Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling.

Interaction of cells with the extracellular matrix (ECM) results in the regulation of cell growth, differentiation and migration by coordinated signal transduction through integrins and growth-factor receptors. Integrins achieve signalling by interacting with intracellular effectors that couple integrins and growth-factor receptors to downstream components. One well-studied effector is focal-adhesion kinase (FAK), but recently another protein kinase, integrin-linked kinase (ILK), has been identified as a receptor-proximal effector of integrin and growth-factor signalling. ILK appears to interact with and be influenced by a number of different signalling pathways, and this provides new routes for integrin-mediated signalling. This article discusses ILK structure and function and recent genetic and biochemical evidence about the role of ILK in signal transduction.     

Relevance of the stroma and epithelial-mesenchymal transition (EMT) for the rheumatic diseases

Epithelial-mesenchymal transition (EMT) is a term applied to the process whereby cells undergo a switch from an epithelial phenotype with tight junctions, lateral, apical, and basal membranes, and lack of mobility into mesenchymal cells that have loose interactions with other cells, are non-polarized, motile and produce an extracellular matrix. The importance of this process was initially recognized from a very early step in embryology, but more recently as a potential mechanism for the progression and spread of epithelial cancers. As the sequence of morphological changes has become understood in molecular terms, diseases characterized by alterations in stromal elements and fibrosis are being considered as examples of EMT. This review will focus on the pathogenetic features of immune-mediated renal disease, systemic sclerosis and rheumatoid arthritis that could be explained by EMT.     

Integrin linked kinase (ILK) expression and function in vascular smooth muscle cells

