Background

TMEM16A (also known as anoctamin 1) was identified as a Ca2+-activated chloride channel (CaCC) in 2008 [1,2,3], and is the first member of the ten-member family of “Transmembrane protein 16” (abbreviated as TMEM16). Besides TMEM16A, TMEM16B and TMEM16F have been found to function as CaCCs [4,5,6]. TMEM16F can also function as a Ca2+-dependent phospholipid scramblase [7, 8], and Ca2+-activated nonselective cation channel [9]. However, controversies exist among other members of the TMEM16 family regarding whether they are CaCCs or Ca2+-dependent lipid scramblases [7, 10]. The Ca2+-dependent lipid scrambling function of TMEM16 family members have been implicated in the regulation of membrane trafficking, the release of extracellular vesicle, and the modulation of cell-cell communication [11].

As a CaCC, TMEM16A is activated by intracellular Ca2+, and the current is characterized by voltage-dependent activation, strong outward rectification, and a deactivating tail current on depolarization at low intracellular Ca2+ concentrations, and voltage-independent activation and linear current-voltage relationship at high [Ca2+]i [12]. Based on the crystal structure of a TMEM16 family member from the fungus Nectria haematococcaten (nhTMEM16), a conserved Ca2+-binding site located within the membrane has been identified [13]. Ca2+-dependent properties of TMEM16A such as rectification, activation and deactivation kinetics, and rundown can be well explained by the presence of Ca2+ binding site within the membrane [14]. However, because nhTMEM16 is a scramblase, not an ion channel, the location of the pore in the TMEM16A channel remains unclear. A “double-barrel” pore architecture of TMEM16A channel has been recently proposed [15, 16].

TMEM16A is widely expressed in many cells including secretory epithelia [1, 17,18,19], airway and vascular smooth muscle cells [18, 20,21,22], vascular endothelium [23, 24], interstitial cells of Cajal [25, 26], and nociceptive neurons [27,28,29]. TMEM16A regulates many cellular functions, such as fluid secretion in secretory epithelia, smooth muscle contraction, gut mobility, cell volume regulation, apoptosis, and pain (reviewed in [30,31,32,33]). In addition, TMEM16A dysfunction contributes to many diseases such as cancer, hypertension, gastrointestinal motility disorders, and cystic fibrosis [31, 34,35,36]. Recently, growing evidence has shown that TMEM16A is overexpressed in many tumors (Table 1). However, conflicting results exist regarding the role of TMEM16A in cell proliferation and migration in cancer cells. In addition, it remains unclear how TMEM16A overexpression contributes to tumorigenesis.

Table 1 TMEM16A expression and function in cancers
Full size table

In this review, we examine recent findings in the study of TMEM16A in cancer, and focus on the role of TMEM16A in cancer cell proliferation and migration. We summarize the mechanisms of TMEM16A overexpression, the signaling pathways that are activated by TMEM16A, and potential clinical use of TMEM16A as a prognostic and predictive marker in cancer. Since TMEM16A plays different roles in different cancer cells, we try to develop the idea that TMEM16A regulates cancer cell proliferation and migration via a cell-specific mechanism.

TMEM16A Overexpression in cancer

Before it was identified as a CaCC, TMEM16A had been found to be amplified in oral cancer, head and neck squamous cell carcinoma (HNSCC), gastrointestinal stromal tumor (GIST), breast cancer, and esophageal squamous cell (ESCC) cancer under other names such as FLJ10261, TAOS1 (tumor amplified and overexpressed sequence 1) and DOG1 (discovered on GISTs protein 1) [37,38,39,40,41]. Recently, TMEM16A has been reported to be highly expressed in many human tumors including breast cancer [42, 43], HNSCC [44,45,46,47], colorectal cancer (CRC) [48, 49], ESCC [50], lung cancer [51], hepatocellular carcinoma [52], prostate cancer [53], gastric cancer [54, 55], and glioma [56] (Table 1).

TMEM16A is located on chromosome 11q13, which is frequently amplified in many malignant tumors [57, 58]. Several studies have examined the copy number of TMEM16A in many tumors including breast cancer, HNSCC, and ESCC, and found that gene amplification commonly accounts for TMEM16A overexpression in these cancers (Table 1). To further confirm TMEM16A gene amplification in cancers, we performed bioinformatics analysis to detect TMEM16A gene alterations using the cBioPortal database (cBioPortal for Cancer Genomic). TMEM16A gene amplification accounts for the most alterations, and more frequently occurs in HNSCC, ESCC, breast cancer, and lung cancer than in other tumors (Fig. 1a). Interestingly, many tumors have missense mutations and deletions in the TMEM16A gene. A total of 165 missense mutations have been identified in TMEM16A, and the most frequent mutations are R561L/Q/W, R433Q, and R588G/Q (Fig. 1b). However, the role of these mutations has not been investigated in cancer.

