Integrin-linked Kinase Inhibitor Research Articles

β1 Integrin Signaling Maintains Human Epithelial Progenitor Cell Survival In Situ and Controls Proliferation, Apoptosis and Migration of Their Progeny

β1 integrin regulates multiple epithelial cell functions by connecting cells with the extracellular matrix (ECM). While β1 integrin-mediated signaling in murine epithelial stem cells is well-studied, its role in human adult epithelial progenitor cells (ePCs) in situ remains to be defined. Using microdissected, organ-cultured human scalp hair follicles (HFs) as a clinically relevant model for studying human ePCs within their natural topobiological habitat, β1 integrin-mediated signaling in ePC biology was explored by β1 integrin siRNA silencing, specific β1 integrin-binding antibodies and pharmacological inhibition of integrin-linked kinase (ILK), a key component of the integrin-induced signaling cascade. β1 integrin knock down reduced keratin 15 (K15) expression as well as the proliferation of outer root sheath keratinocytes (ORSKs). Embedding of HF epithelium into an ECM rich in β1 integrin ligands that mimic the HF mesenchyme significantly enhanced proliferation and migration of ORSKs, while K15 and CD200 gene and protein expression were inhibited. Employing ECM-embedded β1 integrin-activating or -inhibiting antibodies allowed to identify functionally distinct human ePC subpopulations in different compartments of the HF epithelium. The β1 integrin-inhibitory antibody reduced β1 integrin expression in situ and selectively enhanced proliferation of bulge ePCs, while the β1 integrin-stimulating antibody decreased hair matrix keratinocyte apoptosis and enhanced transferrin receptor (CD71) immunoreactivity, a marker of transit amplifying cells, but did not affect bulge ePC proliferation. That the putative ILK inhibitor QLT0267 significantly reduced ORSK migration and proliferation and induced massive ORSK apoptosis suggests a key role for ILK in mediating the ß1 integrin effects. Taken together, these findings demonstrate that ePCs in human HFs require β1 integrin-mediated signaling for survival, adhesion, and migration, and that different human HF ePC subpopulations differ in their response to β1 integrin signaling. These insights may be exploited for cell-based regenerative medicine strategies that employ human HF-derived ePCs.

Inactivation of the Hippo tumour suppressor pathway by integrin-linked kinase

One of the hallmarks of cancers is the silencing of tumour suppressor genes and pathways. The Hippo tumour suppressor pathway is inactivated in many types of cancers, leading to tumour progression and metastasis. However, the mechanisms of pathway inactivation in tumours remain unclear. Here we demonstrate that integrin-linked kinase (ILK) plays a critical role in the suppression of the Hippo pathway via phospho-inhibition of MYPT1-PP1, leading to inactivation of Merlin. Inhibition of ILK in breast, prostate and colon tumour cells results in the activation of the Hippo pathway components MST1 and LATS1 with concomitant inactivation of YAP/TAZ (Yes-associated protein/transcriptional co-activator with PDZ-binding motif) transcriptional co-activators and TEAD-mediated transcription. Genetic deletion of ILK suppresses ErbB2-driven YAP/TAZ activation in mammary tumours, and its pharmacological inhibition suppresses YAP activation and tumour growth in vivo. Our data demonstrate a role for ILK as a multiple receptor proximal regulator of Hippo tumour suppressor pathway and as a cancer therapeutic target.

3: Integrin-linked kinase modulates LPS-induced TNFα production via regulation of p65 phosphorylation at serine 536 during NF-kB activation

Integrin-Linked Kinase (ILK) is a ubiquitously expressed protein that regulates cell responses to a wide variety of extracellular stimuli, however its role in the innate immune system is largely unknown. We have studied its role in mediating pro-inflammatory signalling in response to LPS. Pharmacologic or genetic inhibition of ILK in MEFS and macrophages selectively blocks LPS-induced production of the pro-inflammatory cytokine TNF-α. Our results have shown that ILK is required for TLR-induced NF-κB activation and transcriptional induction of TNF-α, through an ILK-PI3K axis. We have demonstrated that the modulation of LPS-induced TNF-α synthesis by ILK does not involve the classical NF-κB pathway since IkBα degradation and p65 nuclear translocation are both unaffected by ILK inhibition. Instead, ILK is involved in a non-classical activation of NF-κB signalling by modulating the phosphorylation of p65 at ser536. Furthermore, ILK-mediated non-classical NF-κB activation through p65 ser536 phosphorylation also exists in a host-pathogen interaction model using Helicobacter pyroli infection in macrophages and gastric cancer cells. Our findings implicate ILK as a critical regulatory molecule for NF-κB- mediated proinflammatory signalling pathway, which is vital for innate immune responses against pathogenic microorganisms.

