Emerging roles for multifunctional ion channel auxiliary subunits in cancer

Graphical abstract


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 Ca 2+ ,K + ,Na + and Cl − channels and the growing evidence supporting a link to cancer.

Ca 2+ channels
Ca 2+ channels regulate a multitude of cellular processes; accordingly, much research has focused on various Ca 2+ channels in cancer, including voltage-gated Ca 2+ channels (VGCCs) [15], STIM and Orai [16], and TRP channels [17]. In terms of Ca 2+ channel auxiliary subunits however, only VGCC auxiliary subunits have received notable attention thus far. VGCCs are transmembrane complexes responsible for the inward Ca 2+ 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 aC a 2+ -conducting α 1 subunit (Ca v 1-3.x) associated with multiple auxiliary subunits (α 2 δ 1-4 , β 1-4 , γ 1-8 ), with the exception of Ca v 3.x, which can form a T-type Ca 2+ channel in the absence of an associated auxiliary subunit ( Fig. 1) [ 20]. A Ca v 1/2 subunit is joined at the membrane by an α 2 δ-, β-, and potentially a γ-subunit, although γ-subunits are not always precipitated with Ca v α [21]. Ca v α 1 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.

Ca V β
The VGCC β-subunits are cytoplasmic proteins that interact with the α 1 DI-DII intracellular linker region [22][23][24]. β-subunit binding enhances membrane expression of α 1 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 v 2.2, resulting in a higher proportion of Ca v 2.2 at the plasma membrane [25,27]. However, membrane targeting of the DI-DII linker of Ca v 2.2 via an inserted palmitoylation motif still results in ER retention and degradation, leading to the hypothesis that Ca v β subunits are required for correct folding, and thus membrane insertion, of functional α 1 subunits [28]. The impact on electrophysiological properties of α 1 subunits by Ca v βs is complex. In general, Ca v βs increase current density and regulate activation/inactivation kinetics. For instance, disruption of the Ca v β 3 -Ca V 2.2 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 v β 2 enhances the current density more than Ca v β 3 , potentially through increased membrane expression as Ca v β 2a , unlike Ca v β 3 , contains a palmitoylation site [30]. Additionally, forced membrane localisation of Ca v β 3 using the N-terminal Lyn sequence enhanced the current density relative to WT-Ca v β 3 [30]. The complexity arises in the differential sensitivity to PIP 2 -mediated modulation of different Ca v βs [ 30,31], competition for α 1 -binding between Ca v β subunits [32], the spectrum of functionally-distinct Ca v β splice variants [33,34], and the opposing impacts on α 1 -function by the different domains within the Ca v β protein [35].
Ca v βs are functional independent of direct α 1 association. All Ca v βs demonstrate nucleus localisation, Ca v β 4 particularly within nucleoli, and gene expression regulation [36][37][38][39]. All Ca v βs also contain a Src homology 3 domain capable of regulating endocytosis via interaction with dynamin and can interact with small GTPases [40,41]. Ca v βs show subunit-specific function as well, for instance Ca v β 1 is expressed in muscle progenitor cells (MPCs) earlier than Ca v 1.1, where it regulates proliferation and directly suppresses myogenin expression. Accordingly, Ca v β 1 knockout mice demonstrate impaired muscle development [36,42]. Similarly, Ca v β 2 is required for ventricle cell proliferation and heart development in zebrafish, although pharmacological VGCC inhibition caused a similar phenotype, suggesting Ca v β 2 may be functioning in an α1-dependent manner [43]. Ca v β 2 is also required for depolarisation-induced c-Fos and meCP2 activation, which intriguingly was shown to be independent of Ca 2+ influx [37]. Ca v β 4 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 v β 4 was inhibited when co-expressed with Ca v 1.1 and only upon depolarisation and the presence of extracellular Ca 2+ did Ca v β 4 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 V α 1 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 v β 1 expression is upregulated in colon cancer [50], Ca v β 2 mutations are seen in bladder cancer [51] and increased Ca v β 3 expression is observed in patients with recurrent non-small cell lung tumours compared to recurrence-free patients [52]. Furthermore, expression of Ca v β 1 and Ca v β 3 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 v β RNA expression as a high-risk prognostic marker [50][51][52].  offered additional pathophysiological justification for increased Ca v β 2 expression in cancer, by observing an enrichment in mutations of genes, including CACNB2 which encodes Ca v β 2 , involved in NCAM-mediated neurite outgrowth [51].

