Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, AE1.

We identified 11 human pedigrees with dominantly inherited hemolytic anemias in both the hereditary stomatocytosis and spherocytosis classes. Affected individuals in these families had an increase in membrane permeability to Na and K that is particularly marked at 0 degrees C. We found that disease in these pedigrees was associated with a series of single amino-acid substitutions in the intramembrane domain of the erythrocyte band 3 anion exchanger, AE1. Anion movements were reduced in the abnormal red cells. The 'leak' cation fluxes were inhibited by SITS, dipyridamole and NS1652, chemically diverse inhibitors of band 3. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na and K fluxes in the oocytes, and the induced Cl transport was low. These data are consistent with the suggestion that the substitutions convert the protein from an anion exchanger into an unregulated cation channel.

The potassium-chloride cotransporter 2 promotes cervical cancer cell migration and invasion by an ion transport-independent mechanism.

K(+)-Cl(-) cotransporters (KCCs) play a fundamental role in epithelial cell function, both in the context of ionic homeostasis and also in cell morphology, cell division and locomotion. Unlike other ubiquitously expressed KCC isoforms, expression of KCC2 is widely considered to be restricted to neurons, where it is responsible for maintaining a low intracellular chloride concentration to drive hyperpolarising postsynaptic responses to the inhibitory neurotransmitters GABA and glycine. Here we report a novel finding that KCC2 is widely expressed in several human cancer cell lines including the cervical cancer cell line (SiHa). Membrane biotinylation assays and immunostaining showed that endogenous KCC2 is located on the cell membrane of SiHa cells. To elucidate the role of KCC2 in cervical tumuorigenesis, SiHa cells with stable overexpression or knockdown of KCC2 were employed. Overexpression of KCC2 had no significant effect on cell proliferation but dramatically suppressed cell spreading and stress fibre organization, while knockdown of KCC2 showed opposite effects. In addition, insulin-like growth factor 1 (IGF-1)-induced cell migration and invasiveness were significantly increased by overexpression of KCC2. KCC2-induced cell migration and invasion were not dependent on KCC2 transport function since overexpression of an activity-deficient mutant KCC2 still increased IGF-1-induced cell migration and invasion. Moreover, overexpression of KCC2 significantly diminished the number of focal adhesions, while knockdown of KCC2 increased their number. Taken together, our data establish that KCC2 expression and function are not restricted to neurons and that KCC2 serves to increase cervical tumourigenesis via an ion transport-independent mechanism.

In vitro reconstitution of eukaryotic ion channels using droplet interface bilayers.

The ability to routinely study eukaryotic ion channels in a synthetic lipid environment would have a major impact on our understanding of how different lipids influence ion channel function. Here, we describe a straightforward, detergent-free method for the in vitro reconstitution of eukaryotic ion channels and ionotropic receptors into droplet interface bilayers and measure their electrical activity at both the macroscopic and single-channel level. We explore the general applicability of this method by reconstitution of channels from a wide range of sources including recombinant cell lines and native tissues, as well as preparations that are difficult to study by conventional methods including erythrocytes and mitochondria.

Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism.

Hereditary forms of iron-deficiency anemia, including animal models, have taught us much about the normal physiologic control of iron metabolism. However, the discovery of new informative mutants is limited by the natural mutation frequency. To address this limitation, we have developed a screen for heritable abnormalities of red blood cell morphology in mice with single-nucleotide changes induced by the chemical mutagen ethylnitrosourea (ENU). We now describe the first strain, fragile-red, with hypochromic microcytic anemia resulting from a Y228H substitution in the ferrireductase Steap3 (Steap3(Y288H)). Analysis of the Steap3(Y288H) mutant identifies a conserved motif required for targeting Steap3 to internal compartments and highlights how phenotypic screens linked to mutagenesis can identify new functional variants in erythropoiesis and ascribe function to previously unidentified motifs.

B0AT1 Amino Acid Transporter Complexed With SARS-CoV-2 Receptor ACE2 Forms a Heterodimer Functional Unit: In Situ Conformation Using Radiation Inactivation Analysis.

