Voltage-gated ion channels in cancer cell proliferation
Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K+, Ca++, Cl−, Na+. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC pharmacological manipulation.
Keywords: cancer, cell proliferation, voltage gated ion channel, membrane potential
Cancers continue to be a leading cause of mortality and morbidity worldwide [1]. While extensive research over the years has resulted in better diagnosis and treatment options, there still is a significant dearth of effective, affordable and safe therapeutics that can improve the prognosis and the life style of affected patients [2,3]. A vast number of studies have significantly contributed to the current understanding of molecular mechanisms contributing to cancers which ranges from specific genetic mutations, to intricate biochemical and molecular pathways [4,5]. Albeit the complexity and heterogeneity of the disease, uncontrolled cell proliferation of individual cancer cells is essential for initiation and further progression of the disease [6,7]. More recently, ion channels have been recognized to play an important role in cancer pathology as they are capable of regulating key events in cell proliferation and therefore, cancer initiation and progression [8,9,10]. Interestingly, aberrant expression of specific ion channels and activity has been associated with various stages of cancer and increased aggressiveness. Furthermore, in vitro and preclinical studies have revealed that pharmacological manipulation of channel activity offers protection against several cancers. Ion channels therefore offer a novel strategy that can be potentially utilized to treat cancers. In this article, we review the unique role of a specific class of ion channels, the voltage-gated ion channels, in regulating cell proliferation and therefore their contribution to development and progression of cancer.
Cell proliferation in normal cells is a complex, well synchronized event that is stringently regulated by a number of ions, molecules and proteins associated with the cell cycle machinery including Ca++, ATP, cyclins, cyclin dependent kinases and many other cell cycle regulators [11,12]. A cell cycle can be distinguished into phases (Figure 1), namely, the G0 phase, comprised mainly of the non-proliferating cells, G1 phase with cells getting primed for DNA replication, followed by the S phase with cells undergoing DNA replication, leading to the G2 phase, where the cells are getting ready to undergo mitosis/cell division. Finally, the mitosis (M) phase results in complete division of the cells with daughter cells ensuing that individually continue the process of cell cycle [11,12]. One of the most significant and dynamic factors that regulates cell cycle is the membrane potential (Vm; Voltage membrane) [13,14]. Vm (also called transmembrane potential) is an electrical charge that is created by the discrepancy in ionic concentration between the intracellular and extracellular environment. Ion channels and ion transporters play a fundamental role in generating Vm as they are selectively permeant to ions that can cross the membrane according to chemical and/or electrical gradient. As a result of their activity, the Vm of a resting cell is negative. The cells are said to be depolarized (Figure 1) when the Vm is altered to relatively less negative state, whereas the cells are said to be hyperpolarized, when the membrane potential is moved to more negative values than the resting membrane potential [15].
A number of studies have reported that cells with a much hyperpolarized resting potential, such as muscle cells and neurons, typically show little or no mitotic activity, while proliferating cells, particularly cancer cells, have a depolarized membrane potential in comparison to normal cells [11,13,15,16,17,18]. In the seminal studies conducted in sarcoma cells by Clarence D. Cone Jr., it was observed that Vm underwent a transient hyperpolarization before entering mitosis, followed by a rapid depolarization through the M Phase suggesting that Vm varies through the cell cycle progression [2]. Further, it was observed that lowering the Vm to a hyperpolarized state similar to that of neurons, contributed to a mitotic block in proliferating CHO cells, while a sustained depolarization could induce DNA synthesis and mitosis in mature neurons [16,17,18]. In MCF-7, a breast cancer cell line, it has been observed that the Vm during a cell cycle progression correlates with the transition in each phase, such that, the pharmacological arrest of MCF-7 cells in G1/S or G2/M transition enriches cells with hyperpolarized Vm while cells arrested in the G0/G1 and M Phases had enriched cells with depolarized Vm. [19]. Similarly, in neuroblastoma cell lines, cell cycle progression was observed to correlate with hyperpolarized Vm in G1-S transition and depolarized Vm at the M phase [20]. Thus, progression of the cell cycle is accompanied by rhythmic oscillation of the Vm accompanied by transient hyperpolarization and depolarization [14,18,20,21,22,23,24]. Voltage-gated ion channels (VGICs) are a distinct group of ion channels that are selectively permeable to Na+, K+, Ca++ or Cl− and respond to changes in the membrane potential [25,26,27].
