, there were nochanges in p-ERK5 or ERK5 (Supplementary Fig. S6c).

It is well known that RAS oncogenic signaling can activateERK1/2. HRAS, in particular, has been associated withSCCHN development (29, 30). In fact, no mutations in KRASor NRAS were observed in 2 recent genomic studies of

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Figure 2. TMEM16A knockdownresults in significant growthinhibition of SCCHN tumorxenografts in vivo. TMEM16Aknockdown in vivo led to smallertumors (A and B; �, P < 0.05).Xenografts generated fromTMEM16A shRNA cells showeddecreased proliferation (asdetermined by Ki-67 staining) andreduced pERK1/2 (C and D;�, P < 0.05). Representative imagesshowing TMEM16A, pERK1/2, andKi-67 staining are shown (n ¼ 6).Inset image shows �10magnification. hpf, high-powerfield.

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SCCHN (30, 31). We therefore evaluated the presence ofHRAS, KRAS, and NRAS mutations in the cell lines used inthis study. T24 cells harbor the activating HRASG12V muta-tion, however, HEK-293T, EPC1, and UM-SCC1 cells did notharbor mutant HRAS, KRAS, or NRAS (data not shown).Taken together, these data suggest that the impact ofTMEM16A on ERK1/2 signaling is independent of activatingHRAS mutations.Next, we explored the association between Tmem16a and

cyclinD1 in oral cavity tissues obtained from wild-type andTmem16a�/� embryos. As expected, Tmem16a levels werereduced in knockout mouse tissues as compared with wild-type and heterozygous mice (Fig. 5C). Interestingly, cyclinD1 was also decreased in tissues derived from knockoutmice, but not in their heterozygous or wild-type littermates.We observed intense nuclear cyclin D1 staining in the

proliferative mesoderm of wild-type mice but not in theTmem16a�/� littermates (Supplementary Fig. S6a). We fur-ther explored this finding by measuring mRNA expression ofTmem16a and Ccnd1 in these tissues. As expected, knockouttissues had significantly lower levels of cyclin D1 (Supple-mentary Fig. S6b). This finding suggested that TMEM16Amay be influencing cell proliferation on a fundamental leveland occurs in vivo.

We postulated that if TMEM16A affects proliferation byactivating MEK/ERK, inhibition of MEK/ERK should abro-gate TMEM16A-induced growth. Indeed, treatment witheither UO126 or dominant-negative ERK1/2 led to a com-plete abrogation of TMEM16A-induced growth (Fig. 5D andE). Similar data were observed with the specific ERK inhib-itor AZD6244 and with siRNA against ERK1/2 (Supplemen-tary Fig. S5c and S5d). Treatment with TMEM16A shRNA

Figure 3. TMEM16Aoverexpression (OE) induces in vivotumor growth. Overexpression ofTMEM16A markedly promotedtumor growth in the T24 xenograftmodel (A and B). TMEM16A-overexpressing cells showedincreased proliferation (asdetermined by Ki-67 staining)and pMAPK staining (C and D;��, P < 0.01). Increased levels ofp-ERK1/2 are also found in tissuederived from tumor explants (D).Inset image shows �10magnification. OE3 and OE7 refer toindividual clones. Ctl, control; hpf,high-power field.

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reversed the activation of MEK and ERK1/2 inducedby TMEM16A overexpression (Fig. 5F). This observationsuggests that TMEM16A overexpression directly impactsERK1/2 activation.

Recent data suggests that ERK activation may impact thechloride conductance of TMEM16A (15). Similarly, muta-tions in the putative pore forming domain can impact thewhole-cell chloride conductance in forced overexpressionexperiments (32). We sought to determine if the putativepore-forming region of TMEM16A was necessary for activa-tion of ERK1/2. Forced overexpression of a mutant versionof TMEM16A (TMEM16A-K610A) that has been describedto display significantly abrogated chloride conductance(15) did not induce ERK1/2 or phospho-ERK1/2 (Fig. 6A).We verified that TMEM16A-K610A was trafficked to thecell membrane using biotinylation experiments (data notshown). Our data raise the intriguing possibility that

TMEM16A affects ERK activation by modulating intracellu-lar chloride levels. Unfortunately, manipulation of intracel-lular chloride levels can itself induce changes in the expres-sion of ion channels. Therefore, further work is necessary todissect the effect of intracellular chloride on ERK activation.

