The Ca²⁺-activated chloride channel ANO1/TMEM16A: An emerging therapeutic target for epithelium-originated diseases?

Abstract

Anoctamin 1 (ANO1) or TMEM16A gene encodes a member of Ca²⁺ activated Cl^– channels (CaCCs) that are critical for physiological functions, such as epithelial secretion, smooth muscle contraction and sensory signal transduction. The attraction and interest in ANO1/TMEM16A arise from a decade long investigations that abnormal expression or dysfunction of ANO1 is involved in many pathological phenotypes and diseases, including asthma, neuropathic pain, hypertension and cancer. However, the lack of specific modulators of ANO1 has impeded the efforts to validate ANO1 as a therapeutic target. This review focuses on the recent progress made in understanding of the pathophysiological functions of CaCC ANO1 and the current modulators used as pharmacological tools, hopefully illustrating a broad spectrum of ANO1 channelopathy and a path forward for this target validation.

Graphical abstract

The abnormal expression or dysfunction of Ca²⁺ activated Cl^– channel anoctamin 1 (ANO1) is involved in pathological phenotypes and diseases, suggesting that pharmacological modulation of ANO1 may serve as therapeutic potential and strategy.

1. Introduction

Ca²⁺-activated chloride channels (CaCCs) are a heterogeneous group of Cl^– channels that can be synergistically activated by intracellular calcium and voltage. The CaCCs are found in almost all species ranging from invertebrates to mammals, and the ubiquitous expression of CaCCs indicates a variety of functions important for physiology, including regulation of epithelial Cl^– secretion, excitability of neuronal and cardiac cells, smooth muscle contraction and nociception1, 2, 3.

A subtype of CaCCs was first described in Xenopus oocytes nearly 40 years ago and its molecular identity was debated until in 2008 when three independent laboratories reported that anoctamin-1 (ANO1) or transmembrane protein 16A (TMEM16A) underlies the molecular basis of a subgroup of CaCCs4, 5, 6. The Ano1/Tmem16a gene encodes a 986-amino-acid protein that belongs to anoctamin family consisting of 10 members (ANO1–ANO10) in mammals. ANO1, as a CaCC, is primarily expressed in epithelial cells, smooth muscle cells and sensory neurons. ANO2, also as a CaCC, is expressed in the olfactory sensory neurons⁷, photoreceptor synaptic terminals⁸, hippocampal pyramidal neurons⁹, thalamocortical neurons¹⁰, and inferior olive neurons¹¹ in the brain. Other ANOs family members including ANO6 and ANO7 were onetime considered to be CaCCs¹², but evidence shows that ANOs3–7 neither generate Ca²⁺-activated Cl^– currents nor traffic into membrane, indicating that they are endoplasmic reticulum proteins¹³. More studies reveal that ANOs3–7 and ANO9 are linked to the Ca²⁺-dependent membrane phospholipid scramblases that are responsible for translocation of phospholipids14, 15, 16. It is generally accepted that ANO1 and ANO2 are two members of the CaCC subfamily, whereas ANO3–ANO10 have been debated for their functions as CaCCs or Ca²⁺-dependent membrane phospholipid scramblases, or other physiological proteins^17,18. In addition, an integral membrane protein bestrophin-1 (BEST1) encoded by the BEST1 gene also functions as a CaCC. BEST1 is predominantly expressed in retinal pigment epithelium¹⁹, non-neuronal tissue, peripheral and central neurons^20,21.

ANO1 was initially thought to have eight transmembrane domains, possessing multiple protein isoforms generated by alternative splicing of four segments (a, b, c, and d) located at the C-terminus and the first intracellular loop⁵. ANO1 splice variant lacking segment b, for instance, increases the calcium sensitivity, whereas deletion of the segment c decreases apparent Ca²⁺ sensitivity and increases voltage-dependent activity of ANO1 channel^22,23. The X-ray structure of a Ca²⁺-activated lipid scramblase ANO1 from fungus Nectria haematococca (nhANO1) shows a homodimer with each subunit containing a ten-transmembrane α-helices²⁴. Several critical amino acid residues for Ca²⁺ sensitivity and ion selectivity are subsequently found^25,26. Recently, a high-resolution cryo-EM structure reveals an overall structure of mouse ANO1 similar to nhANO1^27,28, exhibiting two Ca²⁺ binding sites within the inner vestibule of the pore, and Ca²⁺ binding triggering conformational changes of α-helix rendering the pore conductive (Fig. 126, 27, 28, 29). The mechanisms underlying ANO1 activation and modulation are beginning to emerge. It has been shown that calmodulin, protons, cell volume and thermal stimuli can regulate the channel activation^30,31, and phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates the activation and desensitization of ANO1^32,33.

Figure 1.

The molecular structure of ANO1/TMEM16A channel. (A) The Ca²⁺-bound structure of mANO1 channel in dimer (chains A and B), and 2 yellow filled circles for Ca²⁺ in each monomer containing 10 transmembrane α helices. (B) Ca²⁺ binding sites formed by residues N650, N651, E654 from α6, E702, E705 from α7, and E734, D738 from α8^28,27. (C) Residues critical for ion selectivity including R515 from α3, N546, D554 from α4, N591, V599 from α5, K603, R621 from α5–6 linker, S639 from α6, and Q709, F716 from α7^26,28. (D) Putative binding sites, R515 from α3, K603, R621 from α5–6 linker, and R788 from α8, for ANO1 inhibitors NTTP and 1PBC²⁶; and N650 from α6, A697, E705 from α7, and L746 from α8 for ANO1 activator GRb1²⁹. The structure is regenerated based on the cryo-EM structure of ANO1 channel (PDB 5OYB)²⁷. The residue number labeling is based on the sequence of mTMEM16A (ac) isoform (UniProt Q8BHY3.2).

ANO1 channel appears to be preferentially activated by local rise of intracellular Ca²⁺ release from the endoplasmic reticulum (ER) Ca²⁺ stores, which may represent a general mechanism of ANO1 activation³⁴. ANO1 expression is regulated by multiple signaling cascade pathways such as mitogen-activated protein kinase (MAPK), nuclear factor κB (NF-κB) and transforming growth factor-β (TGF-β) in pathological functions³⁵. Several recent reviews have substantially covered the molecular basis, structure and pathophysiological functions of ANO1^17,35, 36, 37. In this review we will mainly focus on the aspect of channelopathies and pharmacological validation of ANO1 as an emerging therapeutic target.

2. Pharmacological modulation of ANO1 channel

Validating ANO1 as a therapeutic target requires specific modulators that can serve as essential tools for understanding the channel pharmacology. Up to now, many ANO1 modulators have been reported, and unfortunately most of them are lack of potency and efficacy. Therefore, there exists a great need for discovery of more selective and potent modulators, inhibitors in particular, which can be used for ANO1 target validation.

