3611

ILK

The role of integrin-linked kinase (ILK) in transforming growth factor beta (TGFbeta)-mediated epithelial to mesenchymal transition was investigated. A stable transfection of dominant-negative ILK results in the prevention of TGFbeta-mediated E-cadherin delocalization. TGFbeta-mediated phosphorylation of Akt at Ser-473 was inhibited by dominant-negative ILK and PI3K inhibitors, LY294002 and wortmannin. Treatment with TGFbeta stimulated induction of Akt and ILK kinase activity in HaCat control cells. This increased ILK activity by TGFbeta was lowered by PI3K inhibitor, LY294002. In addition, PI3K inhibitor, dominant-negative Akt, and dominant-negative ILK could not block TGFbeta-mediated C-terminal phosphorylation of Smad2. Taken together, these data suggest that PI3K-ILK-Akt pathway that is independent of the TGFbeta-induced Smad pathway is required for TGFbeta-mediated epithelial to mesenchymal transition.

PURPOSE: To evaluate the expression and location of integrin-linked kinase (ILK) within the mouse lens and to characterize the role of this protein during mouse lens epithelial cells (LEC) differentiation in vitro. METHODS: Transcription levels of ILK mRNA were determined by RT-PCR in cultured cells and lens tissue. ILK protein was detected by immunoblotting, immunocytochemistry, immunohistochemistry, and immunoprecipitation. A role for ILK in the outgrowth of LEC from dissected mouse lens explants was determined by the use of ILK short interfering RNA (siRNA). Affinity-purified polyclonal anti-recombinant human ILK IgG was prepared and characterized for these experiments. A comparison of several anti-ILK antibodies was performed by immunoblotting, immunoprecipitation, and ELISA. RESULTS: ILK was transcribed in LEC and lens fiber cells in vivo. ILK protein was expressed in the differentiating LEC at the equatorial region of the lens and, to a lesser extent, within the cortical and nuclear fiber cells. LEC in vitro produced copious ILK, which exhibited a filamentous pattern throughout the cytoplasm. The expression of ILK was increased during epithelial-mesenchymal-transition (EMT) of LEC from lens explants, whereas inhibition of ILK by siRNA delayed expression of the EMT markers smooth muscle alpha-actin and fibronectin. CONCLUSIONS: Analysis of ILK expression, localization, and activity in the mouse lens and cultured LEC is substantially facilitated by the generation of a multi-functional, polyclonal, affinity-purified anti-ILK antibody. Expressed in most tissues and cells lines, ILK is unexpectedly restricted to the equatorial LEC and differentiated fiber cells of the mouse lens. The occurrence of ILK expression with LEC differentiation is consistent with the positive regulatory function of ILK, which is revealed in a model of EMT in vitro. This is the first study to show the expression of ILK in the lens and its unique distribution pattern within cultured lens epithelia.

PINCH-1 is an adaptor protein that binds to the integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays a critical role in mediating tubular epithelial-to-mesenchymal transition (EMT). To determine whether PINCH-1 is also involved in the EMT process, we investigated its regulation and function during TGF-beta1-stimulated EMT. TGF-beta1 induced PINCH-1 mRNA and protein expression in human proximal tubular epithelial cells in a time-dependent fashion, an effect that was largely dependent on intracellular Smad signaling. Overexpression of PINCH-1 suppressed epithelial markers E-cadherin and ZO-1 and increased fibronectin expression and extracellular assembly, whereas knockdown of PINCH-1 via small interfering RNA reduced TGF-beta1-mediated fibronectin expression and partially restored E-cadherin. PINCH-1 formed a ternary complex with ILK at the focal adhesion sites of tubular epithelial cells. Treatment with an ILK inhibitor or disruption of the ILK/PINCH-1 interaction by overexpressing a dominant-negative N-terminal ankyrin domain of ILK resulted in reduced fibronectin deposition, indicating that the ability of PINCH-1 to stimulate EMT is ILK-dependent. In a mouse model of obstructive nephropathy, PINCH-1 expression increased in a time-dependent manner, suggesting that it may play a role in EMT and renal fibrosis in vivo. We conclude that PINCH-1, through its interaction with ILK, plays an important role in regulating TGF-beta1-mediated EMT and could be a potential future therapeutic target to prevent progression of renal disease.