α 2 δ
The Ca V α 2 δ subunit has a unique structure compared to other auxiliary subunits. The translated polypeptide is proteolytically cleaved into two separate proteins, α 2 and δ, which remain coupled by a disulphide bond [53]. The α 2 segment is extracellular while the δ-subunit remains associated with the membrane via a GPI-anchor [54]. α 2 δ and Ca V β subunits can both induce surface expression of α 1 , but also function synergistically to maximise α 1 surface expression and Ca 2+ current [26,55,56]. Preventing proteolytic cleavage of the α 2 δ 1 proprotein reduces both Ca v 2.2 surface expression and presynaptic Ca 2+ influx in hippocampal neurons [57] and site-directed mutagenesis of either cysteine residue involved in the disulphide interaction, which results in a dissociation of α 2 , reduces the whole-cell Ca 2+ current [53]. Similarly, digestion of the GPI anchor of α 2 δ 3 , by prokaryotic Fig. 1. Voltage-gated Ca 2+ channel auxiliary subunits. Voltage-gated Ca 2+ channels (VGCCs) are composed of a conducting α1 subunit accompanied and functionally modulated by Ca v β, α 2 δ and Ca v γ 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 v β modulates Ca 2+ influx via binding the DI-DII linker of α1. Ca v βs are also involved in regulating gene expression and endocytosis [22,[36][37][38]40,44]. α 2 δ subunits are extracellular proteins that remain associated to the membrane via a GPI-anchor [54]. α 2 δ subunits are involved in synaptogenesis [65]. Ca v γ subunits are four-pass transmembrane proteins also involved in cervical ganglion neurite outgrowth and synaptogenesis [108,109].
More is known about the involvement of α 2 δ subunits in cancer compared to the other Ca v auxiliary subunits. Increased α 2 δ 1 expression occurs in both ovarian and hepatocellular tumour-initiating cells and correlates with decreased overall survival and a shorter progressionfree survival in clinical ovarian samples [69][70][71]. Zhao et al. developed a monoclonal antibody against α 2 δ 1 , 1B50-1 [71]. Sorting of a 1B50-1positive 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 α 2 δ 1 to be involved in maintaining cell viability and spheroid formation, via increasing Ca 2+ influx through Ltype and N-type Ca 2+ channels and MAPK signalling [71]. In non-small cell lung cancer cells, α 2 δ 1 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 α 2 δ 1 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 α 2 δ 1 is restored [75].
The involvement of α 2 δ 2 in cancer is complex, as α 2 δ 2 can be both oncogenic and tumour suppressive [76,77]. α 2 δ 2 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 α 2 δ 2 induces apoptosis via mitochondrial cytochrome-c release and subsequent caspase activation [77]. In contrast, α 2 δ 2 overexpression occurs in prostate tumours [76] and in insulin-secreting pancreatic adenomas, where elevated intracellular Ca 2+ is known to stimulate β-cell proliferation [83]. Furthermore, α 2 δ 2 overexpression in prostate cancer cells induces tumourigenesis and angiogenesis in mice, which is treatable by administering the α 2 δ 2 inhibitor, gabapentin [76].