Besides their classical role in excitable cells, which include generating action potentials in neurons or contraction in muscles, voltage-gated ion channels also play vital roles in non-excitable cells including maintenance of cellular homeostasis by controlling ion transport, fluids, volume regulation and as well as proliferation.
Voltage-gated K+ channels (VGKC; Kv), as the name suggests, are selectively permeable to K+ ions and comprised of a large family of heterogeneous groups of ion channels forming 12 subfamilies (Kv1–Kv12). Activities of these proteins are regulated by the changes in the membrane potential and are critical for governing the resting membrane potential in both excitable and non-excitable cells [28]. Several VGKC have been found aberrantly expressed in different cancer tissues but not in the tissues from which they generated. In addition, the alteration of VGKC activity exhibited a strong impact on several aspects of cancer biology ranging from the arrest of cell proliferation to the inhibition of motility. In what follows, we discuss the roles of several VGKC that have been involved in cancer.
The voltage sensitive ether à-go-go potassium (EAG 1) channel, also referred to as Kv10.1, encoded by KCNH1 gene has been particularly investigated for its role in cancer cell proliferation. The normal expression of Kv10.1 is limited to the brain; however, aberrant expression of this channel has been reported in a number of tumors and cancer cell lines, and it has been associated with poor prognosis [29,30,31,32,33,34,35]. Although the mechanism linking Kv10.1 activity to proliferation is still not clear, it appears clear that Kv10.1 plays a fundamental role in cancer biology. This conclusion is centered mainly on data showing that the inhibition of Kv10.1 current activity not only reduces the proliferation of cancer cells and tumors in in vitro and in vivo models [36,37,38,39], but also overexpression of this channel alone is able to increase cell proliferation and render the cells tumorigenic [29]. More recently, EAG2 (Kv10.2), an isoform of Kv10.1 has been reported to play a key role in a medulloblastoma progression. A knock-down of Kv10.2 results in tumor inhibition in mouse models of medulloblastoma and the tumor suppression has been attributed to inhibition of mitosis via cell volume regulation.
The human ether à go-go-related gene 1 (hERG1/KCNH2) encodes for the Kv11.1 channel that is expressed in several organs in normal adults, including in heart, brain and hematopoietic cells [40,41,42]. Interestingly, it has been observed that, expression Kv11.1 is selectively upregulated in a variety of cancers [43] and several studies have shown that Kv11.1 channel can be a potential novel biomarker for cancer invasion and survival. Inhibition of Kv11.1 results in reduced proliferation of cancer cell lines and activation of an apoptotic event resulting in cell death [44,45,46,47].
Interestingly, changes in protein levels of cyclin E2 and p21Waf1/Cip1 are Ca++ dependent [48]. Therefore, changes of Kv channel current activity can alter Ca++ homeostasis which can result in activation or inactivation of signaling that is critical for cell proliferation [49].
Interestingly, Kv-related oncogenicity is not necessarily only associated with ionic conduction as it has been shown that the voltage-dependent gating of EAG can control the Mitogen Activated Protein Kinase signaling pathways (MAP kinase) and cell proliferation by a mechanism that is independent from K+ flux through the channel [50]. A possible explanation for this event is that the morphological changes occurring to the channel during gating might expose a specific domain that serves as a docking/activator of the MAP kinase signaling. Therefore, the channel exhibits also functions of scaffolding. Moreover, it was observed that, a mutation in EAG1 that eliminates ion permeation failed to abolish xenograft tumor formation and vascularization by transfected cells possibly via increasing HIF—1apla activity [51]. Similarly, non-conducting mutants of Kv1.3 channels retained their pro-proliferative effects on vascular smooth muscle cells [52]. These provide an interesting example of how ion channels can play important roles in normal and/or cancer cell biology independently from their ability to transport ions across the membranes.