BACKGROUND: Increasing evidence suggests that connective tissue growth factor (CTGF) is involved in the epithelial-to-mesenchymal transition (EMT). The exact intracellular events that drive this process, however, are not fully understood. In this study, we investigated the role of integrin-linked kinase (ILK) in mediating CTGF-induced EMT. METHODS: The expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin upon the stimulation by recombinant human CTGF (rhCTGF) in cultured human tubular epithelial cell line (HK-2) was detected by real-time RT-PCR and Western blot. Subsequently, the role of ILK was determined by using ILK siRNA. RESULTS: rhCTGF increased the mRNA expression of alpha-SMA significantly in a dose- and time-dependent manner, while E-cadherin mRNA decreased in a dose- and time-dependent manner. alpha-SMA protein was up-regulated after stimulation by 5 ng/ml CTGF for 96 h, and increased further after stimulation by 50 ng/ml. An immunocytochemical study showed that alpha-SMA was initially detectable at 48 h, and increased further at 72 h, while there was almost no alpha-SMA immunostaining observed in the control group at the same time point. E-cadherin protein was also down-regulated in a dose-dependent manner. Transfection of HK-2 cells with ILK-siRNA significantly attenuated rhCTGF-induced alpha-SMA induction and E-cadherin repression. CONCLUSION: Our study suggested that ILK mediated the effect of EMT in proximal tubular epithelial cells stimulated by CTGF.CI - (c) 2007 S. Karger AG, Basel.

Integrin-linked kinase (ILK) is an intracellular serine/threonine protein kinase that regulates cell adhesion, survival, and epithelial-to-mesenchymal transition (EMT). In this study, we investigated the kinase activity of ILK during tubular EMT induced by TGF-beta1 and examined the therapeutic potential of an ILK inhibitor in obstructive nephropathy. TGF-beta1 induced a biphasic activation of ILK in renal tubular epithelial cells, with rapid activation starting at 5 min and the second wave of activation peaking at 24 h; the latter paralleled the induction of ILK protein expression. Pharmacologic inhibition of ILK with small-molecule inhibitor QLT-0267 abolished TGF-beta1-induced phosphorylation of Akt and glycogen synthase kinase-3beta, suppressed cyclin D1 expression, and largely restored the expression of E-cadherin and zonula occludens 1. Inhibition of ILK also blocked TGF-beta1-mediated induction of fibronectin, Snail1, plasminogen activator inhibitor 1, and matrix metalloproteinase 2. In a mouse model of obstructive nephropathy, administration of QLT-0267 inhibited beta-catenin accumulation; suppressed Snail1, alpha-smooth muscle actin, fibronectin, vimentin, and type I and type III collagen expression; and reduced total tissue collagen content. Inhibition of ILK did not affect kidney structure or function in normal mice. These findings suggest that increased ILK activity mediates EMT and the progression of renal fibrosis. Pharmacologic inhibition of ILK signaling may hold therapeutic potential for fibrotic kidney diseases.

AIMS: epithelial-mesenchymal transition (EMT) has been known to play a significant role in tumour progression. Integrin-linked kinase (ILK) has been recently added to the growing list of EMT regulators that control some aspect of carcinogenesis. The aim was to study ILK expression and its relevance to EMT markers in human basal cell carcinoma (BCC). METHODS AND RESULTS: Paraffin-embedded tissue sections from 100 human BCC cases were processed by immunohistochemistry for the expression of ILK, E-cadherin, Snail, beta-catenin and alpha-smooth muscle actin (alpha-SMA). ILK overexpression was observed in 100% of cases and strongly correlated with tumour invasion and infiltrative BCC. Loss of membranous E-cadherin was found in 71% of cases while nuclear immunoreactivity for E-cadherin was also observed in 90% of the tumours. Snail, nuclear beta-catenin and alpha-SMA expression was detected in 100%, 99% and 97% of tumours, respectively. Aberrant expression of E-cadherin, nuclear beta-catenin and alpha-SMA correlated with BCC tumour invasion. Interestingly, there was a significant correlation between ILK expression and all the EMT markers examined. CONCLUSIONS: ILK overexpression in BCC is implicated in tumour progression probably through the induction of an EMT-related molecular profile. Nuclear localization of E-cadherin in BCC is also associated with aggressive tumour features.

