Role of ANO1 in tumors and tumor immunity

Abstract

Dysregulation of gene amplification, cell-signaling–pathway transduction, epigenetic and transcriptional regulation, and protein interactions drives tumor-cell proliferation and invasion, while ion channels also play an important role in the generation and development of tumor cells. Overexpression of Ca²⁺-activated Cl- channel anoctamin 1 (ANO1) is shown in numerous cancer types and correlates with poor prognosis. However, the mechanisms involved in ANO1-mediated malignant cellular transformation and the role of ANO1 in tumor immunity remain unknown. In this review, we discuss recent studies to determine the role of ANO1 in tumorigenesis and provide novel insights into the role of ANO1 in the context of tumor immunity. Furthermore, we analyze the roles and potential mechanisms of ANO1 in different types of cancers, and provide novel notions for the role of ANO1 in the tumor microenvironment and for potential use of ANO1 in clinical applications. Our review shows that ANO1 is involved in tumor immunity and microenvironment, and may, therefore, be an effective biomarker and therapeutic drug target.

Introduction

Chloride channels are critical for the regulation of transepithelial transport, membrane excitability, and cell volume (Chou et al. 1995; Hartzell et al. 2005). In epithelial cells, calcium-activated chloride channels (CaCCs) play an important role in epithelial secretion (Hartzell et al. 2005), responding to hypotonic stress and leading to regulatory volume decrease (RVD) (Furst et al. 2002; Jentsch 2016). These channels participate in the modulation of cellular volume following osmotic perturbations. In 2008, three studies used different methods to demonstrate that anoctamin 1 (ANO1), also known as anoctamin (ANO), is a calcium-activated chloride channel (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008) in the ANO family, which has 10 members, ANO1–ANO10, in mammals (Brunner et al. 2016). ANO1 is also known as transmembrane protein 16A (TMEM16A), discovered on GIST 1 (DOG1), oral cancer overexpressed (ORAOV2), and tumor amplified and overexpressed sequence (TAOS2) (West et al. 2004; Huang et al. 2002).

Oncogenes, which directly or indirectly promote cancer proliferation and progression, are drivers of tumorigenesis (Crottes and Jan 2019; Wang et al. 2017). Multiple studies have described a correlation between the expression of ion channels and tumorigenesis (Bose et al. 2015; Becchetti et al. 2013; Schwab et al. 2012; Cuddapah and Sontheimer 2011; Davies et al. 2004). Recent studies have shown that ANO1 is upregulated in numerous diseases and tumor types, and affects the process of carcinogenesis (Ji et al. 2019). However, ANO1 plays different roles in different, and even in same, tumor types, although the mechanisms by which ANO1 affects tumors are unclear (Qu et al. 2014). This review summarizes the current knowledge on ANO1 in the context of different types of cancer, and provides an outlook on the future of ANO1 as a potential biomarker and therapeutic target for the diagnosis and treatment of patients with cancer.

Structure and function of ANO1

The ANO1 gene contains 26 exons and is located on the CCND1-EMS1 segment of the human chromosome 11q13 (Huang et al. 2002, 2006). The ANO1 membrane protein encoded by ANO1 has a molecular weight of 114 kDa and contains 10 transmembrane structures, 986 amino acids, and an intracellular amino and carboxy terminus (Brunner et al. 2016).

ANO1 exists as a dimer with each subunit composed of 10 transmembrane (TM) segments. Unlike other transmembrane proteins, the helical structure of ANO1 dimers is not symmetrical. This asymmetry may be caused by the high hydrophilicity of the transmembrane subunits. In the membrane, dimer interfaces are relatively small, and the spiral arrangement forms a broad cavity (called the dimer-chamber) between the two subunits; this dimer-chamber is thought to be filled with lipids (Brunner et al. 2016).

ANO1 has four subtypes: a, b, c, and d; among these, b, c, and d are produced by selective exons. ANO1 may function as a homodimer, but whether the dual subunit structure constitutes a single-channel or a two-channel structure requires further study. Expression of the ANO1 subtypes in tissues has tissue-determining properties. For example, ANO1 with a shorter amino terminus is expressed in the stomach tissues of patients with gastroparesis caused by diabetes; the amino-acid terminus is related to the calcium-ion binding site and dimer formation. Changes in the topology of ANO1 reduce its sensitivity to calcium ions, and alter the structure of phosphorylation- and calmodulin-binding sites (Kane Dickson et al. 2014; Pang et al. 2014). These changes increase the complexity of CaCCs, resulting in different biological properties and regulatory mechanisms. This may also partly explain why ANO1 plays different roles in the development of different types of cancer.

ANO1 is upregulated in various tissues of the human body, including bronchial epithelium (Veit et al. 2012), proximal renal tubular epithelium (Iqbal et al. 2012; Svenningsen et al. 2014; Lian et al. 2017), retina (Caputo et al. 2015), dorsal root nerve section (Takayama et al. 2015), submandibular gland (Jung et al. 2013), genital smooth muscle (Harper et al. 2003), fallopian tube, and epididymis tube (Colon-Otero et al. 2017). The main effects of ANO1 include the regulation of: (1) epithelial secretion in the respiratory and intestinal epithelium, biliary epithelial cells, and proximal tubular epithelial cells, (2) secretory glands, including exocrine secretion of the liver, pancreas, salivary, sweat, insulin, and lacrimal glands, (3) smooth muscle contraction (first identified in the smooth muscle cells of the mouse airway, oviduct, and ductus epididymis in 2009, while more recent studies have focused on the role of ANO1 in the pulmonary and cerebral arteries, portal vein, and the thoracic aorta), (4) nociception—ANO1 is mainly expressed in small-diameter dorsal root ganglion neurons, which play important roles in sensory transduction of pain-related pathways, and (5) regulation of cell volume, which is essential for the maintenance of most cellular processes. The cell swells when extracellular osmolality decreases. Cell volume is then restored using a known mechanism of regulatory volume decrease (RVD). ANO1 has been shown to participate in mediating volume-regulated Cl⁻ current and cell volume in certain tissues, and (6) other functions. Recent studies have shown that ANO1 is overexpressed in radial glial cells, which are crucial for cortex development (Hong et al. 2019). Recently, Pabl et al. found that KCNE1 regulates ANO1-mediated activity pertaining to calcium-dependent and voltage-dependent ion channels, and that ANO1-KCNE1 current may be involved in inherited pathologies (Avalos Prado et al. 2021).

Role of ANO1 in tumors

ANO1 has been recently shown to be highly expressed in tumors originating from the epithelium such as gastrointestinal stromal tumor (GIST) (Simon et al. 2013; Berglund et al. 2014), prostate cancer, colorectal cancer (CRC), oral squamous cell carcinoma (Li et al. 2014; Chenevert et al. 2012), head and neck squamous cell carcinoma (SCCHN), lung cancer, stomach cancer (Zeng et al. 2019), breast cancer, hepatocellular carcinoma (HCC), anaplastic thyroid carcinoma (Kim et al. 2019), esophageal squamous cell carcinoma (ESCC) (Shi et al. 2013; Yu et al. 2020), pancreatic ductal carcinoma (Mazzone et al. 2012; Crottes et al. 2019), and ovarian cancer. Table 1 provides a summary of the characteristics of ANO1, including the roles of ANO1 in clinical prognosis and ANO1-mediated mechanisms in cancer.

Table 1.