Ca V γ
The interaction between Ca V γ-subunits and α 1 subunits is less well understood. Ca v γ-subunits were originally identified following immunoprecipitation of the skeletal muscle 1,4-dihydropyridine (DHP) receptor (later known as L-type VGCCs), which yielded γ 1 as a binding partner [93,94]. Following the discovery of Ca V γ 1 , seven more Ca v γsubunits were identified by homology studies [95][96][97][98]. Ca v γ 2 and Ca v γ 3 have been shown to associate with Ca v 2.1 [99], Ca v γ 2-4 to Ca v 2.2 [99] and Ca v γ 6 to Ca v 3.1 [100]. Using cryo-electron microscopy, the γ-subunit was predicted to interact with the Ca v 1.1 voltage-sensing domain (S4) of domain IV [24]. However, the α 1 -γ coupling remains contentious as more recent efforts failed to precipitate a Ca v γ-subunit with Ca v 2. Further, Ca v γ 2 can regulate Ca v 2.2 indirectly, suggesting a direct coupling may not be necessary for Ca v γ-induced channel modulation [21,101]. Ca v γ-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 v γ-subunits negatively regulate VGCC-mediated Ca 2+ 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 v γ-induced regulation of Ca 2+ influx observed at the cellular level is supported by the Stargazer mouse mutant, which lacks Ca v γ 2 and presents with ataxia and absence seizures [104]. Interestingly, a subclass of Ca v γ-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 v γ 7 -induced neurite outgrowth in superior cervical ganglion neurons [108] and Ca v γ 2 -induced synaptogenesis [109].
Aberrant Ca v γ expression is seen in various cancers, including increased Ca v γ 1 in early progressing human epidermal growth factorpositive (HER2+) metastatic breast cancer [110], increased Ca v γ 4 in bladder squamous cell carcinoma [111] and increased Ca v γ 7 in leiomyoma via downregulation of miR-197 [112]. Furthermore, a prediction algorithm using a dataset of 1.7 million cancer mutations identified Ca v γ 3 as a putative oncogene [113]. Similar to Ca v β, the functional role of Ca v γ in cancer is not yet clear. However, a Ca v γ 4 mutation appears in a cluster of mutations involved in MAPK signalling [111], suggesting a possible role in regulation of mitogenesis.
In summary, although Ca v α 1 subunits have an oncogenic role [15], it is not yet clear whether Ca v auxiliary subunits function through Ca v α 1 or have secondary functions in cancer, or both. Given that Ca v β and Ca v γ are both oncogenic but have antagonistic effects on α 1 function, and Ca v α 2 δ can be oncogenic or tumour suppressive, it would seem that the involvement of auxiliary subunit-mediated Ca 2+ 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 α 1 -independent, that could contribute to oncogenesis and tumour progression. All Ca v βs regulate gene expression and interact with small GTPases [36][37][38]40,41,44]. Ca v β 1 and Ca v β 2 are also essential for maintaining proliferation and cellular plasticity during development [36,43]. The TARP family of Ca v γs induce AMPA receptor membrane trafficking [107], a receptor with an emerging involvement in cancer [114,115], and Ca v γ 4 and Ca v γ 7 induce transcellular adhesion and neurite outgrowth respectively [108,109]. α 2 δ 1 is also involved in transcellular adhesion [66]. Furthermore, increased Ca 2+ conductance potentially underpins both the oncogenic function of α 2 δ 1 and α 2 δ 2 [71,83] and the tumour suppressive function of α 2 δ 2 and α 2 δ 3 [77,92].

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 v 1-12.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 v β 1-3 , KChIP1-4, and KCNE1-5 which canonically interact with K v 1, K V 4, and K v 7.1 respectively [119][120][121][122], although K v βs and KCNEs interact with other VGKC αsubunits and K V βs also interact with TRPV1 and K 2 P2.1 [123][124][125][126]. The activity of K v 1 [116,127], K v 4 [128], and K v 7.1 [129] is upregulated in various cancers. However, the expression pattern of VGKC auxiliary subunits in cancer is more complex.

K v β
K v β subunits are cytoplasmic proteins, which form homo-or heterotetramers [130] that are involved in trafficking of K v 1 and K v 4.3 to the cell surface [131][132][133]. Additionally, K v β 2 is involved in targeted axonal trafficking of K v 1.2 and K v β 1 differentially regulates the K v composition in ventricular myocytes [134,135]. K v β 1 and K v β 3 modulate VGKC α-subunits via an N-terminal ball domain, which permits rapid inactivation of delayed-rectifying K v 1 α-subunits [136,137]. K v β 1 also slows deactivation, accelerates slow inactivation and hyperpolarises activation of K v 1.2 [138]. K v β 2 lacks the ability to inactivate delayed-rectifying K v 1 channels, but does hyperpolarise channel activation [139]. K v β 1 and K v β 2 are both expressed in developing rat heart and skeletal muscle and during induced myogenesis of L6E9 cells [140]. Furthermore, deletion of K v β 1 results in aberrant cardiac electrical activity and cardiac hypertrophy in female mice [141]. K v β 2 deletion leads to reduced K v 1.5 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 v βs 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 V β 1 -and K V β 3 -mediated inactivation of K v 1.5 as well as inhibiting K v β 2 -mediated hyperpolarisation of K v 1.5 activation [143,144].
Evidence suggests that K v βs are downregulated in cancer. K v β 1 is downregulated in malignant thyroid carcinomas relative to benign thyroid adenomas [145,146]. The gene encoding K v β 2 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 v β 3 is seen in oral squamous cell cancers relative to adjacent normal tissue [149]. Together, these data suggest K v βs are tumour suppressor genes, but in depth in vitro and in vivo characterisation of K v β in cancer is still currently lacking.
primarily with K v 7; two KCNEs interact with tetrameric K v 7 [ 150]. In vitro studies document a range of effects of KCNEs on K v 7.1. 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 v 7.1 produces voltage-insensitive channels and all KCNEs depolarise the activation voltage of K v 7, with KCNE4 and KCNE5 depolarising activation to a non-physiological membrane potential [151]. K V 7.1 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 3fold 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 v 7.1, 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 v 7.1 complexes, in the apical membrane of non-excitable gastric parietal cells, are essential for maintaining acidification of the stomach, as KCNE2 transforms K v 7.1 to a constitutively open channel that is potentiated by extracellular H + [169]. Luminal K + released by KCNE2-K v 7.1 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 v 7.1 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.