Overexpression of mutant TMEM16A-K610A did not pro-mote anchorage-independent viability compared with vec-tor controls (Fig. 6B–D). To confirm that the effects areindeed directly dependent on TMEM16A expression, werescued TMEM16A shRNA-treated cells with shRNA-resis-tant versions of TMEM16A or TMEM16A-K610A (Supplemen-tary Fig. S7a). The reduction in viability induced byTMEM16A shRNA was rescued by expression of anshRNA-resistant version of TMEM16A, but not resistantTMEM16A-K610A (Fig. 6E). This approach provides strongevidence to show that TMEM16A directly induces the pro-liferative phenotype.

Figure 4. TMEM16A expressionmediates both anchorage-dependentand -independent growth. TMEM16Aknockdown caused a decrease inproliferation (mean � SEM; n ¼ 3;�, P < 0.05; A and B). Representativeimmunoblots are shown. The growthadvantage conferred by TMEM16Aoverexpression was abrogated byshRNA (C; mean � SEM; n ¼ 3,P < 0.05). Immunoblotting confirmsoverexpression (OE) and subsequentknockdown of TMEM16A (C). Softagar colony formation was alsoenhanced by TMEM16Aoverexpression and rescued bysubsequent knockdown (D; mean �SEM; n ¼ 4, P < 0.01).

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Figure 5. Themitogenic phenotype of TMEM16A is associatedwith ERK1/2 activation. TMEM16Aoverexpression inducedphospho-ERK1/2 andcyclin D1 (A).TMEM16A-siRNA led to decreasedphospho-ERK1/2 and cyclin D1. Phospho-Akt levelswere not consistentlymodulated by TMEM16Amanipulation (B). Therelationship between TMEM16A and cyclin D1 in head and neck tissues derived from Tmem16a knockout (�/�) versuswild-type (þ/þ) or heterozygous (þ/�)mice (C). ERK inhibition with UO126 or dominant-negative ERK2 abrogated the growth advantage in overexpressing cells (D and E; mean � SEM; n ¼ 3;��, P < 0.01). Treatment of overexpressing cells with TMEM16A shRNA abrogated the activation MEK and ERK1/2 (F). Ctl, control.






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Figure 6. The putative pore-forming region of TMEM16Amediates ERK1/2 activation and subsequent mitogenic effects. Expression of the TMEM16A-K610Amutant fails to induceERK1/2 and cyclin D1 (A). TMEM16A-K610Adid not increase anchorage-independent viability (B–D;mean�SEM; n¼ 3; ��,P < 0.001).TMEM16A shRNA–induced decrease in growthwas subsequently rescuedwith TMEM16A shRNA–resistant DNA (TMEM16ADNA�), but not with TMEM16A-K610A shRNA–resistant DNA (TMEM16A-K610A DNA�; E; mean � SEM; n ¼ 3, P < 0.05). Ctl, control.

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TMEM16A activates the Ras-Raf-MEK-ERK pathwayThe observation that TMEM16A induces activation of

ERK1/2 led us to interrogate the RAS-RAF-MEK-ERK1/2pathway. Indeed we observed robust activation of RAS-RAF-MEK-ERK pathway upon TMEM16A overexpression(Fig. 7A and B). Densitometric quantitation is shown inSupplementary Fig. S7b. Interestingly, this activation wasindependent of HRAS mutation status, because T24 cellsharbor an activating HRAS mutation, but SCC1 cells do not(data not shown). We next sought to determine if Rasinhibition (using a dominant negative construct) couldabrogate the observed phenotype. Forced overexpression ofa dominant-negative mutant of H-Ras (H-RasN17) abrogatedthe observed activation of MEK, ERK1/2 and also the growthphenotype (Fig. 7C and D).