Regulatory mechanisms and clinical phenotype of ANO1 in tumors

Cancer type	Method of inhibition of ANO1		Role of ANO1 in cancer cells				Overexpression of ANO1			R
	Gene silencing	Inhibitor	Cell lines	P or V	M	Mechanisms	Xenograft	Human tissue/blood	Clinical prognosis and ANO1 overexpression
Breast cancer	shRNA	CaCCinh-A01	ZR75-1, HCC1954, MDA-MB-415	+	NR	11q13 amplification, antiapoptotic function, Chloride Channel Activity, EGFR, CAMKII, Akt	Inhibition of ANO1 can reduced tumor growth	High expression in tissue	Poor prognosis	Britschgi et al. (2013)
	NR	NR	HEK293	No effect	No effect	NR	NR	NR	ANO1 is not necessary for cancer-cell viability	Ubby et al. (2013)
	NR	5f	MCF-7	+	NR	NR	NR	NR	NR	Seo et al. (2018)
	siRNA	T16Ainh-A01 (data not shown)	YMB-1 MCF-7	+	NR	Suppressive effect of HDAC	NR	NR	NR	Sayo (2014)
	NR	NR	NR	NR	NR	NR	NR	High expression in tissue	Combined gene expression of FADD, PPFIA1, and ANO1 was associated with disease-free survival	Eun (2014)
	siRNA	Niflumic acid T16Ainh-A01	YMB-1, MDA-MB-453, MCF-7, Hs578T-Luc, and BT-549	YMB-1( +)	NR	ANO1 may function as a transcriptional regulator of HER2	NR	NR	ANO1 inhibitors have potential in the treatment of BCA patients with resistance to HER2-targeted therapy	Fujimoto (2017)
	NR	NR	MECs	NR	NR	NR	NR	High expression in tissue	ANO1 provides more sophisticated information when diagnosing uncertain cases in the breast	Cheng et al. (2016)
	NR	NR	NR	NR	NR	NR	NR	High expression in tissue	Associated with good prognosis in patients with the PR-positive or HER2-negative following tamoxifen treatment	Wu et al. (2015)
	NR	NR	MCF7, MDA-MB-435S	MCF7( +), MDA-MB-435S(-)	NR	NR	NR	High expression in tissue	Good prognosis in PR + or HER2- breast cancer patients the low expression of Ki67	Wu et al. (2017)
	siRNA	T16Ainh-A01 CaCCinh-A01	SKBR3	+	NR	EGFR/HER2 signaling	NR	NR	ANO1 improves response to biological therapies targeting EGFR/HER family members	Kulkarni et al. (2017)
	siRNA	T16Ainh-A01	YMB‐1, MDA‐MB‐453	NR	NR	Repressed HER2 transcription, PI3K/AKT/mTOR and/or STAT3 signaling	NR	NR	ANO1 is useful for patients with resistance to anti-HER2 therapies	Fujimoto et al. (2018)
	siRNA	T16Ainh-A01	MCF7, MDAMB-231	+	+	Cell cycle, E-cadherin	NR	High expression in tissue	Indicator of poor prognosis and potential therapeutic target	Jun (2018)
	shRNA	T16Ainh-A01	MCF-7, T47D	+	NR	EGFR/STAT3 signaling	Inhibition of ANO1 can reduced tumor growth	High expression in tissue	ANO1 associated with shorter overall survival in ER-positive breast cancer patients without tamoxifen treatment	Wang et al. (2019)
	shRNA	NR	MCF-7, T47D	NR	+	EGFR/STAT3/ROCK1 signaling	NR	High expression in tissue	ANO1 associated with greater lymph-node metastasis and poor survival	Shuya (2021)
HNSCC	shRNA	T16Ainh-A01	UM-SCC1 T24	+	NR	11q13 amplification, Ras-Raf-Mek-ERK1/2	Inhibition of ANO1 can reduced tumor growth	High expression in tissue	Poor prognosis and potential therapeutic target	Duvvuri et al. (2012)
	siRNA	NR	BHY	No effect	+	11q13 amplification, Regulatory Volume Decrease	NR	High expression in tissue	Poor survival	Ruiz et al. (2012)
	siRNA	NA, DCPIB, DIDS, Fluoxetine, CFTRinh172	HEp-2, SCC-25	No effect	+	NR	NR	High expression	Distal metastasis	Ayoub et al. (2010)
	shRNA	NR	UM-SCC1	NR	–	Promoter hypermethylation	Reduction in ANO1 led to the formation of smaller tumors in xenograft models, while increasing metastatic development	High expression in primary tumor and low expression in metastatic tumor	ANO1 promotes primary tumor growth and decreases motility	Shiwarski et al. (2014)
	NR	NR	NR	NR	NR	NR	NR	High expression	No correlation with clinical parameter; affects patient’s survival depending on tumor’s site	Rodrigo et al. (2015)
	shRNA	CaCCinh-A01	93-VU-147 T SCC90 FaDu PE/CA-PJ34	+	NR	Promoter hypermethylation	NR	High expression in HPV-negative HNSCC	Decreased survival in HPV-negative HNSCC	Dixit et al. (2015)
	shRNA	CaCCinh-A01	Te11	+	NR	11q13 amplification EGFR,		NR	Enhanced sensitivity to Geftinib	Bill et al. (2015)
	shRNA	CaCCinh-A01	Tell	+	NR	11q13 amplification, antiapoptotic function, Chloride Channel Activity EGFR, CAMKII, Akt	Inhibition of ANO1 can reduced tumor growth	High expression in tissue	Poor Prognosis	Britschgi et al. (2013)
	siRNA	T16Ainh-A01 CaCCinh-A01	Cal-33, OSC19 UM-SCC-1 FaDu	OSC19( +)	NR	EGFR/HER2 signaling	ANO1-overexpressing tumors were ~ 2–threefold higher than control tumors	NR	Increase the response to antibody-mediated EGFR/HER family-targeted biologic therapies in HNSCC	Kulkarni et al. (2017)
	shRNA	NFA, T16Ainh-A01	SW620, HCT116 LS174T	+	+	MAPK signaling cell cycle	NR	NR	NR	Sui et al. (2014)
	shRNA	Oxaliplatin 5-FU	HCT116 HT29	NR	NR	Apoptosis, EGFR signaling	NR	High expression in tissue	F. nucleatum involved in the development of colon cancer chemoresistance via ANO1 pathway	Pei (2019)
ESCC	siRNA	NR	KYSE30 KYSE510	+	NR	NR	NR	High expression in tissue	Correlated with lymph-node metastasis and advance clinical stage	Shi et al. (2013)
	NR	NR	NR	NR	NR	NR	NR	High expression in tissue	A valuable prognostic IHC panel for ESCC	Yu et al. (2020)
Prostate cancer	siRNA	T16Ainh-A01, CaCCinh-A01	PC3 LnCap	+	NR	NR	NR	High expression in tissue	NR	Cha et al. (2015)
	siRNA	Niflumic acid	PC-3 LNCaP	+	NR	Epigenetic regulation	NR	NR	NR	Matsuba et al. (2014)
	NR	5f	PC3	+	NR	NR	NR	NR	NR	Seo et al. (2018)
	shRNA	DIDS	LNCaP PC-3	+	+	NR	Inhibition of ANO1 can reduced tumor growth	High expression in tissue	Inhibiting ANO1 may be useful in pharmacological intervention in prostate cancer	Liu et al. (2012)
	NR	Idebenone,	PC3, CFPAC-1	NR	NR	NR	NR	NR	Idebenone, a novel ANO1 inhibitor, has potential for use in cancer therapy	Seo et al. (2015)
	shRNA siRNA	CaCCinh-A01, T16Ainh-A01, Ani9	PC-3	+	_	TNF-α signaling	Silencing of ANO1 inhibits xenograft tumor growth	NR	NR	Song et al. (2018)
Gastric cancer	shRNA	NR	AGS BGC823	No effect	+	TGF-β signaling	NR	High expression in tissue	An independent prognostic factor	Liu et al. (2015)
	siRNA	NR	AGS BGC823	NR	+	MiR-381 regulate TGF-β signaling by targeting ANO1	High expression due to low expression of miR-381	High expression due to low expression of miR-381	pPoor prognosis and EMT	Cao et al. (2017)
	siRNA	NR	AGS, SGC7901	NR	+	SP1 increased ANO1 transcription, recruited MLL1 to the ANO1 promoter region, facilitated H3K4 trimethylation	Loss of ANO1 resulted in inhibition of tumor metastasis	High expression in tissue	Tumor-node-metastasis stage	Zeng et al. (2019)
Hepatocellular carcinoma	siRNA	NR	SMMC-7721	+	+	MAPK signaling	Inhibition of ANO1suppress tumorigenicity in vivo	High expression in tissue	NR	Deng et al. (2016)
Glioma	siRNA	NR	U87MG U251 SHG44 U118	+	+	Nuclear factor-κB signaling	NR	High expression in tissue	NR	Liu et al. (2014)
Lung cancer	shRNA	NR	GLC82 NCI-H520	+	+	NR	Inhibition of ANO1 can reduced tumor growth	High expression in tissue	NR	Jia et al. (2015)
Pancreatic adenocarcinoma	NR	T16Ainh-A01	CFPAC-1	+	NR	NR	NR	NR	NR	Mazzone et al. (2012)
	siRNA	T16Ainh-A01, CaCCinh-A01, NS3728	BxPC-3, AsPC-1, Capan-1	No effect	+	Ion channel for volume changes	NR	NR	NR	Sauter et al. (2015)
	shRNA	NR	AsPC-1	NR	+	Ligand-dependent EGFR signaling, EGF-induced store-operated calcium entry and phosphoproteome remodeling	NR	High expression in tissue	Biomarkers that contribute to the pathological typing of pancreatic cancer	David (2019)
Gastrointestinal stromal tumor	shRNA	T16Ainh-A01	GIST-T1 GIST-882	No effect	NR	NR	ANO1 knockdown inhibits growth of GIST xenografts	NR	NR	Simon et al. (2013)
	NR	T16Ainh-A01	GIST48	Small effects	NR	Inhibition of ANO1 has pro-apoptotic effects	NR	NR	NR	Berglund et al. (2014)
	NR	NR	NR	NR	NR	NR	NR	High expression in PBMCs	Monitoring recurrence and evaluating therapeutic efficacy of imatinib for GISTs	Li et al. (2016)
	NR	NR	NR	NR	NR	NR	NR	High expression in tissue and PBMCs	Combined analysis with ANO1 mRNA and conventional tumor markers can increase the diagnostic ability for GISTs	Li et al. (2019)
	NR	T16inh-A01 CaCCinh-A01	GIST-T1 GIST882	+	NR	Cell cycle	NR	NR	NR	Robin (2019)
Oral squamous cell carcinoma	shRNA	DIDS	scc-25	+	NR	NR	NR	High expression in tissue	ANO1 expression was higher in metastatic tumors	Li et al. (2014)
Salivary gland carcinoma	NR	NR	NR	NR	NR	NR	NR	High expression in tissue	NR	Jacinthe (2012)
Ovarian cancer	siRNA	NR	SKOV3 ES-2 Caov-3	SKOV3( +)	SKOV3( +)	PI3K-Akt signaling	Inhibition of ANO1 can reduced tumor growth	High expression in tissue and PBMCs	ANO1 upregulation is correlated with pathologic stage and differentiation	Liu et al. (2019)
Anaplastic thyroid carcinoma	siRNA	T16Ainh-A01	SNU-80 KTC	NR	+	NR	NR	High expression in tissue	NR	Kim et al. (2019)
Colorectal cancer	shRNA	NR	SW620, HCT116 LS174T	+	+	MAPK signaling, Cell cycle	NR	NR	NR	Sui et al. (2014)
	siRNA	NR	DLD-1 HCT116	NR	NR	Regulated by miR-132	NR	High expression in tissue	NR	Mokutani et al. (2016)
	siRNA	NR	HCT116	+	+	NR	NR	High expression in tissue	Advanced tumor stage and lymph-node metastasis	Park et al. (2019)
	siRNA	NR	SW480	NR	+	EGFR signaling, Regulated by miR-144	NR	High expression in tissue	Poor differentiation and advanced tumor node metastasis stage	Jiang et al. (2019)
	shRNA	NR	HCT116, HT29	NR	NR	NR	NR	NR	F. nucleatum might be involved in the chemoresistance of colon cancer via ANO1 pathway	Pei (2019)
	NR	NR	NR	NR	NR	NR	NR	High expression in tissue	Predictive factor of lymph-node metastasis	Hongxia (2021)