Integrin-linked kinase (ILK) is a multifunctional serine/threonine kinase in cytoplasm. Recent studies showed that cancer patients with increased ILK expression had low survival, poor prognosis and increased metastasis. Although the causes of ILK overexpression remain to be fully elucidated, accumulating evidence suggests that its oncogenic capacity derives from its regulation of several downstream targets that provide cells with signals that promote proliferation, survival and migration. However, the mechanisms underlying tumor metastasis by ILK is still not fully understood. epithelial-mesenchymal transition (EMT) is a critical event of cancer cells that triggers invasion and metastasis. We recently reported that knockdown of ILK inhibited the growth and induced apoptosis in human bladder cancer cells. Therefore, we postulate that ILK might involve in EMT. Here we further investigate the function of ILK with RNA interference in bladder cancer cells. Knockdown of ILK impeded an EMT with low Vimentin, Snail, Slug and Twist as well as high E-cadherin expression in vivo and vitro. In addition, we found that knockdown of ILK inhibited cell proliferation, migration and invasion as well as changed cell morphology, adhesion and rearranged cytoskeleton in vitro. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3beta, increased expression of nm23-H1, as well as reduced expression of MMP-2 and MMP-9 in vivo and vitro. Furthermore, downregulation of ILK could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. Finally, the effects of ILK siRNA on bladder cancer cell phenotype and invasiveness translate into suppression for tumorigenesis and metastasis in vivo. Taken together, our findings highlight that ILK signaling pathway plays a novel role in the development of bladder cancer through regulating EMT. ILK could be a promising diagnostic marker and therapeutic target for bladder cancer.

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein possibly involved in biological functions other than the inhibition of RNase A and angiogenin activities. We have previously shown that RI can inhibit growth and metastasis in some cancer cells. epithelial-mesenchymal transition (EMT) is regarded as the beginning of invasion and metastasis and has been implicated in the metastasis of bladder cancer. We therefore postulate that RI regulates EMT of bladder cancer cells. We find that the over-expression of RI induces the up-regulation of E-cadherin, accompanied with the decreased expression of proteins associated with EMT, such as N-cadherin, Snail, Slug, vimentin and Twist and of matrix metalloprotein-2 (MMP-2), MMP-9 and Cyclin-D1, both in vitro and in vivo. The up-regulation of RI inhibits cell proliferation, migration and invasion, alters cell morphology and adhesion and leads to the rearrangement of the cytoskeleton in vitro. We also demonstrate that the up-regulation of RI can decrease the expression of integrin-linked kinase (ILK), a central component of signaling cascades controlling an array of biological processes. The over-expression of RI reduces the phosphorylation of the ILK downstream signaling targets p-Akt and p-GSK3beta in T24 cells. We further find that bladder cancer with a high-metastasis capability shows higher vimentin, Snail, Slug and Twist and lower E-cadherin and RI expression in human clinical specimens. Finally, we provide evidence that the up-regulation of RI inhibits tumorigenesis and metastasis of bladder cancer in vivo. Thus, RI might play a novel role in the development of bladder cancer through regulating EMT and the ILK signaling pathway.

epithelial-to-mesenchymal transition (EMT) is a process for fully differentiated epithelial cells to undergo a phenotypic change to fibroblasts via diverse intracellular signaling pathways. While the pivotal role of fibroblasts in renal fibrosis is widely accepted, their origin remains undefined. In addition, although a large number of studies have provided evidence of EMT in human kidney diseases, specific signaling pathways leading to EMT have not yet been discovered in humans. To evaluate the origin of interstitial fibroblasts and signaling pathways involved in the EMT process, we analyzed the differential expression of EMT-related molecules in paraffin-fixed sections from 19 human fibrotic kidneys and 4 control kidneys. In human fibrotic kidneys, tubular epithelial cells (TECs) with intact tubular basement membrane (TBM) showed loss or down-regulation of an epithelial marker (E-cadherin), de novo expression of mesenchymal markers (vimentin and fibronectin), and significant up-regulation of inducers and mediators controlling the EMT process (transforming growth factor-beta1 (TGF-beta1), p-Smad2/3, beta1-integrin, p38 mitogen-activated protein kinase (MAPK), WNT5B and beta-catenin) in the areas of interstitial inflammation and fibrosis, compared with their expression in control kidneys. In conclusion, the type II EMT process in humans is thought to be an adaptive response of TECs to chronic injury and is regulated by interconnections of TGF-beta/Smad, integrin/integrin-linked kinase (ILK) and wnt/beta-catenin signaling pathways.

