PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of amjtrLink to Publisher's site
 
Am J Transl Res. 2010; 2(4): 345–355.
Published online 2010 June 18.
PMCID: PMC2923859

The emerging role of KCl cotransport in tumor biology

Abstract

The electroneutral KCl cotransport carried out by the KCl cotransporter family (KCC) plays a significant role in the ionic and osmotic homeostasis of epithelial cells. Here we review the emerging importance of KCl cotransport in epithelial carcinogenesis and tumor malignant behaviors. The malignant transformation of cervical epithelial cells is associated with the differential expression of volume-sensitive KCC isoforms. The loss-of-function KCC mutant cervical cancer cells exhibit inhibited cell growth accompanied by decreased activities of the cell cycle regulators and matrix metalloproteinase. Additionally, insulin-like growth factor-1 (IGF-1) stimulation of KCl cotransport plays an important role in IGF-1 signaling to promote growth and spread of gynecological cancers. IGF-1 upregulates KCC3 and KCC4 which are differentially required for cancer cell proliferation and invasiveness. KCC3 overexpression downregu-lates E-cadherin/β-catenin complex formation by inhibiting the transcription of E-cadherin gene and accelerating the proteosome-dependent degradation of β-catenin protein. That therefore promotes the epithelial-mesenchymal transition of cervical cancer cells, and thereby stimulating tumor progression. Moreover, epidermal-growth factor (EGF) and IGF-1 stimulate the membrane recruitment of KCC4 at lamellipodia through myosin Va-actin trafficking route. KCC4 functions as a membrane scaffold forthe assembly of signal complexes via the association with the actin-binding protein, ezrin. The molecular studies of surgical specimens suggest that the expression of KCC3, KCC4, and their stimulators, EGF or IGF-1, exhibit a close association with the clinical outcome of cancer patients. Therefore, KCC3, KCC4, EGF, and IGF-1 may be a panel of biomarkers to predict cancer patient outcome.

Keywords: KCl cotransport, proliferation, migration, metastasis, cervical cancer, ovarian cancer

Introduction of KCl cotransport

As originally described in red blood cells (RBCs), KCl cotransport is the electroneutral symport of K+ and Cl across the plasma membrane that can be activated by cell swelling [1, 2] and sulf-hydryl-alkylating reagent N-ethylmaleimide [3, 4]. KCl cotransport is necessary for the cell volume regulation, transepithelial ion transport, and maintenance of intracellular Cl concentration [4]. The KCl cotransport activity is mediated by the four homologous SLC12 gene family of electroneutral cation-chloride cotransporters, namely KCC1 [5], KCC2 [6], KCC3 [7-9], and KCC4 [8] (SLC12A4-7, respectively). The four KCC isoforms are 65-75% identical in protein sequences. The phylogenetic analyses revealed that four KCC isoforms fall into two subgroups that KCC1 pairs with KCC3 whereas KCC2 pairs with KCC4 [8]. The four KCC isoforms share a common protein structure with a central core of twelve hydrophobic transmembrane domains flanked by two hydrophilic N- and C-terminal intracellular domains [8]. A large extracellular loop between 5th and 6th transmembrane domains that conserves three identical N-linked glycosylation sites has also been predicted from the published sequences of four KCC isoforms [8]. However, these transporters differ in amino acid residues within key transmembrane domains and in the distribution of putative phos-phorylation sites within the N- and C-terminal cytoplasmic domains.

KCl cotransport plays a significant role in ionic and osmotic homeostasis of RBC and epithelial cells [4, 10]. The activities of KCC1, KCC3, and KCC4 are osmotically sensitive and participate in the cell volume regulation [11]. The neuronal-specific isoform KCC2 exhibits the constitutive KCl cotransport activity under isotonic conditions and is therefore important for the maintenance of intraneuronal Cl concentration [12, 13]. Regulation of KCl cotransport is likely complicated; however studies in RBCs have identified several regulators of KCl cotransport activity [4, 14]. Under physiological conditions, KCl cotransport activity can be stimulated by cell swelling, low intracellular pH, high partial pressure of oxygen, and urea [14]. Pharmacological activators include the thiol-alkylating reagent N-ethylmaleimide (NEM) and the oxidizing reagents (e.g. H2O2, hydroxylamine, and NO). The intracellular signaling cascades of phosphorylation/dephosphorylation events also play important roles in regulating KCl cotransport activity [15, 16]. KCl cotransport is stimulated by kinase inhibition (e.g. thiol reagents, stauro-sporine, and genistein) and inactivated by phos-phatase inhibition (e.g. okadaic acid and calycu-lin) [10, 15, 16]. Protein phosphatase PP1Aand PP2A and serine-threonine kinase WNK family are implicated in modulating the dephosphorylation and phosphorylation of KCC isoforms, respectively [17, 18]. Furthermore, a recent study based on the biochemical analyses of KCC3 polypeptides has identified two key phosphorylation sites in the intracellular C-terminal tail [19]. These two phosphorylation sites are conserved among four KCC isoforms and are critical for regulating KCl cotransport activity. Surface expression of KCC3 displays high phosphorylation levels of these two sites under isotonic conditions in which KCl cotransport activity is low. In contrast, these two sites are rapidly dephosphorylated in response to hypotonic stress in parallel with increased KCl cotransport activity.