α1β1 Integrin/Rac1-Dependent Mesangial Invasion of Glomerular Capillaries in Alport Syndrome

Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. L-NAME salt-induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2-deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2-null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1β1-dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology.

Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines

Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA-MB-468 and PC-3 cells. Thus, we hypothesize that ILK might bestow growth advantage and metastatic potential in the course of tumor progression.

Integrin-Linked Kinase Inhibition Attenuates Permeability of the Streptozotocin-Induced Diabetic Rat Retina

Integrin-linked kinase (ILK), as a multi-functional regulator, has been associated with diabetic retinopathy (DR). In this study, we investigated whether inhibition of ILK could result in therapeutic effects. Diabetes mellitus's rats were induced by streptozotocin (STZ) injection. After 1 weeks induction, rats were injected intraperitoneally daily with ILK inhibitor, QLT0267, at 5 mg/kg. Then, the rats were examined by 4, 8, and 12 weeks after first STZ injection. We found that QLT0267 treatment could not only lower ILK level in retina at as early as 3 weeks after the onset of diabetes but also attenuate retina permeability, which was measured by Evan's blue. Maximum effect was found in 11 weeks treatment. Meanwhile, QLT0267 did not disturbed blood glucose concentration. Furthermore, QLT0267 inhibited Akt (Ser473) activation and reduced expression of HIF1α and VEGF which were evaluated by western blot, real time PCR, and immunohistochemistry. We conclude that ILK may be a new target for DR.

Abstract 3958: T315, a novel integrin-linked kinase inhibitor, suppresses hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer.

Abstract The epithelial-to-mesenchymal transition (EMT) is an early event in metastasis that involves the loss by epithelial cells of many of their distinctive epithelial characteristics and the acquisition of mesenchymal properties. EMT can be induced in cancer cells by several factors, such as hypoxia and TGF-β1, leading to an aggressive and malignant phenotype. In prostate cancer therapy, EMT can be a major clinical challenge as it can contribute to tumor recurrence, therapy resistance, and metastasis. Recently, the integrin-linked kinase (ILK) has been identified as an important protein involved in the process of EMT by inducing the protein expression and activation of Snail, one of the major EMT markers in various cancer cells. The objective of this study was to evaluate the ability of T315, a novel ILK inhibitor developed in our laboratory, to block hypoxia-induced EMT in prostate cancer cells and to validate the role of ILK in hypoxia-induced EMT. Based on our results, the protein expression level of ILK was induced by hypoxia as well as EMT examined by efficiently decreasing the protein expression level of E-cadherin and increasing those of vimentin, Snail, and Zeb1 in PC-3 cells. This ILK induction was due to transcriptional regulation by HIF1α which also could be induced by positively regulating ILK promoter. To elucidate the mechanism of hypoxia-induced EMT in prostate cancer, we used T315 to show its ability to downregulate PKB/Akt and mTOR activities and decrease HIF1α expression. These results showed that hypoxia-induced EMT is driven by HIF1α/ILK positive loop in prostate cancer. Meanwhile, we also demonstrated that YB-1, a DNA/RNA binding protein, plays as a transcriptional factor to negatively regulate the expression of Foxo3a which has been showed as an EMT-related protein to regulate Snail and E-cadherin expression. Our results showed T315 could increase Foxo3a expression by inhibition of YB-1 protein expression. On the other hand, according to a previous study, GSK3β-mediated Snail phosphorylation altered the nuclear sequestration of Snail and caused Snail to undergo proteasomal degradation. Our results also showed that T315 could cause GSK3β dephosphorylation, increase the phosphorylation status of Snail and promote Snail degradation. More importantly, we performed functional assays to observe the inhibition of hypoxia-induced EMT in prostate cancer. T315 showed its ability to inhibit hypoxia-induced cell motility dose-dependently in migration, invasion and 3D culture assays. In conclusion, these results indicate that the inhibition of ILK can block hypoxia-induced EMT in prostate cancer cells as reflected by changes in molecular markers and cell behavior, and that this inhibition can be achieved by treatment with the novel small molecule agent T315, which may have therapeutic benefits for prostate cancer patients with subsequent metastasis. Citation Format: Chih-Chien Chou, Su-Lin Lee, Samuel K. Kulp, Ching-Shih Chen. T315, a novel integrin-linked kinase inhibitor, suppresses hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3958. doi:10.1158/1538-7445.AM2013-3958

Abstract 2631: IL-6 promotes ICAM-1 expression and cell motility in human osteosarcoma.