P proliferation, M migration, PBMCs peripheral blood mononuclear cells, GIST gastrointestinal stromal tumor, HDAC histone deacetylase, N not mentioned, +  effect, – no effect, R reference

ANO1 and head and neck squamous cell carcinoma

Squamous cell carcinoma of head and neck (SCCHN) ranks sixth in the incidence of malignant tumors with a 5-year survival rate of < 50% (Siegel et al. 2021). Because SCCHN shows rapid metastasis and poor prognosis, there is an urgent need to develop new drug targets and biomarkers for the treatment and monitoring of patients with SCCHN. Under the combined action of gene expression, transcription, methylation, and signaling pathways, ANO1 has many clinical manifestations in the context of SCCHN (Rodrigo et al. 2015). Ruiz et al. found that ANO1 expression is amplified in SCCHN, and is accompanied by amplified expression of the 11q13 gene, which can promote the proliferation of cancer cells. Ruiz et al. also found that overexpression of ANO1 is correlated with poor clinical outcome in patients with SCCHN. Additionally, Duvvuri et al. found that ANO1 can induce cancer-cell proliferation via activation of extracellular signal-regulated kinase (ERK) 1/2 (Duvvuri et al. 2012), and that ANO1 interacts with epidermal growth factor receptor (EGFR) to form a functional complex that promotes EGFR signaling in SCCHN (Bill et al. 2015). ANO1 also affects tumor proliferation and metastasis via a methylation promoter (Shiwarski et al. 2014; Finegersh et al. 2017). Shiwarski et al. demonstrated that ANO1 can promote cell migration via volume regulation depending on channel characteristics (Ruiz et al. 2012). Consequently, Madalena et al. found that combined inhibition of ANO1 and casein kinase 2 (CK2) expression by niclosamide and silmitasertib synergistically inhibits the growth of cal33 head and neck cancer cells, and that this combined inhibition shows weaker cytotoxicity than inhibition of ANO1 or CK2 expression alone. This result shows that silmitasertib combined with the ANO1 inhibitor niclosamide can be used to inhibit tumor growth in patients with SCCHN (Pinto et al. 2020). Moreover, Artemis et al. found that ANO1 can modulate the expression of MCL1 and p27Kip1, both of which are involved in SCCHN tumorigenesis via regulation of the cell cycle (Filippou et al. 2012). These results indicate that ANO1 plays an important role in SCCHN tumorigenesis and can be used as a drug target or predictive marker to monitor responses to EGFR-targeting agents in anti-SCCHN therapy.