2.1. Inhibitors

Some broad-spectrum blockers, such as NFA, FFA, DIDS, NPPB and 9AC, have been used as tools for understanding functions of CaCCs (Table 138, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84). These small molecules can block endogenous CaCCs in Xenopus laevis oocytes^39,43,45,85 and are also known to non-specifically modulate other channels, such as inhibition of volume regulated anion channel (VRAC) and Kv4 by NFA and DIDS and activation of Ca²⁺ activated K⁺ channel by NFA and FFA or potentiation of TRPV1 channel by DIDS^2,38. CaCC_inh-A01 was identified to inhibit CaCC current in 2008 from screen of 50,000 compounds using human intestinal epithelial HT29 cell that highly express endogenous CaCCs without obvious effects on intracellular Ca²⁺, Ca²⁺/calmodulin-dependent protein kinase (CAMK) or cystic fibrosis transmembrane conductance regulator (CFTR)⁴⁹. CaCC_inh-A01 as an ANO1 inhibitor has been commonly used in many investigations for the role of ANO1 in interstitial cells of cajal (ICC), cardiac fibroblast and rod bipolar cells of retina86, 87, 88, and channelopathies, including cancer, hypertension, nociception, diarrhea and high glucose induced renal cyst growth^51,53, 54, 55. CaCC_inh-A01 is shown to inhibit cancer cell proliferation through increasing the ubiquitination of ANO1 and facilitating ER-associated, proteasomal degradation of ANO1, while other ANO1 inhibitors, such as T16A_inh-A01 and digallic acid, are shown to have no effect on cancer cell proliferation89, 90, 91. Similarly, CaCC_inh-A01 inhibits proliferation of cardiac fibroblast, but not another ANO1 inhibitor T16A_inh-A01⁸⁷. CaCC_inh-A01 reduces upregulation of ANO1 expression and attenuates brain infarct size and neurological deficits after ischemic stroke whereas T16A_inh-A01 shows no effect⁵⁶. That ANO1 protein expression is reduced by CaCC_inh-A01 but not T16A_inh-A01 may partially explain why the two ANO1 inhibitors show different effects on cell proliferation and ischemic stroke. In addition, intrathecal injection of CaCC_inh-A01 reduces tactile allodynia and thermal hyperalgesia and also decreases ANO1 upregulation after spinal nerve injury⁵⁴.

Table 1.

Pharmacology of CaCC ANO1/TMEM16A inhibitors.

Inhibitor	Structure	IC50 (μmol/L)	Test assay	Selectivity	Effect	Ref.
NFA		7–37	Xenopus oocytes	KCa channel (+)	Anti-cancer,	38, 39, 40, 41, 42
			mANO1-CHO	Kv4 channel (−)	Anti-asthma
				VRAC (−)
				ANO6 (−)
				[Ca2+]i (+)
FFA		14–35	Xenopus oocytes	KCa channel (+)	NR	38,39
			mANO1-CHO	[Ca2+]i (+)
NPPB		22–68	Xenopus oocytes	K+ channel (−)	Anti-cancer,	38,39,43,44
			mANO1-CHO	[Ca2+]i (+)	Antinociception
				VRAC (−)
DIDS		11–550	Xenopus oocytes	Kv4 channel (−)	Anti-cancer,	38,39,44, 45, 46, 47
			mANO1-CHO	VRAC (−)	Antinociception
				[Ca2+]i (+)
				TRPV1 (+)
				xBest2a (/)
Dichlorophen		5.5	ANO1-HEK293	ANO2 (−)	Anti-asthma	48
				CFTR (/)
				ENaC (/)
Benzbromarone		10	ANO1-HEK293	ANO2 (−)	Anti-asthma	48
				CFTR (/)
				ENaC (/)
CaCCinh-A01		1–7.8	HT-29	hBest1 (–, 7 μmol/L)	Anti-cancer,	38,47,49, 50, 51, 52, 53, 54, 55, 56
			hNO1-FRT	ANO2 (−)	Anti-hypertension,
			mANO1-CHO	CFTR (/)	Anti-diarrhea,
				VRAC (−)	Antinociception,
				[Ca2+]i (/)	Anti-high glucose induced renal cyst growth,
				xBest2a (/)	Anti-ischemic stroke induced BBB intergrity
T16Ainh-A01		1	hANO1-HEK293	ANO2 (−)	Anti-cancer,	38,47,50,52,54,57, 58, 59, 60
				CFTR (/)	Anti-hypertension,
				[Ca2+]i (/)	Anti-Asthma,
				hBest1 (/)	Antinociception,
				VGCC (–, 50 nmol/L)	Anti-eosinophilic esophagitis,
				xBest2a (/)
MONNA		1.27	hANO1-HEK293	VRAC (−)	Anti-cancer,	47,61, 62, 63, 64, 65
			xANO1 (0.08 μmol/L)	ANO2 (−)	Anti-itch,
				mBest1 (/)	Anti-hypertension,
				hBest1 (/)	Anti-polyfertilization
				mCLC2 (/)
				hCFTR (/)
				xBest2a (/)
Ani9		0.08	hANO1-FRT	ANO2 (/)	Anti-cancer,	47,57,65,66
				CFTR (/)	Anti-polyfertilization
				ENaC (/)
				VRAC (/)
				ANO6 (slower activation)
				xBest2a (/)
10bm		0.03	hANO1-FRT	ANO2 (–, 0.4 μmol/L)	NR	67
				CFTR (/)
Ani9-5f		0.02	hANO1-FRT	ANO2 (/) CFTR (/)	Anti-cancer	68
Dimer trans-ε-viniferin (TV)		1.1	HT-29	CFTR (−)	Anti-diarrhea	69
Tetramer, γ-2-viniferin (RV)		12.3	HT-29	CFTR (−)	Anti-diarrhea	69
Niclosamide		0.1–2.4	hANO1-HEK293T	CFTR (/)	Anti-asthma	42,66,70
				ANO6 (−)
				[Ca2+]i (−)
Tannic acid		6–25	hANO1-FRT	CFTR (/)	Anti-nociception,	38,50,62,71, 72, 73
			mANO1-CHO	ENaC (/)	Anti-cancer,
				hBest1 (–, 15 μmol/L)	Anti-asthma
				ANO2 (−)
Eugenol		150	hANO1-FRT T84 cell	CFTR (/)	Anti-diarrhea,	74, 75, 76
				[Ca2+]i (/)	Local analgesia
				Nav1.8 (−)
				TRPV1 (−)
				Kv1.5 (−)
				VGCC (−)
Galangin		4.5–9.7	mANO1-CHO	NR	Anti-cancer	77,78
Luteolin		9.37	hANO1-FRT	ANO2 (–, 120 μmol/L)	Anti-cancer	77, 78, 79
			mANO1-CHO	L-type Ca2+ channel (−)
				[Ca2+]i (/)
Quercetin		13.7	mANO1-CHO	NKCC1 (+)	Anti-cancer,	78,80
				Na+-K+-ATPase (+)	Attenuation of GI tract motility,
				CFTR (+)	Increasement of intestinal Cl– secretion
				K+ channel (+)
				TRPM7 (−)
Idebenone		9.2	hANO1-FRT	ANO2 (−)	Anti-cancer,	81,82
				CFTR (/)	Anti-renal cyst
				[Ca2+]i (/)
Plumbagin		12.46	hANO1-FRT	CFTR (−)	Anti-cancer,	81,83
				K+ channel (/)	Anti-diarrhea
				Na+-K+-ATPase (/)
				[Ca2+]i (/)
Avermectins		0.15–1.32	mANO1-CHO	NR	Anti-cancer	84

+, activation effect; –, inhibitory effect; /, no effect; NR, no report; NKCC, Na⁺/K⁺/Cl^– co-transporter.