Vascular smooth muscle cell (SMC) migration and proliferation contribute to arterial wound repair and thickening of the intimal layer in atherosclerosis, restenosis and transplant vascular disease. These processes are influenced by cell adhesion to molecules present in the extracellular matrix, and regulated by the integrin family of cell-surface matrix receptors. An important signaling molecule acting downstream of integrin receptors is integrin-linked kinase (ILK), a serine/threonine kinase and scaffolding protein. ILK has been implicated in cancer cell growth and survival through modulation of downstream targets, notably Akt and glycogen synthase kinase-3β (GSK3β). Evidence also exists to establish ILK as a molecular adaptor protein linking integrins to the actin cytoskeleton and regulating actin polymerization, and this function may not necessarily depend upon the kinase activity of ILK. ILK has been implicated in anchorage-independent growth, cell cycle progression, epithelial-mesenchymal transition (EMT), invasion and migration. In addition, ILK has been shown to be involved in vascular development, tumor angiogenesis and cardiac hypertrophy. Despite the documented involvement of integrin signaling in vascular pathologies, the function of ILK has not been well characterized in the SMC response to vascular injury. This brief review summarizes and puts into context the current literature on ILK expression and function in the vascular smooth muscle cell.Key words: smooth muscle cell, migration, extracellular matrix, atherosclerosis, cytoskeletonA large body of research is dedicated to elucidating the mechanisms by which smooth muscle cells (SMCs) contribute to thickening of the arterial wall in pathologies such as atherosclerosis and restenosis. After arterial injury and during neointimal hyperplasia, SMCs undergo a phenotypic switch characterized by the transition from a quiescent to an active/synthetic phenotype, and they begin to synthesize an abundant extracellular matrix.¹ In turn, interactions between cells and the matrix govern the process of neointimal thickening.² Cell surface integrin receptors play important roles in signaling proliferative and migratory cellular responses during arterial wound repair. Integrin-linked kinase (ILK) is an important downstream mediator of integrin signaling, yet little is known of its function in the arterial response to injury.Integrin-linked kinase (ILK) was originally identified as a serine-threonine kinase binding to the cytoplasmic domain of β₁- and β₃-integrin subunits.³ ILK functions to activate Akt and inhibit glycogen synthase kinase-3β (GSK3β),^4–6 and has been implicated in cancer cell growth and survival through modulation of these downstream targets. Given its role in anchorage-independent growth, survival and cell cycle progression,⁷ epithelial-mesenchymal transition (EMT), and invasion and migration,^8,9 it is often suggested that ILK be targeted for cancer treatment.¹⁰ ILK is also involved in vascular development^11,12 and tumor angiogenesis.^13,14Concurrent studies in model organisms and cell cultures point to a role for ILK as a molecular scaffold linking integrins to the actin cytoskeleton and regulating actin polymerization.^15–17 Furthermore, this scaffolding function may be independent of the kinase activity of ILK. In C. elegans, genetic ablation of pat-4/ilk (ILK homologue) leads to severe adhesion defects, muscle detachment and embryonic lethality.¹⁵ However PAT-4/ILK does not phosphorylate GSK3β in C. elegans.¹⁵ Similarly, in Drosophila melanogaster, loss of function mutants for ILK resulted in severe embryonic muscle-attachment defects and detachment of F-actin from the cell membrane, and the muscle attachment defect was rescued by expressing a kinase-deficient ILK.^15,17 Finally, tissue-specific conditional knockout of ILK in mouse chondrocytes results in defects in the skeleton,^18,19 and inhibition of cell adhesion, spreading and cytoskeletal assembly in chondrocytes in culture.¹⁸ These deficiencies were not attributable to impaired Akt or GSK3β signaling. In fact, the importance of ILK kinase function appears to be cell type-dependent. Inhibition of ILK activity in transformed cells resulted in a decrease in Akt phosphorylation and apoptosis, but had no effect in non-transformed cell types including vascular SMCs, thus calling into question the importance of ILK as a kinase in non-cancerous cell types.²⁰We have studied the function of ILK in vascular smooth muscle cell wound repair and found that ILK acted as a scaffolding protein at focal adhesion sites.²¹ In our experiments, immunostaining of cultured SMCs revealed co-localization of ILK and paxillin at focal adhesions, a finding which is consistent with a previous study.²² Several proteins such as PINCH1, parvins and paxillin interact directly with ILK to facilitate its localization to focal adhesions and coordinate actin organization and cell spreading.^23–25 Overexpression of an ILK-binding-deficient PINCH protein in tracheal SMCs led to decreased recruitment of ILK and PINCH to focal adhesions, and decreased association between ILK, paxillin and vinculin.²⁶We hypothesized that ILK acting as a scaffolding protein might regulate the SMC response to vascular injury. To study this, we examined ILK using in vitro models mimicking vascular injury. Silencing ILK expression with siRNA decreased cell adhesion to fibronectin, and accelerated cell proliferation and wound closure.