The SARS-CoV-2 receptor, angiotensin-converting enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared with all other human tissues. Enterocyte ACE2 is coexpressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2:B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2:B0AT1] heterodimer pair constitutes a physiological "functional unit." This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on radiation target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size molecular weight 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2:B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2:B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance understanding the physiology of B0AT1 interaction with ACE2 in the gut, and thereby contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul postacute sequelae of SARS-CoV-2.

Structural and functional changes in the membrane and membrane skeleton of red blood cells induced by peroxynitrite.

The changes in passive ion permeability of the red blood cell membrane after peroxynitrite action (3 microM-3 mM) have been studied by biophysical (using radioisotopes of rubidium, sodium and sulphur (sulphate)) and electrophysiological methods. The enhancement of passive membrane permeability to cations (potassium and sodium ions) and the inhibition of anion flux through the anion exchanger in peroxynitrite-treated red blood cells were revealed. In patch-clamp experiments the whole-cell conductance after peroxynitrite (80 microM) treatment of red blood cells increased 3-3.5-fold with a shift in the reversal potential from -7.0+/-1.5 mV to -4.3+/-0.9 mV (n=7, p=0.005). The addition of cobalt and nickel ions to red blood cell suspensions before peroxynitrite treatment had no effect on the peroxynitrite-induced cation flux but zinc ions in the same condition decreased cation flux about 2-fold. Using atomic force microscopy methods we revealed an increase in red blood cell membrane stiffness and the membrane skeleton complexity after peroxynitrite action. We conclude that the peroxynitrite-induced water and ion imbalance and reorganization in membrane structure lead to crenation of red blood cells.

Dengue fever activates the L-arginine-nitric oxide pathway: an explanation for reduced aggregation of human platelets.

In patients with Dengue fever, a viral inflammatory syndrome, haemorrhage is a significant pathological feature, yet the underlying mechanisms remain unclear. Nitric oxide (NO) is an important regulator of platelet function, inhibiting aggregation, recruitment and adhesion to the vascular endothelium. We have investigated whether changes in the activity of the L-arginine-NO pathway in human platelets may account for increased bleeding in patients with Dengue fever. A total of 16 patients with Dengue fever and 18 age-matched healthy volunteers participated in the study. Collagen induced platelet aggregation in a dose-dependent manner in both Dengue patients and controls, but the degree of platelet aggregation was significantly reduced in the patient group. Elevated rates of L-arginine transport in Dengue fever patients were associated with enhanced NO synthase activity and elevated plasma fibrinogen levels. The present study provides the first evidence that Dengue fever is associated with increased L-arginine transport and NO generation and reduced platelet aggregation.

Characterization of sulphate transporters in isolated bovine articular chondrocytes.

Uptake of SO(4) (2-) by articular chondrocytes is an essential step in the pathway for sulphation of glycosaminoglycans (GAGs), with mutations in SO(4) (2-) transport proteins resulting in abnormalities of skeletal growth. In the present study, the transporters mediating SO(4) (2-) transport in bovine articular chondrocytes have been characterized. Expression of candidate transporters was determined using RT-PCR, while SO(4) (2-) transport was measured in radioisotope flux experiments. RT-PCR experiments showed that bovine articular chondrocytes express three transporters known to transport SO(4) (2-): AE2 (SLC4a2), DTDST (SLC26a2), and SLC26a11. Other transporters--NaS-1 (SLC13a1), SAT-1 (SLC26a1), DRA (SLC26a3), SLC26a6 (PAT1), SLC26a7, SLC26a8 (Tat-1), and SLC26a9--were, however, not detected. In functional experiments, SO(4) (2-) uptake was temperature-sensitive, inhibited by 60% by DIDS (50 microM) and exhibited saturation kinetics, with a K(m) value of 16 mM. Uptake was also inhibited at alkaline extracellular pH. In further experiments, a K(i) value for DIDS inhibition of SO(4) (2-) efflux of 5 microM was recorded. A DIDS-sensitive component of SO(4) (2-) efflux persisted in solutions lacking Cl(-) ions. These data are interpreted as evidence for the preferential operation of carrier-mediated exchange of SO(4) (2-) for Cl(-), while an alternative SO(4) (2-)-OH(-) exchange mode is also possible.