KChIP
Ca 2+ -sensing K v channel interacting proteins (KChIPs) are involved in K V 4 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 v 4.2/3 Ntermini [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 Ca 2+ stimulation, DREAM tetramers dissociate from DNA allowing gene transcription [173]. Despite KChIP3 being the first Ca 2+ -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 Ca 2+ -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.

BK channels
Large conductance Ca 2+ -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 Ca 2+ ([Ca 2+ ] i ) [ 181]. BK channels can be stimulated by depolarisation or increased [Ca 2+ ] i alone, however the required membrane potential (V 1/2 = 168 mV at [Ca 2+ ] i =0 )o r [Ca 2+ ] i (EC 50 ≥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 doublepass membrane proteins. BKβ 1 and BKβ 2 increase Ca 2+ sensitivity [187], BKβ 2 hyperpolarises and accelerates channel activation [188], BKβ 3 depolarises channel activation [188] and BKβ 4 hyperpolarises channel activation whilst simultaneously inhibiting channel opening at low [Ca 2+ ] i but enhancing activation at high [Ca 2+ ] i [189]. BKγ subunits hyperpolarise BK channel activation [190]. BKγ 1 hyperpolarises channel activation to such an extent (−140 mV in LNCaP prostate cancer cells) that BK channels open without the need for increased [Ca 2+ ] 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 BKγ 1 implicated. There are conflicting reports on the involvement of BKγ 1 (also known as LRRC26 and CAPC) in cancer. BKγ 1 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]. BKγ 1 is also upregulated in many breast and prostate cancer cell lines and breast, prostate, colon and pancreatic samples [192,193]. However, BKγ 1 is frequently methylated in triple-negative breast cancer specimens and cell lines and siRNA knockdown of BKγ 1 in the triple-negative HCC70 breast cancer cell line enhances anchorage-independent growth, invasion, migration, and NF-κB activity [194]. Similarly, knockdown of BKγ 1 expression enhances anchorage-independent growth in LNCaP cells and overexpression of BKγ 1 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, BKγ 1 expression is lowest in poorly differentiated and highly invasive prostate and breast cancer lines [195]. Thus, BKγ 1 appears to have oncogenic and tumour-suppressive function depending on the cancer type. At this stage, the mechanism by which BKγ 1 performs these functions in cancer cells is unclear. BK channels may thus perform multiple functions in cancer cells, dependent on, or independent of, BKγ 1 .

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 ir 6.x) and Gprotein gated (G-protein inwardly rectifying K + channels-GIRKs; K ir 3.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 Gprotein-mediated inhibitory neurotransmitter signalling, such as GABA signalling [204,205].
Certain K ir channels are regulated by auxiliary subunits. K ir 6 binds sufonylurea receptors (SUR) 1 or 2 in an octameric conformation (tetrameric K ir 6 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 ir 6.2, is upregulated in cervical cancer biopsies [213]. In addition, the effectiveness of glibenclamide at inhibiting proliferation correlates with the K ir 6.2 expression of the cell line tested, suggesting proliferation is dependent on SUR and K ir 6.2 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.

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).