Fig. 1
figure 1

The alterations of the TMEM16A gene in cBioPortal database. a TMEM16A gene was examined in 29 studies with >100 human cancer samples and >5% gene alterations. The copy number alteration (CNA) occurs more frequently in cancer. b TMEM16A missense mutations identified in cBioPortal database. A total of 165 missense mutations are shown. The most frequent mutations are R561L/Q/W, R433Q, and R588G/Q

Full size image

Several studies have reported that 11q13 amplification is associated with poor prognosis in patients with malignant tumors [57, 58]. Consistent with the idea, Ruiz et al. found that 11q13 gene amplification correlated with TMEM16A expression in human HNSCC cancer, and TMEM16A overexpression was associated with poor overall survival in HNSCC patients [45]. In addition, Ayoub et al. reported that TMEM16A gene amplification and protein overexpression were associated with distant metastasis in patients with papillomavirus (HPV)-negative HNSCC [46]. Similarly, Bristschgi et al. reported that 11q13 amplification resulted in a higher TMEM16A expression in human breast cancer than in non-11q13-amplified tumors, and TMEM16A gene amplification and protein overexpression correlated with poor prognosis [42]. Shi et al. found that TMEM16A gene amplification and protein overexpression was associated with lymph node metastasis and advanced clinical stage in patients with ESCC [50].

Consistent with the results from the human tumor samples, TMEM16A has been found to be highly expressed in many cell lines with 11q13 amplification, including ZR75–1, HCC1954, and MDA-MB-415 breast cancer cell lines, UM-SCC1, BHY, and Te11 HNSCC cell lines, and FaDu, KYSE30 and KYSE510 ESCC cell lines [42, 44, 50] (Table 1). Knockdown of TMEM16A in cancer cells with 11q13 amplification results in a decrease in cell proliferation and an inhibition in xenograft tumor growth [42, 44, 50, 59]. These studies indicate that TMEM16A is critical for cell proliferation and tumor growth in 11q13-amplified tumors.

Although TMEM16A gene amplification is responsible for TMEM16A overexpression in many tumors, it is clearly not the only mechanism for TMEM16A expression. For example, in breast cancer, 11q13 amplification only occurs in approximately 15% of breast cancer patients, but TMEM16A overexpression occurs in >78% human breast cancer samples [42, 43]. Similarly, TMEM16A overexpression was more pervasive than gene amplification in human gastric cancer samples [54]. In contrast, in HNSCC, TMEM16A gene amplification was more frequently detected than protein expression [45, 47]. Therefore, other mechanisms that regulate TMEM16A expression must exist.

Multiple regulatory mechanisms of TMEM16A overexpression in cancer

In non-tumor cells, TMEM16A expression is regulated by many signaling pathways under physiological and pathological conditions. For example, in the airway epithelial cells, IL-4 induces TMEM16A upregulation, which is important for goblet cell differentiation [2, 60]. In human aortic smooth muscle cells, myocardin promotes TMEM16A expression by forming a complex with serum response factor (SRF) on the TMEM16A promoter, and angiotensin II inhibits TMEM16A expression via Kruppel-like factor 5, which competes with SRF to interact with myocardin [61]. In endothelial cells, angiotensin II increases TMEM16A expression [23]. In pulmonary arterial smooth muscle cells, chronic hypoxia increases TMEM16A expression [62]. In human lung epithelial A549 cells, TMEM16A expression is upregulated after lipopolysaccharide treatment [63]. Therefore, it appears that TMEM16A expression is controlled by various molecules and stimuli and the regulatory mechanisms varies in different cells. Here, we summarize the regulatory mechanisms of TMEM16A expression in cancer cells, and TMEM16A expression is controlled via transcriptional regulation, epigenetic regulation, and microRNAs (Fig. 2).