²¹ However, silencing ILK in wounded SMCs did not attenuate the increase in Akt and GSK3β phosphorylation observed after wounding.²¹ Nonetheless, we observed rearrangement of focal adhesions and stress fibers in ILK-silenced SMCs, which may have contributed to the reduced adhesion to fibronectin and enhanced cell migration and proliferation. Thus it seems that the scaffolding role of ILK may be more important for focal adhesion dynamics and remodeling in SMCs than the kinase function of ILK. These results were also surprising because they imply that ILK functions to inhibit cell growth and motility, unlike several studies which have suggested that ILK signals to increase these processes.^7,8,10To address in vivo arterial wound repair, we studied ILK expression after balloon catheter injury of the rat carotid artery. Following balloon injury, SMCs undergo a process of dedifferentiation which includes enhanced proliferation and migration from the media to the intima. We found that ILK protein expression was dramatically decreased in the media during the SMC proliferative and migratory responses.21 The rapid decrease in ILK protein expression is consistent with the effects of silencing ILK in cultured SMCs. We propose that the decrease in ILK following injury facilitates the rearrangement of focal adhesions, altering cell adhesion to facilitate SMC migration and proliferation. The decrease in ILK expression in SMCs following injury may be related to the transition of these cells to a de-differentiated state. A recent study has shown that increased ILK expression correlates with cell differentiation in the luminal layers of the epithelium in the esophagus, colon and intestines when compared to the basal layers.²⁷ ILK was also prominent in more differentiated areas of malignant tumors. In our studies, we noted an increase in ILK expression in the layers of the intima closest to the vascular lumen. This was consistent with findings in another recent study reporting increased ILK protein expression in the intima of balloon-injured rat carotid arteries in vivo and in the developing intima of human saphenous veins cultured ex vivo.²⁸ We suggest that ILK is upregulated here in coincidence with the re-establishment of SMC quiescence.In addition to maintaining stable cell adhesion to matrix, in the quiescent differentiated SMC, ILK may function to mediate contraction and aid the cell in exerting force on surrounding extracellular matrix fibers. In SMCs, ILK is localized to myofilaments, and promotes cell contraction by directly phosphorylating myosin light chain (MLC) or myosin light chain phosphatase (MLCP).^9,29,30 Alternatively, ILK may activate smooth-muscle contraction indirectly via phosphorylation and activation of MLCP inhibitors including CPI-17 and PHI-1.²⁹ Consistent with a role for ILK in mediating contraction, stimulation of tracheal SMCs with acetycholine recruits ILK and PINCH to the cell membrane, and overexpression of an ILK-binding-deficient mutant PINCH attenuated the localization of ILK at adhesion sites, and attenuated actin polymerization, the activation of the actin nucleation initiator N-WASP, and the development of tension.²⁶ ILK has also been identified as a key regulator of cardiac myocyte contractility.³¹ Likewise, ILK is required in the skeletal muscle of zebrafish for integrin-matrix adhesion to maintain the stability of muscle fibres.³² Mice with a skeletal muscle-specific deletion of ILK develop muscular dystrophy and detachment of muscle cells from basement membranes.³³ ILK mutants also showed displacement of several focal adhesion proteins and reorganization of the actin cytoskeleton.³⁴Our results after silencing ILK expression differ somewhat from a previous study of ILK in vascular SMCs. Overexpression of wild- type ILK in SMCs increased cell migration in response to stromal derived factor-1 or angiotensin II, while overexpression of a kinase-dead mutant of ILK (E359K) suppressed SMC migration in Boyden chamber assays.³⁵ In contrast to this study, we have shown the effects of inhibiting endogenous ILK by siRNA. ILK-induced quiescence of SMC may require tight regulation of intracellular ILK levels such that both its suppression and its upregulation promote cell motility.Taken together, these studies reveal that the functions of ILK are broader and more complex than originally thought. This molecule has the potential to function as an adapter protein regulating cytoskeletal assembly and signal transduction from focal adhesion sites, as a protein kinase activating several signaling axes, and as a regulator of the mitotic spindle.^36,37 The breadth of ILK function in regulating cell-matrix interactions, cytoskeletal organization and cell signaling is of great importance to normal development and disease progression. Functional studies using both kinase-deficient ILK variants and ILK siRNA will allow researchers to specifically attribute cellular behaviors to the proposed functions of ILK, and to determine their relative importance in different cells and pathologies. Based on our studies using injury models mimicking cellular events in occlusive vascular disease, we propose that ILK functions to maintain SMCs in a stationary, contractile phenotype in the normal artery. Following arterial injury, decreased ILK expression facilitates the reorganization of focal adhesions and the actin cytoskeleton, allowing for more efficient SMC migration and proliferation to establish a thickened neointima.     