ANO1 and breast cancer

Breast cancer, which accounts for 11.6% of all cancers, is one of the most common malignancies and is the leading cause of cancer-related death in women worldwide (Bray et al. 2018). ANO1 is involved in breast cancer tumorigenesis (Fujimoto et al. 2017, 2018; Cheng et al. 2016; Kulkarni et al. 2017; Luo et al. 2021). Bae et al. found that ANO1 can regulate the expression of MMP9, β-catenin, snail, E-cadherin, and cyclin D1 to promote the proliferation of breast cancer cells (Bae et al. 2018). Bae et al. have also shown that ANO1 can regulate the breast cancer-cell cycle, and secretion of intracellular and extracellular matrix proteins, to promote the growth and metastasis of breast cancer. Additionally, Bristschgi et al. found that amplified expression of the ANO1 gene and ANO1 protein are correlated with poor outcome in patients with breast cancer, and that ANO1 overexpression is associated with poor prognosis in other cancers, possibly via apoptosis and activity of the ANO1 channel (Britschgi et al. 2013).

ANO1 is associated with clinical prognosis in breast cancer patients with human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER) positive status (Wu et al. 2015, 2017). Moreover, our recent meta-analysis found that ANO1 is significantly correlated with HER2 positive, but not with PR- or ER-positive, breast cancer (Zhang et al. 2021). These results indicate that ANO1 is an effective indicator in breast cancer prognosis and represents a drug target in the treatment of patients with breast cancer.

ANO1 and gastrointestinal stromal tumors (GIST)

Gastrointestinal stromal tumors (GIST) often occur in the gastrointestinal tract and abdomen, and are usually KIT-positive on immunohistochemical staining. Most GISTs arise in the stomach, accounting for approximately 60–70% of all GIST cases. GISTs have an insidious onset, and most patients are asymptomatic; as a result, almost 50% of patients are in middle and late stages when they are diagnosed (Xu et al. 2018). ANO1 expression has been used as an immunohistochemical indicator of pathology in the diagnosis of GIST (Lee et al. 2010). However, studies continue to examine the role of ANO1expression in GIST. A previous study has shown that silencing ANO1 expression can increase that of insulin-like growth factor binding protein 5 (IGFBP5), which can promote the progression of GIST (West et al. 2004). Another study demonstrated that inhibition of ANO1 expression exerts a pro-apoptotic effect in early apoptotic imatinib-resistant cells, indicating that ANO1 can decrease cancer-cell apoptosis (Dailey et al. 2015). Recent studies have shown that ANO1 is highly expressed in the peripheral blood mononuclear cells (PBMCs) of patients with GIST, and that expression of ANO1 is reduced following surgery, demonstrating that ANO1 may be a promising in vitro biomarker in cancer diagnosis (Li et al. 2016). In our previous study, we have also shown that the expression of ANO1 in PBMCs is consistent with ANO1 expression in GIST tissues. Furthermore, our results also indicate that compared with that of tumor markers currently in clinical use, ANO1 shows a higher diagnostic efficacy in GIST diagnosis, and, combined with other tumor markers, can improve the sensitivity and specificity of GIST diagnostic tools (Li et al. 2019). In summary, ANO1 is a promising drug target and in vitro diagnostic marker for GIST therapy and diagnosis, respectively.

ANO1 and ovarian cancer

Ovarian cancer ranks third in female reproductive system malignancies, and the mortality rate of ovarian cancer ranks first in gynecological malignancies. Worldwide, the incidence of ovarian cancer is 9.1/100,000 and 5.0/100,000 in the developed and developing countries, respectively. In 2017, approximately 22,440 women in the United States developed ovarian cancer, and 14,080 of these patients died of ovarian cancer, showing a mortality rate of 62% (Siegel et al. 2018). The etiology of epithelial ovarian cancers remains elusive, although ion channels are known to be involved in ovarian cancer tumorigenesis (Frede et al. 2013^). Recently, we have shown for the first time that ANO1 is upregulated in different types of epithelial ovarian cancer cells and tissues. ANO1 overexpression is associated with the International Federation of Gynecology and Obstetrics (FIGO) staging, and with a poor grade in ovarian cancer tissues. Additionally, the amplification of ANO1 in the blood of patients with ovarian cancer has been shown to decrease after surgery, which is consistent with the previous studies on GIST. To the best of our knowledge, we were the first to show that ANO1 contributes to the carcinogenesis of ovarian cancer (Liu et al. 2019). Suppression of ANO1 overexpression can decrease the proliferation of ovarian cancer cells, indicating that ANO1 is a drug target for ovarian cancer therapy. Furthermore, the detection of ANO1 in blood represents a promising method for the surveillance of ovarian cancer (Liu et al. 2019).

ANO1 and colorectal cancer (CRC)

Colorectal cancer (CRC) is one of the most common malignancies, and the third most deadly cancer, worldwide (Siegel et al. 2021). Despite significant advances in the diagnosis and treatment of CRC over the past decade, the survival rate of patients with advanced CRC remains low due to tumor recurrence, distant metastasis, and lack of diagnostic markers. Studies have shown that ANO1 is correlated with poor differentiation, advanced tumor node metastasis stage, and TNM stage in CRC (Lu et al. 2019; Li et al. 2021). Mechanism studies have shown that knockdown of ANO1 is accompanied by a shift in phospho-ERK1/2, cyclin D1, and phospho-MEK expression in CRC (Sui et al. 2014). Recent studies have shown that in colon cancer, ANO1 expression is greatly affected by transcriptional regulation, and that both miR-144 and miR-132 affect cell-signaling pathways by regulating ANO1 expression (Jiang et al. 2019; Mokutani et al. 2016). Moreover, miR-9 can suppress the epithelial–mesenchymal transition (EMT)-mediated metastasis of CRC by regulating ANO1 expression (Park et al. 2019). These studies provide new avenues for therapeutic molecular targeting in CRC. Additionally, a logistic regression model has shown that amplified expression of ANO1 mRNA is an important predictor of metastasis in CRC (Li et al. 2021). This finding indicates that ANO1 mRNA represents a useful biomarker for predicting and diagnosing CRC.

ANO1 and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly aggressive primary liver cancer. Worldwide, 750,000 people die annually of liver cancer, with China accounting for 51% of these cases (Siegel et al. 2017; Chen et al. 2016). Studies have shown that ANO1 is overexpressed in HCC tissues. Suppression of ANO1 expression can inhibit HCC cell growth and progression by inactivating ERK1/2 signaling without affecting apoptosis (Deng et al. 2016). Conversely, Chuantao et al. found that overexpression of ANO1 stimulates HCC cell growth and progression by suppressing apoptosis and expression of the PI3K/AKT-MAKP signaling pathway (Zhang et al. 2020a). These findings indicate that ANO1 is a promising therapeutic target in the treatment of patients with HCC.