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 v 1.1-1.9) and auxiliary β-subunits (Na v β 1 -Na v β 4 ). Na v βs are single pass transmembrane glycoproteins that bind Na v α covalently, in the case of Na v β 2 and Na v β 4 [219,220], or non-covalently, in the case of Na v β 1 and Na v β 3 [221][222][223]. I Na is responsible for propagation of action potentials and mutations in Na v βs underlie certain types of epilepsy [224] and cardiac arrhythmia [225]. Na v β 1-3 trafficN a v α to the cell surface [226][227][228] and all Na v βs increase I Na [229][230][231]. Na v βs induce other changes in Na v α gating kinetics, including accelerated recovery from inactivation [232,233] and accelerated inactivation [230,234]. Na v βs 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 v βs are also cell adhesion molecules, owing to the presence of an extracellular immunoglobulin loop [237][238][239][240], which permits Na V β-mediated neurite outgrowth [241][242][243][244]. Na V β1 plays an important role in regulating neuronal migration in CNS development, particularly in the cerebellum [14,245], and Na V β 2 promotes dendritic expansion during hippocampal development via a Na v α-independent mechanism [243]. Na V β subunits are also substrates for proteolytic processing by secretases [246,247] and evidence suggests that the cleaved intracellular domain of Na V β 2 shuttles to the nucleus to regulate expression of α-subunit genes [248].
Emerging evidence suggests that Na v βs play diverse functional roles in cancer. Na v β 1 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 v β 1 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 v β 1 -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 v β 1 [249]. In vitro, MDA-MB-231-Na v β 1 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 v β 1 cells show decreased in vitro motility and proliferation compared to MDA-MB-231 cells and knockdown of endogenous Na v β 1 in the MCF-7 breast cancer cell line increases cell migration [251]. Similarly, Na v β 1 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 v β 1 ) [ 253]. Na v β 1 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 v β 2 also appears to be oncogenic. Na v β 2 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 v β 2 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 v β 2 in vitro, overexpression of Na v β 2 in LNCaP cells inhibits tumour growth, compared to LNCaP cells, when implanted into mice, suggesting the functional contribution of Na v β 2 might be site or stage-specific during cancer progression [255].
Unlike Na v β 1 and Na v β 2 ,N a v β 3 and Na v β 4 are considered tumoursuppressive. SCN3B (encoding for Na v β 3 ) expression is strongly upregulated by p53 following DNA damage and Na v β 3 expression induces apoptosis and suppresses colony formation in osteosarcoma and glioblastoma cell lines [256]. Na v β 4 expression is downregulated in thyroid and high-grade breast cancer and is associated with favourable survival [231,257]. Downregulation of Na v β 4 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 v β 4 [231]. Furthermore, loss of Na v β 4 increases Na v α-independent RhoA-mediated cancer cell migration and invasion [231]. Na v β 4 also suppresses invasion in cervical cancer cells [252]. Na v βs are structurally very similar and generally have a broadly comparable effect increasing I Na , so it is intriguing that Na v β 1 and Na v β 2 are oncogenic, whereas Na v β 3 and Na v β 4 are tumour-suppressive. Additionally, both Na v β 1 and Na v β 4 were investigated using the same breast cancer cell, MDA-MB-231, so the endogenous VGSC subunit expression accompanying the Na v β-subunit is comparable [231,249]. Both Na v β 1 and Na v β 4 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].

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 Ca 2+ -sensitive Cl − channel (CaCC) subfamilies possess auxiliary subunits with a robust link to cancer (Fig. 4A, B).

Ca 2+ -sensitive Cl − channels
Four single membrane-pass auxiliary subunits of CaCCs have been identified (known as Ca 2+ -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 voltagedependence at steady-state, which is abolished following an increase in [Ca 2+ ] i [296]. Increased [Ca 2+ ] 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 Fig. 3. Voltage-gated Na + channel auxiliary subunits. Voltage-gated Na + channels (VGSCs) contain a conducting Na v α subunit and auxiliary Na v β subunits. Na v α 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 v βs function as cell adhesion molecules via an extracellular immunoglobulin domain [238,239,332]. Na v βs also induce neurite outgrowth and migration [245] and the intracellular domain of Na v β 2 has putative transcription regulation function [248].

Conclusion
Many ion channel auxiliary subunits are upregulated, e.g. Ca v βs, or downregulated, e.g. K v βs, 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 v β 1 and α 2 δ 1 (Fig. 5). However, others, e.g. CLCAs, Na V β 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 nonconducting signalling. For example, α 2 δ 1 -and α 2 δ 2 -induced Ca 2+ 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 Ca 2+ store replenishment by interacting with Orai1 and STIM1 [305].
influx may promote hepatocellular carcinoma cell sphere formation and pancreatic adenoma proliferation respectively [71,83]. Other examples include Na V α-dependent, Na V β 1 -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 nonconducting roles, e.g. regulation of transcription, proliferation and differentiation by Ca v β 1 and KChIP3 [36,172]. Various auxiliary subunits also function as adhesion molecules in cancer cells, e.g. GlialCAM, CLCAs and Na v βs [ 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.

Conflicts of interest statement
The authors declare that they have no conflicts of interest.  5. Involvement of ion channel auxiliary subunits in different stages of tumour progression. A number of different ion channel auxiliary subunits are up-or downregulated 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.