Emerging roles for multifunctional ion channel auxiliary subunits in cancer

Graphical abstract

Highlights

• •Ion channels consist of conducting and non-conducting (auxiliary) subunits.
• •Auxiliary subunits regulate ion conductance and have non-conducting roles.
• •Ion channels control diverse cellular processes and are aberrantly expressed in cancer.
• •Auxiliary subunits play major roles in cancer cells, including regulating adhesion, migration, invasion and gene expression.

Abstract

Several superfamilies of plasma membrane channels which regulate transmembrane ion flux have also been shown to regulate a multitude of cellular processes, including proliferation and migration. Ion channels are typically multimeric complexes consisting of conducting subunits and auxiliary, non-conducting subunits. Auxiliary subunits modulate the function of conducting subunits and have putative non-conducting roles, further expanding the repertoire of cellular processes governed by ion channel complexes to processes such as transcellular adhesion and gene transcription. Given this expansive influence of ion channels on cellular behaviour it is perhaps no surprise that aberrant ion channel expression is a common occurrence in cancer. This review will focus on the conducting and non-conducting roles of the auxiliary subunits of various Ca²⁺, K⁺, Na⁺ and Cl⁻ channels and the burgeoning evidence linking such auxiliary subunits to cancer. Several subunits are upregulated (e.g. Ca_vβ, Ca_vγ) and downregulated (e.g. K_vβ) in cancer, while other subunits have been functionally implicated as oncogenes (e.g. Na_vβ₁, Ca_vα₂δ₁) and tumour suppressor genes (e.g. CLCA2, KCNE2, BKγ₁) based on in vivo studies. The strengthening link between ion channel auxiliary subunits and cancer has exposed these subunits as potential biomarkers and therapeutic targets. However further mechanistic understanding is required into how these subunits contribute to tumour progression before their therapeutic potential can be fully realised.

1. Introduction

Ion channels are heteromeric membrane protein complexes which permit transmembrane ion conduction. Several ion channels, e.g. K⁺ channels and voltage-gated Na⁺ channels (VGSCs), are notable for regulating membrane potential in excitable cells [1], but an expanding repertoire of other cellular processes, such as proliferation, differentiation [2], cell volume control and migration [3,4], are also known to be influenced by ion channels. Owing to their extensive impact on cellular function, it is no surprise that ion channel dysregulation is a common characteristic in cancer [5]. Ion channels are often multimeric, with ion-conducting subunits accompanied by non-conducting auxiliary subunits [6]. Auxiliary subunit-mediated modulation of the conducting subunit is well established but increasing evidence has unveiled a multitude of non-conducting roles for these proteins as well [[7], [8], [9], [10], [11], [12], [13], [14]]. An emerging field has focused on investigating auxiliary subunits in cancer, which, like the conducting subunits, are often aberrantly expressed and could represent novel therapeutic targets. In this review, we dissect the conducting and non-conducting roles of the auxiliary subunits of Ca²⁺, K⁺, Na⁺ and Cl⁻ channels and the growing evidence supporting a link to cancer.

2. Ca²⁺ channels

Ca²⁺ channels regulate a multitude of cellular processes; accordingly, much research has focused on various Ca²⁺ channels in cancer, including voltage-gated Ca²⁺ channels (VGCCs) [15], STIM and Orai [16], and TRP channels [17]. In terms of Ca²⁺ channel auxiliary subunits however, only VGCC auxiliary subunits have received notable attention thus far. VGCCs are transmembrane complexes responsible for the inward Ca²⁺ current seen in excitable cells following depolarisation, however VGCCs are also expressed in other non-excitable cell types, e.g. osteoblasts and osteoclasts [18,19]. VGCCs are composed of a Ca²⁺-conducting α₁ subunit (Ca_v1-3.x) associated with multiple auxiliary subunits (α₂δ_1-4, β_1-4, γ_1-8), with the exception of Ca_v3.x, which can form a T-type Ca²⁺ channel in the absence of an associated auxiliary subunit (Fig. 1) [20]. A Ca_v1/2 subunit is joined at the membrane by an α₂δ-, β-, and potentially a γ-subunit, although γ-subunits are not always precipitated with Ca_vα [21]. Ca_vα₁ subunits have an oncogenic influence in cancer [15]. Research into Ca_v auxiliary subunits in cancer is a growing field, but it appears Ca_v auxiliary subunits have both oncogenic and tumour-suppressive effects.

Fig. 1.

Voltage-gated Ca²⁺ channel auxiliary subunits. Voltage-gated Ca²⁺ channels (VGCCs) are composed of a conducting α1 subunit accompanied and functionally modulated by Ca_vβ, α₂δ and Ca_vγ subunits [20]. α1 consists of four domains (domains I-IV), each consisting of six segments (S1-S6). The voltage-sensing domain is found within S4 of each domain and the pore consists of the P-loop found between S5-6 of each domain. Ca_vβ modulates Ca²⁺ influx via binding the DI-DII linker of α1. Ca_vβs are also involved in regulating gene expression and endocytosis [22,[36], [37], [38],40,44]. α₂δ subunits are extracellular proteins that remain associated to the membrane via a GPI-anchor [54]. α₂δ subunits are involved in synaptogenesis [65]. Ca_vγ subunits are four-pass transmembrane proteins also involved in cervical ganglion neurite outgrowth and synaptogenesis [108,109].

2.1. Ca_Vβ

The VGCC β-subunits are cytoplasmic proteins that interact with the α₁ DI-DII intracellular linker region [[22], [23], [24]]. β-subunit binding enhances membrane expression of α₁ subunits [25,26], however the mechanism by which this occurs has not yet been elucidated. It is thought that β-subunit binding prevents ER retention and the subsequent degradation of Ca_v2.2, resulting in a higher proportion of Ca_v2.2 at the plasma membrane [25,27]. However, membrane targeting of the DI-DII linker of Ca_v2.2 via an inserted palmitoylation motif still results in ER retention and degradation, leading to the hypothesis that Ca_vβ subunits are required for correct folding, and thus membrane insertion, of functional α₁ subunits [28]. The impact on electrophysiological properties of α₁ subunits by Ca_vβs is complex. In general, Ca_vβs increase current density and regulate activation/inactivation kinetics. For instance, disruption of the Ca_vβ₃-Ca_V2.2 interaction by a small molecule inhibitor results in a decrease in current density and a depolarised shift in the voltage threshold of activation and inactivation [29]. In comparison, Ca_vβ₂ enhances the current density more than Ca_vβ₃, potentially through increased membrane expression as Ca_vβ_2a, unlike Ca_vβ₃, contains a palmitoylation site [30]. Additionally, forced membrane localisation of Ca_vβ₃ using the N-terminal Lyn sequence enhanced the current density relative to WT- Ca_vβ₃ [30]. The complexity arises in the differential sensitivity to PIP₂-mediated modulation of different Ca_vβs [30,31], competition for α₁-binding between Ca_vβ subunits [32], the spectrum of functionally-distinct Ca_vβ splice variants [33,34], and the opposing impacts on α₁-function by the different domains within the Ca_vβ protein [35].