Since the identification of CaCC_inh-A01, several ANO1 inhibitors have been screened out using the iodide-sensitive yellow fluorescent protein (YFP)-based high throughput screening (HTS) assay^50,65,67. T16A_inh-A01 was reported to inhibit human ANO1 current expressed in Fisher rat thyroid (FRT) cells with an IC₅₀ of 1 μmol/L and had no effect on CFTR current⁵⁰. However, our previous study showed that T16A_inh-A01 at 10 μmol/L only inhibits mouse ANO1 current expressed in CHO cells about 28% at +80 mV³⁸, and it may because that the efficacy of T16A_inh-A01 on ANO1 inhibition dependents on splice variants of ANO1 and intracellular calcium⁹². Nevertheless, T16A_inh-A01 has been used as a pharmacological probe to investigate the role of ANO1 in pancreatic ductal adenocarcinoma cells⁹³, and also physiological and pathological functions of ANO1 in different tissues, including contraction of vesical smooth muscle, iodide release from thyrocyte, initial waveform modulation in retinal rod bipolar cell and melatonin secretion in pineal glands^52,88,94, 95, 96 (Table 1). T16A_inh-A01 attenuates the interleukin-13 (IL-13) induced increase of ANO1 expression and secretion of mucin in human nasal polyp epithelial cells from chronic rhinosinusitis patients, cultured human bronchial epithelial cells and goblet cells in the guinea pig asthma model^59,97, suggesting that T16A_inh-A01 may be useful for treatment of hypersecretion in asthma and other inflammatory airway diseases. In addition, several studies show that ANO1 inhibitor T16A_inh-A01 exerts therapeutic effect on cancer, neuropathic pain and eosinophilic esophagitis^58,98, 99, 100.

MONNA, the most potent blocker of anthranilic acid derivatives, blocks xANO1 and hANO1 with IC₅₀ of 0.08 and 1.27 μmol/L, respectively, without any effects on CFTR, CLC2 and BEST1⁶¹. Similar to ANO1 inhibitors CaCC_inh-A01 and T16A_inh-A01, MONNA concentration-dependently inhibits agonist-induced rodent vesical constriction through hyperpolarization of vesical smooth muscle cells^52,63. MONNA also inhibits chloroquine-induced action potential discharge at itch nerve terminals and bouts of scratching⁶⁴, suggesting a role of ANO1 in pruritus. MONNA has also been used to investigate the role of ANO1 in blocking poly-fertilization⁴⁷.

Another small molecule Ani9 was also identified to inhibit hANO1 expressed in FRT cells with an IC₅₀ of 0.08 μmol/L using YFP-HTS assay (Table 1). Ani9 belongs to acetamides and shows a relatively high selectivity on ANO1 over ANO2 without inhibitory effects on CFTR, VRAC, epithelial sodium channels (ENaC) and intracellular Ca²⁺ signaling at concentration of 30 μmol/L⁶⁵, whereas Ani9 can inhibit inward current and slow the time-dependent activation of ANO6⁶⁶. The Ani9 derivative 5f shows more potent inhibitory effect on ANO1 with an IC₅₀ of 20 nmol/L without activating on ANO2⁶⁸. Ani9 as a novel potent and selective ANO1 inhibitor has also been used in several investigations for the physiological and pathological functions of ANO1, including hypertension, polyspermy block and cancer^47,57,63. As one of the 2-acylamino-cycloalkylthiophene-3-carboxylic acid arylamides, 10bm shows a potent inhibition on ANO1 current with an IC₅₀ of 30 nmol/L and exhibits dose-dependent inhibition on isometric smooth muscle contraction. The 10bm compound also inhibits ANO2 with an IC₅₀ of 0.4 μmol/L without effect on CFTR⁶⁷. At present, synthesized ANO1 inhibitors are only used as tools for preclinical studies (Table 1).

Many natural products from diverse plants have been found to inhibit ANO1. Tannic acid presented in the green tea and red wine was identified as a blocker for both ANO1 and ANO2 without effect on CFTR or ENaC, showing an inhibitory effect on arterial smooth muscle contraction and intestinal Cl⁻ secretion⁷¹. Like the synthesized small molecule ANO1 inhibitors, tannic acid has also been used as a pharmacological tool for investigations of the biophysical feature and physiological functions of ANO1101, 102, 103. Series of flavonoids were recently identified to inhibit ANO1, exerting anticancer effects^77,78. Natural products or synthetic analogues of natural products, including idebenone with anticancer activity⁸¹, plumbagin with antidiarrhea⁸³ and matrine with anti-lung adenocarcinoma activity¹⁰⁴ have been shown to inhibit ANO1.

Several clinical drugs are recently found to have inhibitory effect on ANO1, including clarithromycin, benzbromarone, niclosamide, nitazoxanide and avermectins. Clarithromycin is a clinical antibiotic that was reported to decrease IL-13 induced ANO1 expression in goblet cells from asthma models¹⁰⁵. Benzbromarone, a clinical drug for gout treatment, shows anti-asthmatic effect through inhibiting IL-13 induced mucin secretion and methacholine induced airway smooth muscle (ASM) contraction via the inhibition of ANO1^70,106. Niclosamide and nitazoxamide are clinical anthelmintics that as potent ANO1 inhibitors can block ASM depolarization and contraction⁷⁰. Avermectins are a type of macrocyclic lactones that are widely used as pharmaceuticals against roundworms in humans and animals and also for crop protection. Several avermectins, including avermectin B1, ivermectin, doramectin, selamectin and moxidectin, exhibit inhibitory effects on cancer cell proliferation and migration through inhibition of ANO1⁸⁴. These drugs may have repurposing potential for dysfunctional ANO1 related diseases, such as asthma and hypertension.

It is noticeable that many compounds can act on ANO1 with limited selectivity, which imposes a big challenge for validation of ANO1 as a therapeutic target. The reasons for lack of identifying specific ANO1 modulators can be in multiple folds including chemical designs without guidance of target structure, use of an indirect YFP fluorescence-based HTS assay, and compound screens against cells expressing endogenous xANO1 in oocytes (for MONNA)⁶¹, hANO1 in HT29 cells (for CaCC_inh-A01)⁴⁹, or heterogeneous hANO1 in FRT cells (for T16A_inh-A01, Ani9 and 10bm)^50,65,67. In addition, ANO1 and ANO2 subunits share 62% of sequence⁴, which also presents the selectivity challenge. The recent solved cryo-EM structure of ANO1 may help understand the biophysical features and physiological functions of the channel and also can greatly assist to develop more specific ANO1 modulators^27,28. A pharmacophore model based on three-dimensional quantitative structure–activity relationship (3D-QSAR) for predicting best target and compound interactions also appears to be a promising tool for virtual screening and enhancing rational design for novel potent and specific ANO1 modulators¹⁰⁷.