Despite RBC has been the well-studied model for understanding the cellular physiology of KCl cotransport, most functional studies of KCC were done on KCl cotransport fluxes without knowing the physiological and molecular details of individual KCC isoforms. Knockout mouse models and human diseases which link to the mutation of specific KCC genes have greatly advanced our knowledge of the physiological functions and pathological defects of individual KCC isoforms. The molecular characterization, physiological functions, and pathological defects of KCC isoforms are described as follows.

KCC1 (SLC12A4)

KCC1, along with KCC2, are two first KCC isoforms which were cloned and identified [5]. The human KCC1 gene is on chromosome 16q22 and encodes for a protein product of 1085 amino acids that has highest identity (75%) with KCC3 among three isoforms. KCC1, ubiquitously detected in mammalian cells and tissues analyzed by Northern blot analyses, is considered as the “housekeeping” transport mechanism in cell volume regulation and transepithelial ion transport [20, 21]. The structure-function relationship study on mammalian KCC1 revealed that both the C-terminal and the membrane-proximate region of N-terminal domain are necessary for KCl cotransport function [22]. The removal of the N-terminal 117 amino acids from KCC1 produced a dominant-negative loss-of-function phenotype for the function of all four KCC isofroms in many types of cells [22-24].

Despite human KCC1 is expressed in every cell and tissue tested so far, the specific cellular and physiological function of KCC1 have remained unclear due to the absence of a KCC1-associated human disease. Pathological activation of KCl cotransport in RBC is thought to contribute to the dehydration and deformation of sickle blood cells and is implicated in the sickle cell disease [25-27]. KCC1 has therefore been proposed as a modifier gene in sickle cell disease [21]. A recent study has shown that the KCl cotransport activity in RBCs is undiminished in KCC1 knockout mice, suggesting other unknown functions of KCC1 in RBCs [28].

KCC2 (SLC12A5)

The human KCC2 gene is on chromosome 20q13 and encodes for a protein product of 1116 amino acids [6, 12, 13]. KCC2, a neuronal specific KCC isoform, is unique in mediating KCl cotransport constitutively under isotonic conditions; whereas the other three KCC isoforms are only active in response to the cell swelling by hypotonic stress. KCC2 displays a distinct C-terminal insertion rich in prolines and negatively charged amino acids that confers the constitutive isotonic activity [29, 30]. This region encompasses two predicted PEST [Proline/E (glutamate)/Serine/Threonine] sequences that are completely unique to KCC2. Depending on the chemical concentration gradients of K+ and Cl, this transporter can operate as a net efflux or influx pathway [6, 12].

The expression of KCC2 is detected in the developing synapse and in adult neurons throughout the central nervous system, especially in neurons of the cortex, hippocampus, and the cere-bellar granular cell layer [6]. The physiological function of KCC2 in the mature brain is to lower the intraneuronal Cl concentration below its electrochemical equilibrium and thereby facilitating the maturation of inhibitory γ-aminobutyric acid responses. It is crucial in promoting the synaptic inhibition, controlling the central nervous system excitability, and inhibiting the postsynaptic potential. Moreover, several genetic and functional evidences have shown that KCC2 is necessary for the development of central nervous system. Complete deletion of KCC2 gene in mice causes neonatal death due to the severe motor deficit that also abolishes the respiration [31]. Mice with partial deletion of KCC2 die from general seizure and respiratory failure by the postnatal stage [32]. Furthermore, adult heterozygous mice show elevated susceptibility for epileptic seizure and increased resistance to the anticonvulsant drug, suggesting the involvement in the pathology of human epilepsy.

In addition to the developmental role in the inhibitory synapses, KCC2 is crucial for the maturation of dendritic spines and functional excitatory synapses [33]. Evidences form electro-physiological and immunefluorescent studies have revealed that neurons lacking KCC2 expression develop immature filopodia-like dendritic protrusions in parallel with a reduction in active synapses. Moreover, the morphogenic role of KCC2 in dendrite maturation is mediated by the specific structural interaction between KCC2 with the actin-binding ezrin/radixin/moesin protein, 4.1N, rather than the KCl co-transport activity of KCC2.

KCC3 (SLC12A6)

The human KCC3 gene is located on chromosome 15q13 and is colocalized with the gene for myoclonal epilepsy [8, 9]. The mRNA transcripts of KCC3 are abundant in muscle, brain, spinal cord, kidney, heart, pancreas, and placenta [8, 9]. Two main 5'-splice variants of KCC3, KCC3a and KCC3b, have been identified [33]. The two KCC3 variants have different N-terminal ends due to the deletion at exon 1 in the shorter KCC3b. Both KCC3 variants contain 5 potential N-linked glycosylation sites in the extracellular loop between the 5th and 6th transmembrane domain and share about 77% amino-acid identity with KCC1 and 73% with KCC2. The predicted KCC3a protein is longer by 50 amino acids and encompasses several unique phosphorylation sites that are absent in the N-terminus of KCC3b. The distribution of KCC3a is more widespread than that of KCC3b and is particularly abundant in the kidney [35].