Two functionally distinct organic osmolyte pathways in Plasmodium gallinaceum-infected chicken red blood cells.

Red cells infected with the human malaria parasite Plasmodium falciparum have an increased permeability to a range of small, structurally unrelated solutes via a malaria-induced pathway. We report here a similar pathway present in parasitised red cells from chickens infected with the avian malaria parasite, Plasmodium gallinaceum. Parasitised cells showed a marked increase in the rate of influx of sorbitol (76-fold) and, to a lesser degree, taurine (3-fold) when compared with red cells from uninfected chickens. Pharmacological data suggest that both sorbitol and taurine are transported via a single malaria-induced pathway, which is sensitive to inhibition by 5-nitro-2-(3-phenylpropylamino)benzoic acid (IC(50) approximately 7 microM). The malaria-induced pathway differed in its inhibition by a range of anion channel inhibitors when compared to the endogenous, volume-activated osmolyte pathway of chicken red cells. There were also differences in the selectivity of sorbitol and taurine by the two permeation routes. The data presented here are consistent with the presence of two distinct organic solute pathways in infected chicken red cells. The first is an endogenous volume-activated pathway, which is not activated by the parasite and the second is a malaria-induced pathway, similar to those that are induced by other types of malaria in other host species.

The new permeability pathways: targets and selective routes for the development of new antimalarial agents.

The malaria parasite, Plasmodium falciparum, spends part of its complex life cycle within the red blood cells of a human host. During this time, the parasite alters the permeability of the red blood cell's plasma membrane to allow the uptake of nutrients, the removal of "waste" and volume and ion regulation of the infected cell. The increased permeability is due to the induction of new permeability pathways (NPP), which are obvious chemotherapeutic antimalarial targets and/or selective routes for drugs, which target the internal parasite. This review covers our present understanding of the NPP, the methods used to screen for putative inhibitors of the NPP, the current repertoire of NPP inhibitors and the problems that need to be addressed to realise the potential of the NPP as antimalarial targets. In addition, the review will cover the use of the NPP as specific drug delivery routes.

Anion exchanger isoform 2 operates in parallel with Na(+)/H(+) exchanger isoform 1 during regulatory volume decrease of human cervical cancer cells.

Intracellular pH (pH(i)) homeostasis was investigated in human cervical cancer SiHa cells undergoing regulatory volume decrease (RVD) to determine which transport systems were involved. Using isoform-specific primers, mRNA transcripts of Na(+)/H(+) exchanger isoform 1 (NHE1) and isoform 3 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) and the results confirmed by Western immunoblotting. From anion exchanger isoforms 1-3 (AE1-3), only the mRNA transcript of AE2 was identified by RT-PCR and the identity was confirmed by digestion with a specific restriction endonuclease. SiHa cells loaded with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and resuspended in isotonic media showed a stable pH(i). In contrast, a gradual internal acidification took place following resuspension in hypotonic media. The NHE inhibitors, HOE694 (10 microM) and amiloride (1 mM), showed a similar potency in enhancing the rate and extent of the hypotonicity-induced internal acidification. The absence of extracellular Na(+) also substantially enhanced the acidification during RVD. These results suggest that internal acidification during RVD is mainly compensated by the operation of NHE1. Extracellular Cl(-) was critically necessary for the pH(i) acidification during RVD. The hypotonicity-induced acidification was significantly attenuated by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, a concentration inhibiting more than 90% AE activity. This indicates that AE2 mediates a net Cl(-) influx with compensating HCO(3)(-) efflux during RVD. We conclude that AE2 operates in parallel with NHE1 to regulate pH(i) during RVD of human cervical cancer cells.

Plasmodium falciparum-induced channels.