Cell-specific role of ANO1 in tumors

A previous study found that upregulation of ANO1 expression is correlated with good clinical outcome after treatment using tamoxifen in patients with PR-positive or HER2-negative breast cancer (Wu et al. 2015). Moreover, Ubby et al. reported that various ANO1 isoforms are expressed in breast cancer, and that overexpression of these ANO1 isoforms in HEK-293 cells does not promote HEK-293 cell proliferation and migration. Whether ANO1 is involved in apoptosis in HCC remains debated (Ruiz et al. 2012; Sui et al. 2014; Deng et al. 2016; Ayoub et al. 2010; Jia et al. 2015; Liu et al. 2014; Lee et al. 2016; Ubby et al. 2013). Although numerous studies have shown that ANO1 is involved in tracheal mucus secretion and pulmonary artery smooth muscle contraction, recent studies have shown that treatment using an ANO1 agonist potentiator ETX001 or inhibitor Ani9 is ineffective against mucus secretion in the trachea or against the contraction of pulmonary artery smooth muscle. These conflicting results indicate that ANO1 expression shows tissue and cell specificity (Danahay et al. 2020). ANO1 can interact with different protein networks in different cells (Wang et al. 2017). Allosteric regulation changes the topology of ANO1 expression, reduces sensitivity to calcium ions, and consequently alters the phosphorylation and calmodulin-binding sites (Kane Dickson et al. 2014; Pang et al. 2014). These changes increase the complexity of CaCCs, resulting in different biological properties and regulatory mechanisms. This may also partly explain why ANO1 plays different roles in tumor progression.

Raquel et al. found that three ANO1 inhibitors—Ani9, benzbromarone, and niclosamide—decrease the expression of ANO1 and increase Ca²⁺ levels in different cell types. This phenomenon may occur, because overexpressed ANO1 is more accessible to intracellular Ca²⁺, which can cause spontaneous activity (Centeio et al. 2020). The different cell-specific roles of ANO1 in proliferation and invasion may explain the different roles of tumor cells expressing ANO1 in proliferation and migration, which ultimately affects clinical outcomes. However, in the process of tumor development, the frequency of gene mutations caused by factors such as heredity, age, or other diseases is increased, and the ability of immune cells to eliminate tumor cells is decreased, which also affects clinical outcomes.

ANO1-mediated mechanisms in tumors

ANO1 is involved in the progression of tumors via multiple mechanisms, which are summarized in Fig. 1.

Fig. 1.

ANO1-mediated mechanism of action in cancer cells. ANO1 promotes cancer-cell growth in a multi-pronged approach. Increases in intracellular calcium-ion concentration activate the activity of the ANO1 channel, which directly stimulates the Akt, CAMKII, and NF-κB signaling pathways. ANO1 can also act directly or indirectly on EGFR to activate the Akt and ERK signaling pathways. These signaling pathways jointly promote the proliferation and invasiveness of cancer cells. However, ANO1 can also promote gene amplification and protein expression in cancer cells by regulating methylation and transcriptional processes. Additionally, the activity of the ANO1 channel not only regulates the balance between water and chloride ions inside and outside the cell, but also interacts with numerous cytoskeletal proteins. Together, these effects regulate changes in cell shape to facilitate cellular deformation and transfer of cells

ANO1 can accompany or directly initiate gene amplification

Cancer proliferation is closely associated with the amplification of the human chromosome 11q13, which expresses cyclinD1, ORAOV1, FGF19, ANO1, and PPFIA1 (Huang et al. 2002; Bautista and Theillet 1998; Choi et al. 2014). The 11q13 gene locus includes ANO1. Numerous studies have shown that ANO 1 amplification is accompanied by that of the 11q13 gene in SCCHN and ESCC, and in breast and lung cancer. R561L/Q/W, R433Q, and R588G/Q are the most frequent mutations in the 165 missense mutations found in ANO1; the role of these mutations in cancer, however, requires further study (Wang et al. 2017). Additionally, ANO1 combines with EGFR to form a polymer, which acts as a trans-acting factor that can directly activate the amplification of 11q13 (Bill et al. 2015).

Although ANO1 gene is amplified in numerous tumor types, while the amplification of ANO1 gene and ANO1 expression in tissues are not always consistent. Overexpression of the ANO1 protein is more common than that of the ANO1 gene in human breast cancer samples (Britschgi et al. 2013; Wu et al. 2015). Similarly, gene amplification changes after metastasis even in the same tumor. For example, the 11q13 gene shows a 10.4–30% amplification rate from recurrent and metastatic SCCHN (Morris et al. 2017) to TCGA in primary SCCHN (Cerami et al. 2012). Interestingly, we recently found that the degree of ANO1 amplification in the PBMCs of patients with GIST is consistent with tissue expression levels. This inconsistency between gene amplification and protein expression may be related to cell-specific roles of ANO1 in the tumors discussed above, indicating that gene amplification is not the only factor leading to tumorigenesis.

ANO1 and transcription regulation

Transcription regulation plays an important role in tumorigenesis. A previous study has shown that expression of the ANO1 can be regulated during transcription by initiator elements (INRs) contained in the ANO1 gene (Mazzone et al. 2015). Cha et al. described that the promoter section of ANO1 contains three presumptive binding regions targeting the androgen response element to initiate transcription in prostate epithelial cells (Liu et al. 2012). This finding suggests that ANO1 is involved in the transcription process of prostate cancer and that ANO1 may be involved in carcinogenesis.

ANO1 and epigenetic regulation

Methylation can inhibit the initiation of gene promoters, which play a critical role in tumorigenesis (Jones et al. 2016). Hypermethylation inactivates gene expression, while hypomethylation stimulates gene transcription. The ANO1 promoter contains CpG islands, which frequently undergo methylation, demonstrating that regulation of ANO1 expression may occur via methylation (Shiwarski et al. 2014; Mazzone et al. 2015). Dixit et al. found that promoter hypomethylation in ANO1 results in increased upregulation of ANO1 expression in HPV-negative SCCHN compared with that in HPV-positive SCCHN (Dixit et al. 2015). Furthermore, Shiwarski et al. found that methylation of the ANO1 promoter can inhibit metastasis in SCCHN (Shiwarski et al. 2014). Epigenetic regulation of gene expression can also be mediated by histone deacetylase (HDAC), which deacetylates lysine residues in histones and is involved in cancer progression (Zhang et al. 2017). Matsuba et al. found that a small molecule inhibitor targeting HDAC not only reduces ANO1 expression, but also decreases cancer-cell viability (Matsuba et al. 2014). Similarly, Wanitchakool et al. found that HDAC inhibition can decrease ANO1 overexpression and suppress cell growth in SCCHN (Wanitchakool et al. 2014). These studies indicate that HDAC inhibition can inhibit cell growth by decreasing the expression levels of ANO1, and thus, pharmacological intervention targeting HDAC is a promising potential approach in anti-tumor therapy. However, the mechanisms driving HDAC-mediated regulation of ANO1 transcription require further study.

ANO1 and microRNAs

MicroRNAs (miRNAs), which are small noncoding RNA molecules, can decrease gene expression by suppressing the 3′ UTR of mRNAs; hence, miRNAs may be involved in the tumorigenesis of human cancers (Rupaimoole and Slack 2017). Mokutani et al. have shown that the 3′ UTR of ANO1 mRNA contains a complementary region for miR-132, and that miR-132 can directly regulate the expression of ANO1 mRNA (Mokutani et al. 2016). Additionally, ANO1 overexpression is accompanied by a decrease in that of miR-132 and is associated with poor prognosis in patients with CRC (Mokutani et al. 2016). Furthermore, a decreased expression of miR-381 can downregulate ANO1 overexpression in gastric cancer (Cao et al. 2017). These results show that decreased miRNA expression can regulate ANO1 overexpression in human cancers, indicating that miRNAs may be a promising biomarker or therapeutic target in the diagnosis, prediction, and treatment of cancer.