Little is known about the cellular and physiological properties of KCC3 proteins. The expression of KCC3 is correlated with the myelinization in the rodent central nervous system, suggesting a role in the physiology of myelinization [35]. KCC3 also plays an important role in regulating cell growth and proliferation [23, 36, 37]. Moreover, the functional defects of KCC3 knockout mice have provided clues about the physiological functions of KCC3. KCl cotransport activity in RBCs is decreased in KCC3 knockout mice, and almost completely abolished in mice lacking KCC1 and KCC3, indicating that KCl cotransport activity of mouse RBC is largely mediated by KCC3 [28]. Loss ofKCC3 also causes arterial hypertension, slowly progressive deafness, peripheral neurodegeneration and reduced seizure threshold in mice [34, 38, 39]. Furthermore, several genetic and functional evidences have shown that loss-of-function mutations in human KCC3 gene cause a rare autosomal disease, the agenesis of the corpus callosum with peripheral neuropathy (ACCPN) [34]. The syndrome of ACCPN includes the severe sensorimo-tor neuropathy associated with mental retardation, dysmorphic features, and complete or partial agenesis of the corpus callosum. Indeed, the neurodegenerative phenotypes of KCC3 knockout mice, including locomotor deficit, peripheral neuropathy, and the sensorimotor gating deficit, are consistent with certain features of ACCPN. However, the pathophysiological properties of KCC3 underlying this neurological deficit is not yet clear. The axonal and periax-onal swelling proceeding the peripheral neurodegeneration of KCC3 knockout mice implicates that KCC3-mdiated cell volume regulation is critical for the development and maintenance of peripheral axon and myelin [40].

KCC4 (SLC12A7)

The human KCC4 gene is located on chromosome 5p15 and encodes for a protein product of 1083 amino acids which shares high identity (about 75%) with KCC2 [8]. However, unlike KCC2, KCC4 does not mediate the constitutive KCl cotransport under isotonic conditions but the swelling-activated cotransport [11]. Like KCC1, the mRNA transcripts of KCC4 are widely expressed in various tissues. The protein expression of KCC4 is particularly robust in the kidney and is localized at the basolateral membrane of type-A intercalated cells and proximal tubules [18, 41]. It was therefore postulated that the K+ and Cl efflux-mediated by KCC4 play an important role in the salt absorption of the distal convoluted tubule [41]. KCC4 may have some undetermined functions in the central nervous system as the significant expression of KCC4 has also been demonstrated in cranial nerves, spinal cord and peripheral nerves [42]. Moreover, mice with KCC4 gene disruption exhibit deafness and renal tubular acidosis [43]. The renal tubular acidosis phenotype may result from the defect in the acid secretion of intercalated cells. The impaired K+ recycling from outer hair cells into supporting Deiter's cells may lead to the degeneration of outer hair cells of the cochlear and thereby the deafness. The physiological functions and pathological defects of four KCC isoforms are summarized in Table 1.

Table 1
The physiological functions and pathological defects of KCC isoforms

KCl cotransport activity affects the tumor invasion and proliferation

The emerging importance of KCl cotransport in the epithelial carcinogenesis and tumor malignant behaviors has been established from several studies based on the models of gynecological cancers over the past decade. KCl cotransport is one of the major K+ and Cl flux pathways in epithelial cells and plays a significant role in the ionic and osmotic homeostasis [4, 10]. Homeostasis of cell volume is a fundamental property of mammalian cells. Cell proliferation and migration are accompanied by the dynamics in cell shape and volume. The metabolism, mitosis, proliferation, and migration of cancer cells are usually more active than those of normal cells. The close linkage between cell volume regulation, cell proliferation, and cell migration leads to an obvious question that is “Do cancer cells develop an advantage in cell volume regulation that benefits them in tumor malignant behaviors?”

It was first tested if the malignant transformation of human epithelial cells is associated with the differential expression of volume-sensitive KCC isoforms. The molecular identification and functional flux studies demonstrated that the malignant transformation of cervical epithelial cells is accompanied by the differential expression of volume-sensitive KCl cotranposrt activities [44]. Cervical carcinogenesis is accompanied by the up-regulation of mRNA transcripts of KCC1, KCC3 and KCC4. Moreover, the growth and invasion of cervical cancer cells are strongly correlated with the expression and activity of KCC [23]. Functional assays of KCl cotransport activation by osmotic swelling, staurosporine, and NEM in human cervical cancer cells indicate that the removal of N-terminal 117 amino acids from KCC1 produces a dominant-negative loss-of-function phenotype for KCl cotransport and thereby elevating intracellular Cl concentration. Alteration of intracellular Cl concentration affects the activity of retinoblastoma (Rb) protein and cdc2 kinase, two key cell cycle regulators controlling the progression from G1 into S phase and from G2 into M phase, respectively [36]. The loss-of-function KCC mutant cervical cancer cells exhibit inhibited cell growth accompanied by decreased activity of Rb protein and cdc2 kinase. Reduced cellular invasiveness is in parallel by decreased expression of αvβ3 and α6β4 integrins, accompanied by inhibited activity of matrix metalloproteinase (MMP)-2 and -9. Inhibition of tumor growth in severe combined immunodeficient mice confirms the critical role of KCl cotransport in promoting cervical cancer growth and invasion. Taken together, KCl cotransport plays a crucial role in the proliferation and invasion of cancer cells as well as in the epithelial carcinogenesis. The mechanisms by which KCl cotransport affects the tumor invasion and proliferation are summarized in Figure 1.