Integrin-linked kinase (ILK), a ubiquitously expressed and evolutionally conserved serine/threonine kinase, has been shown to be aberrantly overexpressed and activated in diversified types of human malignancies, including colorectal cancer (CRC). However, the potential role of ILK in cancer cell migration and invasion remains to be elucidated. In this study, we introduced the human ILK gene into a low ILK-expressing human CRC cell line SW480. Cell migration and invasion were evaluated by the wound healing assay and transwell invasion assay, respectively. The epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot analysis or immunofluorescence. We found that enforced overexpression of ILK in SW480 cells dramatically promoted their migratory and invasive ability in vitro. Furthermore, SW480 cells stably overexpressing ILK underwent EMT, as indicated by mesenchymal morphology, decreased expression of E-cadherin, and increased expression of vimentin, Snail, and Slug. Finally, the nuclear factor (NF)-kappaB inhibitor BAY 11-7028 or NF-kappaB p65 small interfering RNA significantly restored the reduced E-cadherin level in ILK-overexpressing cells, suggesting that ILK-mediated down-regulation of E-cadherin is dependent on NF-kappaB activation. Overall, our study demonstrates a pivotal role of ILK in EMT and metastasis, and suggests novel therapeutic opportunities for the treatment of CRC.CI - Copyright (c) 2013 Elsevier GmbH. All rights reserved.

Renal cell carcinoma (RCC) is the most common malignancy in the kidney. Antiangiogenic targeted therapies inhibit the progression of RCC, but have limited impacts on invasion or metastasis of tumor cells. Integrin-linked kinase (ILK) is a serine/threonine kinase implicated in the regulation of cell growth/survival, cell-cycle progression, epithelial-mesenchymal transition (EMT), invasion/migration, and angiogenesis. However, the role of ILK in RCC has not been evaluated. We investigated the role of ILK on cancer progression and metastasis and the therapeutic potential of ILK inhibition in RCC. Our investigation reveals that ILK is expressed at a low level in normal cells and low-stage RCC cells and is highly expressed in advanced and metastatic cells. Caki-1, a metastatic RCC cell line, showed higher expression of molecular EMT markers, including Snail and Zeb1, but decreased activity of GSK3beta. Knockdown of ILK using small interference (si)-ILK minimally inhibited tumor proliferation and cell-cycle progression was not significantly affected. However, ILK knockdown suppressed the formation of stress fibers and focal adhesions and impeded phenotypic EMT markers, including cell migration and invasion, in Caki-1 and UMRC-3 cells. Finally, in vivo knockdown of ILK suppressed the progression, invasion, and metastasis of primary RCC in nude mice by downregulation of EMT markers (Snail, Zeb1, vimentin, and E-cadherin). Our results show that ILK may be essential for invasion and metastasis in RCC and regulates vimentin and E-cadherin expression by regulating the EMT-related transcription factors Snail and Zeb1. These results suggest that ILK may be a potential target in RCC.CI - (c)2015 American Association for cancer Research.

Twist, a highly conserved basic Helix-Loop-Helix transcription factor, functions as a major regulator of epithelial-mesenchymal transition (EMT) and tumor metastasis. In different cell models, signaling pathways such as TGF-beta, MAPK/ERK, WNT, AKT, JAK/STAT, Notch, and P53 have also been shown to play key roles in the EMT process, yet little is known about the signaling pathways regulated by Twist in tumor cells. Using iTRAQ-labeling combined with 2D LC-MS/MS analysis, we identified 194 proteins with significant changes of expression in MCF10A-Twist cells. These proteins reportedly play roles in EMT, cell junction organization, cell adhesion, and cell migration and invasion. ECM-receptor interaction, MAPK, PI3K/AKT, P53 and WNT signaling were found to be aberrantly activated in MCF10A-Twist cells. Ingenuity pathways Analysis showed that integrin beta1 (ITGB1) acts as a core regulator in linking integrin-linked kinase (ILK), Focal-adhesion kinase (FAK), MAPK/ERK, PI3K/AKT, and WNT signaling. Increased Twist and ITGB1 are associated with breast tumor progression. Twist transcriptionally regulates ITGB1 expression. Over-expression of ITGB1 or Twist in MCF10A led to EMT, activation of FAK/ILK, MAPK/ERK, PI3K/AKT, and WNT signaling. Knockdown of Twist or ITGB1 in BT549 and Hs578T cells decreased activity of FAK, ILK, and their downstream signaling, thus specifically impeding EMT and cell invasion. Knocking down ILK or inhibiting FAK, MAPK/ERK, or PI3K/AKT signaling also suppressed Twist-driven EMT and cell invasion. Thus, the Twist-ITGB1-FAK/ILK pathway and their downstream signaling network dictate the Twist-induced EMT process in human mammary epithelial cells and breast cancer cells.CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved.