Ca_vβs are functional independent of direct α₁ association. All Ca_vβs demonstrate nucleus localisation, Ca_vβ₄ particularly within nucleoli, and gene expression regulation [[36], [37], [38], [39]]. All Ca_vβs also contain a Src homology 3 domain capable of regulating endocytosis via interaction with dynamin and can interact with small GTPases [40,41]. Ca_vβs show subunit-specific function as well, for instance Ca_vβ₁ is expressed in muscle progenitor cells (MPCs) earlier than Ca_v1.1, where it regulates proliferation and directly suppresses myogenin expression. Accordingly, Ca_vβ₁ knockout mice demonstrate impaired muscle development [36,42]. Similarly, Ca_vβ₂ is required for ventricle cell proliferation and heart development in zebrafish, although pharmacological VGCC inhibition caused a similar phenotype, suggesting Ca_vβ₂ may be functioning in an α1-dependent manner [43]. Ca_vβ₂ is also required for depolarisation-induced c-Fos and meCP2 activation, which intriguingly was shown to be independent of Ca²⁺ influx [37]. Ca_vβ₄ regulates cell proliferation in vitro [44], downregulates Wnt signalling via sequestration of the Wnt pathway effector TCF4 [39], and regulates gene expression via various interacting partners [45,46]. Interestingly, the nuclear localisation of Ca_vβ₄ was inhibited when co-expressed with Ca_v1.1 and only upon depolarisation and the presence of extracellular Ca²⁺ did Ca_vβ₄ interact with its nuclear signalling partner, B56δ [45].

Owing to its role in driving cellular functions such as proliferation and migration, it is perhaps no surprise that Ca_Vα₁ expression is increased in various cancers [[47], [48], [49]]. However, much research has also been dedicated to evaluating the involvement of Ca_v auxiliary subunits in cancer. Ca_vβ₁ expression is upregulated in colon cancer [50], Ca_vβ₂ mutations are seen in bladder cancer [51] and increased Ca_vβ₃ expression is observed in patients with recurrent non-small cell lung tumours compared to recurrence-free patients [52]. Furthermore, expression of Ca_vβ₁ and Ca_vβ₃ are included in proposed high-risk gene signatures that correlate with decreased patient survival in colon and recurring non-small cell lung cancer [50,52]. However, the aforementioned studies are largely limited to statistical observations based on tissue sequencing data that identified altered Ca_vβ RNA expression as a high-risk prognostic marker [[50], [51], [52]]. Chen et al. (2016) offered additional pathophysiological justification for increased Ca_vβ₂ expression in cancer, by observing an enrichment in mutations of genes, including CACNB2 which encodes Ca_vβ₂, involved in NCAM-mediated neurite outgrowth [51].

2.2. α₂δ

The Ca_V α₂δ subunit has a unique structure compared to other auxiliary subunits. The translated polypeptide is proteolytically cleaved into two separate proteins, α₂ and δ, which remain coupled by a disulphide bond [53]. The α₂ segment is extracellular while the δ-subunit remains associated with the membrane via a GPI-anchor [54]. α₂δ and Ca_Vβ subunits can both induce surface expression of α₁, but also function synergistically to maximise α₁ surface expression and Ca²⁺ current [26,55,56]. Preventing proteolytic cleavage of the α₂δ₁ proprotein reduces both Ca_v2.2 surface expression and presynaptic Ca²⁺ influx in hippocampal neurons [57] and site-directed mutagenesis of either cysteine residue involved in the disulphide interaction, which results in a dissociation of α₂, reduces the whole-cell Ca²⁺ current [53]. Similarly, digestion of the GPI anchor of α₂δ₃, by prokaryotic phosphatidylinositol-phospholipase C, results in a release of the α₂δ from the membrane and a decreased Ca²⁺ current [54]. Both these results suggest an intact α₂δ subunit is required at the membrane to induce and sustain the α₂δ-mediated regulation of α₁ subunits. In addition to its role in trafficking, α₂δ has been proposed to stabilise α₁ at the membrane by reducing internalisation and in targeting α₁ to detergent-resistant membranes [54,58]. Phenotypes of α₂δ knockout mice have been very informative, both α₂δ₁ and α₂δ₃ have thus been implicated in neuropathic pain, with α₂δ₁-overexpressing mice demonstrating hyperalgesia [59] and α₂δ₃ -knockout mice demonstrating an enhanced insensitivity to pain [60]. Mice deficient in α₂δ₂, the isoform found overwhelmingly in cerebellar Purkinje neurons, present with seizures and ataxia [61]. Gabapentin, used in the treatment of epilepsy and neuropathic pain, preferentially binds to α₂δ_1/2 and lowers α₂δ surface expression, demonstrating that the α₂δ auxiliary subunit is a druggable target [[62], [63], [64]]. All α₂δ subunits are involved in synaptogenesis, but potentially through different mechanisms [65]. α₂δ₁ promotes cortical synaptogenesis, independently of Ca²⁺ influx, through binding to secreted astrocytic thrombospondin in the postsynaptic membrane and promoting actin remodelling via Rac-1 [66], whereas loss of α₂δ₄ causes impaired retinal synaptogenesis, which correlates with a decrease in presynaptic Ca_v1.4 [67,68].

More is known about the involvement of α₂δ subunits in cancer compared to the other Ca_v auxiliary subunits. Increased α₂δ₁ expression occurs in both ovarian and hepatocellular tumour-initiating cells and correlates with decreased overall survival and a shorter progression-free survival in clinical ovarian samples [[69], [70], [71]]. Zhao et al. developed a monoclonal antibody against α₂δ₁, 1B50-1 [71]. Sorting of a 1B50-1-positive subpopulation of Hep-11 cells, a hepatocellular carcinoma (HCC) cell line, resulted in a subset of cells that initiated tumour formation in all implanted mice, whereas the 1B50-1-negative subpopulation failed to form any tumours. Furthermore, 62/86 of HCC samples were 1B50-1-positive compared to 0/6 normal tissue samples. in vivo experimentation demonstrated that administering 1B50-1 reduced tumour volume following implantation of two HCC cell lines and increased survival, especially when co-administered with doxorubicin, compared to doxorubicin or 1B50-1 alone. Lastly, in vitro work in the same study demonstrated α₂δ₁ to be involved in maintaining cell viability and spheroid formation, via increasing Ca²⁺ influx through L-type and N-type Ca²⁺ channels and MAPK signalling [71]. In non-small cell lung cancer cells, α₂δ₁ expression confers radioresistance in vitro, by enhancing the DNA repair response, and chemoresistance in vivo, potentially through MAPK signalling [72,73]. In addition, various miRNAs that are downregulated in cancer target α₂δ₁ expression, including hsa-miR-208a-3p and hsa-miR-1207-5p in medulloblastoma [74], and miR-107 in chronic myeloid leukaemia (CML) [75]. Overexpressing miR-107 promotes differentiation in CML cell lines, which is reversed when expression of α₂δ₁ is restored [75].

