Integrin-linked Kinase Activity Research Articles

Integrin-linked kinase-frizzled 7 interaction maintains cancer stem cells to drive platinum resistance in ovarian cancer

BackgroundPlatinum-based chemotherapy regimens are a mainstay in the management of ovarian cancer (OC), but emergence of chemoresistance poses a significant clinical challenge. The persistence of ovarian cancer stem cells (OCSCs) at the end of primary treatment contributes to disease recurrence. Here, we hypothesized that the extracellular matrix protects CSCs during chemotherapy and supports their tumorigenic functions by activating integrin-linked kinase (ILK), a key enzyme in drug resistance.MethodsTCGA datasets and OC models were investigated using an integrated proteomic and gene expression analysis and examined ILK for correlations with chemoresistance pathways and clinical outcomes. Canonical Wnt pathway components, pro-survival signaling, and stemness were examined using OC models. To investigate the role of ILK in the OCSC-phenotype, a novel pharmacological inhibitor of ILK in combination with carboplatin was utilized in vitro and in vivo OC models.ResultsIn response to increased fibronectin secretion and integrin β1 clustering, aberrant ILK activation supported the OCSC phenotype, contributing to OC spheroid proliferation and reduced response to platinum treatment. Complexes formed by ILK with the Wnt receptor frizzled 7 (Fzd7) were detected in tumors and correlated with metastatic progression. Moreover, TCGA datasets confirmed that combined expression of ILK and Fzd7 in high grade serous ovarian tumors is correlated with reduced response to chemotherapy and poor patient outcomes. Mechanistically, interaction of ILK with Fzd7 increased the response to Wnt ligands, thereby amplifying the stemness-associated Wnt/β-catenin signaling. Notably, preclinical studies showed that the novel ILK inhibitor compound 22 (cpd-22) alone disrupted ILK interaction with Fzd7 and CSC proliferation as spheroids. Furthermore, when combined with carboplatin, this disruption led to sustained AKT inhibition, apoptotic damage in OCSCs and reduced tumorigenicity in mice.ConclusionsThis “outside-in” signaling mechanism is potentially actionable, and combined targeting of ILK-Fzd7 may lead to new therapeutic approaches to eradicate OCSCs and improve patient outcomes.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1392.

Abstract 5808: Integrin-linked kinase regulates Wnt transcriptional activity in platinum resistant ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the most lethal gynecological cancer, characterized by chemo-resistance and fatal tumor recurrence after primary treatment. The metastatic progression is mediated by exfoliation of tumor cells into the peritoneal cavity, the formation of compact OC spheroids, and their implantation onto the overlying peritoneal surface. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components, such as fibronectin (FN), that stiffen the tumor stroma, promote the aberrant activation of integrins, and recruit the adaptor protein integrin-linked kinase (ILK). ILK drives cytoskeletal assembly and modulates key processes, including survival, invasion, and stemness. However, the functional role of ILK in chemoresistance of ovarian cancer stem cells (OCSCs) remains incompletely understood. We tested the hypothesis that the formation of FN-integrin complexes at the cell membrane promotes survival of OCSCs and supports OC spheroid formation by activating ILK and downstream oncogenic signaling. In OCSCs compared with non-CSC, ILK, FN, and integrin β1 mRNA expression levels were increased (P<0.001) and further enriched in OC spheroids compared with monolayers (P<0.01). ILK blockade with the specific inhibitor cpd-22 suppressed spheroid proliferation and tumor initiating capacity in xenograft mouse model. Furthermore, key oncogenic signaling pathways, in particular decreased β-catenin signaling essential to sustaining cancer cell stemness, were altered in OC spheroids treated with cpd-22. Of the genes examined, expression of Wnt receptor Frizzled 7 (Fzd7) was the mostly highly downregulated in OC spheroids treated with cpd-22, indicating a direct correlation between ILK activation and Wnt signaling. By using co-immunoprecipitation and proximity ligation assay, we demonstrated that ILK co-localizes with Fzd7 in OC spheroids. Mechanistically, treatment of OC spheroids with the combination of carboplatin and ILK-Fzd7 blockade decreased β-catenin signaling, inhibited phospho-Akt at Ser473 and increased levels of cleaved-caspase-3 compared to single agent alone, resulting in apoptosis. By regulating β-catenin signaling, Fzd7 expression and ILK activation are essential for OCSCs survival. Targeting Fzd7-ILK clusters may represent a new therapeutic approach to eradicate OCSC and ultimately improve patient outcomes. Citation Format: Rula Atwani, Virginie Lazar, George Earl Sandusky, Salvatore Condello. Integrin-linked kinase regulates Wnt transcriptional activity in platinum resistant ovarian cancer stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5808.