Pharmacologic inhibition of TMEM16A induces cancercell deathTo validate TMEM16A as a potential target in epithelial

malignancies, we wanted to determine if small-molecule inhi-bition of TMEM16A could inhibit tumor cell proliferation.

Therefore, we treated UM-SCC1 and T24 cells with a novelTMEM16A inhibitor (T16A-inh01; refs. 33, 34). T16A-inh01induced a dose-dependent reduction in cell viability (Supple-mentary Fig. S8a and S8B). To determine whether combinedinhibition of TMEM16A and MAPK had an additive effect oncell viability, we treated cells with the MEK/ERK inhibitorUO126 alone or in combination with TMEM16A shRNA.TMEM16A knockdown resulted in a modest decrease in cellproliferation, however, we observed an additive effect withMAPK inhibition (Supplementary Fig. S8c and S8d). Thechemical structure of T16A-inh01 is shown in SupplementaryFig. S8e. We did not observe additive inhibitory effects on ERKphosphorylation. These data show that TMEM16A and MAPKinhibition may act in an additive fashion to retard tumor cellproliferation, suggesting that ERK1/2may not uniquely controlTMEM16A-induced cell growth.

DiscussionThere is accumulating evidence that chloride channels

influence tumor growth and progression (35–37), howeverthe mechanism(s) by which this occurs remains unclear.Calcium-activated chloride channels (CaCCs) are a uniquesubset of chloride channels that play important roles inmany fundamental physiologic processes (38, 39). Recently,ANO1/TMEM16A was described as a bona fide CaCC (5–7).However, it remains controversial whether TMEM16A isitself a functional CaCC, or forms a subunit within theprotein complex that facilitates CaCC activity (7, 40, 41).Recent reports suggest that CaCCs can both promote andretard tumor cell proliferation (16, 17). These contradictoryreports suggest that improved understanding of the impactof CaCC regulation and activation on cell proliferation mayhelp to define whether these molecules can serve as a futuretherapeutic target.

TMEM16A is frequently overexpressed in several tumorsincluding squamous cell carcinoma of the head and neck,esophageal cancer, and gastrointestinal stromal tumors (GIST;refs. 4, 18, 42, 43). To date, the exact role(s) that TMEM16Aplays in tumor development/progression remains unclear.Ayoub and colleagues have recently reported that TMEM16Aoverexpression facilitates cell motility and may contribute tothe development of metastases (44); however, no mechanismwas proposed to explain this phenotype.

This report provides the first mechanistic link betweenTMEM16A expression and cell proliferation in human cancer.Our data show that TMEM16A expression directly impactscellular proliferation. TMEM16A also cooperates with onco-genic H-RAS to induce focus formation in immortalized MEFs.Taken together, these data strongly suggest that TMEM16Amay function as a proto-oncogene, and that its overexpressiondrive tumor growth.

It has recently been shown that TMEM16A channel activityis linked to ERK1/2 activation and that the ERK inhibitorUO126 can inhibit TMEM16A channel function (15). Our datasuggest that TMEM16A activates the RAS-RAF-MEK-ERK1/2pathway, thereby influencing cellular proliferation. However,this activation does not occur when a hypomorphic mutant

C

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Figure 7. TMEM16A expression activates the Ras-Raf-MEK-ERK1/2pathway. Activation of Ras, B-Raf, and C-Raf was observed in T24 andUM-SCC1 cells after TMEM16A overexpression (A and B). Activation ofthe pathway was abrogated by forced expression of dominant-negativeRas (C). Forced expression of dominant-negative Ras abrogated thegrowth advantage conferred by TMEM16A overexpression (D).