The increase in K channel activity corresponding to a transient hyperpolarization observed during the cell cycle seems to be essential for cell cycle progression and proliferation [53,54,55]. The transient hyperpolarization could serve as a potential drive from passive Ca+ influx, which is essential for initiating cell proliferation [56,57,58]. However, the aberrant or selective increase in Kv channels in tumors or cancer cell lines is poorly understood.
The increase in expression and activity of Kv channels in cancer cells can be attributed to genetic factors, as in the case of genomic amplification observed for Kv10.1 resulting in its overexpression in some cancers [35,59]. Epigenetic mechanisms including DNA methylation and histone acetylation has been implicated in the altered expression of several ion channels [60,61,62]. The altered expression of Kv channels could also be attributed to tumorigenic mechanisms including, for example, mutations in p53 gene and a number of oncogenic stimuli such as epidermal growth factor [63].
Interestingly, altered Kv channel activities can also be related to steroid hormone-activated signaling that does not involve transcription of the channel. For example, several groups have reported that Kv11.1 channel activity can be modulated by a non-genomic steroid hormone receptor-mediated mechanism [64,65,66,67,68] suggesting that Kv11.1 can play a major role in hormone-driven cellular response in cancer biology.
Given the changes in the membrane potential during cell cycle progression, the possible role of Kv channels in regulating cell cycle and proliferation has been extensively investigated. Interestingly, the expression and activity of certain Kv channels seem to be regulated differentially through the course of cell cycles both in normal and cancer cells. For example, increase in Kv channel density including that of Kv1.3, Kv10.1 (EAG1), Kv10.2 or Kv11.1 (HERG) has been observed to increase through M and G1 contributing a transient repolarization and a subsequent decrease in activity during the S and G2 phases in various cell lines [69,70,71,72,73].
It has been well established that ion channels work in concert as the activity of one type of channel can determine dramatic changes of ionic flux of another ion channel. This phenomenon occurs independently from the cell type and suggests that ion channels can self-regulate their function according to the cellular need. For example, neurons can accommodate overexpression of a specific voltage-gated member of the shaker family of ion channel by inducing expression of a member of another ion channel family such as the HCN channel [74]. This event appears to be independent from the shaker channel current activity and results in activating a compensatory mechanism that allow cells to preserve homeostasis. Therefore, oncogenic properties of ion channels are observed or amplified in cells that do not have the necessary tools to compensate overexpression of a specific ion channel.
Kv1.3 belongs to the shaker family of voltage-gated K+ ion channels. Kv1.3 is known to function in conjunction with KCa3.1, a Ca++ activated potassium channel in physiologic lymphocyte activation and proliferation. However, expression of Kv1.3 channel in non-excitable cells revealed a strong inhibition of protein tyrosine kinases suggesting that changes in membrane potential can affect kinase-dependent signaling [75]. An increase in activity of both Kv1.3 and KCa3.1 has been implicated in several cancer types and inhibition of these channels also inhibits cell proliferation [76]. Mitochondrial Kv1.3 have been reported to play important roles in cancer cell apoptosis [77]. Inhibition of mitochondrial Kv1.3 using cell permeant inhibitors mediates apoptosis and inhibits tumor growth in vivo. The apoptosis mediated by my Kv1.3 inhibitors seems to be mediated via both extrinsic or intrinsic pathways suggesting that Kv1.3 inhibition could be helpful in treating cancers with deregulated apoptotic signaling pathways [78,79]. Similarly, it has been reported that mitochondrial Kv1.5 activity and expression is suppressed in various cancer cell lines including, glioblastoma, breast cancer cell lines, and bringing the levels of Kv1.5 back to normalcy results in cell growth inhibition via [80] apoptosis.