2.2. Activators

During the past almost 40 years since the identification of CaCC in 1982¹⁰⁸, only a few activators of ANO1 have been reported. Two ANO1 activators Eact and Fact are the first discovered using an HTS approach¹⁰⁹. Eact and Fact are two different classes of chemicals that activate ANO1 through different mechanisms (Table 2^66,109, 110, 111, 112, 113, 114, 115, 116, 117). Eact is an activator that induces ANO1 current in the absence of intracellular Ca²⁺ with an EC₅₀ of 3 μmol/L. Several studies indicate that Eact might indirectly activate ANO1 through an intracellular Ca²⁺ increase^110,111. Fact is a potentiator that increases ANO1 current in a Ca²⁺-dependent manner with an EC₅₀ of 6 μmol/L. INO-4995 is an 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester directly activates overexpressed ANO1 current but without effect on endogenous ANO1 currents in Xenopus oocytes, human airways and colonic cells¹¹².

Table 2.

Pharmacology of CaCC ANO1/TMEM16A Activators.

Activator	Structure	EC50 (μmol/L)	Test assay	Selectivity	Effect	Ref.
Eact		3	hANO1-FRT	TRPV1 (+) TRPV4 (+) [Ca2+]i (+) ANO6 (+)	Pain, Smooth muscle contraction	66,109, 110, 111
Fact		6	hANO1-FRT	NR	NR	109
INO-4995		NR	hANO1-HEK293	NR	NR	112
Resveratrol		48	mANO1-HEK293T	NR	Smooth muscle contraction	113
GRb1		38.4	mANO1-HEK293	NR	Smooth muscle contraction	114
Cinnamaldehyde		9.7	ANO1-HEK293	NR	Smooth muscle contraction	66
Chitosan oligosaccharides		74.5 μg/mL	mANO1-HEK293	NR	Smooth muscle contraction	115
ETX001	NR	116 nmol/L	hANO1-FRT sANO1-CHO	[Ca2+]i (/)	Anti-CF	116
Melittin	C131H229N39O31	0.5–2	hANO1-HEK293	ANO6 (+) PLA2 (+)	Phospholipid scrambling through ANO6 but not ANO1	66,117

+, activation effect; /, no effect; NR, no report.

A small molecule ANO1 potentiator ETX001 is recently shown to increase ANO1 current with an EC₅₀ of 116 nmol/L without effect on intracellular Ca²⁺ signaling¹¹⁶ (Table 2). ETX001 enhances fluid secretory response in human bronchial epithelial cells from cystic fibrosis (CF) patients, and increases airway mucus clearance in sheep CF models. Several compounds from traditional Chinese medicines, including resveratrol¹¹³, ginsenoside Rb1 (GRb1)¹¹⁴, GRb2²⁹, cinnamaldehyde¹¹⁸ and chitosan oligosaccharides¹¹⁵, were recently reported to activate ANO1 channel although their mechanisms of action remain elusive. Activators of ANO1 can be useful for validating ANO1 as a therapeutic target for treatment of disorders associated with ANO1 hypofunction, including Sjogren's syndrome, cystic fibrosis lung disease, and dry eye syndrome.

It should be mentioned that the relatively wide distribution of ANO1 may impose a liability concern or a challenge in developing organ- or tissue-specific therapy. Nevertheless, development of targeted drug delivery systems such as tissue-specific or selective organ targeting nanoparticles and controlled release of therapeutic agents may circumvent potential ANO1-originated complications¹¹⁹. In addition, target-related complications can also be minimized thought means of different drug formulations, such as topical preparations, sublingual, buccal and rectal administrations.

3. Pathological functions and related diseases

Since ANO1 was identified as a member of CaCCs in 2008, its investigations have been focused on the distribution and expression in numerous organs and tissues, and its roles in pathological conditions and diseases (Fig. 2^{51,98,120,121}).

Figure 2.

Distribution of ANO1/TMEM16A in different tissues and its role in diseases. (A) In epithelial cells, ANO1 activation contributes to electrolyte and music secretion. Activation of GPCRs causes an increase in intracellular cAMP and Ca²⁺, further inducing Cl⁻ secretion through the activation of CFTR and ANO1. A crosstalk between CFTR and ANO1-dependent secretions also occurs for regulation of secretory signaling in the airway epithelia. ANO1 high expression or hyperactivity can cause inflammatory diseases such as asthma and diarrhea. (B) Outward flow of Cl⁻ through the activation of ANO1 in smooth muscle cells causes depolarization and smooth muscle contraction. ANO1 high expression or hyperactivity is responsible for asthma diarrhea, and hypertension. (C) In DRG sensory neurons, activation of ANO1 by intracellular Ca²⁺ or heat causes Cl⁻ efflux and increases neuronal excitability for induction of neuropathic pain. The functional coupling between TRPV1 and ANO1 is also involved in nociception. (D) In cancer cells, ANO1 upregulation promotes cell proliferation and migration, whereas ANO1 downregulation induces apoptosis through multiple signaling pathways, including EGFR/MAPK signaling pathway⁹⁸, CaMKII/MAPK signaling pathway⁵¹, TGF-β signaling pathway¹²⁰ and NF-κB signaling pathway¹²¹. Pharmacological activation of ANO1 by activators or potentiators may serve as a therapeutic strategy for treatment of CF, dry mouth and dry eye syndromes, and inhibition of ANO1 by inhibitors may be beneficial for ANO1 related channelopathies including asthma, diarrhea, hypertension, neuropathic pain and cancers.

3.1. Epithelial diseases

Epithelial cells are a group of tightly compressed cells that line the inside of all organs and function as a barrier between the inside and outside of an organ. Chloride ion is important for transepithelial secretion, whereas intracellular Ca²⁺ is one of two major signals to modulate Cl^– secretion^122,123, indicating an important role of Ca²⁺ activated Cl^– channels in regulating epithelial secretion. ANO1 is observed in many epithelial tissues, including but not limited airway, bronchus, salivary glands, pancreatic ductal cells and intestinal epithelium^1,4,124. Numerous literatures not only identify native ANO1 expressions in multiple epithelia tissues but also demonstrate the pathological functions of ANO1 important for the process of fluid and electrolyte secretion^5,37,125, 126, 127. Here, we review several epithelial diseases involved in ANO1 dysfunction.

3.1.1. Asthma

Asthma is a prevalent inflammatory airway disease characterized by chronic inflammation, remodeling, and excessive constriction of the airway. The mechanism underlying asthma is complex and treatment for asthma is still challenging¹²⁸. Hypersecretion of airway epithelium and hypercontraction of airway smooth muscle are two major contributions to asthma¹²⁹. The Ca²⁺ activated Cl^– channel ANO1 as an anion channel plays a critical role in airway transepithelial mucus function. Mucus secreted by goblet cells is composed of water and mucus protein, which requires anion channel activity to instill Cl^– and HCO₃^– for ensurance of proper salination, hydration and pH of the mucus gel layers¹³⁰. ANO1 is found robustly expressed in epithelial goblet cells and involved in mediating native Ca²⁺ dependent Cl^– current¹³¹. ANO1 is also highly permeable to HCO₃^– at higher intracellular Ca²⁺ levels, likely contributing to mucus release by providing a secretory pathway for HCO₃^– that is an essential ingredient in mucus^132,133. Downregulation of ANO1 markedly decreases epithelial mucus secretion^126,127, suggesting ANO1 inhibition beneficial for asthma. ANO1 expression is upregulated by cytokines IL-4, IL-13 and T helper type 2 that are asthmatic biomarkers, and upregulated ANO1 colocalizes to the apical plasma membrane of goblet cells along with the mucin MUC5AC^48,97,131. In addition, ANO1 is also found abundantly expressed in the smooth muscle cells and plays a role in airway hyperreactivity⁴⁸. ANO1 expression is up-regulated both in epithelial cells and airway smooth muscle cells from asthmatic human patients and rodent models^41,48,127. These investigations suggest that hyperactivity of ANO1 may underlie the pathogenesis of asthma, and inhibition of ANO1 channel activity may represent a therapeutic strategy for mucus hypersecretion in asthma.