Abstract Osteosarcoma is characterized by a high malignant and metastatic potential. Interleukin-6 (IL-6) is a multifunctional cytokine that is associated with the disease status and outcomes of cancers. However, the effect of IL-6 on migration activity in human osteosarcoma cells is mostly unknown. Here we found that IL-6 increased the migration and expression of intercellular adhesion molecule-1 (ICAM-1) in human osteosarcoma cells. Transfection of cells with ICAM-1 siRNA reduced IL-6-mediated cell migration. We also found that expression of IL-6 was significantly greater in human osteosarcoma tissues than in normal bone. The integrin-linked kinase (ILK)/Akt/AP-1 pathway was activated after IL-6 treatment, and IL-6- induced expression of ICAM-1 and migration activity was inhibited by the specific inhibitor and siRNA of ILK, Akt, and AP-1 cascades. In addition, over-expression of IL-6 shRNA inhibited the migratory ability and ICAM-1 expression in osteosarcoma cells. Taken together, these results indicate that IL-6 and IL-6 receptor interaction occurs through ILK and Akt, which in turn activates AP-1, resulting in the activations of ICAM-1 and contributing the migration of human osteosarcoma cells. Citation Format: Yi-Chin Fong, Chih Hsin Tang. IL-6 promotes ICAM-1 expression and cell motility in human osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2631. doi:10.1158/1538-7445.AM2013-2631

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38(MAPK) are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38(MAPK) is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38(MAPK) is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK-p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK-p38β signaling complexes, playing a key role in bladder cancer cell migration.

Integrin-Linked Kinase Regulates Interphase and Mitotic Microtubule Dynamics

Integrin-linked kinase (ILK) localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

IL-6 promotes ICAM-1 expression and cell motility in human osteosarcoma

Osteosarcoma is characterized by a high malignant and metastatic potential. Interleukin-6 (IL-6) is a multifunctional cytokine that is associated with the disease status and outcomes of cancers. However, the effect of IL-6 on migration activity in human osteosarcoma cells is mostly unknown. Here we found that IL-6 increased the migration and expression of intercellular adhesion molecule-1 (ICAM-1) in human osteosarcoma cells. Transfection of cells with ICAM-1 siRNA reduced IL-6-mediated cell migration. We also found that expression of IL-6 was significantly greater in human osteosarcoma tissues than in normal bone. The integrin-linked kinase (ILK)/Akt/AP-1 pathway was activated after IL-6 treatment, and IL-6-induced expression of ICAM-1 and migration activity was inhibited by the specific inhibitor and siRNA of ILK, Akt, and AP-1 cascades. In addition, over-expression of IL-6 shRNA inhibited the migratory ability and ICAM-1 expression in osteosarcoma cells. Taken together, these results indicate that IL-6 and IL-6 receptor interaction occurs through ILK and Akt, which in turn activates AP-1, resulting in the activations of ICAM-1 and contributing the migration of human osteosarcoma cells.

Abstract 3887: A screen for new cancer specific drugs that target centrosome clustering

Abstract Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain extra centrosomes. Such cancer cells then achieve bipolar division by clustering centrosomes into two functional poles. Our lab has previously shown that inhibition of Integrin-linked kinase, which is a signaling and scaffold protein in focal adhesion and is also a centrosomal protein, results in scattered centrosomes and multipolar spindles, which leads to cancer specific cell death (Fielding et al., 2011, 30:521-534, Oncogene). A major problem with clinically used anti-mitotic drugs, such as taxol, is their toxicity in normal cells. Our goal is to develop new drugs that act specifically on cancer cells through targeting extra centrosomes without affecting normal cells. To discover new drugs, we established a high-content screen that automatically detects cells with de-clustered centrosomes. BT-549, a breast cancer cell line, was used for the screen, since it is known to effectively cluster centrosomes. The Canadian Chemical Biology Network enabled us to test chemical libraries predicted to have drug-like properties. For screening, cells were grown in 96 well plates, treated with test compounds for 5 hours, and subjected to immunofluorescence to examine centrosome arrangement in mitotic cells. Images were taken and analyzed with an automated fluorescence imager, Cellomics Array Scan VTI. We have thus screened over 6000 compounds from which we identified 18 hits. Fifteen of these were confirmed to inhibit centrosome clustering in the cells cultured under standard conditions, and induce an arrest in mitosis. Three compounds are structurally similar, suggesting a common structural motif as preventing centrosome clustering. To compare the effects of these drugs on normal and cancer cells, the viability of several breast and other cancer cell lines, an immortalized model of normal human mammary epithelial cells (MCF10A), and freshly isolated primary normal human mammary epithelial cells were examined after two days of drug treatment using the MTT assay. These comparisons identified some compounds that selectively reduced the viability of cancer cells, but not normal mammary epithelial cells suggesting their potential for cancer-specific therapy. These small molecules are currently being tested in orthotopic and xenograft models, and will be studied for target identification and mechanism of action for further drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3887. doi:1538-7445.AM2012-3887