Although periostin was confirmed to facilitate the pathogenesis of endometriosis by enhancing the migration, invasion, and adhesion of human endometrial stromal cells (ESCs), its effect on the endometrial epithelial cells (EECs) is still unknown. The current study aimed to determine whether periostin enhanced the epithelial-mesenchymal transition (EMT) of EECs. EECs were isolated from 12 women with endometriosis. The migration and invasion abilities of EECs were evaluated by transwell assays. expressions of proteins were detected by western blot. After treatment with periostin, the migration and invasion abilities of EECs were enhanced. Additionally, E-cadherin and keratin were downregulated while N-cadherin and vimentin were upregulated in EECs. Simultaneously, levels of ILK, p-Akt, slug, and Zeb1 were all upregulated in EECs. After silencing the expression of ILK in EECs, levels of p-Akt, slug, Zeb1, N-cadherin, and vimentin were downregulated while E-cadherin and keratin were upregulated. Although periostin weakened the above effects in EECs after silencing the expression of ILK, it failed to induce the EMT of EECs. Thus, periostin enhanced invasion and migration abilities of EECs and facilitated the EMT of EECs through ILK-Akt signaling pathway. Playing a pivotal role in the pathogenesis of endometriosis, periostin may be a new clinical therapy target for endometriosis.

OBJECTIVE: To determine whether emodin facilitates the mesenchymal-epithelial transition (MET) of endometrial stromal cells (ESCs) as well as to explore the mechanism through which emodin favored the MET of ESCs. METHODS: Cell viability was tested by methyl thiazolyl tetrazolium assay. Cell migration and invasion abilities were detected by transwell assays. Levels of integrin-linked kinase (ILK) and epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot. RESULTS: Upregulated ILK and increased abilities of migration and invasion were confirmed in the eutopic and ectopic ESCs (EuSCs and EcSCs), especially in the EcSCs. After treated with emodin, the expression of ILK was statistically downregulated in EcSCs, resulting in the MET and decreased migration and invasion abilities of EcSCs. Additionally, silencing of the ILK gene in EcSCs also achieved the above-mentioned effects, which were strengthened by emodin. Furthermore, exogenous expression of ILK in control ESCs (CSCs) resulted in the EMT and increased abilities of migration and invasion of CSCs, which can be abrogated by emodin. Besides, exogenous expression of ILK also abrogated the effects of emodin on CSCs. CONCLUSION: Emodin inhibits the migration and invasion abilities of human ESCs by facilitating the MET through targeting ILK.CI - (c) The Author(s) 2016.

BACKGROUND: Integrin-linked kinase (ILK) is a multifunctional adaptor protein which is involved with protein signalling within cells to modulate malignant (cancer) cell movement, cell cycle, metastasis and epithelial-mesenchymal transition (EMT). Our previous experiment demonstrated that ILK siRNA inhibited the growth and induced apoptosis of bladder cancer cells as well as increased the expression of Ribonuclease inhibitor (RI), an important cytoplasmic protein with many functions. We also reported that RI overexpression inhibited ILK and phosphorylation of AKT and GSK3beta. ILK and RI gene both locate on chromosome 11p15 and the two genes are always at the adjacent position of same chromosome during evolution, which suggest that ILK and RI could have some relationship. However, underlying interacting mechanisms remain unclear between them. Here, we postulate that RI might regulate ILK signaling pathway via interacting with ILK. METHODS: Co-immunoprecipitation, GST pull-down and co-localization under laser confocal microscope assay were used to determine the interaction between ILK and RI exogenously and endogenously. Furthermore, we further verified that there is a direct binding between the two proteins by fluorescence resonance energy transfer (FRET) in cells. Next, The effects of interplay between ILK and RI on the key target protein expressions of PI3K/AKT/mTOR signaling pathway were determined by western blot, immunohistochemistry and immunofluorescence assay in vivo and in vitro. Finally, the interaction was assessed using nude mice xenograft model. RESULTS: We first found that ILK could combine with RI both in vivo and in vitro by GST pull-down, co-immunoprecipitation (Co-IP) and FRET. The protein levels of ILK and RI revealed a significant inverse correlation in vivo and in vitro. Subsequently, The results showed that up-regulating ILK could increase cell proliferation, change cell morphology and regulate cell cycle. We also demonstrated that the overexpression of ILK remarkably promoted EMT and expressions of target molecules of ILK signaling pathways in vitro and in vivo. Finally, we found that ILK overexpression significantly enhanced growth, metastasis and angiogenesis of xenograft tumor; Whereas, RI has a contrary role compared to ILK in vivo and in vitro. CONCLUSIONS: Our findings, for the first time, directly proved that the interplay between ILK and RI regulated EMT via ILK/PI3K/AKT signaling pathways for bladder cancer, which highlights the possibilities that ILK/RI could be valuable markers together for the therapy and diagnosis of human carcinoma of urinary bladder.

epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite the anticancer capabilities of emodin observed in many cancers, including EOC, the underlying molecular mechanism remains to be elucidated. A crucial link has been discovered between the acquisition of metastatic traits and the epithelial-mesenchymal transition (EMT). The present study aimed to determine whether emodin could inhibit the EMT of EOC cells and explore the underlying mechanism. The CCK-8 assay and transwell assay showed that emodin effectively repressed the abilities of proliferation, invasion, and migration in A2780 and SK-OV-3 cells. The Western blot showed that emodin upregulated epithelial markers (E-cadherin and Claudin) while it downregulated mesenchymal markers (N-cadherin and Vimentin) and transcription factor (Slug) in a dose-dependent fashion. After transfection of siRNA-Slug, both Slug and N-cadherin were downregulated in EOC cells while E-cadherin was upregulated, which was intensified by emodin. Besides, emodin decreased the expression of ILK, p-GSK-3beta, beta-catenin, and Slug. Transfection of siRNA-ILK also achieved the same effects, which was further strengthened by following emodin treatment. Nevertheless, SB216763, an inhibitor of GSK-3beta, could reverse the effects of emodin except for ILK expression. These findings suggest that emodin inhibited the EMT of EOC cells via ILK/GSK-3beta/Slug signaling pathway.

OBJECTIVE: Adenomyosis is a benign gynecological disease, characterized by the malignant biological behaviors of invasion and metastasis. ILK plays an important role in intercellular adhesion and triggers the process of EMT. In this study, we investigated the role of ILK-induced EMT in the pathogenesis of adenomyosis. METHODS: ILK and EMT markers including E-cadherin, N-cadherin and Vimentin have been detected with Immunohistochemistry(IHC), RT-PCR and Western Blot, in normal endometrium, matched eutopic and ectopic endometrium respectively. Primary endometrial cells were isolated in order to observed the morphology features, as well as the change of invasiveness. RESULTS: Hyper-activation of ILK were detected in the adenomyosis lesions, along with the typical aberrant expression of EMT markers. Furthermore, comparing with ESCs, the EuSCs showed a more invasive and dynamic phenotype. CONCLUSIONS: ILK-induced EMT is a novel mechanism in the pathogenesis of adenomyosis and may be a potential therapeutic agent for adenomyosis.CI - Copyright (c) 2018 The Authors. Published by Elsevier Inc. All rights reserved.

BACKGROUND/AIM: epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critically implicated in cancer metastasis and chemoresistance. Herein, we investigated integrin-linked kinase (ILK)'s role in human colon cancer (CRC) progression and chemoresistance in relation to EMT and CSC markers. PATIENTS AND METHODS: expression of ILK, EMT and CSC markers were evaluated by immunohistochemistry in 149 CRC samples. We also generated colon cancer cells resistant to 5-FU and oxaliplatin and studied the effect of ILK inhibition on drug response by MTT assay and on EMT and CSC markers' expression. RESULTS: ILK expression in human CRC correlates with EMT and CSC markers and is associated with metastasis and chemoresistance. ILK inhibition increases sensitivity of resistant cells to 5-FU and oxaliplatin and reduces the levels of EMT and CSC markers in 5-FU resistant cells. CONCLUSION: ILK overexpression in human CRC associates with EMT and CSC traits, contributing to tumor progression and chemoresistance.CI - Copyright(c) 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Phyllodes tumors (PTs) of the breast constitute an uncommon group of mammary fi broepithelial lesions with ambiguous biologic behavior. Recent evidence suggests that epithelial mesenchymal transition (EMT), a driving force of cancer progression is implicated in PTs pathogenesis. Integrin-linked kinase (ILK), a focal adhesion kinase, has been implicated in cancer and EMT and represents a novel cancer therapeutic target. In this study, we aimed to investigate ILK and EMT markers expression in phyllodes breast tumors in relation to tumor grade. expression of ILK and EMT markers E-cadherin, beta-catenin, Nu-cadherin, vimentin, Snail, ZEB1 and Twist was evaluated by immunohistochemistry in paraffin-embedded tissue sections from 96 human phyllodes breast tumors (48 benign, 27 borderline, 21 malignant). Cytoplasmic and nuclear immunopositivity of ILK were observed in both the epithelial and the stromal component of phyllodes breast tumors and were significantly higher with increasing tumor grade. An EMT-related expression profile consisting of decreased membranous and increased nuclear/cytoplasmic immunoreactivity of E-cadherin and beta-catenin and increased expression of N-cadherin, vimentin, Snail, ZEB1 and Twist was observed in tumor epithelial and stromal component and was significantly associated with malignant phyllodes breast tumor histopathology. Interestingly, there was a significant correlation of ILK expression with all of the EMT markers examined. Our results suggest that EMT significantly contributes to phyllodes tumor pathogenesis and originally implicate ILK and ZEB1 in phyllodes tumors malignant phenotype.