The Big Potassium (BK) channel encoded by the KCNMA1 respond to changes in membrane potential and/or increased intracellular calcium. BK has been found abundantly expressed in meningiomas and gliomas in which they presented a higher sensitivity to calcium [81,82,83]. This suggests that this channel can contribute to cell proliferation by responding promptly to the small changes of intracellular calcium during cell cycle.
Voltage-gated Ca++ (CaV) channels are critical mediators of Ca++ influx in excitable cells, where they are responsible for the control of neurotransmitter and hormone secretion, gene expression, muscle contraction, and many other physiological processes [84,85]. Gain of function mutations associated with neurological diseases and pathologies can lead to excessive activation of CaV channels, resulting in acute or chronic states of excitotoxicity [84]. In non-excitable cells, CaV channels have been linked to modulate and control cell proliferation and attracted interest as potential targets for anti-cancer therapy [86].
CaV channels belong to the superfamily of voltage-gated ion channels and share significant homology with both voltage-gated K+ and Na+ channels. CaV channels are classified as either high voltage-activated or low voltage-activated channels, and open upon membrane depolarization to mediate inward Ca++ influx [84,85]. The group of high voltage-activated channels is comprised of dihydropyridine-sensitive CaV1 (L-type) channels and three subtypes of CaV2 (P/Q-, N− and R-type) channels, while CaV3 (T-type) channels constitute the group of low voltage-activated CaV channels. Functional diversity of CaV channels is conferred both by the subtype α1 subunit and by extensive alternative splicing.
Both CaV1 and CaV3 channels have been implicated in the proliferative pathophysiology of various cancers, and their contributions are reviewed below.
Most evidence for the involvement of CaV1 channels stems from studies on adrenal aldosterone-producing adenomas (APAs) and prostate cancers. Specifically, mutations in the CACNA1D gene encoding the CaV1.3 channel were recently identified in APAs [87]. The mutations (Gly403 and Ile770) caused a gain-of-function phenotype at the single channel level, resulting in activation of the channel at less depolarizing membrane potentials [87]. Interestingly, the same authors also report de novo germline mutations in two children with a previously unrecognized syndrome of primary aldosteronism and neuromuscular abnormalities [87]. In addition, up-regulation of CACNA1D expression has been linked to prostate cancer progression [88]. Specifically, CACNA1D up-regulation and concomitant increases in CaV1.3 protein levels were identified in tissue biopsies from subjects with malignant prostate cancer compared with non-malignant controls [88]. CaV1.3 functional involvement in LNCaP cells was shown by both pharmacologic blockade of the channel and genetic approaches knocking down CACNA1D expression, both of which resulted in a decrease in androgen-stimulated Ca++, and suppressed the associated androgen receptor transactivation and cell growth [88]. Knock-down of the CACNA1C-encoded CaV1.2 channel did not show any effects [88]. Additional evidence for a pathologic CaV1.3 up-regulation is based on pharmacologic studies with the cyclooxygenase-2 inhibitor, celecoxib, which exerts chemopreventive effects against multiple cancers, including prostate cancer [89]. These effects appear to be mediated at least in part by down-regulation of androgen receptors [89]. However, it is tempting to speculate that down-regulation is the result of celecoxib’s inhibitory effects on CaV1 channel-mediated L-type Ca++ currents [89,90].