The mechanism for ANO1 involving in asthma is unclear. A recent investigation shows that the GPCR/ANO1/VGCC axis contributes to the bronchial hyperresponsiveness in asthma¹³⁴. The asthmatic inflammatory mediators, including 5-HT, histamine and thromboxane A2 binding to G-protein coupled receptors (GPCRs), activate IP₃R to release Ca²⁺ from sarcoplasmic reticulum for activation of ANO1 and voltage gated calcium channel (VGCC) that leads to Ca²⁺ influx and subsequently depolarize the membrane potential and cause airway contraction both ex vivo ASM and in vivo asthmatic animals¹³⁴. Downregulation of ANO1 by either pharmacological inhibitor T16A_inh-A01 or knockdown can inhibit the inflammatory mediator-induced Ca²⁺-activated Cl^– current and ASM contractile response¹³⁴. Several reports show the involvement of ANO1 in mucus production or secretion^48,59,127, but the mechanistic insights into mucus production and secretion in airways and validation of the role ANO1 in asthma are required.

Huang and his colleges⁴⁸ adopted a HTS assay and identified benzbromarone, a clinical drug for gout treatment, as an ANO1 blocker. Benzbromarone shows anti-asthmatic effect through inhibiting IL-13-induced mucin secretion and methacholine-induced ASM contraction via inhibition of ANO1 function^70,106. In addition, several ANO1 inhibitors, such as NFA⁴¹, tannic acid⁷³, T16A_inh-A01⁵⁹, and niclosamide⁷⁰ have been shown to exert anti-asthma effects. However, the anti-asthma effects observed using those ANO1 inhibitors need to be further confirmed in some degree because of their relatively low potency and poor selectivity.

3.1.2. Diarrhea

Diarrhea is characterized by an increase in the frequency of bowel movements with loose and watery stools and major causes of infectious secretory diarrhea include bacteria, viruses and parasites¹³⁵. Intestinal Cl^– channels are important for regulation of excessive mucus secretion induced by bacteria/virus-secreted enterotoxins through increasing apical membrane permeability, suggesting an involvement of Ca²⁺ activated Cl^– channel ANO1 in pathology of diarrhea. There is an increasing evidence that ANO1 plays an important role in the regulation of intestinal epithelial secretion and the pathogenesis of diarrhea based on the following observations: 1) ANO1 is expressed in intestinal epithelia and ANO1 gene knockdown reduces Ca²⁺ activated Cl^– secretion induced by muscarinic agonist carbochol¹²⁶; 2) Ca²⁺-dependent Cl⁻ secretion increases through enhancement of ANO1 expression or activation of ANO1 current in the distal colon in response to the rotavirus nonstructural glycoprotein NSP4 that causes infantile gastroenteritis¹³⁶; and 3) ANO1 expression is upregulated in animal models of diarrhea¹³⁷.

The increased activity of gastrointestinal (GI) tract is another important cause for diarrhea. ICC are mesenchymal cells located within the muscle layers in the GI tract and myenteric ICC serve as a pacemaker generating the bioelectrical slow wave potential that leads to contraction of GI smooth muscles in the GI tract¹³⁸. Recent studies have found that ANO1 is also highly expressed in the ICC and mediates the slow wave current in the ICC^4,139,140. The intracellular Ca²⁺ rise from intracellular stores and VGCC in the ICC causes the generation and propagation of pacemaker potential that is also amplified by activation of ANO1 channels¹³⁸, suggesting that inhibition of ANO1 may reduce intestinal mobility for diarrhea. Indeed, pharmacological inhibition or gene silencing of ANO1 blocks slow wave in intestinal smooth muscle^141,142. It is noted that several ANO1 inhibitors including T16A_inh-A01 and CaCC_inh-A01 exhibit different potency on blocking gastric and small intestinal slow waves. For instance, CaCC_inh-A01 blocks slow waves in the murine stomach at 5 μmol/L and the small intestine at more than 30 μmol/L¹⁴³. The mechanism underlying these different sensitivities is not entirely clear, but several probabilities including other channels contributions, different splice variants and the local Ca²⁺ concentrations may contribute to these differences¹³⁸. Nevertheless, both GI epithelium and ICC are synergistically involved in the pathogenesis of diarrhea and downregulation of ANO1 in either type of the two cells may help mitigate diarrhea.

Rotavirus has been known to cause severe diarrhea in infants and young children, whereas red wine extracts with alcohol-free and CaCC_inh-A01 can prevent intestinal fluid loss in a neonatal mouse model of rotaviral diarrhea through inhibition of ANO1-mediated Ca²⁺-activated Cl^– secretion⁵³. However, red wine extracts and CaCC_inh-A01 show no obvious effect on cholera toxin-induced diarrhea or CFTR current in cultured cells or intestinal absorption, suggesting the important role of ANO1 in diarrhea⁵³. A recent study demonstrates that glucose and NSP4 synergistically increase the expression of ANO1 and Ca²⁺-activated Cl^– secretory in the mouse model of diarrhea¹³⁷. Suppressing ANO1 expression in apical membranes of colonic epithelium decreases Ca²⁺-activated Cl^– secretion in chemical dextran sulfate sodium-induced chronic colitis in mice¹⁴⁴. There are also observations that inhibition of ANO1 by non-specific ANO1 inhibitors including eugenol⁷⁴, shikonin¹⁴⁵, plumbagin⁸³, resveratrol dimer trans-ε-viniferin (TV) and tetramer γ-2-viniferin (RV)⁶⁹ can reduce water content in stools, but those observations should be confirmed with specific ANO1 inhibitors.

3.1.3. Cystic fibrosis

CF is an inherited disease of airway obstruction caused by mucus hypersecretion, mucus plugging and bronchoconstriction¹⁴⁶. The dysfunction of CFTR Cl^– channel is considered to be a major cause for CF as defective CFTR Cl^– channel mutations are identified in CF patients¹⁴⁷. It has been shown that small molecules can rescue dysfunctional CFTR mutation through increasing the number or open probability of CFTR Cl^– channels. Unfortunately, these CFTR correctors or potentiators only exhibit limited efficacy for CF patients because of their multiple and different mutation sites of CFTR¹⁴⁸. Therefore, an alternative strategy is CFTR-independent approach for treatment of CF by increasing the activity of ANO1 channel to promote mucus secretion¹⁴⁹. This hypothesis is supported by observations that: 1) ANO1 is abundantly expressed in the airway goblet cells and upregulated in inflammatory conditions¹³¹; 2) overexpression of ANO1 in CF human bronchial epithelia suppresses proinflammatory cytokine IL-8 secretion¹⁵⁰; 3) specific knockdown of ANO1 in respiratory airways eliminates Ca²⁺-and cAMP-activated Cl^– secretion¹⁵¹; 4) Ano1 gene knockout mouse exhibits abnormal trachea morphology, and also mucus obstruction and defective mucociliary clearance that presenting with a CF-like lung phenotyp^125,152.