epithelial-mesenchymal transition (EMT) and migration play an important role in tumor progression, and lipoxin (LX), the 'stop signal' for inflammation, has been studied in basic research for its anti-inflammatory or inflammatory pro-resolving properties. Here, in the in vitro experiment, we showed that LXA4 could inhibit the EMT and migration in phorbol myristate acetate (PMA) or activated conditioned medium (ACM)-stimulated Hep3B cells by downregulation of integrin-linked kinase (ILK), a pseudokinase in cytoplasm and these effects were via inhibiting the phosphorylation of Akt and GSK3beta. Morover, LXA4 could not affect the EMT and migration of PMA-stimulated Hep3B cells by knockdown of ILK. In the in vivo experiment, BML-111 (the analog of LXA4) could inhibit the EMT and metastasis of hepatocarcinoma cells. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3beta, decreased expression of MMP-2 and MMP-9. These results showed that LXA4 could be a possible candidate for liver cancer therapy, and blocking ILK axis would be an effective drug target.CI - Copyright (c) 2018 Elsevier Inc. All rights reserved.

epithelial-mesenchymal transition (EMT) plays a significant part in the pathogenesis of endometriosis by facilitating the migration and invasion abilities of cells. Integrin-linked kinase (ILK) increases the cell migration and invasion abilities by inducing the EMT. Eutopic and control endometrial stromal cells (EuSCs and CSCs) were isolated and cultured. Cell migration and invasion abilities were detected by transwell assays. Levels of proteins were detected by Western blot. EuSCs showed higher levels of ILK, N-cadherin, vimentin and stronger migration and invasion abilities. After transfection of siRNA-ILK, E-cadherin and keratin levels were increased while N-cadherin and vimentin levels were decreased in EuSCs. Besides that, the migration and invasion abilities of EuSCs were significantly decreased after transfection of siRNA-ILK. On the contrary, levels of ILK, N-cadherin and vimentin were increased while levels of E-cadherin and keratin were decreased simultaneously after transfecting CSCs with pEGFP-C1-ILK. Simultaneously, the migration and invasion abilities of CSCs were increased after transfection of pEGFP-C1-ILK. Our study verified that high expression of ILK enhanced the migration and invasion abilities of ESCs by facilitating the EMT. Given that ILK played crucial roles in the pathogenesis of endometriosis, it may be considered as a promising targeted therapy for endometriosis.

OBJECTIVE: To investigate the role of integrin-linked kinase (ILK) on renal tubular epithelial-mesenchymal transition and the regulatory effect of urokinase on LIK expression in mice with obstructive nephropathy. METHODS: Normal male mice were randomly divided into sham-operated group (n=20), unilateral ureteral obstruction (UUO) group (n=28), and UUO with urokinase treatment group (uPA, n=28), and UUO was induced surgically in the latter two groups. The mice were sacrificed on days l, 3, 7 and 14 after the surgery, and renal interstitial fibrosis (RIF) was graded according to the result of Masson staining. The expression of ILK in the renal tissues of the rats was examined by immunofluorescence staining and Western blotting, and the expression of E-cadherin was detected by immunohistochemistry. RT-PCR was used to examine the mRNA expressions of ILK, E-cadherin and alpha-smooth muscle actin (alpha-SMA). RESULTS: The expressions of ILK mRNA and protein were significantly increased in UUO group, but significantly decreased by treatment with uPA (P<0.05). The expression of alpha-SMA mRNA level was significantly increased, while E-cadherin decreased in mice with UUO on day 3 after the surgery. Treatment with uPA significantly inhibited such effects (P<0.05). CONCLUSION: ILK plays an important role in renal interstitial fibrosis by mediating epithelial-mesenchymal transition. Urokinase attenuates renal tubulointerstitial fibrosis in mice with UUO possibly by inhibiting ILK expression and preventing tubular epithelial-mesenchymal transition.