ANO1 inhibits tumor-cell apoptosis

The suppression of apoptosis is essential for carcinogenesis (Hanahan and Weinberg 2011; Kunzelmann et al. 2019); however, the mechanisms underlying inhibition of apoptosis in cancer remain largely elusive. Ion channels are involved in tumorigenesis and cell-cycle regulation, particularly in chloride channels (Hartzell et al. 2005; Okada et al. 2004), and suppression of ANO1 expression has been shown to stimulate cancer-cell apoptosis (Lian et al. 2017; Wanitchakool et al. 2014; Godse et al. 2017; Song et al. 2018; Guan et al. 2016). N.R. Godse, et al. found that in HNSCC cells, overexpression of ANO1 is associated with tumor growth, increased Erk1/2 activity, reduced expression of the pro-apoptotic protein Bim, and decreased apoptosis (Godse et al. 2017). A recent study has shown that cell death stimulated by ANO1 knockdown does not cause necrosis, indicating that ANO1 is a key factor in the regulation of cellular apoptosis (Kulkarni et al. 2017). Y. Song et al. also found that in PC-3 prostate cancer cells, suppression of ANO1 inhibits cell viability, leads to upregulated levels of TNF-alpha, and induces apoptosis; however, upregulation of TNF-alpha signaling via inhibition of ANO1 expression results in increased levels of phosphorylated Fas-associated protein (Song et al. 2018). These studies demonstrate that ANO1 is involved in carcinogenesis via inhibition of apoptosis.

Signaling pathways activated by ANO1 in cancer

Signaling pathways are important factors in tumorigenesis and are promising treatment targets in anti-tumor therapy. Table 1 summarizes previous studies that have demonstrated that ANO1 can regulate EGFR, CAMKII, PI3K/Akt, and ERK1/2 signaling in breast cancer, SCCHN, and ESCC. ANO1 expression can also affect gastric and prostate cancer by regulating the TGF-β signaling pathway. Our recent findings show that ANO1 plays an important role in ovarian cancer by regulating PI3K/Akt signaling. These results indicate that ANO1 may directly stimulate the phosphorylation of EGFR, CAMKII, Akt, and TGF-β (Britschgi et al. 2013; Yano et al. 1998; Schmitt et al. 2012; Cipolletta et al. 2010; Jiang et al. 2018), and may activate various signaling pathways via depolarization caused by an imbalance in ion homeostasis (Qu et al. 2014; Habela et al. 2009; Chatterjee et al. 2012; Contreras-Vite et al. 2016). Figure 1 illustrates the possible mechanisms driving ANO1-mediated activity in cancer cells.

ANO1 and EGFR signaling

EGFR-mediated signaling is involved in inhibition of cancer-cell growth and angiogenesis (Steelman et al. 2016). A. Bill et al. found that ANO1 expression can stimulate EGFR phosphorylation in SCCHN (Bill et al. 2015). Furthermore, Britschgi demonstrated that inhibition of ANO1 expression can suppress EGFR phosphorylation in breast cancer and ESCC cells (Britschgi et al. 2013). ANO1 can form a functional complex with EGFR. This complex serves to initiate transcription, thereby regulating cancer proliferation in SCCHN cells (Bill et al. 2015). These findings demonstrate that ANO1 can activate transcriptional expression in cancer cells by enhancing EGFR expression and phosphorylation.

ANO1 and CAMKII signaling

ANO1-mediated activation of CAMKII signaling is involved in tumorigenesis (Britschgi et al. 2013). Recently, Cabrita et al. demonstrated that ANO1 can combine with IP3R to increase ATP-induced release of compartmentalized Ca²⁺ from endoplasmic reticulum storage in HeLa cells (Cabrita et al. 2017). These findings indicate that Ca²⁺ release activates the CAMKII pathway, which promotes the proliferation of cancer cells.

ANO1 and mitogen-activated protein kinase (MAPK) signaling

The physiological roles of the MAPK signaling pathway include promotion of cellular proliferation and involvement in the stress and inflammatory responses, cellular differentiation, functional synchronization, transformation, and apoptosis. MAPK signaling is also involved in tumorigenesis and the development of tumor cells (Dhillon et al. 2007). Recently, Duvvuri et al. demonstrated that upregulation of ANO1 expression can activate the Ras–Raf–MEK–ERK1/2 signaling pathways in the bladder and in SCCHN cells (Siegel et al. 2021). Similarly, suppression of ANO1 expression can hinder the phosphorylation of ERK1/2 and MEK, and decrease their signaling, in colorectal cancer cells (Duvvuri et al. 2012). Additionally, ERK1/2 signaling can be stimulated by upregulation in ANO1 expression via CaMKII- and EGFR-mediated signaling in breast cancer cells (Britschgi et al. 2013). The suppression of ANO1 expression can also inhibit ERK1/2 and p38 phosphorylation in hepatoma cells (Deng et al. 2016). These results show that ANO1 stimulates the MAPK/ERK1/2 signaling pathway in different types of cancer, indicating that ANO1-mediated regulation of MAPK/ERK1/2 signaling is a potentially useful drug target in anti-cancer therapy.

ANO1 and nuclear factor κB (NFκB) signaling

NF-κB, which is expressed in nearly all animal cells, participates in the cellular response to external stimuli such as that induced by cytokines, radiation, heavy metals, and viruses; NF-κB thereby plays key roles in the cellular immune response. Dysregulation of NF-κB signaling can cause autoimmune diseases, chronic inflammation, and numerous types of cancer (Hoesel and Schmid 2013; Mirzaei et al. 2021). NF-κB is involved in the initial response to harmful stimulation. The numerous known activators of the NF-κB pathway include interleukins, adhesion molecules, chemokines, and colony-stimulating factors (Mirzaei et al. 2021; Yi et al. 2021). These factors can also activate the TNF-α signaling pathway to participate in the progression of prostate cancer (Mazzone et al. 2015; Song et al. 2018). Similarly, ANO1 can also be regulated by interleukins, including IL-13 and IL-4, in inflammatory tracheal and esophageal diseases (Qin et al. 2016; Kang et al. 2017; Zhang et al. 2015; Vanoni et al. 2020). A recent study found that ANO1 participates in the infection caused by respiratory syncytial virus, indicating that ANO1 can interact with cytokines in the immune response (Pearson et al. 2021). Liu et al. found that overexpression of ANO1 activates NFκB signaling and contributes to glioma progression in vitro (Liu et al. 2014). These results suggest that ANO1 is directly involved in the stimulation of NF-κB signaling and may contribute to the inflammatory response.

ANO1 and PI3K signaling

PI3K/Akt signaling is a critical therapeutic target in anti-cancer therapy (Bast et al. 2009). Studies have shown that silencing of ANO1 expression significantly suppresses the phosphorylation of CAMKII, EGFR, Akt, and ERK1/2 in breast cancer, HNSCC, and ESCC cells (Britschgi et al. 2013). Recently, we have shown that decreasing upregulated ANO1 expression can inhibit the growth of ovarian cancer cells via PI3K/Akt signaling (Fig. 1). ANO1 upregulation activates the phosphorylation of CAMKII and EGFR, which subsequently stimulates Akt signaling (Yano et al. 1998; Schmitt et al. 2012; Jiang et al. 2018). ANO1 can also directly stimulate the activation of Akt signaling via depolarization caused by an imbalance in ion homeostasis (Habela et al. 2009; Chatterjee et al. 2012; Contreras-Vite et al. 2016). These findings indicate that ANO1 is involved in the progression of carcinogenesis via regulation of the PI3K-Akt signaling pathway.