Negative regulation of type I interferon signaling by integrin-linked kinase permits dengue virus replication.

Dengue virus (DENV) infection can induce life-threatening dengue hemorrhagic fever/dengue shock syndrome in infected patients. DENV is a threat to global health due to its growing numbers and incidence of infection in the last 50 years. During infection, DENV expresses ten structural and nonstructural proteins modulating cell responses to benefit viral replication. However, the lack of knowledge regarding the cellular proteins and their functions in enhancing DENV pathogenesis impedes the development of antiviral drugs and therapies against fatal DENV infection. Here, we identified that integrin-linked kinase (ILK) is a novel enhancing factor for DENV infection by suppressing type I interferon (IFN) responses. Mechanistically, ILK binds DENV NS1 and NS3, activates Akt and Erk, and induces NF-κB-driven suppressor of cytokine signaling 3 (SOCS3) expression. Elevated SOCS3 in DENV-infected cells inhibits phosphorylation of STAT1/2 and expression of interferon-stimulated genes (ISGs). Inhibiting ILK, Akt, or Erk activation abrogates SOCS3 expression. In DENV-infected mice, the treatment of an ILK inhibitor significantly reduces viral loads in the brains, disease severity, and mortality rate. Collectively, our results show that ILK is a potential therapeutic target against DENV infection.

Tissue transglutaminase activates integrin-linked kinase and β-catenin in ovarian cancer

Ovarian cancer (OC) is the most lethal gynecological cancer. OC cells have high proliferative capacity, are invasive, resist apoptosis, and tumors often display rearrangement of extracellular matrix (ECM) components, contributing to accelerated tumor progression. The multifunctional protein tissue transglutaminase (TG2) is known to be secreted in the tumor microenvironment, where it interacts with fibronectin (FN) and the cell surface receptor integrin β1. However, the mechanistic role of TG2 in cancer cell proliferation is unknown. Here, we demonstrate that TG2 directly interacts with and facilitates the phosphorylation and activation of the integrin effector protein integrin-linked kinase (ILK) at Ser246. We show that TG2 and p-Ser246-ILK form a complex that is detectable in patient-derived OC primary cells grown on FN-coated slides. In addition, we show that coexpression of TGM2 and ILK correlates with poor clinical outcome. Mechanistically, we demonstrate that TG2-mediated ILK activation causes phosphorylation of glycogen synthase kinase-3α/β, allowing β-catenin nuclear translocation and transcriptional activity. Furthermore, inhibition of TG2 and ILK using small molecules, neutralizing antibodies, or shRNA-mediated knockdown blocks cell adhesion to the FN matrix, as well as the Wnt receptor response to the Wnt-3A ligand, and ultimately, cell adhesion, growth, and migration. In conclusion, we demonstrate that TG2 directly interacts with and activates ILK in OC cells and tumors and define a new mechanism that links ECM cues with β-catenin signaling in OC. These results suggest a central role of TG2–FN–integrin clusters in ECM rearrangement and indicate that downstream effector ILK may represent a potential new therapeutic target in OC.

The kinase activity of integrin-linked kinase regulates cellular senescence in gastric cancer

The activity of integrin-linked kinase (ILK) in cancerous cells is often oncogenic and associated with malignant properties, such as uncontrolled cell cycle progression and evasion from senescence. However, the role of ILK in cellular senescence in gastric cancer (GC) has not been previously examined. We generated single-cell clones of ILK knock-out using CRISPR-Cas9 in human GC lines with mesenchymal or epithelial histology. Cells with no residual ILK expression exhibited strong cellular senescence with diminished clathrin-mediated endocytosis, Surprisingly, ILK loss-induced cellular senescence appeared to be independent of its function in integrin signaling. The low dose of CPD22, a small molecule inhibitor of ILK activity-induced senescence in three GC cell lines with different histologies. Furthermore, senescent cells with ILK depletion transfected with N-terminal truncated ILK mutant remaining catalytic domains displayed the reduction of senescent phenotypes. RNA sequencing and cytokine array results revealed the enrichment of multiple pro-inflammatory signaling pathways in GC lines in the absence of ILK. Our study identified the important role and the potential mechanism of ILK in the cellular senescence of cancerous epithelial cells. The inhibition of ILK activity using small molecule compounds could have a pro-senescent effect as a therapeutic option for GC.