Figure 1
Schematic diagram indicating the mechanisms by which KCl cotransport activity affects the tumor invasion and proliferation. KCl cotransport activity is involved in cell volume regulation, matrix metalloprotease (MMP) activity, and integrin activation, ...

The cooperation between KCl cotransport and certain growth factors has also been implicated in modulating the proliferation and invasive migration of cancer cells. Overexpression of specific growth factors significantly enhances the invasive and metastatic properties of cancer cells, which poses serious problems to the successful treatment of neoplastic disease. Insulin-like growth factor 1 (IGF-1) has been implicated in promoting malignant phenotypes, such as mitogenesis, invasiveness, metastasis and antiapoptosis, in most types of cancer [45, 46]. The good correlation between the expression level of IGF-1 and that of KCC polypeptides in the surgical specimens of cervical cancers, ovarian cancers, and breast cancers suggest a likely autocrine or paracrine regulation of IGF-1 on KCl cotransport activity and expression in vivo [47, 48]. Studies in the cell line models indicate that IGF-1 increases the expression and activity of KCl cotransport through the differentially activation of the downstream phosphophatidy-linostiol 3-kinase (PI3K) and extracellular signal–regulated kinase-1/2 (Erk1/2) MAP kinase signaling pathways. Pharmacological inhibition and genetic modification of KCl cotransport activity demonstrate that KCl cotranpsort is necessary for IGF-1-induced cancer cell invasiveness and proliferation. Furthermore, IGF-1 upregulates KCC3 and KCC4 which are differentially required for tumor cell proliferation and invasiveness [36, 48]. In summary, the activation of KCl cotransport by IGF-1 plays an important role in IGF-1 signaling to promote growth and spread of gynecological cancers.

The important role of KCC3 in epithelial-mesenchymal transition (EMT)

Cancer development and progression is a multistep process that involves the uncontrolled growth of tumor cells, detachment from the primary tumor, migration through the extracellular matrix, invasion through the basal membrane, and ultimately cancer metastasis. The contribution of individual KCC isoforms in cancer development and progression has been addressed in recent studies [27, 48]. Real-time reverse transcription-PCR on samples collected by laser microdissection indicates that KCC3 is the most abundant isoform in primary tumors of cervical carcinoma and ovarian cancer [24, 49]. The expression level of KCC3 is closely correlated with tumor size of cervical carcinoma, an indicator of tumor growth in vivo. In addition, KCC3 overexpression enhances cervical cancer proliferation in vitro. These are consistent with the previous study showing that KCC3 plays an important role in cell growth regulation via the modulation of cell cycle progression [36].

Overexpression of KCC3 and the consequent upregulation of KCl cotransport activity also benefit cancer cells in the epithelial-mesenchymal transition (EMT) [49], a key event occurring in cancer development and progression. Acquisition of EMT in cancer cells is associated with disrupted epithelial integrity, increased invasive migration, and cancer metastasis [50-52]. Studies in cervical cancer cell lines demonstrate that the characteristics of EMT appear in KCC3-overexpressed tumor cells, including the elongated cell shape, increased cell scattering, downregulated epithelial markers (E-cadherin and β-catenin), and upregulated mesenchymal marker (vimentin) [49]. The blockade of KCC activity in tumor cells leads to the increase in both the abundances and the association of E-cadherin and β-catenin in cell-cell junctions. Some cellular functions are enhanced by KCC3 overexpression, such as increased invasiveness and proliferation, and weakened cell-cell association. KCC3 overexpression decreases the mRNA level of E-cadherin. The promoter activity assays of various regulatory sequences reveal that KCC3 expression is a potent negative regulator for human E-cadherin gene expression. The pro-teosome inhibitor restores the decreased protein abundance of β-catenin by KCC3 overexpression. In the surgical specimens of cervical carcinoma, the decreased expressions of E-cadherin as well as the loss of cell integrity are accompanied by the increased KCC3 abundance. Meanwhile, vimentin begins to appear at the invasive front and becomes significantly expressed in the tumor nest. Taken together, KCC3 downregulates E-cadherin/β-catenin complex formation by inhibiting the transcription of E-cadherin gene and accelerating the proteosome-dependent degradation of β-catenin protein. The disruption of E-cadherin/β-catenin complex formation promotes EMT, thereby facilitating tumor progression (summarized in Figure 2). The close linkage between KCC3 abundance, cell proliferation, invasiveness, and EMT suggests that the expression and activity of KCC3 may serve as a selective advantage for cancer cells in malignant behaviors.