Effect of ANO1 channel characteristics on tumors

How does ANO1 contribute to the process of cancer-cell migration? Ion channel currents and cell volume regulation can partly explain the relationship between ANO1 and the migration of cancer cells (Schwab et al. 2012; Davies et al. 2004). Previous studies indicate that ANO1 expression can be stimulated by hypotonic cell swelling, which requires an increase in intracellular Ca²⁺ concentration (Almaca et al. 2009). This increase in intracellular Ca²⁺ concentration activates ANO1 channel activity. Driven by the activity of ATP pumps, K⁺ and Cl⁻ flow out of the cell, which reduces intracellular penetrability. Under pressure, intracellular H₂O also flows out of the cell, resulting in a rapid reduction in the volume of the cellular tail. The head of the cell, under the control of ANO1, causes ions to flow inward, causing the cell head to become enlarged due to the inflow of H₂O. This morphological change in cells promotes the migratory and invasive properties of cancer cells (Schroeder et al. 2008; Pang et al. 2014; Cha et al. 2015). These findings indicate that ANO1 can regulate the balance between water and ions in cells, and that cell morphology also changes with alterations in the water and ion levels. These changes in cell morphology can adapt to the migration of cancer cells and in response to epithelial-to-mesenchymal transition (EMT). Additionally, studies have shown that depolarization can activate signaling pathways such as PI3K-Akt. These results indicate that characteristics of the ANO1 channel play a critical role in the growth of tumor cells.

ANO1 and cytoskeletal proteins

Under the influence of the tumor microenvironment, vascular endothelial cells contract when cancer cells migrate, which is conducive to the spread of cancer cells (Allison Stewart et al. 2017). Studies have shown that ANO1 can interact with various cytoskeletal proteins, such as ezrin, radixin, and moesin, via continuous polymerization and depolymerization of ANO1 and cytoskeletal proteins, this activity regulates morphological changes in cancer cells and enhances cancer-cell adhesion, which promotes the movement and migration of cancer cells. The bridging effect between cancer-cell membrane and the cytoskeleton is induced by this protein family, which also promotes the movement and migration of cancer cells (Shiwarski et al. 2014; Wanitchakool et al. 2014). As described above, alterations in the cellular water and ion balance lead to changes in cancer-cell morphology. These findings indicate that ANO1 participates in the morphological changes of cancer cells via interactions between ANO1 and cytoskeletal proteins, which promotes cancer-cell metastasis.

Clinical prognosis and diagnosis using ANO1

Studies have shown that upregulation of ANO1 expression is correlated with poor clinical outcomes, and that ANO1 plays a prognostic role in patients with SCCHN (Shiwarski et al. 2014; Dixit et al. 2015; Reddy et al. 2016). Upregulation of ANO1 expression has also been correlated with poor clinical outcomes in gastric and esophageal cancer (Yu et al. 2020; Liu et al. 2015), and in CRC (Li et al. 2021; Sui et al. 2014). Taken together, these findings indicate that ANO1 plays diagnostic and prognostic roles in the context of cancer.

A recent meta-analysis demonstrated that ANO1 is a prognostic marker in SCCHN (Reddy et al. 2016), while recent studies have shown that ANO1 is upregulated in breast cancer tissues and colorectal cancer (Li et al. 2021; Wang et al. 2019). Additionally, suppression of ANO1 has been shown to activate responses to EGFR/HER2-targeted treatment in SCCHN cells (Cipolletta et al. 2010). Ayoub et al. have shown that ANO1 expression is correlated with metastasis in patients with SCCHN (Duvvuri et al. 2012). Hence, the upregulation of ANO1 expression can be used to monitor treatment response in patients with breast or SCCHN cancer following the administration of EGFR/HER2 inhibitors.

Li et al. demonstrated that ANO1 expression is upregulated in the circulating tumor cells (CTCs) of patients with recurrent GIST. ANO1 overexpression is correlated with poor clinical outcome (Li et al. 2016), while suppression of ANO1 overexpression in patients with GIST promotes favorable responses to imatinib therapy. These findings suggest that ANO1 expression can be used as a marker for monitoring recurrence and effects of imatinib therapy in patients with GIST. In our recent study, we found that ANO1 can be amplified in the PBMCs of patients with ovarian cancer, and that ANO1 expression is reduced following surgery. Another study has shown that expression of ANO1 mRNA is amplified in the PBMCs of patients with GIST, which is consistent with the above-described results obtained in patients with ovarian cancer (Liu et al. 2019). We further explored the diagnostic potential of ANO1 compared with that of CA199, CEA, and CA724, and found that ANO1 combined with other clinical biomarkers shows the best diagnostic efficiency in GIST diagnosis (Li et al. 2019; Liu et al. 2019). Recent studies have used bioinformatics to construct a predictive model composed of four genes, including ANO1, to be used in prognosis and monitoring chemoresistance in pancreatic cancer. This model will aid in designing treatment strategies for patients with pancreatic cancer and in targeted drug development (Lin et al. 2021).

The above findings indicate that ANO1 can be explored as a biomarker for the diagnosis, prediction, and prognosis of various types of cancer. Additionally, the in vitro diagnostic method of detecting ANO1 expression in PBMCs of patients with cancer appears promising, but the amplification mechanism of ANO1 expression requires further study and preclinical validation.

Development and application of ANO1 inhibitors

Inhibitors targeting ANO1 are currently used only in preclinical studies. Among the numerous inhibitors of ANO1 expression, few have shown confirmed efficacy (Liu et al. 2021). In this review, we list some of the ANO1 inhibitors that may warrant further exploration as ANO1-targeting anti-tumor drugs.

CaCCinh-A01, a broad-spectrum blocker inhibiting CaCC expression, was first discovered in human intestinal epithelial HT29 cells (Fuente et al. 2008). CaCCinh-A01 has been frequently used in therapy against numerous diseases including diarrhea, cancer, nociception, hypertension, and renal cysts (Bose et al. 2015; Britschgi et al. 2013; Ko et al. 2014; Kraus et al. 2016). CaCCinh-A01 can suppress cancer-cell growth by decreasing the ubiquitination and driving the proteasomal degradation of ANO1 (Bill et al. 2014; Frobom et al. 2019). These findings indicate that decreasing ANO1 overexpression is a promising strategy for treating diseases such as tumors and cardiovascular pathologies, but the mechanisms, scope, and side effects of this therapy need to be further explored.

T16Ainh-A01, a specific inhibitor of ANO1 expression, was first identified in Fisher rat thyroid cells (Namkung et al. 2011). T16Ainh-A01 is used as a pharmacological tool to explore the effect of ANO1 expression in cancer cells (Sauter et al. 2015; Zhang et al. 2015; Kondo et al. 2017), suggesting that T16Ainh-A01 is a candidate ANO1-targeted drug for anti-cancer therapy.

Ani9, another small molecule inhibitor of ANO1 expression, has also been used to suppress ANO1 overexpression in different diseases. Ani9 is currently used to examine the roles of ANO1 in the physiology and pathology of cancer (Song et al. 2018; Seo et al. 2018).