New Perspectives on the Role of Integrin-Linked Kinase (ILK) Signaling in Cancer Metastasis.

Simple SummaryToday, the vast majority of deaths from cancer are due to cancer metastasis. Metastasis requires that cancer cells escape from the initial tumor, travel through blood vessels, and form new tumors in distant host tissues. Integrin-linked kinase (ILK) is overexpressed by many types of cancer cells and provides both structural and signaling functions that are important for successful metastasis. Here, we discuss recent findings that show how ILK is involved in promoting physical changes important for cell motility and invasion, and how ILK relays signals to other machinery components during metastasis, including interactions with components of the immune system and communication between cancer cells and normal cells, to affect the process of metastasis. We also discuss the contribution of ILK to therapeutic resistance and examine efforts to target ILK for the treatment of metastatic disease.Cancer metastasis is a major barrier to the long-term survival of cancer patients. In cancer cells, integrin engagement downstream of cell-extracellular matrix (ECM) interactions results in the recruitment of cytoskeletal and signaling molecules to form multi-protein complexes to promote processes critical for metastasis. One of the major functional components of these complexes is Integrin Linked Kinase (ILK). Here, we discuss recent advances in our understanding of the importance of ILK as a signaling effector in processes linked to tumor progression and metastasis. New mechanistic insights as to the role of ILK in cellular plasticity, epithelial mesenchymal transition (EMT), migration, and invasion, including the impact of ILK on the formation of invadopodia, filopodia-like protrusions (FLPs), and Neutrophil Extracellular Trap (NET)-induced motility are highlighted. Recent findings detailing the contribution of ILK to therapeutic resistance and the importance of ILK as a potentially therapeutically tractable vulnerability in both solid tumors and hematologic malignancies are discussed. Indeed, pharmacologic inhibition of ILK activity using specific small molecule inhibitors is effective in curtailing the contribution of ILK to these processes, potentially offering a novel therapeutic avenue for inhibiting critical steps in the metastatic cascade leading to reduced drug resistance and increased therapeutic efficacy.

Abstract 995: Tissue transglutaminase regulates integrin-linked kinase and beta-catenin signaling in ovarian cancer cells

Abstract Ovarian cancer (OC) is the most lethal gynecological cancer characterized by an enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and rearrangement of extra-cellular matrix (ECM) components. In OC, the interaction between fibronectin (FN) and the β1 integrins activates the adaptor protein integrin linked kinase (ILK) that acts as central hub integrating the mechanical forces with the activation of various signaling pathways, leading to cell survival, proliferation, and migration. ILK overexpression was correlated with OC progression and its functional inhibition blocked tumorigenicity by inactivation of glycogen synthase kinase-3α/β (GSK-3α/β) and the β-catenin-TCF/LEF1 transcriptional activity, thus pointing towards the direct involvement of ILK as a modulator of the crosstalk between ECM and the canonical Wnt signaling. However, the connection between integrins and β-catenin remains poorly understood. The multifunctional protein tissue transglutaminase (TG2) with enzymatic and scaffold functions is an important molecule secreted in the tumor microenvironment where it modulates oncogenic signaling by interacting with FN and integrins. Previously, we demonstrated that TG2/FN/β1 integrin clusters regulated β-catenin activation, correlating with OC progression. Here, we investigated whether TG2-FN interaction is essential to activate the integrin-ILK axis, thereby stabilizing β-catenin. First, we demonstrate that TG2-expressing OC cells plated on FN matrix increase the pool of active p-ILKSer246. Confocal microscopy shows that TG2 co-localizes with p-ILKSer246 in OC cells and that the presence of FN matrices is instrumental in the formation of active TG2/p-ILKSer246 clusters. Further, proximity ligation assay analysis reveals that each protein of the ternary TG2/FN/β1 integrin complex actively engages with p-ILKSer246 in primary ovarian tumor cells, supporting the functional relevance of TG2-mediated outside in transduction signals in vivo. Second, we show that TG2-mediated ILK activation mechanistically amplifies the response of OC cells to Wnt-3A treatment, leading to the inhibitory phosphorylation of the GSK-3α/β at Ser21/9, which in turn allows β-catenin nuclear translocation and transcriptional activity. Finally, we evaluate whether the TG2-ILK complex has functional outcomes. In the presence of an active TG2/p-ILKSer246 axis, Wnt-3a treatment efficiently increases proliferation and migration in OC cells compared to control, whereas the inhibitory anti-TG2 blocking antibody (clone 4G3) directed against the FN-binding domain of TG2 and ILK inhibition by the small molecule cpd-22 drastically reduce the Wnt-3A impact on the OC cell proliferative and migratory capacity. In sum, here we demonstrate that TG2 directly activates ILK and define a new mechanism which links ECM cues with β-catenin signaling in OC. Citation Format: Salvatore Condello, Rula Atwani, Daniela Matei. Tissue transglutaminase regulates integrin-linked kinase and beta-catenin signaling in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 995.