Figure 2
KCl cotransporter KCC3 downregulates E-cadherin/β-catenin complex formation to promote EMT that is important for cervical cancer cell invasiveness. The oncogenic growth factors such as IGF-1 and VEGF are known to stimulate the activity and expression ...

The important role of KCC4 in cancer invasion and metastasis

The important role of KCC4 in tumor malignant behavior, such as cancer cell invasiveness and tumor metastasis has been identified in a recent study [24]. In surgical specimens of metastatic cervical and ovarian cancers, both mRNA transcripts and protein levels of KCC4 are the most abundant one among that of four KCC isoforms. The significant association between KCC4 abundance and prognostic factors of cervical cancer patients, such as parametrium invasion, pelvic lymph node metastasis, and the recurrence-free or overall survival rates, suggest the important role of KCC4 in cancer metastasis. Studies based on the in vitro cell line model indicate that cancer cell invasiveness is enhanced by KCC4 overexpreesion but attenuated by KCC4 knockdown. Furthermore, the reduced cellular invasive migration is associated with decreased MMP-2 activity. These indicate that cancer cells benefit from KCC4 overexpression in the invasive migration and possibly in the tumor metastasis.

The novel mechanisms by which specific growth factors modulate the biosynthesis and functions of KCC4, thereby promoting cancer cell invasiveness, have been highlighted. IGF-1 and EGF are known to be overexpressed in most types of cancer tissues and notably contribute to cancer invasiveness and metastasis [46, 53-55]. In the metastatic cancer tissues, KCC4 colocalizes with IGF-1 or EGF, indicating a likely in vivo stimulation on KCC4 functions by growth factors. Chronic stimulation with IGF-1 upregulates the de novo proteins synthesis of KCC4 that is important for IGF-1 signaling to promote invasive migration of gynecologic cancer cells [48]. Moreover, IGF-1 and EGF stimulate the rapid recruitment of KCC4 from a presumably inactive cytoplasmic pool, such as endoplasmic reticu-lum and Golgi, to the active plasma membrane target one alongthe actin cytoskeleton. PI3K activation is the dominant signal controlling KCC4 trafficking. Throughout the trafficking process, KCC4 is incorporated into the specialized membrane microdomain, lipid rafts, that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. Like KCC2, surface expression of KCC4 exhibits a structural role in the organization of signaling complex in addition to ion transport. KCC4 and ezrin, an actin-binding protein which functions as the membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either a pharmacologic inhibitor or a dominant-negative loss-of-function mutant profoundly suppresses the IGF-1-induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Taken together, KCC4 can function as a plasma membrane scaffold protein with ezrin in the assembly of cytoskeletal reorganization complex. The complex of KCC4 and ezrin may be important for regulating the integrity of cortical cytoskeleton and maintaining cell adhesion, which are required for cellular invasive migration (illustrated in Figure 3).

Figure 3
Motor-dependent trafficking of KCC4 is important for cancer cell invasion. (A) The recruitment of KCC4 from the inactive storage pool in the cytosol to the active membranous target pool at the lamellipodia is sensitive to IGF-1 and EGF stimulation. PI3K ...

Potential clinical implication of KCC

Despite the significant improvement in both diagnostic and therapeutic modalities for the treatment of cancer patients, tumor metastasis and cancer recurrence still represent the major cause of mortality [56, 57]. It is important to identify target genes that are consistently upregulated in metastatic and recurrent cancers as the reliable biomarker to predict occurrence, progression or prognosis of human cancers. The previous studies have highlighted the important role of KCl cotransport family in tumor development and progression and have provided the rationale for its application in predicting clinical prognosis of cancer patients. For example, KCC polypeptides and its stimulator, IGF-1 protein, are scanty in normal cervical epithelial and noncancerous stromal tissues, but are abundantly expressed in adjacent cervical cancer tissues [47, 48]. In addition, IGF-1 and KCC colocalize in the surgical specimens of breast cancer, cervical cancer and ovarian cancer, suggesting the cooperation between IGF-1 and KCC in tumor progression. Moreover, the expression of IGF-1 and KCC in surgical specimens shows good linear correlation that in turn corresponds well to tumor size, the in vivo indicator of tumor progression. The close association between the expression of individual KCC isoforms and clinical outcome of cancer patients has also been investigated. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluores-cent stainings with different KCC isoform antibodies indicated that KCC3 is abundant in cervical carcinoma whereas KCC4 in metastatic cervical and ovarian cancer tissues [24, 49]. In the normal or noncancerous cervix, KCC3 protein expression is weak in both normal squamous epithelial and noncancerous stromal tissues [49]. In striking contrast, KCC3 protein is abundant in cervical carcinoma and in the tumor nest invaded deeply into stromal tissues. The expression level of KCC3 mRNA in tumor tissues is closely correlated with tumor size. Like KCC3, KCC4 protein is nearly undetectable in noncancerous cervical squamous epithelial tissues [24]. In contrast, primary cancerous tissues of cervix clearly express KCC4 protein at different levels. Additionally, KCC4 and its stimulators, EGF and IGF-1, are colocalized in the metastatic cancer tissues, suggesting the cooperation between KCC4 and EGF or IGF-1 in tumor metastasis. Higher KCC4 expression in tumor tissues is correlated with higher risk of tumor metastasis and cancer recurrence. Compared with low-grade KCC4 expression, primary tumors with high-grade KCC4 expression present the significantly higher percentage of parametrium invasion and pelvic lymph node metastasis, which are two major poor prognostic factors for early-stage cervical cancer. Consistently, increased KCC4 expression is associated with the poor clinical outcome, including increased percentage of cancer recurrence. Taken together, the findings from molecular qualitative and quantitative studies of KCC in surgical specimens suggest that the expression of KCC3, KCC4, and their stimulators, EGF or IGF-1, exhibit a close association with clinical outcome of cancer patients. Therefore, KCC3, KCC4, EGF, and IGF-1 may be a panel of promising diagnostic bio-markers to predict cancer patient outcome.