Proteinuria is a primary clinical symptom of a large number of glomerular diseases that progress to end-stage renal failure. Podocyte dysfunctions play a fundamental role in defective glomerular filtration in many common forms of proteinuric kidney disorders. Since binding of these cells to the basement membrane is mediated by integrins, we determined the role of integrin-linked kinase (ILK) in podocyte dysfunction and proteinuria. ILK expression was induced in mouse podocytes by various injurious stimuli known to cause proteinuria including TGF-beta1, adriamycin, puromycin, and high ambient glucose. Podocyte ILK was also found to be upregulated in human proteinuric glomerular diseases. Ectopic expression of ILK in podocytes decreased levels of the epithelial markers nephrin and ZO-1, induced mesenchymal markers such as desmin, fibronectin, matrix metalloproteinase-9 (MMP-9), and alpha-smooth muscle actin (alpha-SMA), promoted cell migration, and increased the paracellular albumin flux across podocyte monolayers. ILK also induced Snail, a key transcription factor mediating epithelial-mesenchymal transition (EMT). Blockade of ILK activity with a highly selective small molecule inhibitor reduced Snail induction and preserved podocyte phenotypes following TGF-beta1 or adriamycin stimulation. In vivo, this ILK inhibitor ameliorated albuminuria, repressed glomerular induction of MMP-9 and alpha-SMA, and preserved nephrin expression in murine adriamycin nephropathy. Our results show that upregulation of ILK is a convergent pathway leading to podocyte EMT, migration, and dysfunction. ILK may be an attractive target for therapeutic intervention of proteinuric kidney diseases.

BACKGROUND: Overexpression of connective tissue growth factor (CTGF) in alveolar type II epithelial (AT II) cells disrupts alveolar structure, causes interstitial fibrosis, and upregulates integrin-linked kinase (ILK). Whether CTGF-ILK signaling induces epithelial to mesenchymal transition (EMT) in AT II cells is unknown. METHODS: Transgenic mice with targeted overexpression of CTGF in AT II cells were generated utilizing the surfactant protein C (SP-C) gene promoter and doxycycline-inducible system. AT II cells were isolated from 4-wk-old CTGF-overexpressing (CTGF+) mice and control littermates, and cultured on Matrigel. Cells were transfected with ILK siRNA, and cell morphology and expression of cell differentiation markers were analyzed. RESULTS: The AT II cells from the control lungs grew in clusters and formed alveolar-like cysts and expressed SP-C. In contrast, the cells from CTGF+ lungs were spread and failed to form alveolar-like cysts. These cells expressed higher levels of CTGF, alpha smooth muscle actin (alpha-SMA), fibronectin and vimentin, the mesenchymal markers, suggesting EMT-like changes. Transfection with ILK siRNA not only dramatically attenuated ILK expression, but also decreased alpha-SMA expression as well as reversed cell morphological changes in CTGF+ AT II cells. CONCLUSION: Overexpression of CTGF induces EMT in mouse primary AT II cells and this is mediated by ILK.

TGF-beta1 induces epithelial-mesenchymal transition (EMT) and autophagy in a variety of cells. However, the role of autophagy in TGF-beta1-induced EMT has not been clearly elucidated and the underlying mechanisms are unclear. In the present study, we found that TGF-beta1 induced both autophagy and EMT in mouse tubular epithelial C1.1 cells. Inhibition of autophagy by 3-methyladenine or siRNA knockdown of Beclin 1 reduced TGF-beta1-induced increase of vimentin and decreased E-cadherin expression. In contrast, rapamycin-associated enhancement of TGF-beta1-induced autophagy increased EMT of C1.1 cells. Serum rescue inhibited autophagy followed by reversal of EMT. Blocking of autophagosome-lysosomal but not proteosomal degradation reduced the decrease of E-cadherin, demonstrating a role for autophagy in degradation of E-cadherin during EMT. Autophagy promoted the activation of Src and Src-associated phosphorylation of beta-catenin at Y-654 leading to pY654-beta-catenin/p-Smad2 complex formation. Chromatin immunoprecipitation assay demonstrated binding by the pY654-beta-catenin/p-Smad2 complex to ILK promoter thus increasing ILK expression. Taken together, our results demonstrate that TGF-beta1-induced autophagy links beta-catenin and Smad signaling to promote EMT in C1.1 cells through a novel pY654-beta-catenin/p-Smad2/ILK pathway. The pathway delineated links disruption of E-cadherin/beta-catenin-mediated cell-cell contact to induction of EMT via upregulation of ILK.CI - Copyright (c) 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.