To survive within a red blood cell, the malaria parasite alters dramatically the permeability of the host's plasma membrane (allowing the uptake of essential nutrients and the removal of potentially hazardous metabolites). The pathway(s) responsible for the increased permeability have been proposed as putative chemotherapeutic targets and/or selective routes for antimalarial agents that target the internal parasite. This review covers our current understanding of this parasite-induced phenomenon in Plasmodium falciparum-infected human red blood cells. In particular, recent electrophysiological studies, using the patch-clamp technique, are reviewed.

Oxygen dependence of K(+)-Cl- cotransport in human red cell ghosts and sickle cells.

KCC activity in normal human red cells (containing haemoglobin A, HbA, and termed HbA cells) is O2-dependent, being active in oxygenated cells but inactive in deoxygenated ones. The mechanism for O2 dependence is unknown but a role for Hb has been suggested. In this paper, we address two main questions. First, do membrane ghosts prepared from HbA cells retain an O2-sensitive KCC activity? Second, how is the response of KCC to changes in O2 tension altered in sickle cell patients heterozygous for HbS and HbC? We found that substantial Cl(-)-dependent K+ influx, indicative of KCC activity, was present in both pink (5-10% normal Hb complement) and white (no measurable Hb) ghosts when equilibrated with air. KCC responded to deoxygenation in pink ghosts only (86 +/- 10% inhibition, mean+/-S.E.M., n = 3), whilst KCC activity in white ghosts remained high (23 +/- 8% inhibition). Results indicate that pink ghosts retain an O2-dependent KCC activity but that this is lost in white ghosts. Second, HbSC-containing red cells showed sickling (88 +/- 3%) when deoxygenated, together with activation of the deoxygenation-induced cation pathway (Psickle) and the Gardos channel. KCC activity, however, was elevated in oxygenated HbSC cells, but inhibited by deoxygenation. Thus Hb polymerisation and sickling could be dissociated from the abnormal response of KCC to deoxygenation observed in HbS-containing red cells. These preparations provide a useful system with which to study the components involved in O2-sensitive membrane transport and why it is perturbed in certain pathological conditions (such as sickle cell disease and oxidant toxicity).

The emerging role of KCl cotransport in tumor biology.

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/beta-catenin complex formation by inhibiting the transcription of E-cadherin gene and accelerating the proteosome-dependent degradation of beta-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.

Motor protein-dependent membrane trafficking of KCl cotransporter-4 is important for cancer cell invasion.

The KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I-induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I- or EGF-dependent cancer spread.

Deoxygenation-induced non-electrolyte pathway in red cells from sickle cell patients.

Red cells from patients with sickle cell disease contain HbS rather than the normal HbA (here termed HbS cells). On deoxygenation, HbS cells exhibit a distinctive solute permeability pathway, P(sickle), activated stochastically, and partially inhibited by DIDS and dipyridamole. It is often referred to as a cation channel although its permeability characteristics remain vague and its molecular identity is unknown. We show that, in contrast to normal red cells, a proportion of HbS cells underwent haemolysis when deoxygenated in isosmotic non-electrolyte solutions. Haemolysis was stochastic: cells unlysed after an initial deoxygenation pulse showed lysis when harvested, reoxygenated and subsequently exposed to a second period of deoxygenation. O(2) dependence of haemolysis was similar to that of P(sickle) activation. Haemolysis was accompanied by high rates of sucrose influx, and both haemolysis and sucrose influx were inhibited by DIDS and dipyridamole. Sucrose influx was only detected as ionic strength was reduced below 80 mM. These findings are consistent with the postulate that deoxygenation of HbS cells, under certain conditions, activates a novel non-electrolyte pathway. Their significance lies in understanding the nature of the deoxygenation-induced permeability in HbS cells, together with its relationship with novel pathways induced by a variety of manipulations in normal red cells.

The effect of deoxygenation on whole-cell conductance of red blood cells from healthy individuals and patients with sickle cell disease.

Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed P(sickle), contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na(+) and K(+), but was less permeable to NMDG(+) and Cl(-). Conductance to Ca(2+) was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 microM), Zn(2+) (100 microM), and by Gd(3+) (IC(50) of approximately 2 microM). It therefore shares some properties with P(sickle). These findings represent the first electrical recordings of single HbS cells and will facilitate progress in understanding altered red blood cell cation transport characteristics of SCD.

IGF-1 upregulates electroneutral K-Cl cotransporter KCC3 and KCC4 which are differentially required for breast cancer cell proliferation and invasiveness.

The cellular function of electroneutral K-Cl cotransport (KCC) is to regulate epithelial ion transport and osmotic homeostasis. Here we investigate the mechanisms by which insulin-like growth factor 1 (IGF-1) cooperates with KCC to modulate breast cancer biology. IGF-1 stimulates KCC activity of MCF-7 breast cancer cells in a dose- and time-dependent manner. Increased KCC3 and KCC4 abundances contribute to IGF-1-enhanced KCC activity. Endogenous cellular invasiveness was modestly attenuated by KCC4-specific siRNA and the residual invasiveness was much less sensitive to IGF-1 stimulation. KCC3 knockdown significantly reduced basal growth rate and almost abolished IGF-1-stimulated cell proliferation. Consistently, MCF-7 cells obtained advantage in cell proliferation and invasiveness by overexpression of KCC3 and KCC4, respectively. Blockade of gene transcription by actinomycin D abolished IGF-1-mediated increase in KCC3 and KCC4 mRNA, indicating that IGF-1 increases KCC abundance through the regulation of KCC genes. IGF-1 treatment triggered phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) cascades which were differentially required for IGF-1-stimulated biosynthesis of KCC3 and KCC4. Loss-of-function mutations in KCC significantly inhibited the development and progression of xenograft tumor in SCID mice. The expression level of IGF-1 and KCC polypeptides in the surgical specimens showed a good linear correlation, suggesting autocrine or paracrine IGF-1 stimulation of KCC production in vivo. Among patients with early-stage node-negative breast cancer, disease-free survival (DFS) and overall survival (OS) curves were significantly different based on IGF-1 and KCC expression. Thus, we conclude that KCC activation by IGF-1 plays an important role in IGF-1 receptor signaling to promote growth and spread of breast cancer cells.

KCl cotransporter-3 down-regulates E-cadherin/beta-catenin complex to promote epithelial-mesenchymal transition.

The potassium chloride cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. Here, we investigate if KCC is involved in the regulation of epithelial-mesenchymal transition (EMT), a critical cellular event of malignancy. E-cadherin and beta-catenin colocalize in the cell-cell junctions, which becomes more obvious in a time-dependent manner by blockade of KCC activity in cervical cancer SiHa and CaSki cells. Real-time reverse transcription-PCR on the samples collected from the laser microdissection indicates that KCC3 is the most abundant KCC isoform in cervical carcinoma. The characteristics of EMT appear in KCC3-overexpressed, but not in KCC1- or KCC4-overexpressed cervical cancer cells, including the elongated cell shape, increased scattering, down-regulated epithelial markers (E-cadherin and beta-catenin), and up-regulated mesenchymal marker (vimentin). Some cellular functions are enhanced by KCC3 overexpression, such as increased invasiveness and proliferation, and weakened cell-cell association. KCC3 overexpression decreases mRNA level of E-cadherin. The promoter activity assays of various regulatory sequences confirm that KCC3 expression is a potent negative regulator for human E-cadherin gene expression. The proteosome inhibitor restores the decreased protein abundance of beta-catenin by KCC3 overexpression. In the surgical specimens of cervical carcinoma, the decreased E-cadherin amount was accompanied by the increased KCC3 abundance. Vimentin begins to appear at the invasive front and becomes significantly expressed in the tumor nest. In conclusion, KCC3 down-regulates E-cadherin/beta-catenin complex formation by inhibiting transcription of E-cadherin gene and accelerating proteosome-dependent degradation of beta-catenin protein. The disruption of E-cadherin/beta-catenin complex formation promotes EMT, thereby stimulating tumor progression.