Acknowledgments

This work was partly supported by National Science Council, Taiwan and Department of Health, Executive Yuan, Taiwan.

Glossary

Abbreviations

EGF
epidermal growth factor
EMT
epithelial-mesenchymal transition
IGF-1
insulin-like growth factor-1
KCC
potassium chloride cotrans-porter
MMP
matrix metalloproteinase
RBC
red blood cell.

References

1. Dunham PB, Stewart GW, Ellory JC. Chloride-activated passive potassium transport in human erythrocytes. Proc Natl Acad Sci USA. 1980;77:1711–1715. [PubMed]
2. Lauf PK. Evidence for chloride-dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost, Op-sanus tau. J Comp Physiol. 1982;146:9–16.
3. Lauf PK, Theg BE. A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes. Biochem Biophys Res Commun. 1980;92:1422–1428. [PubMed]
4. Lauf PK, Adragna NC. K-Cl cotransport: properties and molecular mechanism. Cell Physiol Biochem. 2000;10:341–354. [PubMed]
5. Gillen CM, Brill S, Payne JA, Forbush B., 3rd Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family. J Biol Chem. 1996;271:16237–16244. [PubMed]
6. Payne JA, Stevenson TJ, Donaldson LF. Molecular characterization of a putative K-Cl cotransporter in rat brain. A neuronal-specific isoform. J Biol Chem. 1996;271:16245–16252. [PubMed]
7. Hiki K, D'Andrea RJ, Furze J, Crawford J, Woollatt E, Sutherland GR, Vadas MA, Gamble JR. Cloning, characterization, and chromosomal location of a novel human K+-Cl- cotransporter. J Biol Chem. 1999;274:10661–10667. [PubMed]
8. Mount DB, Mercado A, Song L, Xu J, George AL, Jr, Delpire E, Gamba G. Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family. J Biol Chem. 1999;274:16355–16362. [PubMed]
9. Race JE, Makhlouf FN, Logue PJ, Wilson FH, Dunham PB, Holtzman EJ. Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter. Am J Physiol. 1999;277:C1210–C1219. [PubMed]
10. Adragna NC, Di Fulvio M, Lauf PK. Regulation of K-Cl cotransport: from function to genes. J Membr Biol. 2004;201:109–137. [PubMed]
11. Mercado A, Song L, Vazquez N, Mount DB, Gamba G. Functional comparison of the K+-Cl cotransporters KCC1 and KCC4. J Biol Chem. 2000;275:30326–30334. [PubMed]
12. Payne JA. Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation. Am J Physiol. 1997;273:C1516–C1525. [PubMed]
13. Song L, Mercado A, Vázquez N, Xie Q, Desai R, George AL, Jr, Gamba G, Mount DB. Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter. Brain Res Mol Brain Res. 2002;103:91–105. [PubMed]
14. Lauf PK, Bauer J, Adragna NC, Fujise H, Zade-Oppen AMM, Ryu KH, Delpire E. Erythrocyte K-Cl cotransport: properties and regulation. Am J Physiol. 1992;263:C917–C932. [PubMed]
15. Flatman PW, Adragna NC, Lauf PK. Role of protein kinases in regulating sheep erythrocyte K -Cl cotransport. Am J Physiol. 1996;271:C255–C263. [PubMed]
16. Jennings ML, al-Rohil N. Kinetics of activation and inactivation of swelling-stimulated K+/Cl transport. The volume-sensitive parameter is the rate constant for inactivation. J Gen Physiol. 1990;95:1021–1040. [PMC free article] [PubMed]
17. Kahle KT, Rinehart J, de Los Heros P, Louvi A, Meade P, Vazquez N, Hebert SC, Gamba G, Gimenez I, Lifton RP. WNK3 modulates transport of Cl in and out of cells: implications for control of cell volume and neuronal excitability. Proc Natl Acad Sci U S A. 2005;102:16783–16788. [PubMed]
18. Garzón-Muvdi T, Pacheco-Alvarez D, Gagnon KB, Vázquez N, Ponce-Coria J, Moreno E, Delpire E, Gamba G. WNK4 kinase is a negative regulator of K+-Cl cotransporters. Am J Physiol Renal Physiol. 2007;292:F1197–F1207. [PubMed]
19. Rinehart J, Maksimova YD, Tanis JE, Stone KL, Hodson CA, Zhang J, Risinger M, Pan W, Wu D, Colangelo CM, Forbush B, Joiner CH, Gulcicek EE, Gallagher PG, Lifton RP. Sites of regulated phosphorylation that control K-Cl cotransporter activity. Cell. 2009;138:525–536. [PMC free article] [PubMed]