Several studies demonstrate that ANO1 has a functional cross talk with CFTR through PSD-95/Dlg/AO-1 proteins^151,153. Such an interaction between ANO1-mediated Ca²⁺-activated Cl^– secretion and CFTR-mediated cAMP-dependent Cl^– secretion in airway epithelial cells strongly overlaps through a cAMP sensor protein termed exchange protein directly activated by cAMP and Ca²⁺-sensitive adenylate cyclase type 1¹⁵⁴. ANO1 expression and activity are deficient in CF patients and upregulation of ANO1 can improve mucus dynamics in CF mice¹⁵⁵. These lines of evidence support the notion that ANO1 may represent an alternative therapeutic target to circumvent CFTR dysfunction in the airway epithelia of CF patients.

Some CFTR-independent drug candidates have been developed for potential CF therapy. Denufosol, a P2Y2 receptor agonist, promotes airway epithelial chloride secretion through activating CaCCs with P2Y receptors¹⁵⁶. Unfortunately, denufosol failed in phase III trial because of its short half-life in vivo^147,156,157. Recently, an ANO1 activator, ET000516-A-2 from pre-clinical study is expected to be evaluated in CF patients¹⁴⁷. Another novel ANO1 potentiator ETX001 developed by Enterprise Therapeutics (Brighton, UK) was recently reported that it can enhance fluid secretion and improve mucociliary clearance in both primary CF bronchial epithelial cells and sheep model of CF-like airway disease¹¹⁶. These two candidates may bring us an exciting prospect for potential development of alternative CF therapy in the clinic.

3.1.4. Other epithelial diseases

A recent study shows that ANO1 is overexpressed in mouse pancreatic tissue of acute pancreatitis model¹⁵⁸. ANO1 promotes the pathogenesis of acute pancreatitis through activating the IP₃R/Ca²⁺/NF-κB/IL-6 pathway, and inhibition of ANO1 by T16A_inh-A01 reduces the pancreatic damage in acute pancreatitis mice¹⁵⁸. ANO1 is also likely involved in other epithelial diseases, such as polycystic kidney disease (PKD), diabetic nephropathy and pulmonary fibrosis. The PKD is characterized by multiple bilateral renal cysts that gradually enlarge and can lead to a decline in renal function. ANO1 is found in human kidney and renal epithelial cell lines, and its expression is upregulated in the forskolin induced renal cyst model, autosomal dominant PKD patients and high-fat diet/streptozotocin-induced diabetic nephropathy mice^55,159,160, indicating a role of ANO1 in kidney disease.

ANO1 promotes renal cyst growth via induction of Cl^– secretion and proliferation of cyst lining epithelium¹⁵⁹. Inhibition of ANO1 by pharmacological inhibitors or gene knockdown significantly decreases glucose dependent cyst growth, reduces nephron numbers and also causes albuminuria and tubular damage^55,159,161. Mechanistically, ANO1 drives the growth of renal cysts through enhancing Ca²⁺ release from IP₃R sensitive Ca²⁺ stores¹⁶² and lipid peroxidation also promotes renal cyst growth through activating ANO1⁸². In diabetic nephropathy, ANO1 deletion alleviates renal injury in diabetic mice through increasing nephrin expression, reducing the expression level of apoptosis related factors and also suppressing the activation of P38/JNK signaling pathway¹⁶⁰.

On the contrary, ANO1 activation aggravates renal injury by activating P38/JNK signaling pathway to promote podocyte apoptosis in diabetic nephropathy mice¹⁶⁰ and exacerbates inflammation via activating the TGF-β-SMAD3 pathway¹⁶³. Downregulation of ANO1 by shRNA can inhibit apoptosis and promote the proliferation of lung fibroblasts in mouse model of idiopathic pulmonary fibrosis¹⁶³. These observations suggest that inhibition of ANO1 may hold therapeutic potential for kidney and other epithelium-originated diseases.

3.2. Cancers

Epithelial cancer is also known as carcinoma that arises from epithelial tissues. Prior to identification as a CaCC, ANO1 was known as TMEM16A first described in 2003¹⁶⁴. ANO1 was also named as DOG1 (gastrointestinal stromal tumor 1), TAOS2 (tumor amplified and overexpressed sequence 2) and ORAOV2 (oral cancer overexpressed 2) because of its overexpression in these cancers³⁵. Investigations from ours and others show that ANO1 is overexpressed and involved in the pathogenesis of cancers especially originated from epithelial cancers, such as gastrointestinal stromal tumor (GIST)¹⁶⁵, head and neck squamous cell carcinoma (HNSCC)¹⁶⁶, prostate cancer⁴⁶, lung cancer¹⁶⁷, colon cancer⁹⁰, ovarian cancer¹⁶⁸, breast cancer⁵¹, liver cancer¹⁶⁹, gastric cancer¹²⁰, esophageal cancers¹⁷⁰, pancreatic adenocarcinoma¹⁷¹, salivary gland carcinoma¹⁷² and glioblastoma¹²¹ (Table 3^{40,46,51,57,62,91,93,98,120,121,}165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187).

Table 3.

ANO1 modulation, expression and function in cancer cell lines, xenograft tumors and human cancer tissues.