Fig. 2
figure 2

TMEM16A expression is upregulated via transcriptional regulation, epigenetic regulation and microRNAs in cancer. TMEM16A upregulation is induced by IL-4 and IL-13 [64, 65], which bind to their receptors and subsequently activate JAK/STAT6 signaling. STAT6 binds to the TMEM16A promoter and increases the transcription of the TMEM16A gene. Testosterone (T) induces TMEM16A upregulation by binding to the androgen receptor (AR), which subsequently increases the transcription of the TMEM16A gene [71]. Histone deacetylase (HDAC) inhibitors reduce TMEM16A expression in breast and prostate cell lines [77]. Promoter hypomethylation contributed to TMEM16A overexpression in HPV-negative HNSCC [75] and promoter hypermethylation results in decreased TMEM16A expression in metastatic lymph node tissues [74]. miR-132 and miR-381 binds to the 3′ UTR of TMEM16A mRNA, resulting in TMEM16A downregulation [49, 55]. Downregulation of miR-132 and miR-318 contributes to TMEM16A in patients with colorectal cancer [49] and gastric cancer [55]

Full size image

Transcriptional regulation

Bioinformatics analyses show that the promoter region of the TMEM16A gene lacks TATAT box sequences, but contains many INRs (initiator elements) and/or TSSs (transcriptional start sites), suggesting that TMEM16A expression can be regulated by diverse transcription factors [64]. The TMEM16A promoter region contains a signal transducer and activator of transcription 6 (STAT6) binding site [64], which mediates TMEM16A upregulation induced by IL-4 and IL-13 [64, 65]. Zhang et al. reported that the expression of TMEM16A and MUC5AC was increased in nasal epithelial cells from patients with chronic rhinosinusitis [66]. IL-13 stimulated MUC5AC expression in human airway and nasal epithelial cells, and this effect was blocked by TMEM16A inhibitors, suggesting that TMEM16A might mediate IL-13-induced mucin secretion [65, 66]. These studies suggest that TMEM16A may play an important role in airway inflammation diseases. It is well known that IL-4 and IL-13 play an important role in cancer development [67,68,69,70]. To date, it remains unclear whether TMEM16A can be regulated by IL-4 and IL-13 in cancer cells. Future studies are required to demonstrate whether TMEM16A upregulation by IL-4 and IL-13 is involved in tumorigenesis.

The transcriptional regulation of TMEM16A expression has been demonstrated in testosterone-induced prostate hyperplasia by Cha et al. [71]. They found that the promoter region of the TMEM16A gene contains three putative binding sites for androgen response element (ARE), which mediates testosterone-induced TMEM16A upregulation in prostate epithelial cells. The testosterone-induced TMEM16A upregulation was blocked by small interfering RNAs (siRNAs) against the androgen receptor, which binds to the ARE region and subsequently promotes gene transcription. This study implies that TMEM16A upregulation induced by testosterone may contribute to the progression of prostate cancer.

Epigenetic regulation

DNA methylation of the target gene promoter plays an important role in the epigenetic regulation of genes that are essential for tumorigenesis [72, 73]. Promoter hypermethylation can repress gene expression, whereas promoter hypomehtylation can result in active transcription of the gene. TMEM16A promoter contains CpG islands, suggesting that DNA may be involved in the regulation of transcription of the TMEM16A gene [64, 74]. Indeed, Dixit et al. reported that TMEM16A expression was higher in HPV-negative HNSCC than in HPV-positive HNSCC, and promoter hypomethylation contributed to the higher expression of TMEM16A in HPV-negative HNSCC [75]. In addition, Shiwarski et al. found that compared with primary HNSCC tumors, methylation of the TMEM16A promoter region was increased in the metastatic lymph node tissue, thus resulting in decreased TMEM16A expression [74]. Promoter methylation-mediated inhibition of TMEM16A expression is believed to drive HNSCC cells from growth to metastic spread [74].

Histone deacetylase (HDAC) plays an important role in epigenetic regulation of gene expression by deacetylating the lysine residues in the histone, and dysregulation of HDACs has been implicated in the pathogenesis of cancer [76]. Matsuba et al. reported that HDAC inhibitors reduced TMEM16A expression and reduced cancer cell viability in breast and prostate cancer cell lines [77]. Wanitchakool et al. reported that HDAC inhibitors decreased TMEM16A expression and inhibited cell proliferation in HNSCC cells [59]. These studies further suggest that HDAC inhibitors may inhibited cell proliferation via downregulation of TMEM16A. However, the molecular mechanisms underlying the epigenetic regulation of TMEM16A transcription by HDAC have not been elucidated yet.