Plants produce many natural products that inhibit ANO1. Tannic acid from green tea and red wine is one such product. Tannic acid can block the expression of both ANO1 and ANO2, and exerts an inhibitory effect on arterial smooth muscle contraction and intestinal Cl- secretion (Namkung et al. 2010). Idebenone, plumbagin, and matrine are other natural compounds that inhibit ANO1 expression (Seo et al. 2015; Yu et al. 2019; Guo et al. 2019). A recent study has identified a novel ANO1 inhibitor, Ani-D2, which is extracted from Mallotus apelta. Ani-D2 exerts anti-cancer effects by decreasing ANO1 expression in prostate cancer cells and oral squamous cell carcinoma (Seo et al. 2020).

Certain clinical drugs have been identified as ANO1 inhibitors (Hara et al. 2019; Miner et al. 2019; Danielsson et al. 2015; Zhang et al. 2020b). Additionally, a recent study found that Fusobacterium nucleatum contributes to colon cancer resistance by regulating ANO1 expression. These findings suggest that using antibiotics to disrupt ANO1 expression may be an effective anti-tumor intervention (Lu et al. 2019). It is also necessary to develop new modulators targeting ANO1. The cryo-EM structure of ANO1, which shows the biophysical and physiologic functions of ANO1, is helpful in exploring specific ANO1 modulators (Paulino et al. 2017; Dang et al. 2017). Furthermore, a model used to develop a pharmacophore that utilizes a three-dimensional quantitative structure–activity relationship (3D-QSAR) identified two ANO1 inhibitors and may be a promising method for screening for other potential ANO1 inhibitors (Lee and Yi 2018).

ANO1 may regulate the tumor immune response

Calcium-activated chloride channel regulator 1 (CLCA1), a secreted modifier of CaCCs, is involved in the regulation of immune responses. CLCA1 binds to biological regions in inflammatory cells to produce cytokines and chemokines targeting lymphatic endothelial and cancer cells to control cancer-cell differentiation, proliferation, and apoptosis (Liu and Shi 2019). Additionally, the ANO1 promoter location includes a signal transducer and activator of transcription 6 (STAT6)-binding region (Mazzone et al. 2015), which can be activated by IL-4 or IL-13 to regulate ANO1 overexpression (Qin et al. 2016; Salomon et al. 2021); indeed, IL-4 and IL-13 are known to be critical in cancer development (Dmitrieva et al. 2016; Suzuki et al. 2015). Another study has shown that IL-6 and EGF can promote amplification of ANO1 expression by activating transcription 3 (STAT3) and PI3K-Akt signaling (Bai et al. 2021). These results indicate that the ANO1 gene or ANO1 protein can be either amplified or overexpressed in cancer cells under the regulation of cytokines, which may be the molecular mechanism underlying the involvement of ANO1 in inflammation and tumor immunity.

Recently, immune infiltration and ANO1 expression was correlated in GISTs, suggesting that ANO1 activity affects immune regulation (Bose et al. 2015; Gasparotto et al. 2020). In the tumor microenvironment, immune and tumor cells interact via cytokines (Hinshaw and Shevde 2019). Dendritic cells, macrophages, and natural killer cells can kill or engulf cancer cells via mechanisms involved in innate immunity and perform antigen presentation. Antigen-presenting cells then present tumor antigens to CD⁸⁺ killer T cells using MHC-1 molecules, resulting in direct killing of tumor cells. CD⁴⁺ helper T cells activated by MHC-2 molecules can activate B cells to produce antibodies, which, upon binding, kill cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation. Immune cells can also kill tumor cells by inducing the release of cytokines, such as interleukins and TNF-α, to activate the relevant signaling pathways and apoptosis. Tumor cells also release cytokines to resist immune clearance; eventually, the escape of tumor cells and killing of tumor cells by immune cells reaches a balance (Hinshaw and Shevde 2019; Wang et al. 2020; Apavaloaei et al. 2020). During the recognition process, the immune and tumor cells form a Ca²⁺-dependent cytotoxic site to induce granzyme-mediated cell death or that mediated by Fas and Fas ligand (death receptors) (Bose et al. 2015). In Fig. 2, we illustrate the hypothetical mechanism underlying ANO1 participation in the immune regulation of the tumor microenvironment. In the tumor microenvironment, immune cells release various cytokines, including IL-4, IL-13, IL-6, and EGF (Bai et al. 2021), which promote ANO1 expression and activation in cancer cells by activating STATA6, STATA3, PI3K-Akt, and other relevant signaling pathways. ANO1 activation then activates the intracellular signaling pathway (Liu et al. 2019) and regulates the balance between water and chloride ions, resulting in changes in intracellular crystal osmotic pressure. Changes in extracellular ion concentration and osmotic pressure in the tumor microenvironment disturb calcium-ion concentration and distribution in immune cells, thereby promoting the amplification and activation of ANO1 in immune cells. However, the imbalance of ions in immune cells and changes in osmotic pressure reduce the proliferation, recognition, and binding ability of immune cells (Bose et al. 2015). These mechanisms indicate that ANO1 is an important target in tumor therapy.

Fig. 2.

Potential roles of ANO1 in the tumor microenvironment. A In the tumor microenvironment, immune cells secrete various cytokines to kill cancer cells. B Cytokines released by immune cells lead to the amplification and activation of ANO1 in cancer cells. Activation of ANO1 subsequently leads to intracellular and extracellular ion imbalance and changes in osmotic pressure. Calcium ion imbalance and changes in osmotic pressure not only induce the amplification of ANO1 in immune cells, but also impair the proliferation and killing ability of immune cells

Previous studies have shown that the ANO1 gene is amplified in the PBMCs of patients with GIST and ovarian cancer (Xu et al. 2018; Liu et al. 2019). We believe that paracrine interaction between cytokines and ion imbalance leads to amplification of ANO1 expression in PBMCs. We found that the diagnostic efficiency of ANO1 is higher than that of other tumor markers in patients with GISTs. Using ANO1 with other diagnostics can improve its diagnostic efficiency in GIST patients (Li et al. 2019^). The discovery of this novel mechanism may lead to new methods for noninvasive diagnosis in vitro. Such approaches would be particularly useful for GIST, which is currently only diagnosed using gastroscopy combined with ultrasound or pathologic examination. However, because our study was conducted at a single center using a small cohort, our results need to be confirmed in future studies.

Conclusion

Our review illustrates the mechanisms underlying ANO1 involvement in tumorigenesis, and clinical application prospects for ANO1. We have also described the potential roles and mechanisms of ANO1 in the tumor microenvironment, which may drive the amplification of ANO1 in the PBMCs of patients with cancer. This review shows that ANO1 may be involved in the process of tumor immunity, and may represent a promising target for tumor diagnosis and immunotherapy (Li et al. 2019; Liu et al. 2018).

Author contributions

ZL conceived the manuscript. HL, ZY, HW, and XH wrote and revised the main text of the manuscript. NW, XS, and SY constructed the figures and tables. All authors have reviewed and approved the final version of the manuscript.

Funding

This research was funded by the General Project of Shandong Provincial Natural Science Foundation (ZR2020MH058) and Qingdao Excellent Young Medical Talent Training Project.

Data availability statement

All authors have agreed to the publication of this manuscript.

Declarations

Conflict of interests

The authors have no conflict of interest to declare.

Institutional review board statement

Not applicable.

Informed consent statement

Not applicable.