(TMEM16A-K610A) is expressed. These data raise the intrigu-ing possibility that the chloride conductance of TMEM16Aimpacts ERK1/2 activation. However, the exact mechanism bywhich this occurs remains unclear.

The availability of small-molecule inhibitors againstTMEM16A provides a novel potential method to inhibit tumorcell growth. The currently available small-molecule inhibitorsof TMEM16A (niflumic acid and NPPB) exhibit off-targeteffects and have been shown to block other chloride channels.However, T16A-inh01 is a novel and potentially more specificTMEM16A inhibitor, and provides amethod to inhibit channelactivity (33, 34, 45). This developmental molecule likely has off-target effects, and therefore definitive conclusions cannot bedefined at this time.

The ubiquitous expression of TMEM16A suggests that theendogenous protein has important physiologic roles that maybe adversely impacted by pharmacologic inhibition. However,TMEM16A is known to undergo alternative splicing, andspecific variants have been isolated fromdiseased tissues (suchas diabetic gastroparesis; ref. 46–48). Recently, mutations inTMEM16A have been described in SCCHN. The existence ofspecificmutations inTMEM16A that have been identified fromwhole-exome sequencing of SCCHN, suggests that tumor-specific targeting may be possible in the future (30).

In conclusion, we have shown that TMEM16Aexpression: (1)is associated with decreased patient survival; (2) modulatesproliferation in vitro and in vivo and induces ERK1/2 activation;(3) the pore-forming region of this molecule is necessary tofacilitate these phenomena; and (4) pharmacologic inhibition(using a novel TMEM16A inhibitor) or siRNA-mediated knock-down of TMEM16A leads to a significant decrease in tumor cellviability by downregulating the TMEM16A-ERK1/2 signaling.In closing, these findings may describe a novel therapeutic role

for TMEM16A in cancer treatment, because TMEM16A mayact as a potential pharmacologic target.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: U. Duvvuri, D.J. Shiwarski, V.W. Lui, S.M. GollinDevelopment of methodology: U. Duvvuri, D.J. Shiwarski, D. Xiao, X. HuangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): U. Duvvuri, D.J. Shiwarski, D. Xiao, C. Bertrand, R.S.Edinger, J.R. Rock, R.D. Harfe, B.J. Henson, R.S. Seethala, A.M. EgloffAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): U. Duvvuri, D.J. Shiwarski, D. Xiao, C. Bertrand, R.S.Edinger, R.S. Seethala, A.M. Egloff, X. Chen, J.R. GrandisWriting, review, and/or revision of the manuscript: U. Duvvuri, D.J.Shiwarski, C. Bertrand, X. Huang, K. Kunzelmann, X. Chen, V.W. Lui, J.R. Grandis,S.M. GollinAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): U. Duvvuri, K. Kunzelmann, R. Schrei-ber, R.S. Seethala, X. ChenStudy supervision: U. Duvvuri, S.M. Gollin

AcknowledgmentsThe authors thank Dr. R.W. Sobol and the University of Pittsburgh Cancer

Institute Lentiviral Facility for the generation of lentiviral particles.

Grant SupportThis work was supported by the Head and Neck Cancer SPORE grant (NIH

P50CA097190 J.R. Grandis), through a Veterans Administration CDA; CPPF (U.Duvvuri) and start-up funds from the Department of Otolaryngology (U. Duv-vuri). The molecular cytogenetic studies were carried out in the University ofPittsburgh Cancer Institute Cytogenetics Facility, supported in part byP30CA047904 to N.E. Davidson/R.B. Herberman/S.M. Gollin. KK was supportedby Deutsche Krebshilfe Projekt 109438.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 8, 2012; revised April 26, 2012; accepted April 28, 2012;published OnlineFirst May 7, 2012.

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2012;72:3270-3281. Published OnlineFirst May 7, 2012.Cancer Res Umamaheswar Duvvuri, Daniel J. Shiwarski, Dong Xiao, et al. Tumorigenesis and Cancer ProgressionTMEM16A Induces MAPK and Contributes Directly to

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