MicroRNAs

MicroRNAs are small, noncoding RNA molecules of ~22 nucleotides that inhibit gene expression by targeting the 3′ UTR of the target mRNAs. MicroRNAs regulate cell proliferation, apoptosis, angiogenesis and invasion, and contribute to tumorigenic processes in human cancers [78]. Recently, Mokutani et al. found that the 3′ UTR of TMEM16A mRNA contained a complementary site for miR-132, and the luciferase reporter assay showed that TMEM16A was the direct target of miR-132 [49]. In addition, TMEM16A overexpression was inversely associated with downregulation of miR-132 in human CRC, and correlated with poor clinical outcomes in patients with CRC [49]. Similarly, Cao et al. found that TMEM16A is the direct target of miR-381, and downregulation of miR-381 was inversely correlated with TMEM16A expression in human gastric cancer tissues [55]. These findings suggest that downregulation of microRNAs may contribute to TMEM16A overexpression in human cancers.

The signaling pathways activated by TMEM16A in cancer

As a CaCC, TMME16A overexpression can result in increased channel function, and opening of TMEM16A chloride channel can lead to changes in intracellular Cl− concentration ([Cl−]i) and membrane potential. This change in [Cl−]i and membrane potential may activate many signaling pathways that are involved in cancer cell proliferation and migration. In addition, as a membrane protein, TMEM16A interacts with several membrane proteins including SNARE proteins that control vesicle trafficking and the ezrin-radixin-moesin (ERM) complex that links membrane proteins with cytoskeleton [79]. It is possible that TMEM16A activates many signaling pathways via its interactome. Here, we summarize the signaling pathways that are activated by TMEM16A in cancer. TMEM16A activates many signaling pathways that participate in cell proliferation, migration, and invasion (Table 1, and Fig. 3).

Fig. 3
figure 3

The signaling pathways that are activated by TMEM16A in cancer. TMEM16A directly interacts with EGFR [81], and promotes EGFR phosphorylation, which activates the AKT/SRC/ERK1/2 signaling [42]. In addition, TMEM16A increases autocrine secretion of EGF in breast cancer cells [42]. TMEM16A directly interacts with IP3R, and increased Ca2+ release from the ER [85]. TMEM16A activates CaMKII by increasing intracellular Ca2+ concentrations, and CaMKII subsequently activates the AKT/SRC/ERK1/2 signaling [42]. TMEM16A also activates the Ras-Raf-Mek-ERK1/2 signaling pathway in UM-SCC1 HNSCC cells and T24 bladder cells [44]. In SMMC-7721 human hepatoma cells, TMEM16A activates the p38 signaling pathway [52]. TMEM16A activates the NFκB signaling pathway and promotes the gene transcription in glioma cells [56]. +, activates the signaling pathway.?, the mechanisms of how TMEM16A activates the signaling pathway are unknown

Full size image

Epidermal growth factor receptor (EGFR) signaling

EGFR is a tyrosine kinase receptor that is overexpressed in many tumors such as HNSCC and breast cancer, and contributes to tumorigenesis [80]. Bill et al. reported that TMEM16A promoted EGFR phosphorylation, and increased the expression in a posttranslational and degradation-independent mechanism in HNSCC cells [81]. In addition, TMEM16A formed a complex with EGFR, and the complex regulated cancer proliferation in HNSCC cells [81]. Activation of EGFR signaling by TMEM16A is further demonstrated by Britschgi et al. showing that TMEM16A knockdown reduced EGFR phosphorylation and subsequently inhibited AKT, SRC, and ERK activation in breast cancer cell lines [42]. Furthermore, they demonstrated that TMEM16A knockdown reduced the autocrine secretion of EGFR ligands, EGF and TGF-α in breast cancer cells, suggesting that TMEM16A can activate EGFR signaling by increasing autocrine secretion of EGFR-ligands [42]. Therefore, TMEM16A activates the EGFR signaling pathway by increasing EGFR expression, phosphorylation, and autocrine EGFR-ligand secretion (Fig. 3).