Cancer type	High expression			Cell assay		Inhibition		Signaling pathways	Ref.
	Cell line	Human tissue	Clinical implication	Proliferation/viability	Migration/invasion	Tool	Xenograft tumor
Breast cancer	ZR75-1, HCC1954, MDA-MB-415	+	Poor Prognosis (+)	+	NR	shRNA/CaCCinh-A01	Tumor growth (−)	11q13 amplification, Cl– channel activity, Apoptosis, EGFR, CAMKII, AKT, MAPK	51
	YMB-1	NR	NR	+	NR	siRNA/NFA	NR	Epigenetic regulation	40
	YMB-1, MDA-MB-453	NR	NR	NR	NR	siRNA/T16inh-A01	NR	AKT, STAT3	173
	SKBR3	NR	Improved response to biological therapies (−)	+	NR	siRNA/T16Ainh-A01 CaCCinh-A01	NR	EGFR, HER2, STAT3	174
	MCF-7, T47D	+	Shorter overall survival in ER + patients (+)	+	NR	shRNA/T16Ainh-A01	Tumor growth (−)	EGFR/STAT3 signaling	175
HNSCC	UM-SCC1, T24	+	Poor prognosis (+)	+	NR	shRNA/T16Ainh-A01	Tumor growth (−)	MAPK, Ki67	176
	HEp-2, SCC-25	+	A marker for distal metastasis (+)	No effect	+	siRNA/NFA, DIDS, Fluoxetine	NR	11q13 amplification	166
	UM-SCC1, T24	High in primary tumor and low in metastatic tumor	A biomarker for metastasis (−)	NR	–	shRNA	Tumor growth (−), Metastatic development (+)	Promoter methylation, E-cadherin	177
	FaDu	NR	Poor prognosis (+)	+	NR	shRNA/CaCCinh-A01	Tumor growth (−)	11q13 amplification, Cl– channel activity, Apoptosis, EGFR, CAMKII, AKT, MAPK	51
	Cal-33, OSC19, UM-SCC-1, FaDu	NR	Increased efficacy of biologic therapies (−)	OSC19 (+)	NR	siRNA/T16Ainh-A01 CaCCinh-A01	ANO1-overexpressing tumors were heavier than control tumors	EGFR, HER2, STAT3	174
	OSC19, FaDu, UM-SCC-1	Positively correlated with tumor size	Recurrence of cancer (+)	+	NR	shRNA	ANO1-overexpressing tumors were greater than control tumors	ERK, BIM, Apoptosis	178
ESCC	KYSE30, KYSE510	+	Lymph node metastasis and advanced clinical stage (+)	+	NR	siRNA	NR	11q13 amplification	170
	KYSE410, KYSE30	+	Poor prognosis (+) Advanced stage (+)	+	+	shRNA	NR	TGF-β signaling, cell cycle	179
Prostate cancer	PC-3, LNCap, RWPE1	+	NR	+	NR	siRNA/T16Ainh-A01, CaCCinh-A01, MONNA, tannic acid	Tumor growth (−)	ERK, AKT	62
	PC-3, LNCaP	NR	NR	+	NR	siRNA/NFA	NR	Epigenetic regulation	40
	LNCaP, PC-3	+	Clinical TNM stage (+) Gleason score (+)	+	+	shRNA/DIDS	Tumor growth (−)	NR	46
	PC-3	NR	NR	+	NR	shRNA siRNA/CaCCinh-A01, T16Ainh-A01, Ani9	Tumor growth (−)	TNF-α signaling, apoptosis	57
Gastric cancer	AGS, BGC823	+	Poor overall survival (+) TNM (+) Lymphnode metastasis (+)	No effect	+	shRNA	NR	TGF-β, E-cadherin	180
	AGS, BGC823	Negatively related with miR381	Poor prognosis (+)	No effect	+	siRNA	NR	Regulated by miR-381, TGF-β, E-cadherin	120
	AGS, SGC7901	+	TNM stage (+)	NR	+	siRNA	Tumor metastasis (−)	Regulated by SP1 through MLL1 and H3K4 trimethylation	181
HCC	SMMC7721	+	NR	+	+	siRNA	Tumorigenicity (−)	MAPK signaling, cell cycle	169
	HepG2, SMMC7721	+	Tumor grade (+)	+	+	shRNA	Tumor growth (−)	PI3K/AKT-MAPK signaling pathway, apoptosis, cell cycle	182
Glioma	U87MG	+	Tumor grade (+)	+	+	siRNA	NR	NF-κB signaling	121
	U251, U87MG	NR	NR	NR	+	shRNA/T16Ainh-A01	NR	CaMKII-β	183
Lung cancer	GLC82, NCI-H520	+	NR	+	+	shRNA	Tumor growth (−)	NR	167
	H1299	NR	NR	+	+	shRNA/T16Ainh-A01	Tumor growth (−)	EGFR/MAPK signaling	98
Pancreatic adenocarcinoma	BxPC-3, AsPC-1, Capan-1	NR	NR	No effect	+	siRNA/T16Ainh-A01, CaCCinh-A01, NS3728	NR	NR	93
	AsPC-1	+	Poor prognosis (+) Biomarker (+)	NR	+	shRNA	NR	Ligand-dependent EGFR signaling	171
GIST	GIST-T1, GIST-882	NR	NR	No effect	NR	shRNA/T16Ainh-A01, NFA, NPPB	Tumor growth (−)	IGFBP-5, no effect on KIT	165
	NR	Cancer and PBMCs	Biomarkers (+) Tumor size (+)	NR	NR	NR	NR	NR	184
	GIST-T1, GIST882	NR	NR	+	NR	T16Ainh-A01 CaCCinh-A01	NR	Cell cycle	91
Salivary gland carcinoma	NR	+	NR	NR	NR	NR	NR	NR	172
Ovarian cancer	SKOV3	Cancer and PBMCs	Pathologic stage and differentiation (+)	+	+	siRNA	Tumor growth (−)	PI3K/AKT signaling	168
Colorectal cancer	SW620	NR	NR	+	+	shRNA	NR	MAPK signaling, cell cycle	185
	DLD-1, HCT116	Liver metastasis cancer tissue	Poor prognosis (+)	+	+	siRNA	NR	Regulated by miR-132	186
	SW480	+	Poor prognosis (+)	NR	+	siRNA	NR	EGFR signaling, regulated by miR-144	187

+, positive effect; –, negative effect; NR, no report. HCC, hepatocellular carcinoma; PBMCs, peripheral blood mononuclear cells; TNM, tumor, lymph nodes and metastasis.

ANO1 upregulation in many kinds of cancers is related to its gene location at chromosome 11q13 that is frequently amplified in many malignant tumors^51,164 and amplification of 11q13 is associated with the increase of ANO1 gene copy numbers¹⁶⁶. Overexpression of ANO1 promotes proliferation and migration in multiple cancer cell lines^51,169,179, and ANO1 upregulation is associated with lower overall survival in patients with breast cancer, pancreatic cancer or gastric cancer^51,171,180 (Table 3). ANO1 mRNA is also highly expressed in the blood of GIST patients¹⁸⁴ and patients with epithelial ovarian cancer, and the expression level of ANO1 mRNA decreases after surgical removal of tumors¹⁶⁸, suggesting that detection of ANO1 gene in blood may serve as a biomarker for early diagnosis of cancer.

Multiple signaling pathways have been shown to be involved in ANO1 modulation in cancer development (Table 3). In breast cancer and HNSCC, downregulation of ANO1 by knockdown or pharmacological inhibition inhibits cancer cell proliferation, induces apoptosis and reduces tumor growth through reducing epidermal growth factor receptor (EGFR) cell signaling pathways⁵¹. Studies have also shown that ANO1 can affect the progression of intestinal cancer, liver cancer, and pancreatic cancer through the EGFR pathway^169,171,187, as well as the modulation of glioma by NF-κB signaling¹²¹. In prostate cancer, ANO1 can regulate TNF-α signaling to contribute to cell growth and apoptosis⁵⁷. In epithelial ovarian cancer, silencing ANO1 can suppress cancer cell proliferation, migration and invasion as well as the growth of xenograft tumors through inactivation of PI3K/AKT cell signaling pathway¹⁶⁸. In gastric cancer, ANO1 overexpression can promote tumor invasion and predict poor prognosis through affecting TGF-β signaling function^120,180, that is also the target for ANO1 regulating cell proliferation, migration and invasion in esophageal squamous cell carcinoma (ESCC)¹⁷⁹. In glioblastoma, ANO1 expression is regulated by CaMKII-β and suppression of CaMKII-β inhibits ANO1 mediated glioblastoma development¹⁸³, and CaMKII also plays a role in ANO1-mediated tumorigenic properties of HNSCC and breast cancer⁵¹. It appears that ANO1 can regulate different signaling pathways in the same type of tumor or the same signaling pathway in different types of tumor.

Inhibition of ANO1 by gene silencing or pharmacological means can suppress cancer cell proliferation and migration, invasion and tumor growth^62,175,181. Small molecules, such as CaCC_inh-A01, T16A_inh-A01, MONNA and Ani9, as well as natural products including avermectins and flavonoids exhibit anti-cancer effects through inhibition of ANO1 activity^57,62,78,84, suggesting that ANO1 may serve as a potential drug target for cancer therapy.