Abstract 324: The integrin-linked kinase (ILK) / Rictor complex is required for TGFβ-1-induced epithelial to mesenchymal transition (EMT)

Abstract Epithelial to mesenchymal transition (EMT) causes fibrosis, cancer progression and metastasis. Integrin-linked kinase is a focal adhesion adaptor and serine/threonine protein kinase that regulates cell proliferation, survival, and EMT. Elucidating the molecular mechanisms necessary for the development and progression of human malignancies is critical to predict the most appropriate targets for cancer therapy. Here we used transforming growth factor beta-1 (TGFβ-1) to promote EMT and migration in mammary epithelial cells. We demonstrate a requirement of ILK activity for TGFβ-1 mediated EMT in mammary epithelial cells. In addition to nuclear translocation of Snail and Slug, TGFβ-1 treatment also induced expression of the mTORC2 component Rictor as well as its phosphorylation on Thr1135. Interestingly, TGFβ-1 treatment also induced an interaction between ILK and Rictor. All of these TGFβ-1-induced processes were significantly suppressed by inhibiting ILK activity or by disrupting the ILK-Rictor complex using siRNA-mediated knockdown. Furthermore, we identified formation of the ILK/Rictor complex formation in cancer but not normal cell types, and this was accompanied by ILK-dependent phosphorylation of Rictor on residue Thr1135. Inhibition of ILK partially reversed the basal mesenchymal phenotype of MDA-MB-231 cells and prevented EMT in MCF10A cells following TGFβ-1 treatment. These data demonstrate a requirement for ILK function in TGFβ-1-induced EMT in mammary epithelial cells and identify the ILK/Rictor complex as a potential molecular target for preventing/reversing EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 324. doi:1538-7445.AM2012-324

154 KNOCKDOWN OF INTEGRIN-LINKED KINASE REDUCES INVASIVE AND METASTATIC POTENTIAL OF RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2012154 KNOCKDOWN OF INTEGRIN-LINKED KINASE REDUCES INVASIVE AND METASTATIC POTENTIAL OF RENAL CELL CARCINOMA Kyung Seok Han, Peter Raven, Shannon Awrey, Estelle Li, Ladan Fazli, Martin Gleave, and Alan So Kyung Seok HanKyung Seok Han Vancouver, Canada More articles by this author , Peter RavenPeter Raven Vancouver, Canada More articles by this author , Shannon AwreyShannon Awrey Vancouver, Canada More articles by this author , Estelle LiEstelle Li Vancouver, Canada More articles by this author , Ladan FazliLadan Fazli Vancouver, Canada More articles by this author , Martin GleaveMartin Gleave Vancouver, Canada More articles by this author , and Alan SoAlan So Vancouver, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.204AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Integrin-linked kinase (ILK) is a serine/threonine kinase implicated in the regulation of cell growth and survival, cell cycle progression, epithelial-mesenchymal transition (EMT), invasion and migration, angiogenesis. The role of ILK has not been evaluated in renal cell carcinoma (RCC). We investigated the role of ILK on cancer progression and metastasis and therapeutic potential of ILK inhibition in RCC. METHODS Baseline expression of ILK was evaluated by immunofluorescence and western blotting in non-cancerous renal tubular cells (HK-2) and RCC cells (UMRC-6, UMRC-3 and Caki-1). Molecular EMT markers were examined by Western blotting. RNAi using siRNA was used to knock down ILK in vitro. After transient transfection, crystal violet assay and cell cycle analysis using FACS were performed to assess the effect of ILK on tumor growth and cell cycle. Confocal microscopy was used to evaluate changes in stress fibers and focal adhesions with phalloidin-rhodamine and vinculin antibody. Scratch assay and Matrigel invasion assay were performed to evaluate changes of EMT phenotypes after knockdown of ILK. RESULTS ILK is relatively less expressed in normal cells (HK-2) and low stage RCC cells (UMRC-6) but highly expressed in advanced and metastatic RCC cells (UMRC-3 and Caki-1). Caki-1, metastatic RCC cells showed higher expressions of molecular EMT markers including Snail, Zeb1 but decreased activity of GSK3β. Knockdown of ILK using si-ILK inhibited tumor proliferation but the inhibition rate was less than 10% and cell cycle progression was not significantly affected by ILK inhibition. However, ILK knockdown suppressed formation of stress fibers and focal adhesions in UMRC-3 and Caki-1 cells and also effectively suppressed phenotypic EMT markers including cell migration and invasion in Caki-1 and UMRC-3 cells. CONCLUSIONS ILK is highly expressed in advanced RCC and its high expression is related to EMT-related protein in RCC. Knockdown of ILK inhibited molecular EMT markers and suppressed cell migration and invasion. These results suggest the therapeutic potential of ILK inhibition on invasion and metastasis in advanced RCC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e63-e64 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kyung Seok Han Vancouver, Canada More articles by this author Peter Raven Vancouver, Canada More articles by this author Shannon Awrey Vancouver, Canada More articles by this author Estelle Li Vancouver, Canada More articles by this author Ladan Fazli Vancouver, Canada More articles by this author Martin Gleave Vancouver, Canada More articles by this author Alan So Vancouver, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role of the integrin-linked kinase (ILK)/Rictor complex in TGFβ-1-induced epithelial–mesenchymal transition (EMT)