20. Holtzman EJ, Kumar S, Faaland CA, Warner F, Logue PJ, Erickson SJ, Ricken G, Waldman J, Kumar S, Dunham PB. Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans. Am J Physiol. 1998;275:F550–F564. [PubMed]
21. Su W, Shmukler BE, Chernova MN, Stuart-Tilley AK, de Franceschi L, Brugnara C, Alper SL. Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation. Am J Physiol. 1999;277:C899–C912. [PubMed]
22. Casula S, Shmukler BE, Wilhelm S, Stuart-Tilley AK, Su W, Chernova MN, Brugnara C, Alper SL. A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity. J Biol Chem. 2001;276:41870–41878. [PubMed]
23. Shen MR, Chou CY, Hsu KF, Hsu YM, Chiu WT, Tang MJ, Alper SL, Ellory JC. KCl cotransport is an important modulator of human cervical cancer growth and invasion. J Biol Chem. 2003;278:39941–39950. [PubMed]
24. Chen YF, Chou CY, Wilkins RJ, Ellory JC, Mount DB, Shen MR. Motor protein-dependent membrane trafficking of KCl cotransporter-4 is important for cancer cell invasion. Cancer Res. 2009;69:8585–8593. [PMC free article] [PubMed]
25. Brugnara C, Bunn HF, Tosteson DC. Regulation of erythrocyte cation and water content in sickle cell anemia. Science. 1986;232:388–390. [PubMed]
26. Lew VL, Bookchin RM. Ion transport pathology in the mechanism of sickle cell dehydration. Physiol Rev. 2005;85:179–200. [PubMed]
27. Joiner CH, Rettig RK, Jiang M, Risinger M, Franco RS. Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes. Blood. 2007;109:1728–1735. [PubMed]
28. Rust MB, Alper SL, Rudhard Y, Shmukler BE, Vicente R, Brugnara C, Trudel M, Jentsch TJ, Hübner CA. Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice. J Clin Invest. 2007;117:1708–1717. [PMC free article] [PubMed]
29. Sallinen R, Tornberg J, Putkiranta M, Horelli-Kuitunen N, Airaksinen MS, Wessman M. Chromosomal localization of SLC12A5/Slc12a5, the human and mouse genes for the neuron-specific K(+)-Cl(−) cotransporter (KCC2) defines a new region of conserved homology. Cytogenet Cell Genet. 2001;94:67–70. [PubMed]
30. Strange K, Singer TD, Morrison R, Delpire E. Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue. Am J Physiol. 2000;279:C860–C867. [PubMed]
31. Hübner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K, Jentsch TJ. Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition. Neuron. 2001;30:515–524. [PubMed]
32. Woo NS, Lu J, England R, McClellan R, Dufour S, Mount DB, Deutch AY, Lovinger DM, Delpire E. Hyperexcitability and epilepsy associated with disruption of the mouse neuronal-specific K-Cl cotransporter gene. Hippocampus. 2002;12:258–268. [PubMed]
33. Li H, Khirug S, Cai C, Ludwig A, Blaesse P, Kolikova J, Afzalov R, Coleman SK, Lauri S, Airaksinen MS, Keinänen K, Khiroug L, Saarma M, Kaila K, Rivera C. KCC2 interacts with the dendritic cy-toskeleton to promote spine development. Neuron. 2007;56:1019–1033. [PubMed]
34. Howard HC, Mount DB, Rochefort D, Byun N, Dupre N, Lu J, Fan X, Song L, Riviere JB, Prevost C, Horst J, Simonati A, Lemcke B, Welch R, England R, Zhan FQ, Mercado A, Siesser WB, George AL, Jr, McDonald MP, Bouchard JP, Mathieu J, Delpire E, Rouleau GA. The K-Cl cotransporter KCC3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum. Nat Genet. 2002;32:384–392. [PubMed]
35. Pearson MM, Lu J, Mount DB, Delpire E. Localization of the K+-Cl cotransporter, KCC3, in the central and peripheral nervous systems: expression in the choroid plexus, large neurons and white matter tracts. Neuroscience. 2001;103:481–491. [PubMed]
36. Shen MR, Chou CY, Hsu KF, Liu HS, Dunham PB, Holtzman EJ, Ellory JC. The KCl cotransporter isoform KCC3 can play an important role in cell growth regulation. Proc Natl Acad Sci USA. 2001;98:14714–14719. [PubMed]
37. Zhang J, Lauf PK, Adragna NC. Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells. Am J Physiol. 2003;284:C674–C680. [PubMed]