Chronic kidney disease induced by an adenine rich diet upregulates integrin linked kinase (ILK) and its depletion prevents the disease progression

Kidney fibrosis is one of the main pathological findings of progressive chronic kidney disease (CKD) although the pathogenesis of renal scar formation remains incompletely explained. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix (ECM) and intracellular signaling pathways, is involved in several pathophysiological processes during renal damage. However, ILK contribution in the CKD progress remains to be fully elucidated. In the present work, we studied 1) the renal functional and structural consequences of CKD genesis and progression when ILK is depleted and 2) the potential of ILK depletion as a therapeutic approach to delay CKD progression. We induced an experimental CKD model, based on an adenine-supplemented diet on adult wild-type (WT) and ILK-depleted mice, with a tubulointerstitial damage profile resembling that is observed in human CKD. The adenine diet induced in WT mice a progressive increase in plasma creatinine and urea concentrations. In the renal cortex it was also observed tubular damage, interstitial fibrosis and progressive increased ECM components, pro-inflammatory and chemo-attractant cytokines, EMT markers and TGF-β1 expressions. These observations were highly correlated to a simultaneous increase of ILK expression and activity. In adenine-fed transgenic ILK-depleted mice, all these changes were prevented. Additionally, we evaluated the potential role of ILK depletion to be applied after the disease induction, as an effective approach to interventions in human CKD subjects. In this scenario, two weeks after the establishment of adenine-induced CKD, ILK was abrogated in WT mice and stabilized renal damage, avoiding CKD progression. We propose ILK to be a potential target to delay renal disease progression.     

Integrin-linked kinase (ILK) binding to paxillin LD1 motif regulates ILK localization to focal adhesions

Paxillin is a focal adhesion adapter protein involved in integrin signaling. Paxillin LD motifs bind several focal adhesion proteins including the focal adhesion kinase, vinculin, the Arf-GTPase-activating protein paxillin-kinase linker, and the newly identified actin-binding protein actopaxin. Microsequencing of peptides derived from a 50-kDa paxillin LD1 motif-binding protein revealed 100% identity with integrin-linked kinase (ILK)-1, a serine/threonine kinase that has been implicated in integrin, growth factor, and Wnt signaling pathways. Cloning of ILK from rat smooth muscle cells generated a cDNA that exhibited 99.6% identity at the amino acid level with human ILK-1. A monoclonal antibody raised against a region of the carboxyl terminus of ILK, which is identical in rat and human ILK-1 protein, recognized a 50-kDa protein in all cultured cells and tissues examined. Binding experiments showed that ILK binds directly to the paxillin LD1 motif in vitro. Co-immunoprecipitation from fibroblasts confirmed that the association between paxillin and ILK occurs in vivo in both adherent cells and cells in suspension. Immunofluorescence microscopy of fibroblasts demonstrated that endogenous ILK as well as transfected green fluorescent protein-ILK co-localizes with paxillin in focal adhesions. Analysis of the deduced amino acid sequence of ILK identified a paxillin-binding subdomain in the carboxyl terminus of ILK. In contrast to wild-type ILK, paxillin-binding subdomain mutants of ILK were unable to bind to the paxillin LD1 motif in vitro and failed to localize to focal adhesions. Thus, paxillin binding is necessary for efficient focal adhesion targeting of ILK and may therefore impact the role of ILK in integrin-mediated signal transduction events.     

Osteoclast‐specific inactivation of the integrin‐linked kinase (ILK) inhibits bone resorption

Bone resorption requires the adhesion of osteoclasts to extracellular matrix (ECM) components, a process mediated by the α_vβ₃ integrin. Following engagement with the ECM, integrin receptors signal via multiple downstream effectors, including the integrin‐linked kinase (ILK). In order to characterize the physiological role of ILK in bone resorption, we generated mice with an osteoclast‐specific Ilk gene ablation by mating mice with a floxed Ilk allele with TRAP‐Cre transgenic mice. The TRAP‐Cre mice specifically excised floxed alleles in osteoclasts, as revealed by crossing them with the ROSA26R reporter strain. Osteoclast‐specific Ilk mutant mice appeared phenotypically normal, but histomorphometric analysis of the proximal tibia revealed an increase in bone volume and trabecular thickness. Osteoclast‐specific Ilk ablation was associated with an increase in osteoclastogenesis both in vitro and in vivo. However, the mutant osteoclasts displayed a decrease in resorption activity as assessed by reduced pit formation on dentin slices in vitro and decreased serum concentrations of the C‐terminal telopeptide of collagen in vivo. Interestingly, compound heterozygous mice in which one allele of Ilk and one allele of the β₃ integrin gene were inactivated (ILK^+/?; β) also had increased trabecular thickness, confirming that β₃ integrin and Ilk form part of the same genetic cascade. Our results show that ILK is important for the function, but not the differentiation, of osteoclasts. J. Cell. Biochem. 110: 960–967, 2010. © 2010 Wiley‐Liss, Inc.     