The involvement of α₂δ₂ in cancer is complex, as α₂δ₂ can be both oncogenic and tumour suppressive [76,77]. α₂δ₂ was initially identified as a potential tumour suppressor gene as it is encoded by CACNA2D2, which is absent in the 3p21.3 chromosomal deletion commonly observed in lung and breast cancer [78]. Similarly, CACNA2D2 is deleted in cervical carcinoma [79], is commonly methylated in head and neck squamous cell carcinoma [80], is downregulated in lung squamous cell carcinoma via miR-205 [81], and its expression correlates with improved survival in patients with lung adenocarcinoma [82]. Functionally, in vitro experiments using various non-small cell lung cancer cell lines have demonstrated that overexpression of α₂δ₂ induces apoptosis via mitochondrial cytochrome-c release and subsequent caspase activation [77]. In contrast, α₂δ₂ overexpression occurs in prostate tumours [76] and in insulin-secreting pancreatic adenomas, where elevated intracellular Ca²⁺ is known to stimulate β-cell proliferation [83]. Furthermore, α₂δ₂ overexpression in prostate cancer cells induces tumourigenesis and angiogenesis in mice, which is treatable by administering the α₂δ₂ inhibitor, gabapentin [76].

Conversely, α₂δ₃ is considered a tumour suppressor gene, as downregulation or deletion is seen in nasopharyngeal cancer [84], breast cancer [85], oesophageal squamous cell carcinoma [86,87], gastric cancer [88,89], lung cancer [90] and cholangiocarcinoma [91]. Mice implanted with cancer cells overexpressing α₂δ₃ show a decreased tumour volume, compared to implanted control cells, in nasopharyngeal cancer [84], oesophageal cancer [87] and glioma [92] models. The consensus mechanism points towards an inhibition of motility and invasion by α₂δ₃, and induction of apoptosis through an increase in intracellular Ca²⁺, leading to mitochondria-induced apoptosis [84,87,92].

2.3. Ca_Vγ

The interaction between Ca_Vγ-subunits and α₁ subunits is less well understood. Ca_vγ-subunits were originally identified following immunoprecipitation of the skeletal muscle 1,4-dihydropyridine (DHP) receptor (later known as L-type VGCCs), which yielded γ₁ as a binding partner [93,94]. Following the discovery of Ca_Vγ₁, seven more Ca_vγ-subunits were identified by homology studies [[95], [96], [97], [98]]. Ca_vγ₂ and Ca_vγ₃ have been shown to associate with Ca_v2.1 [99], Ca_vγ_2-4 to Ca_v2.2 [99] and Ca_vγ₆ to Ca_v3.1 [100]. Using cryo-electron microscopy, the γ-subunit was predicted to interact with the Ca_v1.1 voltage-sensing domain (S4) of domain IV [24]. However, the α₁-γ coupling remains contentious as more recent efforts failed to precipitate a Ca_vγ-subunit with Ca_v2. Further, Ca_vγ₂ can regulate Ca_v2.2 indirectly, suggesting a direct coupling may not be necessary for Ca_vγ-induced channel modulation [21,101]. Ca_vγ-subunit mRNA is expressed in skeletal muscle (γ_1,6,7) and brain (γ_2-8) as well as other tissues such as kidney, liver, colon, testis and lung [98]. Functionally, Ca_vγ-subunits negatively regulate VGCC-mediated Ca²⁺ influx by decreasing channel expression and current amplitude [102], hyperpolarising the voltage threshold of inactivation, accelerating channel inactivation [103], and increasing the time taken for recovery from inactivation [96]. Ca_vγ-induced regulation of Ca²⁺ influx observed at the cellular level is supported by the Stargazer mouse mutant, which lacks Ca_vγ₂ and presents with ataxia and absence seizures [104]. Interestingly, a subclass of Ca_vγ-subunits, γ_2/3/4/5/8 (known as transmembrane AMPA receptor regulatory proteins [TARPs]), which localise to the brain [105], interact with ionotropic AMPA receptors and induce membrane localisation [106,107]. Other functions of γ-subunits include Ca_vγ₇-induced neurite outgrowth in superior cervical ganglion neurons [108] and Ca_vγ₂-induced synaptogenesis [109].

Aberrant Ca_vγ expression is seen in various cancers, including increased Ca_vγ₁ in early progressing human epidermal growth factor-positive (HER2+) metastatic breast cancer [110], increased Ca_vγ₄ in bladder squamous cell carcinoma [111] and increased Ca_vγ₇ in leiomyoma via downregulation of miR-197 [112]. Furthermore, a prediction algorithm using a dataset of 1.7 million cancer mutations identified Ca_vγ₃ as a putative oncogene [113]. Similar to Ca_vβ, the functional role of Ca_vγ in cancer is not yet clear. However, a Ca_vγ₄ mutation appears in a cluster of mutations involved in MAPK signalling [111], suggesting a possible role in regulation of mitogenesis.

In summary, although Ca_vα₁ subunits have an oncogenic role [15], it is not yet clear whether Ca_v auxiliary subunits function through Ca_vα₁ or have secondary functions in cancer, or both. Given that Ca_vβ and Ca_vγ are both oncogenic but have antagonistic effects on α₁ function, and Ca_vα₂δ can be oncogenic or tumour suppressive, it would seem that the involvement of auxiliary subunit-mediated Ca²⁺ influx in cancer is tumour type/stage-specific, dependent on the expression profile of other subunits, or subordinate to a secondary function of the auxiliary subunit. Ca_v auxiliary subunits have functions, potentially α₁-independent, that could contribute to oncogenesis and tumour progression. All Ca_vβs regulate gene expression and interact with small GTPases [[36], [37], [38],40,41,44]. Ca_vβ₁ and Ca_vβ₂ are also essential for maintaining proliferation and cellular plasticity during development [36,43]. The TARP family of Ca_vγs induce AMPA receptor membrane trafficking [107], a receptor with an emerging involvement in cancer [114,115], and Ca_vγ₄ and Ca_vγ₇ induce transcellular adhesion and neurite outgrowth respectively [108,109]. α₂δ₁ is also involved in transcellular adhesion [66]. Furthermore, increased Ca²⁺ conductance potentially underpins both the oncogenic function of α₂δ₁ and α₂δ₂ [71,83] and the tumour suppressive function of α₂δ₂ and α₂δ₃ [77,92].