3.3. Hypertension

Hypertension or high blood pressure is a common condition in which the long-term force of the blood against the blood vessels is high enough that it may increase the risk of heart diseases and brain stroke. ANO1 is expressed in various smooth muscle cells of arteries and veins^188,189, suggesting a role of ANO1 in regulation of vasoconstriction. Activation of Ca²⁺-activated Cl^– current can lead to Cl^– efflux after Ca²⁺ influx and membrane depolarization, thus resulting in vasoconstriction and increase of blood pressure. Indeed, ANO1 expression and activity are upregulated in murine pulmonary arterial myocytes induced by chronic hypoxia, which contributes to pulmonary hypertension⁶⁰. Similar results were also observed in monocrotaline-induced pulmonary hypertension rats¹⁸⁸, spontaneously hypertension rats⁶³, idiopathic pulmonary arterial hypertension patients^190,191, and high-flow-induced pulmonary arterial hypertension (PAH) rats¹⁹². Conversely, cell-specific knockout of ANO1 reduces blood pressure and attenuates hypertension in mice and spontaneously hypertensive rats193, 194, 195.

A recent study shows that upregulation of ANO1 depolarizes pulmonary artery smooth muscle cells (PASMC) membrane potential, contributing vasoconstriction and the increased pulmonary vascular resistance in PAH rats¹⁹⁰. Conversely, pharmacological inhibition or gene silencing of ANO1 reverse the membrane depolarizes of PASMC. For instance, a specific ANO1 inhibitor MONNA hyperpolarizes the rat coronary artery smooth muscle cell membrane potential and increases coronary flow⁶³. Vascular smooth muscle cells (VSMC) are the stromal cells of the vascular wall and are responsible for regulating arterial tone, and blood pressure. Overexpression of ANO1 in healthy donor PASMC promotes the cell proliferation and produces an idiopathic pulmonary arterial hypertension (IPAH)-like phenotype. Pharmacological inhibition of ANO1 may reverse vasoconstriction and remodeling of pulmonary arteries in IPAH^190,196. These investigations suggest the involvement of ANO1 in VSMC contraction and vascular remodeling for hypertension.

Circulating angiotensin II (Ang II) as a major contributor of the renin–angiotensin system is upregulated during hypertension, and Ang II is frequently used to establish hypertension models. It has been shown that Ang II significantly enhances ANO1 expression in human umbilical vein endothelial cells and endothelial-specific ANO1 knockout significantly reduces Ang II-induced hypertension through ROS signaling pathway, whereas endothelial-specific transgenic ANO1 shows the opposite effect¹⁹⁵. These studies suggest that inhibition of ANO1 function may be beneficial for hypertension, and ANO1 inhibitors, including T16A_inh-A01, MONNA, and Ani9 can inhibit agonist-induced vesical constriction and cause vasorelaxation^63,94.

3.4. Nociception

Nociception is a perception in response to painful or harmful stimuli, such as heat, cold, mechanical and chemical stimulus in the environment. Dorsal root ganglion (DRG) is a cluster of sensory neurons in the dorsal root of spinal nerves responsible for pain signal transmission¹⁹⁷. ANO1 is mainly expressed in small diameter DRG neurons that are intimately involved in nociception^4,198, suggesting a modulatory role of ANO1 in pain sensation. It has been shown that an inflammatory mediator bradykinin, released from damaged tissues or applied exogenously can activate ANO1 through B2 receptors and PLC pathway, subsequently depolarizing membrane potential and markedly stimulating firings in DRG neurons, and pharmacological inhibition of ANO1 attenuates pain behaviors⁴⁴. ANO1 expression is upregulated in the spinal cord and DRG neurons after spinal nerve injury, suggesting the involvement of ANO1 in development of neuropathic pain⁵⁴. Protease activated receptor 2 (PAR2), also known as G-protein coupled receptor 11, has been identified to be involved in the pathogenesis of pain¹⁹⁹. PAR2 and ANO1 are co-localized in DRG neurons, and their expressions are increased in rat pain model of chronic constriction injury²⁰⁰.

ANO1 can be activated by temperatures over 44 °C, and silencing of ANO1 in DRG neurons significantly reduces nociceptive behavior in thermal pain model¹⁹⁸, inflammation and nerve-injury induced hyperalgesia or allodynia^54,201. Thus, downregulation of ANO1 activity may present a potential therapeutic strategy for neuropathic pain. ANO1 as a potential target for pain is evidenced by observations that inhibition of ANO1 by small molecules NPPB, NFA, T16A_inh-A01 or CaCC_inh-A01 reduces capsaicin-induced inward current and action potential firing, and as well as pain-related behaviors^99,100. Again, these ANO1 inhibitors are not specific and a key question such as whether ANO1 directly affects the excitability of nociceptors should be addressed.

3.5. Others

A recent study shows that ANO1 is expressed in mouse brain endothelial cells where ANO1 expression is upregulated after ischemic stroke induced by the middle cerebral artery occlusion⁵⁶. Targeting ANO1 with inhibitor CaCC_inh-A01 or silencing attenuates blood–brain barrier (BBB) breakdown after ischemic stroke through decreasing intracellular adhesion molecule-1 via NF-κB signaling pathway, suggesting that downregulation of ANO1 protects BBB disruption after ischemia stroke. It is supported by another study that ANO1 inhibitor T16A_inh-A01 or siRNA inhibits proliferation and migration of brain capillary endothelial cells that comprise BBB²⁰². It would be interesting to see more studies that are designed to validate ANO1 as a therapeutic target for brain stroke.

4. Summary and perspectives

The CaCC ANO1 channel is expressed in a wide variety of epithelial cells, smooth muscle cells and neurons. Although abnormal expression or dysfunction of ANO1 is involved in the pathology of many diseases, validating ANO1 as a therapeutic target still presents a big challenge. While a significant progress has been made for the distribution, expression, structure and pathophysiological functions of ANO1, there still exists an urgent need for selective modulators of the channel for target validation. Current available ANO1 modulators are also in preclinical stage without any treatments ready for clinical utility, which highlights the much-needed efforts in understanding the channel pharmacology and validation of ANO1 as therapeutic target. The recent structure of ANO1 solved by single-particle cryo-electron microscopy can provide a valuable model for the design of more potent and selective ANO1 modulators that can be used to help validate this emerging target for therapeutic potential of diseases, including cancer, inflammatory epithelial diseases, neuropathic pain and hypertension and cystic fibrosis lung disease.

Acknowledgments

We thank Dr. Xiaowen Tang (School of Pharmacy, Qingdao University Medical College, Qingdao, China) for his help in Fig. 1. This work was supported by grants from the National Natural Science foundation of China (81573410), the Ministry of Science and Technology of China (2018ZX09711001-004-006), Shandong Provincial Medicine and Health Technology Development Plan (2019WS145, China), Medical Research Guidance Plan of Qingdao Municipal Health Committee (2019-WJZD091, China) and Qingdao Excellent Young Medical Talent Training Project.

Author contributions

Yani Liu, Zongtao Liu, and KeWei Wang wrote and edited the manuscript. Yani Liu and KeWei Wang contributed to manuscript revision and discussion of the content.

Conflicts of interest

The authors declare that there is no conflict of interest.