Epithelial-to-mesenchymal transition (EMT) causes fibrosis, cancer progression and metastasis. Integrin-linked kinase (ILK) is a focal adhesion adaptor and a serine/threonine protein kinase that regulates cell proliferation, survival and EMT. Elucidating the molecular mechanisms necessary for development and progression of human malignancies is critical to predict the most appropriate targets for cancer therapy. Here, we used transforming growth factor beta-1 (TGFβ-1) to promote EMT and migration in mammary epithelial cells. We demonstrate a requirement of ILK activity for TGFβ-1-mediated EMT in mammary epithelial cells. In addition to nuclear translocation of Snail and Slug, TGFβ-1 treatment also induced expression of the mammalian target of rapamycin complex 2 component Rictor and its phosphorylation on Thr1135. Interestingly, TGFβ-1 treatment also induced an interaction between ILK and Rictor. All of these TGFβ-1-induced processes were significantly suppressed by inhibiting ILK activity or by disrupting the ILK/Rictor complex using small-interfering RNA-mediated knockdown. Furthermore, we identified ILK/Rictor complex formation in cancer but not in normal cell types, and this was accompanied by ILK-dependent phosphorylation of Rictor on residue Thr1135. Inhibition of ILK partially reversed the basal mesenchymal phenotype of MDA-MB-231 cells and prevented EMT in MCF10A cells after TGFβ-1 treatment. These data demonstrate a requirement for ILK function in TGFβ-1-induced EMT in mammary epithelial cells and identify the ILK/Rictor complex as a potential molecular target for preventing/reversing EMT.

Rhein Inhibits Integrin-Linked Kinase Expression and Regulates Matrix Metalloproteinase-9/Tissue Inhibitor of Metalloproteinase-1 Ratio in High Glucose-Induced Epithelial-Mesenchymal Transition of Renal Tubular Cell

Studies have found overexpressed integrin-linked kinase (ILK) and disturbed matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 (MMP-9/TIMP-1) ratio in diabetic nephropathy epithelial-mesenchymal transition (EMT). However, the underlying mechanisms of EMT and the inhibiting effect of rhein need further understanding. The aim of this study was to investigate the possible regulating effects of ILK towards MMP-9/TIMP-1 ratio in EMT and the inhibiting effect of rhein. The characteristic epithelial marker and mesenchymal marker of EMT were examined by cytoimmunostaining, real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blot. To observe the EMT inhibiting effects of rhein, specific ILK-small interfering RNA (ILK-siRNA) was used as a positive control. The results showed that in high glucose conditions, overexpression of ILK and an abnormal changing of MMP-9/TIMP-1 ratio occurred; ILK inhibition by siRNA could adjust MMP-9/TIMP-1 ratio to near normal. Meanwhile, rhein inhibited the overexpressing ILK and inhibits high glucose-induced EMT; the effect was similar to that of ILK-siRNA. The decreased expression of ILK regulated by rhein contributed to the adjustment of the MMP-9/TIMP-1 ratio. Our data indicates that rhein inhibits high glucose-induced-EMT partially through the inhibition of ILK expression and regulates the MMP-9/TIMP-1 ratio in HK-2 cells. This mechanism may be associated with rhein's effect of ILK suppression.