3. K⁺ channels

K⁺ channels represent an extensive superfamily of channels, many of which have been implicated in regulating key elements of tumour progression [[116], [117], [118]]. Here, we focus on the function and involvement in cancer of the auxiliary subunits of the voltage-gated K⁺ channel (VGKC), BK channel and K_ir channel complexes (Fig. 2A-C). VGKC α-subunits represent a diverse family of forty K⁺-conducting proteins, K_v1-12.x, which conduct an outward K⁺ current in response to depolarisation of the membrane potential. Three classes of VGKC auxiliary subunits have been identified: K_vβ_1-3, KChIP1-4, and KCNE1-5 which canonically interact with K_v1, K_V4, and K_v7.1 respectively [[119], [120], [121], [122]], although K_vβs and KCNEs interact with other VGKC α-subunits and K_Vβs also interact with TRPV1 and K₂P2.1 [[123], [124], [125], [126]]. The activity of K_v1 [116,127], K_v4 [128], and K_v7.1 [129] is upregulated in various cancers. However, the expression pattern of VGKC auxiliary subunits in cancer is more complex.

Fig. 2.

K⁺ channel auxiliary subunits. (A) Voltage-gated K⁺ channels (VGKCs). The conducting subunit, K_vα forms tetramers within the membrane that are accompanied and functionally modulated by four K_vβs (for K_v1), four KChIPs (for K_v4) or two KCNEs (K_v7.1) [[119], [120], [121], [122]]. The function of K_vβ is modulated by pyridine nucleotides [143]. KChIPs are involved in regulating gene expression [173]. (B) Large conductance Ca²⁺-activated K⁺ (BK) channels. BK channels consist of a K⁺-conducting, seven-pass (S0-S6) membrane protein subunit (BKα/Slo) accompanied and modulated by dual-pass BKβ and BKγ [182,187]. S0 of BKα is required for interaction with BKβ, S4 is involved in voltage-sensing, the pore region is formed by the linker of S5-6 and an enlarged C-terminus containing two RCK (regulator of conductance of K⁺) domains sense intracellular Ca²⁺ [330]. (C) Inwardly rectifying K⁺ (K_ir) channels. Tetrameric K_ir6 subunits, containing the K⁺-conducting pore, are functionally regulated at the membrane by 17-pass SUR subunits (1:1 stoichiometry), which confer ATP-sensitivity onto K_ir6 via NBDs (nucleotide binding domains) [196]. K_ir1-4 can be bound and modulated by various C-terminal binding proteins [331].

3.1. K_vβ

K_vβ subunits are cytoplasmic proteins, which form homo- or heterotetramers [130] that are involved in trafficking of K_v1 and K_v4.3 to the cell surface [[131], [132], [133]]. Additionally, K_vβ₂ is involved in targeted axonal trafficking of K_v1.2 and K_vβ₁ differentially regulates the K_v composition in ventricular myocytes [134,135]. K_vβ₁ and K_vβ₃ modulate VGKC α-subunits via an N-terminal ball domain, which permits rapid inactivation of delayed-rectifying K_v1 α-subunits [136,137]. K_vβ₁ also slows deactivation, accelerates slow inactivation and hyperpolarises activation of K_v1.2 [138]. K_vβ₂ lacks the ability to inactivate delayed-rectifying K_v1 channels, but does hyperpolarise channel activation [139]. K_vβ₁ and K_vβ₂ are both expressed in developing rat heart and skeletal muscle and during induced myogenesis of L6E9 cells [140]. Furthermore, deletion of K_vβ₁ results in aberrant cardiac electrical activity and cardiac hypertrophy in female mice [141]. K_vβ₂ deletion leads to reduced K_v1.5 surface expression in coronary arterial myocytes and a reduction in total skeletal muscle volume, potentially mediated through downregulation of Pax7 and upregulation of NEDD4 [133,142]. Interestingly, K_vβs are part of the aldo-keto reductase (AKR) superfamily owing to their C-terminal AKR domain. The AKR domain allows for binding and functional modulation by pyridine nucleotides (NAD and NADP). NADP⁺ inhibits K_Vβ₁- and K_Vβ₃-mediated inactivation of K_v1.5 as well as inhibiting K_vβ₂-mediated hyperpolarisation of K_v1.5 activation [143,144].

Evidence suggests that K_vβs are downregulated in cancer. K_vβ₁ is downregulated in malignant thyroid carcinomas relative to benign thyroid adenomas [145,146]. The gene encoding K_vβ₂ is the most significant site of methylation in non-functional (non-hormone secreting) pituitary adenoma compared to functional (hormone-secreting) adenomas and is one of the genes ablated in the common 1p36.3 chromosome deletion seen in neuroblastoma [147,148]. Methylation of the promoter of the gene encoding K_vβ₃ is seen in oral squamous cell cancers relative to adjacent normal tissue [149]. Together, these data suggest K_vβs are tumour suppressor genes, but in depth in vitro and in vivo characterisation of K_vβ in cancer is still currently lacking.

3.2. KCNE

KCNEs are single-pass transmembrane proteins that interact primarily with K_v7; two KCNEs interact with tetrameric K_v7 [150]. In vitro studies document a range of effects of KCNEs on K_v7.1. For example, KCNE1 and KCNE3 both increase surface expression and current density, while KCNE4 and KCNE5 have no effect on current density [151]. KCNE2 and KCNE3 interaction with K_v7.1 produces voltage-insensitive channels and all KCNEs depolarise the activation voltage of K_v7, with KCNE4 and KCNE5 depolarising activation to a non-physiological membrane potential [151]. K_V7.1 has a well-established role in cardiac rhythm and in regulating osmotic and salt transport across gastrointestinal, cochlear and renal epithelia; this is reflected in Kcne1 knockout mice demonstrating atypical QT intervals, hair cell degeneration, impaired renal fluid, glucose and electrolyte uptake, and faecal Na⁺ and K⁺ wasting [[152], [153], [154], [155]]. Furthermore, mutations in KCNE1 underlie Long QT Syndrome 5 and Jervis and Lange-Nielsen syndrome, a disorder characterised by deafness and cardiac arrhythmia [156,157].

With regard to cancer, KCNE1-3 are expressed in uterine cancer cell lines, in which they influence proliferation [158] and a 5-fold and 3-fold upregulation of KCNE3 and KCNE4 respectively has been reported in gliobastoma datasets [159]. Paradoxical to the upregulation of KCNE1 in uterine cancer cell lines, KCNE1 overexpression in an astroglioma cell line (U87-MG) induces apoptosis and KCNE1 is one of the four genes deleted in the 21q22.12 microdeletion which causes a predisposition to acute myelogenous leukaemia [160,161]. The apoptotic influence of KCNE1 in U87-MG cells is proposed to occur through canonical K⁺ efflux through K_v7.1, inducing decreased cytoplasmic K⁺, a known apoptotic trigger [160,162], whereas KCNE1 induces uterine cancer cell proliferation via modulation of HERG channels [158,163]. HERG channels induce proliferation in a range of cell lines and HERG channel inhibition decreases MAPK phosphorylation and c-fos expression in MDA-MB-435S cells [164]. Out of all the K_v auxiliary subunits however, KCNE2 has the most established link to cancer. KCNE2 downregulation is observed in gastric cancer tissue and gastric cancer cell lines, correlates with gastritis cystica profunda development (preneoplastic condition characterised by large gastric cysts) and is a risk factor in gastric cancer stratification [[165], [166], [167]]. Furthermore, Kcne2 knockout mice display a 6-fold increase in stomach size, an upregulation of Ki67 and Cyclin D1 in gastric mucosa, an increase in the metaplastic marker TFF2, pyloric adenomas and neoplastic invasion compared to wild-type mice [168]. Overexpression of KCNE2 in the SGC7901 gastric cancer cell line reduces proliferation and significantly reduces xenograft tumour volume compared to parental SGC7901 cells [167].