Ca2+/Calmodulin-dependent protein kinase II (CAMKII) signaling

Britschgi et al. also found that EGFR inhibition only partially reduced TMEM16A overexpression-induced cell viability, and EGFR activation partially reversed the inhibitory effect of TMEM16A inhibitors on cell viability in breast cancer cells, suggesting that TMEM16A activates additional signaling pathways that are involved in cell viability [42]. Furthermore, they found that TMEM16A overexpression increased calcium/CAMKII phosphorylation, indicating that TMEM16A overexpression activates calcium-dependent CAMKII signaling. It has been reported that TMEM16A is located in the lipid raft of the plasma membrane in nociceptive neurons, where it is in close proximity to IP3R [82], and is believed to play a role in modulating intracellular Ca2+ levels [83]. In addition, TMEM16A inhibition has been found to reduce intracellular Ca2+ flux from both the plasma membrane and sarcoplasmic reticulum in airway smooth muscle [84]. Recently, Cabrita et al. found that TMEM16A directly interacted with the IP3R, and increased compartmentalized Ca2+ release from the ER store induced by ATP in HeLa cells [85]. TMEM16A inhibitors reduced ATP-induced increase in [Ca2+]i [85], suggesting that Cl− transport through TMEM16A channels may be important for Ca2+ release from the Ca2+ store in cancer cells. In addition, TMEM16A did not interacted with ORAI, and TMEM16A activation was not affected by ORAI inhibitors [85], suggesting that TMEM16A may not regulate ORAI-mediated Ca2+ entry in cancer cells.

Mitogen-activated protein kinase (MAPK) signaling

The MAPK signaling pathways regulate many cellular processes such as proliferation, apoptosis, migration, differentiation, and growth, and play an important role in the development and progression of cancer [86]. Duvvuri et al. found that TMEM16A overexpression activated the Ras-Raf-MEK-ERK1/2 signaling pathway in UM-SCC1 HNSCC cells and T24 bladder cells, and ERK1/2 inhibition reduced TMEM16A-induced cell growth [46], and correlates with poor prognosis in patients with breast cancer [42], gastric cancer [54], and HNSCC [45]. However, it has been reported that TMEM16A overexpression has no effects on cell migration when transfected into HEK293 cells [94]. TMEM16A inhibition reduces cell migration in HNSCC cells [14, 125]. TMEM16A couples to different Ca2+-permeable ion channels that are predominantly expressed in different cells. For example, TMEM16A has been found to be activated by Ca2+ influx via TRPV1 in mouse dorsal root ganglion neurons [28], TRPV4 in the choroid plexus [126], TRPV6 in epithelial principal cells of the rat epididymis [127], TRPC1 in salivary gland cells [128], TRPC2 in rat thyroid cells [129], TRPC6 in cerebral artery myocytes [130], Cav1.4 at the photoreceptor ribbon synapse [131], Cav1.2 in canine ventricular myocytes [132], and store-operated Ca2+ entry in eccrine sweat glands [133]. Therefore, TMEM16A is activated by Ca2+ via different Ca2+-permeable ion channels in a cell-specific manner. However, it remains unclear whether TMEM16A may couple to different Ca2+-permeable ion channels in different cancers. Recently, Cabrita et al. have reported that TMEM16A directly interacts with IP3R in HeLa cells, and is activated by Ca2+ release from the ER via the IP3R, but not via ORAI-mediated Ca2+ influx [85]. This finding suggests that TMEM16A may be primarily activated by IP3R-mediated Ca2+ release from the ER in cancer cells. ** et al. reported a similar finding in small neurons from dorsal root ganglia, showing activation of TMEM16A by IP3R-mediated Ca2+ release, but not by Ca2+ influx via voltage-gated Ca2+ channels [82]. Further studies are required to investigate whether IP3R-mediated Ca2+ release from the ER represents a general mechanism for TMEM16A activation in cancer.

TMEM16A overexpression can be used as a prognostic and predictive marker for clinical outcomes in cancer patients (Table 1). We have previously found that TMEM16A overexpression is associated with good prognosis in PR-positive or HER2-negative breast cancer patients following tamoxifen treatment [43]. Since tamoxifen inhibits TMEM16A currents [1], the beneficial effect of tamoxifen in breast cancer patients may be associated with its inhibition on TMEM16A channel function. In addition, TMEM16A inhibition by T16A-inhA01 and CaCC-inhA01 has been reported to increase responses to EGFR/HER2-targeted therapy in HNSCC cells [110]. Recently, several other TMEM16A inhibitors has been discovered, including MONNA [134], eugenol [135], dehydroandrographolide [136], 9-Phenanthrol [137], Ani9 [138], idebenone [139], and luteolin [111]. Although some TMEM16A inhibitors have been tested in certain cancer cell lines [111, 136, 139], it remains unclear whether these compounds can effectively inhibit cancer growth in vivo, since pharmacological sensitivity of TMEM16A channels may be affected by cellular environment [10, 140, 141]. Both animal and clinical studies are required to investigate the efficacy of a TMEM16A inhibitor on cancer cell growth and metastasis before it can be used for cancer therapy.