Role of the integrin-linked kinase (ILK) in determining neuronal polarity

The establishment of axon-dendrite polarity in mammalian neurons has recently been shown to involve the kinases Akt and GSK-3beta. Here we report the function of the integrin-linked kinase (ILK) in neuronal polarization. ILK distribution is differential: with more of it present in the axonal tips than that in the dendritic tips of a polarized neuron. Inactivation of ILK by chemical inhibitors, a kinase-inactive mutant or siRNAs inhibited axon formation, whereas a kinase hyperactive ILK mutant induced the formation of multiple axons. Biochemical studies indicate that ILK is upstream of Akt and GSK-3beta. Manipulations of multiple intracellular components indicate that ILK is functionally upstream of Akt and GSK-3beta but downstream of PI3K in neuronal polarity. These results reveal a key role of ILK in the formation of neuronal polarity and suggest a signaling pathway important for neuronal polarity.     

Differential susceptibility to epithelial-mesenchymal transition (EMT) of alveolar, bronchial and intestinal epithelial cells in vitro and the effect of angiotensin II receptor inhibition

The generation of myofibroblasts via epithelial-mesenchymal transition (EMT), a process through which epithelial cells lose their polarity and become motile mesenchymal cells, is a proposed contributory factor in fibrosis of a number of organs. Currently, it remains unclear to what extent epithelia of the upper airways and large intestine are susceptible to this process. Herein, we investigated the ability of model cell lines of alveolar (A549), bronchial (Calu-3) and colonic (Caco-2) epithelial cells to undergo EMT when challenged with transforming growth factor-β1 (TGF-β1) and other pro-inflammatory cytokines. Western blot and immunofluorescence microscopy demonstrated that A549 cells readily underwent EMT, as evidenced by a spindle-like morphology, increase in the mesenchymal marker, vimentin, and down-regulation of E-cadherin, an epithelial marker. In contrast, neither Calu-3 nor Caco-2 cells exhibited morphological changes nor alterations in marker expression associated with EMT. Moreover, whilst stimulation of A549 cells enhanced migration and reduced their proliferative capacity, no such effect was observed in epithelial cell lines of the bronchus or colon. In addition, concomitant treatment of A549 cells with telmisartan, an angiotensin II receptor antagonist with antifibrotic properties, was found to reduce cytokine-induced collagen I production and cell migration, although expression levels of vimentin and E-cadherin remained unaltered. Mechanistically, telmisartan failed to inhibit phosphorylation of Smad2/3. Together, these results, using representative in vitro models of the alveolus, bronchus and colon, tentatively suggest that epithelial cell plasticity and susceptibility to EMT may differ depending on its tissue origin. Furthermore, our investigations point to the beneficial effect of telmisartan in partial abrogation of alveolar EMT.     

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells

Background

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.

Methods

Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.

Results

The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.

Conclusions

Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.     