KCNE2-K_v7.1 complexes, in the apical membrane of non-excitable gastric parietal cells, are essential for maintaining acidification of the stomach, as KCNE2 transforms K_v7.1 to a constitutively open channel that is potentiated by extracellular H⁺ [169]. Luminal K⁺ released by KCNE2-K_v7.1 is then recycled back into the parietal cell, in exchange for H⁺, via the H⁺/K⁺ ATPase, resulting in gastric acidification [169,170]. Kcne1 knockout mice demonstrate reduced H⁺ secretion, reduced gastric acidification, gastric hyperplasia and atypical K_v7.1 localisation [170]. However, it is not yet known whether KCNE2 downregulation contributes to gastric cancer progression through a failure to acidify the lumen of the stomach or via its role in regulating tumour cell proliferation.

3.3. KChIP

Ca²⁺-sensing K_v channel interacting proteins (KChIPs) are involved in K_V4 channel modulation. KChIPs increase surface channel density, hyperpolarise the voltage of activation, slow inactivation and accelerate the recovery from inactivation [119,171]. KChIPs were identified by a yeast 2-hybrid screen searching for interaction partners with K_v4.2/3 N-termini [119]. Interestingly, KChIP3 was already known as calsenilin/downstream regulatory element antagonistic modulator (DREAM). KChIP3/DREAM plays a key role in differentiation and apoptosis independently of K⁺ channels [172]. DREAM binds upstream genetic elements (DRE sites) as a tetramer and represses transcription of the downstream gene until upon Ca²⁺ stimulation, DREAM tetramers dissociate from DNA allowing gene transcription [173]. Despite KChIP3 being the first Ca²⁺-sensing transcriptional repressor identified, the other KChIPs are also capable of DRE-site binding [174]. DREAM expression is required for maintenance of human embryonic stem cell pluripotency; DREAM knockdown by siRNA results in an increase in apoptosis and spontaneous differentiation [172]. Potentially independent of its nuclear role, DREAM expression induces Ca²⁺-mediated apoptosis possibly through sequestration of hexokinase I from mitochondria [175,176]. Additionally, DREAM expression induces process outgrowth in pheochromocytoma PC12 cells by RhoA inactivation and induces thrombus formation in anucleate platelets via PI3K stimulation [177,178]. There is currently limited evidence of a role for KChIPs in cancer. However, one study identified KChIP4 gene disruption in a renal cancer cell chromosomal break [179]. In addition, KChIP1 upregulation and KChIP3 downregulation have been shown in glioblastoma multiforme, with KChIP2 upregulation correlating with decreased survival for glioblastoma patients [180]. The involvement of KChIP3/DREAM in regulating differentiation, apoptosis, transcellular adhesion and process outgrowth suggests cancer-expressed or downregulated KChIPs could be a worthwhile subject of further study.

3.4. BK channels

Large conductance Ca²⁺-activated K⁺ (BK) channels are seven membrane-pass K⁺ channels that conduct a particularly large outward K⁺ current synergistically in response to membrane depolarisation and a rise in intracellular Ca²⁺ ([Ca²⁺]_i) [181]. BK channels can be stimulated by depolarisation or increased [Ca²⁺]_i alone, however the required membrane potential (V_1/2 = 168 mV at [Ca²⁺]_i = 0) or [Ca²⁺]_i (EC₅₀ ≥10 μM at resting membrane potential) are out of physiological range [182]. BK channels are expressed in most tissues and are involved in a range of functions, such as learning and memory [183], pain modulation [184] and blood pressure regulation [185]. BK channels are upregulated in glioblastoma primary cells and promote proliferation and invasion [117,186]. BK channel function is modulated by two groups of auxiliary subunits- BKβ_1-4 and BKγ_1-4, both double-pass membrane proteins. BKβ₁ and BKβ₂ increase Ca²⁺ sensitivity [187], BKβ₂ hyperpolarises and accelerates channel activation [188], BKβ₃ depolarises channel activation [188] and BKβ₄ hyperpolarises channel activation whilst simultaneously inhibiting channel opening at low [Ca²⁺]_i but enhancing activation at high [Ca²⁺]_i [189]. BKγ subunits hyperpolarise BK channel activation [190]. BKγ₁ hyperpolarises channel activation to such an extent (−140 mV in LNCaP prostate cancer cells) that BK channels open without the need for increased [Ca²⁺]_i at resting membrane potentials [182].

Despite the extensive involvement of BK channels in a range of physiological processes, the link between BK channel auxiliary subunits and cancer is still very tentative, with thus far only BKγ₁ implicated. There are conflicting reports on the involvement of BKγ₁ (also known as LRRC26 and CAPC) in cancer. BKγ₁ is upregulated in the MDA-MB-456 breast cancer cell line and in metastatic secondary breast cancer tumours compared to the primary tumour of a single patient [191]. BKγ₁ is also upregulated in many breast and prostate cancer cell lines and breast, prostate, colon and pancreatic samples [192,193]. However, BKγ₁ is frequently methylated in triple-negative breast cancer specimens and cell lines and siRNA knockdown of BKγ₁ in the triple-negative HCC70 breast cancer cell line enhances anchorage-independent growth, invasion, migration, and NF-κB activity [194]. Similarly, knockdown of BKγ₁ expression enhances anchorage-independent growth in LNCaP cells and overexpression of BKγ₁ in the triple-negative MDA-MB-231 breast cancer cell line downregulates NF-κB activity and inhibits tumourigenesis and metastasis in nude mice [195]. Furthermore, BKγ₁ expression is lowest in poorly differentiated and highly invasive prostate and breast cancer lines [195]. Thus, BKγ₁ appears to have oncogenic and tumour-suppressive function depending on the cancer type. At this stage, the mechanism by which BKγ₁ performs these functions in cancer cells is unclear. BK channels may thus perform multiple functions in cancer cells, dependent on, or independent of, BKγ₁.