38. Boettger T, Rust MB, Maier H, Seidenbecher T, Schweizer M, Keating DJ, Faulhaber J, Ehmke H, Pfeffer C, Scheel O, Lemcke B, Horst J, Leuwer R, Pape HC, Völkl H, Hübner CA, Jentsch TJ. Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold. EMBO J. 2003;22:5422–5434. [PubMed]
39. Rust MB, Faulhaber J, Budack MK, Pfeffer C, Maritzen T, Didié M, Beck FX, Boettger T, Schubert R, Ehmke H, Jentsch TJ, Hübner CA. Neurogenic mechanisms contribute to hypertension in mice with disruption of the K-Cl cotransporter KCC3. Circ Res. 2006;98:549–556. [PubMed]
40. Byun N, Delpire E. Axonal and periaxonal swelling precede peripheral neurodegeneration in KCC3 knockout mice. Neurobiol Dis. 2007;28:39–51. [PMC free article] [PubMed]
41. Velazquez H, Silva T. Cloning and localization of KCC4 in rabbit kidney: expression in distal convoluted tubule. Am J Physiol. 2003;285:F49–F58. [PubMed]
42. Karadsheh MF, Byun N, Mount DB, Delpire E. Localization of the KCC4 potassium-chloride cotransporter in the nervous system. Neuroscience. 2004;123:381–391. [PubMed]
43. Boettger T, Hübner CA, Maier H, Rust MB, Beck FX, Jentsch TJ. Deafness and renal tubular aci-dosis in mice lacking the K-Cl co-transporter Kcc4. Nature. 2002;416:874–878. [PubMed]
44. Shen MR, Chou CY, Ellory JC. Volume-sensitive KCl cotransport associated with human cervical carcinogenesis. Pfluegers Arch. 2000;440:751–760. [PubMed]
45. Fürstenberger G, Senn HJ. Insulin-like growth factors and cancer. Lancet Oncol. 2002;3:298–302. [PubMed]
46. Shen MR, Hsu YM, Hsu KF, Chen YF, Tang MJ, Chou CY. Insulin-like growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation that is modulated by alphavbe-ta3 integrin signaling. Carcinogenesis. 2006;27:962–971. [PubMed]
47. Shen MR, Lin AC, Hsu YM, Chang TJ, Tang MJ, Alper SL, Ellory JC, Chou CY. Insulin-like growth factor 1 stimulates KCl cotransport, which is necessary for invasion and proliferation of cervical cancer and ovarian cancer cells. J Biol Chem. 2004;279:40017–40025. [PubMed]
48. Hsu YM, Chou CY, Chen HH, Lee WY, Chen YF, Lin PW, Alper SL, Ellory JC, Shen MR. IGF-1 upregulates electroneutral K-Cl cotransporter KCC3 and KCC4 which are differentially required for breast cancer cell proliferation and invasive-ness. J Cell Physiol. 2007;210:626–636. [PubMed]
49. Hsu YM, Chen YF, Chou CY, Tang MJ, Chen JH, Wilkins RJ, Ellory JC, Shen MR. KCl cotransporter -3 down-regulates E-cadherin/beta-catenin complex to promote epithelial-mesenchymal transition. Cancer Res. 2007;67:11064–11073. [PubMed]
50. Lee MY, Chou CY, Tang MJ, Shen MR. Epithelial-mesenchymal transition in cervical cancer: correlation with tumor progression, epidermal growth factor receptor overexpression, and snail up-regulation. Clin Cancer Res. 2008;14:4743–4750. [PubMed]
51. Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 2006;172:973–981. [PMC free article] [PubMed]
52. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol. 2006;7:131–142. [PubMed]
53. Aznavorian S, Murphy AN, Stetler-Stevenson WG, Liotta LA. Molecular aspects of tumour cell invasion and metastasis. Cancer. 1993;71:1368–1383. [PubMed]
54. Renehan AG, Zwahlen M, Minder C, O'Dwyer ST, Shalet SM, Egger M. Insulin-like growth factor (IGF)-I, IGF binding protein-3, and cancer risk: systematic review and meta-regression analysis. Lancet. 2004;363:1346–1353. [PubMed]
55. Brand FX, Ravanel N, Gauchez AS, Pasquier D, Payan R, Fagret D, Mousseau M. Prospect for anti-HER2 receptor therapy in breast cancer. Anticancer Res. 2006;26:463–470. [PubMed]
56. Christofori G. New signals from the invasive front. Nature. 2006;441:444–450. [PubMed]
57. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. [PubMed]

Articles from American Journal of Translational Research are provided here courtesy of e-Century Publishing Corporation