Mutations of GADD45G in rabbits cause cleft lip by the disorder of proliferation,apoptosis and epithelial-mesenchymal transition (EMT)

The cleft lip with or without cleft palate (CL/P) is one of the most common congenital defects in humans. Genome-wide association studies (GWAS) have been widely used for identifying candidate genes, and different genes or chromosomal regions have shown strong evidence for the presence of causal genes in CL/P. To date, two independent GWAS have identified GADD45G as influencing risk for CL/P. However, there is no animal model evidence about GADD45G related to CL/P. Here, we reported the generation of a novel GADD45G mutated rabbit model by CRISPR/Cas9 and CRISPR-based BE4-Gam systems. The homozygous (GADD45G^−/−) while not heterozygous (GADD45G^+/−) pups died after birth due to severe craniofacial defects of unilateral or bilateral cleft lip (CL). Moreover, the disorder of proliferation, apoptosis and epithelial-mesenchymal transition (EMT) were also determined in the medial and lateral nasal processes (MNP and LNP) of the embryonic day 13 (E13) GADD45G^−/− rabbits, which compared with the normal wild type (WT) rabbits. Thus, our study confirmed for the first time that loss of GADD45G lead to CL at the animal level and provided new insights into the crucial role of GADD45G for upper lip formation and fusion.     

Aldose reductase-mediated induction of epithelium-to-mesenchymal transition (EMT) in lens

Cataract is a key factor in the morbidity associated with diabetes. While the pathogenesis of diabetic cataract formation is poorly understood, previous research has identified aldose reductase (ALR2) as a key player. To elucidate a potential role for this enzyme in diabetic cataract formation, we created a series of transgenic mice designed for expression of human ALR2 (AKR1B1) in epithelial and outer cortical fiber cells of the lens. One of the founder lines, designated PAR39, developed an early onset cataract that involved formation of a plaque of cells at the anterior aspect of the lens. These cells appear to separate from the anterior epithelium and undergo a dramatic change that is reminiscent of the epithelial to mesenchymal transition (EMT). We characterized this phenotype in the PAR39 strain by examining rates of cell proliferation and by immunostaining for markers of EMT. Incorporation of the thymidine analog bromodeoxyuridine (BrdU) was used to estimate cell proliferation in two functional areas of the lens epithelium: the mitotically active germinative zone (GZ) and the less proliferative center zone (CZ). Staining cell nuclei with diamido 4',6-diamidino-2-phenylindole (DAPI) was used to establish a total cell count in the demarcated areas. Lens epithelium in PAR39 transgenic mice demonstrated a decrease in the percentage of BrdU/DAPI staining within the GZ as compared to nontransgenic littermate controls (8.1% vs. 10.9%). A similar decrease in BrdU/DAPI was observed in the CZ (0.6% compared to 3.3%). However, cell density was greater within the GZ of PAR39 mice as compared with nontransgenic controls, while it was not significantly different in the CZ among the two groups. Furthermore, cells associated with the epithelial plaque did not stain positive for BrdU, but were strongly positive for alpha-smooth muscle actin, a classical marker for EMT. These findings suggest that ALR2 over-expression is associated with an alteration in the balance between proliferation and apoptosis of epithelial cells in the mouse lens, and that cells associated with epithelial plaques in the PAR39 lens have features in common with cells undergoing EMT.     

Activation of Notch pathway is linked with epithelial-mesenchymal transition in prostate cancer cells

Notch signaling has been reported to play an essential role in tumorigenesis. Several studies have suggested that Notch receptors could be oncoproteins or tumor suppressors in different types of human cancers. Emerging evidence has suggested that Notch pathway regulates cell growth, apoptosis, cell cycle, and metastasis. In the current study, we explore whether Notch-1 could regulate the cell invasion and migration as well as EMT (epithelial-mesenchymal transition) in prostate cancer cells. We found that overexpression of Notch-1 enhanced cell migration and invasion in PC-3 cells. However, downregulation of Notch-1 retarded cell migration and invasion in prostate cancer cells. Importantly, we observed that overexpression of Notch-1 led to EMT in PC-3 cells. Notably, we found that EMT-type cells are associated with EMT markers change and cancer stem cell phenotype. Taken together, we concluded that downregulation of Notch-1 could be a promising approach for inhibition of invasion in prostate cancer cells, which could be useful for the treatment of metastatic prostate cancer.