Interestingly, the CaV1 blocker, nifedipine, has been implicated in the proliferation of certain cancers, however, its effects are likely not mediated by blocking CaV channels [91]. One recent study demonstrated that nifedipine promoted both the proliferation and migration of breast cancer in vitro and in vivo [91], while another CaV1 blocker, verapamil, did not affect proliferation [91]. Similarly, the application of nifedipine in the presence of depolarizing KCl concentrations did not reduce the intracellular Ca++ concentration, suggesting a CaV-independent mechanism [91]. Rather, a mechanism involving down-regulation of miRNA524-5p has been proposed based on in vitro studies in MDA-MB-231 cells [91].
One of the earliest studies implicating low voltage-activated CaV3 channels in cancer progression was derived from studies testing the sensitivity of Ca++ entry pathways to Ni++ in non-excitable cells. Synthesis and testing of novel compounds resulted in the inhibition of Ca++ influx and the concomitant stereoselective inhibition of cell proliferation in cancer cells expressing CaV3.2, but not those devoid of CaV3.2 expression [92]. Specificity for the CaV3.2 subtype was demonstrated by inhibition of proliferation of HEK293 cells transfected with the CACNA1H gene encoding Cav3.2 [92].
Interestingly, the opposite was found in MCF-7, a human breast adenocarcinoma cell line. While overexpression of full-length CaV3.1 suppressed cell proliferation, knockdown of the CACNA1G gene encoding for Cav3.1 promoted cell proliferation [93]. In contrast, CaV3.2 overexpression did not affect cell proliferation [93]. In their study, the authors showed convincing evidence of a differential distribution of CaV3.1 and CaV3.2 channels at plasma membranes of apoptotic and non-apoptotic cells, respectively.
Similarly, CaV3.1 channel expression was present in human laryngeal squamous cell carcinoma tissue and cell lines [94]. Notably, CACNA1G gene knock-down and the CaV3 channel blocker, mibefradil, significantly reduced the rate of cell proliferation in this cancer [94].
However, due to the lack of CaV3 subtype specific pharmacologic blockers and/or agonists, many studies have investigated the group of CaV3 channels as a whole. For instance, in a comprehensive study by investigating the expression profile of CaV channels in human melanocytes and a number of melanoma cell lines and biopsies. It was observed that, CaV3 channels were selectively expressed in melanoma [95]. More importantly, mildly hypoxic conditions reminiscent of the hypoxic environment of skin and the high oxygen demand in metastasis increased CaV3 expression in melanoma [95], suggesting a role for CaV3 channels in proliferation of malignancies. The same authors reported also that clinically relevant CaV3 blockers, mibefradil and pimozide, exerted dual effects on cell viability, specifically a reduction of cell viability and initiation of apoptosis [96]. Notably, a temporal analysis of intracellular signaling events revealed that endoplasmic reticulum stress and inhibition of basal macroautophagy precede activation of apoptotic pathways in both M16 and JG melanoma cells, suggesting subtype-specific mechanisms.
CaV3 channel overexpression was also detected in glioblastoma [97]. Pharmacological inhibition and small interfering RNA-mediated CaV3 channel knockdown resulted in reduced viability and clonogenic potential, as well as the induction of apoptosis [97]. CaV3 blockers effectively reduced phosphorylation of Akt, Rictor and anti-apoptotic proteins in glioblastoma cells, and induced activation of caspases [97]; in contrast, genetic knock-down of CaV1 channels had no effect on cellular proliferation [97].
The same mechanism is likely targeted by endostatin, which has been shown to selectively inhibit CaV3.1 and CaV3.2, but not CaV3.3 or CaV1.2 channels recombinantly expressed in human embryonic kidney (HEK) or Chinese hamster ovary (CHO) cells [98]. Furthermore, endostatin dose-dependently reduced cell proliferation and migration of glioblastoma U87 cells [98].
In prostate cancer, exposure of LNCaP prostate cancer cells to sodium butyrate resulted in morphological and molecular differentiation, including a significant up-regulation of CaV3.2 channels [99]. Furthermore, sodium butyrate yielded detectable fast-inactivating, Ni++-sensitive Ca++ currents, which enhanced cellular proliferation [99].