3.5. K_ir channels

Inwardly-rectifying K⁺ (K_ir) channels are double pass membrane proteins which form tetramers in the membrane [196]. K_ir channels lack a voltage sensor domain. I_Kir is instead dictated by the electrochemical gradient and an increasing intracellular blocking of the pore when the membrane potential (E_m) > E_K, resulting in an inward I_K when E_m < E_K and an outward I_K when E_m > E_k, which is progressively blocked as E_m rises [197]. K_ir channels are therefore important for maintenance of the hyperpolarised resting membrane potential and regulating activity in excitable cells, such as vascular smooth muscle [198], central neurons [199] and cardiomyocytes [200]. Subfamilies of K_ir channels exist that are ATP-sensitive (K_ATP channels; K_ir6.x) and G-protein gated (G-protein inwardly rectifying K⁺ channels- GIRKs; K_ir3.x) [201,202]. K_ATP channels are inhibited by ATP/stimulated by ADP. They function as metabolic sensors, for instance in smooth muscle where K_ATP channels regulate vascular tone [203]. GIRKs facilitate G-protein-mediated inhibitory neurotransmitter signalling, such as GABA signalling [204,205].

Certain K_ir channels are regulated by auxiliary subunits. K_ir6 binds sufonylurea receptors (SUR) 1 or 2 in an octameric conformation (tetrameric K_ir6 plus tetrameric SUR) to form a K_ATP channel [196]. Channel assembly is required before K_ATP is released from the endoplasmic reticulum [206]. SUR subunits impart differential sensitivity to ADP/ATP and are the binding target of sulfonylureas, a common form of treatment for type 2 diabetes mellitus [207,208]. SUR1 is overexpressed in cerebral metastases where it decreases vascular permeability [209]. Resveratrol binds to and inhibits SUR1, inducing apoptosis in HEK293 cells, suggesting a potential pro-survival function of SUR1 [210]. SUR2B expression is present in leiomyoma and metastatic breast cancer cells and glibenclamide, a sulfonylurea targeting SUR proteins, inhibits proliferation in these cells [211,212]. SUR2 expression, along with K_ir6.2, is upregulated in cervical cancer biopsies [213]. In addition, the effectiveness of glibenclamide at inhibiting proliferation correlates with the K_ir6.2 expression of the cell line tested, suggesting proliferation is dependent on SUR and K_ir6.2 activity [213]. Glibenclamide also inhibits proliferation in MDA-MB-231 breast cancer cells, inducing G0/G1 cell cycle arrest through an upregulation of P27 and reduction of cyclin E [212]. Treatment of MDA-MD-231 cells with the K_ATP channel opener, minoxidil, conversely induces proliferation, suggesting K⁺ influx underlies K_ATP-regulated proliferation [212]. Glibenclamide treatment also prevents tumour growth in vivo in Sprague-Dawley rats treated with N-nitroso-N-methylurea [214]. Furthermore, in insulinoma, a pancreatic β-cell cancer characterised by insulin release, which is regulated by K_ATP channels, SUR1 expression is increased [215]. In summary, SUR subunits appear to play an oncogenic role in a K_ir-dependent manner.

4. Na⁺ channels

There is a growing body of evidence supporting a role for Na⁺ channels in regulating various aspects of cancer progression [216,217]. With regard to auxiliary subunits, however, only those of the VGSC have been characterised to date and will therefore be the focus of this section (Fig. 3).

Fig. 3.

Voltage-gated Na⁺ channel auxiliary subunits. Voltage-gated Na⁺ channels (VGSCs) contain a conducting Na_vα subunit and auxiliary Na_vβ subunits. Na_vα consists of four domains (domains I-IV), each containing six segments (S1-S6). The voltage-sensing domain is found within S4 of each domain and the pore consists of the P-loop found between S5-6 of each domain. Na_vβs function as cell adhesion molecules via an extracellular immunoglobulin domain [238,239,332]. Na_vβs also induce neurite outgrowth and migration [245] and the intracellular domain of Na_vβ₂ has putative transcription regulation function [248].

4.1. Voltage-gated Na⁺ channels

VGSCs conduct an inward Na⁺ current in response to membrane depolarisation [218]. VGSCs are composed of a pore-forming α-subunit (Na_v1.1–1.9) and auxiliary β-subunits (Na_vβ₁-Na_vβ₄). Na_vβs are single pass transmembrane glycoproteins that bind Na_vα covalently, in the case of Na_vβ₂ and Na_vβ₄ [219,220], or non-covalently, in the case of Na_vβ₁ and Na_vβ₃ [[221], [222], [223]]. I_Na is responsible for propagation of action potentials and mutations in Na_vβs underlie certain types of epilepsy [224] and cardiac arrhythmia [225]. Na_vβ_1-3 traffic Na_vα to the cell surface [[226], [227], [228]] and all Na_vβs increase I_Na [[229], [230], [231]]. Na_vβs induce other changes in Na_vα gating kinetics, including accelerated recovery from inactivation [232,233] and accelerated inactivation [230,234]. Na_vβs can both positively and negatively shift the voltage of activation [235,236] and inactivation [222,226], possibly dependent on endogenous expression of Na_v subunits and other Na_v-interacting proteins in the experimental system used. Na_vβs are also cell adhesion molecules, owing to the presence of an extracellular immunoglobulin loop [[237], [238], [239], [240]], which permits Na_Vβ-mediated neurite outgrowth [[241], [242], [243], [244]]. Na_Vβ1 plays an important role in regulating neuronal migration in CNS development, particularly in the cerebellum [14,245], and Na_Vβ₂ promotes dendritic expansion during hippocampal development via a Na_vα-independent mechanism [243]. Na_Vβ subunits are also substrates for proteolytic processing by secretases [246,247] and evidence suggests that the cleaved intracellular domain of Na_Vβ₂ shuttles to the nucleus to regulate expression of α-subunit genes [248].

Emerging evidence suggests that Na_vβs play diverse functional roles in cancer. Na_vβ₁ is upregulated in breast cancer samples and is more highly expressed in strongly metastatic, compared to weakly metastatic, prostate cancer cell lines [249,250]. Overexpression of Na_vβ₁ in the MDA-MB-231 breast cancer cell line promotes primary tumour growth and metastasis to multiple organs when grafted into mice, compared to parental MDA-MB-231 cells [249]. The Na_vβ₁-induced increase in primary and secondary tumour growth was accompanied by a decrease in apoptotic cleaved caspase-3 staining, no change in proliferative Ki67 staining, and an increase in endothelial CD31 staining, suggesting increased apoptotic resistance and vascularisation underlie the oncogenic influence of Na_vβ₁ [249]. In vitro, MDA-MB-231-Na_vβ₁ cells demonstrate increased cell-cell adhesion, VGSC-mediated Na⁺ current and neurite-like process outgrowth, which is reversible by inhibiting I_Na [249,251]. Interestingly, MDA-MB-231-Na_vβ₁ cells show decreased in vitro motility and proliferation compared to MDA-MB-231 cells and knockdown of endogenous Na_vβ₁ in the MCF-7 breast cancer cell line increases cell migration [251]. Similarly, Na_vβ₁ is also expressed in cervical cancer cells where it inhibits motility [252]. Furthermore, treatment of mouse melanoma B16F10 cells with the anti-cancer polymethoxyflavone, casticin, inhibits cell migration and invasion and causes a concomitant genomic upregulation of SCN1B (encoding for Na_vβ₁) [253]. Na_vβ₁ therefore appears to have a negative influence on cell behaviour in vitro and potentially induces tumour growth and metastasis through an increase in apoptotic resistance and transcellular adhesion.