Identification and Characterization of a Novel Integrin-Linked Kinase Inhibitor

Integrin-linked kinase (ILK) represents a relevant target for cancer therapy in light of its role in promoting oncogenesis and tumor progression. Through the screening of an in-house focused compound library, we identified N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide (22) as a novel ILK inhibitor (IC(50), 0.6 μM), which exhibited high in vitro potency against a panel of prostate and breast cancer cell lines (IC(50), 1-2.5 μM), while normal epithelial cells were unaffected. Compound 22 facilitated the dephosphorylation of Akt at Ser-473 and other ILK targets, including glycogen synthase kinase-3β and myosin light chain. Moreover, 22 suppressed the expression of the transcription/translation factor YB-1 and its targets HER2 and EGFR in PC-3 cells, which could be rescued by the stable expression of constitutively active ILK. Evidence indicates that 22 induced autophagy and apoptosis, both of which were integral to its antiproliferative activity. Together, this broad spectrum of mechanisms underlies the therapeutic potential of 22 in cancer treatment, which is manifested by its in vivo efficacy as a single oral agent in suppressing PC-3 xenograft tumor growth.

Angiopoietin-2, an Angiogenic Regulator, Promotes Initial Growth and Survival of Breast Cancer Metastases to the Lung through the Integrin-linked Kinase (ILK)-AKT-B Cell Lymphoma 2 (Bcl-2) Pathway

The early onsets of breast cancer metastasis involve cell retention, survival, and resistant to apoptosis and subsequent growth at target vascular beds and tissues in distant organs. We previously reported that angiopoietin-2 (Ang2), an angiogenic regulator stimulates MCF-7 breast tumor metastasis from their orthotopic sites to distant organs through the α(5)β(1) integrin/integrin-linked kinase (ILK)/Akt pathway. Here, by using an experimental tumor metastasis model and in vitro studies, we further dissect the underlying mechanism by which Ang2 promotes the initial growth and survival of MCF-7 breast cancer metastasis in the lung of animals. We show that Ang2 increases cell survival and suppresses cell apoptosis through ILK-induced phosphorylation of Akt1, Akt2, and up-regulation of Bcl-2 in breast cancer cells. Inhibition of ILK, Akt1, and Akt2, and their effector Bcl-2 diminishes Ang2-stimulated breast cancer cell survival and Ang2-attenuated apoptosis in vitro, and initial survival and growth of breast cancer metastasis in the lung of animals. Additionally, siRNA knockdown of endogenous Ang2 in three human metastatic breast cancer cell lines also inhibits phosphorylation of Akt, expression of Bcl-2, and tumor cell survival, migration, and increases cell apoptosis. Since increased expression of Ang2 correlates with elevated potential of human breast cancer metastasis in clinic, our data underscore the importance that up-regulated Ang2 not only stimulates breast cancer growth and metastasis at late stages of the process, but is also critical at the initiating stages of metastases onset, thereby suggesting Ang2 as a promising therapeutic target for treating patients with metastatic breast cancer.

Streptococcus pyogenes M49 Plasminogen/Plasmin Binding Facilitates Keratinocyte Invasion via Integrin-Integrin-linked Kinase (ILK) Pathways and Protects from Macrophage Killing

The entry into epithelial cells and the prevention of primary immune responses are a prerequisite for a successful colonization and subsequent infection of the human host by Streptococcus pyogenes (group A streptococci, GAS). Here, we demonstrate that interaction of GAS with plasminogen promotes an integrin-mediated internalization of the bacteria into keratinocytes, which is independent from the serine protease activity of potentially generated plasmin. α(1)β(1)- and α(5)β(1)-integrins were identified as the major keratinocyte receptors involved in this process. Inhibition of integrin-linked kinase (ILK) expression by siRNA silencing or blocking of PI3K and Akt with specific inhibitors, reduced the GAS M49-plasminogen/plasmin-mediated invasion of keratinocytes. In addition, blocking of actin polymerization significantly reduced GAS internalization into keratinocytes. Altogether, these results provide a first model of plasminogen-mediated GAS invasion into keratinocytes. Furthermore, we demonstrate that plasminogen binding protects the bacteria against macrophage killing.