Na_vβ₂ also appears to be oncogenic. Na_vβ₂ expression is increased in strongly metastatic prostate cancer cell lines relative to weakly metastatic cell lines [254]. Perineural invasion is common in invasive prostate cancer, and LNCaP prostate cancer cells overexpressing Na_vβ₂ demonstrate an increased association with ex vivo murine spinal cord axons and an increase in migration, invasion and growth [254,255]. Despite the invasion-promoting behaviour of Na_vβ₂ in vitro, overexpression of Na_vβ₂ in LNCaP cells inhibits tumour growth, compared to LNCaP cells, when implanted into mice, suggesting the functional contribution of Na_vβ₂ might be site or stage-specific during cancer progression [255].

Unlike Na_vβ₁ and Na_vβ₂, Na_vβ₃ and Na_vβ₄ are considered tumour-suppressive. SCN3B (encoding for Na_vβ₃) expression is strongly upregulated by p53 following DNA damage and Na_vβ₃ expression induces apoptosis and suppresses colony formation in osteosarcoma and glioblastoma cell lines [256]. Na_vβ₄ expression is downregulated in thyroid and high-grade breast cancer and is associated with favourable survival [231,257]. Downregulation of Na_vβ₄ in MDA-MB-231 breast cancer cells with shRNA increases primary tumour growth and metastasis in xenograft mice models, relative to MDA-MB-231 cells overexpressing Na_vβ₄ [231]. Furthermore, loss of Na_vβ₄ increases Na_vα-independent RhoA-mediated cancer cell migration and invasion [231]. Na_vβ₄ also suppresses invasion in cervical cancer cells [252]. Na_vβs are structurally very similar and generally have a broadly comparable effect increasing I_Na, so it is intriguing that Na_vβ₁ and Na_vβ₂ are oncogenic, whereas Na_vβ₃ and Na_vβ₄ are tumour-suppressive. Additionally, both Na_vβ₁ and Na_vβ₄ were investigated using the same breast cancer cell, MDA-MB-231, so the endogenous VGSC subunit expression accompanying the Na_vβ-subunit is comparable [231,249]. Both Na_vβ₁ and Na_vβ₄ inhibit cell migration in vitro and induce neurite outgrowth in developing neurons, thus it is unclear where the functional discrepancy between the two proteins lies [231,241,251,258].

5. Cl⁻ channels

Cl⁻ channels are a family of relatively poorly understood proteins that facilitate transmembrane Cl⁻ transport. Cl⁻ concentration is highest intracellularly and E_Cl ˜-30 to −60 mV, so channels conduct an outward Cl⁻ current at resting membrane potentials that can reverse on depolarisation, although inwardly and outwardly rectifying Cl⁻ channels have been identified [13]. Cl⁻ channels are involved in regulating a range of bodily functions, including renal salt retention [259], synaptic inhibition [260], skeletal muscle contraction [261], smooth muscle tone [262] and sperm motility [263]. Various subfamilies of Cl⁻ exist, but only the voltage-gated Cl⁻ channel (CLC) and Ca²⁺-sensitive Cl⁻ channel (CaCC) subfamilies possess auxiliary subunits with a robust link to cancer (Fig. 4A, B).

Fig. 4.

Cl⁻ channel auxiliary subunits. (A) CLCs are a subfamily of voltage-sensitive Cl⁻ channels and transporters found at the plasma membrane and internal membranes [13]. Barttin modulates ClC-K, GlialCAM modulates ClC-2 and Ostm1 modulates the intracellular ClC-7 transporter [264,266,267]. CLCs are composed of eighteen helical domains and two C-terminal cystathionine-β-synthase (CBS) domains which facilitate dimerization [333]. Depicted is the plasma membrane ClC-2 which interacts with single-pass GlialCAM, the only ClC auxiliary subunit implicated in cancer [264]. GlialCAM can also function as a cell adhesion molecule [268]. (B) Two separate CaCC conducting subunits exist- TMEM16 and Bestrophin. Depicted is eight-pass TMEM16 A which is modulated directly by secreted CLCA1 and indirectly by single-pass CLCA2 [303,305]. CLCA2 stimulates Ca²⁺ store replenishment by interacting with Orai1 and STIM1 [305].

5.1. Voltage-gated Cl⁻ channels

CLCs represent a range of cell surface Cl⁻ channels (ClC-1,2,K) and intracellular Cl⁻ exchangers (ClC-3-7). Some CLCs are regulated by auxiliary subunits; ClC-2 by GlialCAM [264,265], ClC-7 by Ostm1 [266], and ClC-K by Barttin [267]. GlialCAM targets ClC-2 to cell-cell junctions, increases Cl⁻ current (I_Cl), accelerates I_Cl activation, and abolishes ClC-2 inward rectification and pH sensitivity [264]. GlialCAM also functions as a cell adhesion molecule via an extracellular immunoglobulin domain [268,269]. ClC-7 is an intracellular, electrogenic H⁺/Cl⁻ exchanger involved in lysosomal acidification [270]. Interestingly, ClC-7 regulates the trafficking and expression of its auxiliary subunit, Ostm1 [266,271]. Nevertheless, Ostm1 is required to activate ClC-7 function [270]. Barttin traffics ClC-K to the cell surface, resulting in increased I_Cl, and abolishes the voltage-dependence of ClC-K [[272], [273], [274]]. Mutations in the gene encoding Barttin are the cause of Bartter syndrome type IV, characterised by hypokalaemia, blood alkalosis and hypotension [275,276]. Knockin mice with the disease-causing Barttin mutation R8L present with reduced plasma membrane Barttin-ClC-K complexes and transepithelial Cl⁻ transport is impaired in the loop of Henle [277].

GlialCAM (also called HepaCAM) was identified as a putative tumour suppressor gene that is silenced in hepatocellular carcinoma [278]. GlialCAM downregulation is observed in liver, bladder, prostate, kidney, breast, uterus, colon, stomach, and rectal cancer biopsies [269,[278], [279], [280], [281], [282]]. Functionally, when GlialCAM is expressed in the liver carcinoma cell line HepG2, cell motility and adhesion are increased, colony formation is reduced, and proliferation is reduced [278]. Similarly, when expressed in MCF-7 breast cancer cells, GlialCAM increases cell motility and adhesion, decreases proliferation, and induces p53-mediated cellular senescence [279,283]. GlialCAM inhibits proliferation and β-catenin signalling in bladder carcinoma cells [284,285]. Furthermore, in renal carcinoma cells, GlialCAM decreases proliferation, induces cell cycle arrest, and stimulates c-Myc degradation [286]. GlialCAM expression is also sufficient for reducing Notch-mediated invasion and migration in prostate cancer cells [282]. Lastly, GlialCAM stabilises connexin-43 at cell-cell gap junctions [287], connexin-43 being a potential tumour suppressor itself [288,289]. In summary, GlialCAM has a strong anti-proliferative influence when expressed in cancer cells, which could underpin its role as a tumour suppressor.

5.2. Ca²⁺-sensitive Cl⁻ channels

Four single membrane-pass auxiliary subunits of CaCCs have been identified (known as Ca²⁺-activated Cl⁻ channel regulator or Cl⁻ channel accessory [CLCA]1-4) [290,291]. Interestingly, the molecular identities of the conducting subunits were only discovered later and termed Best1-4 and TMEM16 [[292], [293], [294], [295]]. CaCCs demonstrate voltage-dependence at steady-state, which is abolished following an increase in [Ca²⁺]_i [296]. Increased [Ca²⁺]_i also increases I_Cl and accelerates current onset [296]. CaCCs are expressed in epithelia and excitable tissues, where they regulate excitability [297], smooth muscle contraction [298] and fluid secretion [299]. Expression of CLCA1 and CLCA2 in HEK293 cells induces an enlarged and outwardly-rectifying I_CaCC [290,300]. More recent work has demonstrated that the secreted N-terminus of CLCA1, produced following autoproteolysis, is sufficient to stabilise TMEM16 A at the membrane, increasing I_CaCC [[301], [302], [303]]. CLCA1 contains an intrinsic metalloprotease domain in the N-terminus that is thought to be responsible for autoproteolysis and regulating mucus turnover in the colon [304]. Despite CLCA2 enlarging I_CaCC, CLCA2 does not interact directly with TMEM16 or Best1 [305]. Instead, CLCA2 interacts directly with store-operated Ca²⁺ channels, Orai1 and STIM-1, stimulating ER Ca²⁺ replenishment following cytosolic depletion [305].

CLCAs have a well-documented tumour-suppressive role [[306], [307], [308]]. CLCA1 is downregulated in colorectal and pancreatic cancer specimens [306,[309], [310], [311]]. CLCA1 knockdown induces proliferation and inhibits differentiation of caco-2 colorectal cancer cells [311]. Furthermore, CLCA1 overexpression inhibits Wnt signalling and colorectal tumour growth and metastasis in vivo [306]. CLCA2 expression is also decreased in high-grade nasopharyngeal, colorectal, lymphoid and breast cancer specimens compared to low grade samples [307,[312], [313], [314]]. Expression of CLCA2 decreases nasopharyngeal and breast tumourigenesis in vivo [307,312,315]. Similarly, CLCA2 depletion increases the number of circulating prostate tumour cells in mice [316]. At a cellular level, CLCA2 inhibits Wnt signalling [317], decreases invasion [315], inhibits proliferation [312], induces transcellular adhesion [316], inhibits epithelial-to-mesenchymal transition [312,316], induces differentiation [316,318], inhibits focal adhesion kinase [312,319] and induces p53-mediated cellular senescence [320]. The ability of CLCA2 to inhibit cancer cell migration appears to be I_Cl independent, as inhibiting I_Cl has a further anti-migratory effect in cells expressing CLCA2 as well as having an anti-migratory effect in cells not expressing CLCA2 [312]. Ramena et al. observed CLCA2 at cell-cell junctions, interacting with EVA1/ZO-1 or β-catenin [317]. Sequestration of β-catenin at the plasma membrane was therefore suggested as a mechanism for CLCA2-induced inhibition of epithelial-to-mesenchymal transition. CLCA4 expression is decreased in bladder, hepatocellular and breast cancer specimens compared to adjacent normal tissue [308,321,322]. CLCA4 expression also decreases tumourigenicity in mice [321]. Furthermore, CLCA4 depletion induces epithelial-to-mesenchymal transition via PI3K/Akt signalling [308,322]. Despite the abundance of evidence implicating CLCAs as tumour suppressor genes, CLCAs have also been implicated in induction of lung colonization in vivo via adhesive interactions between endothelial CLCA and β₄ integrin expressed on circulating cancer cells [323,324]. Similarly, increased CLCA2 expression is seen in circulating lung adenocarcinoma cells and ovarian cancer cell aggregates [325,326], suggesting CLCAs may potentially be tumour suppressors on the one hand, and metastasis-promoting on the other.

6. Conclusion

Many ion channel auxiliary subunits are upregulated, e.g. Ca_vβs, or downregulated, e.g. K_vβs, in tumours and thus may represent novel cancer biomarkers. in vitro and in vivo experimentation has further implicated various auxiliary subunits in tumour formation and progression, such as Na_vβ₁ and α₂δ₁ (Fig. 5). However, others, e.g. CLCAs, Na_Vβ_3/4, may function as tumour suppressors. Clearly, it is important from a treatment perspective to understand the mechanistic function of ion channel auxiliary subunits, including the extent that they contribute to cancer progression through potentiating ion conductance or via non-conducting signalling. For example, α₂δ₁- and α₂δ₂-induced Ca²⁺ influx may promote hepatocellular carcinoma cell sphere formation and pancreatic adenoma proliferation respectively [71,83]. Other examples include Na_Vα-dependent, Na_Vβ₁-mediated process outgrowth and the extent of glibenaclamide-induced inhibition of SUR2-mediated cancer cell proliferation correlating with the mRNA expression of Kir6.2 [213,249]. Validating the contribution of ion conductance to the oncogenic function of these auxiliary subunits would provide a potential therapeutic target, as many ion channel inhibitors are already in clinical use and could be repurposed [[327], [328], [329]]. On the other hand, numerous auxiliary subunits many regulate cancer progression via non-conducting roles, e.g. regulation of transcription, proliferation and differentiation by Ca_vβ₁ and KChIP3 [36,172]. Various auxiliary subunits also function as adhesion molecules in cancer cells, e.g. GlialCAM, CLCAs and Na_vβs [254,278,316]. Further work is required to fully delineate the diverse functional contributions of these subunits to carcinogenesis, tumour progression and metastasis, and understand their potential as novel therapeutic targets.

Fig. 5.

Involvement of ion channel auxiliary subunits in different stages of tumour progression. A number of different ion channel auxiliary subunits are up- or down-regulated in cancer cells promoting proliferation, reducing apoptosis and differentiation. Other auxiliary subunits have been shown to regulate angiogenesis, invasion, and metastasis, thus promoting tumour progression. Finally, ion channel auxiliary subunits may also play a role in chemo/radioresistance, underscoring the potential importance of these proteins in relation to therapeutic intervention.

Conflicts of interest statement

The authors declare that they have no conflicts of interest.