Platelets drive fibronectin fibrillogenesis using integrin αIIbβ3.

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

Laminin 411 mediates endothelial specification via multiple signaling axes that converge on β-catenin.

SummaryThe extracellular matrix (ECM) provides essential cues to promote endothelial specification during tissue development in vivo; correspondingly, ECM is considered essential for endothelial differentiation outside of the body. However, systematic studies to assess the precise contribution of individual ECM proteins to endothelial differentiation have not been conducted. Further, the multi-component nature of differentiation protocols makes it challenging to study the underlying mechanisms by which the ECM contributes to cell fate. In this study, we determined that Laminin 411 alone increases endothelial differentiation of induced pluripotent stem cells over collagen I or Matrigel. The effect of ECM was shown to be independent of vascular endothelial growth factor (VEGF) binding capacity. We also show that ECM-guided endothelial differentiation is dependent on activation of focal adhesion kinase (FAK), integrin-linked kinase (ILK), Notch, and β-catenin pathways. Our results indicate that ECM contributes to endothelial differentiation through multiple avenues, which converge at the expression of active β-catenin.

The integrin beta1 modulator Tirofiban prevents adipogenesis and obesity by the overexpression of integrin-linked kinase: a pre-clinical approach in vitro and in vivo

BackgroundObesity is caused by the enlargement of the white adipose tissue (WAT) depots, characterized by the hypertrophic enlargement of malfunctioning adipocytes within WAT which increases the storage of triglycerides (TG) in the lipid droplets (LD). Adipogenesis pathways as well as the expression and activity of some extracellular matrix receptors integrins are upregulated. Integrinβ1 (INTB1) is the main isoform involved in WAT remodeling during obesity and insulin resistance-related diseases. We recently described Integrin Linked Kinase (ILK), a scaffold protein recruited by INTB1, as an important mediator of WAT remodeling and insulin resistance. As the few approved drugs to fight obesity have brought long-term cardiovascular side effects and given that the consideration of INTB1 and/or ILK modulation as anti-obesogenic strategies remains unexplored, we aimed to evaluate the anti-obesogenic capacity of the clinically approved anticoagulant Tirofiban (TF), stated in preclinical studies as a cardiovascular protector.MethodsFully differentiated adipocytes originating from C3H10T1/2 were exposed to TF and were co-treated with specific INTB1 blockers or with siRNA-based knockdown ILK expression. Lipid-specific dyes were used to determine the TG content in LD. The genetic expression pattern of ILK, pro-inflammatory cytokines (MCP1, IL6), adipogenesis (PPARγ, Leptin), thermogenesis (UCP1), proliferation (PCNA), lipid metabolism (FASN, HSL, ATGL), and metabolite transporters (FABP4, FAT, AQP7) were detected using quantitative PCR. Cytoskeletal actin polymerization was detected by confocal microscopy. Immunoblotting was performed to detect INTB1 phosphorylation at Thr788/9 and ILK activity as phosphorylation levels of protein kinase B (AKT) in Ser473 and glycogen synthase kinase 3β (GSK3β) at Ser9. TF was intraperitoneally administered once per day to wildtype and ILK knockdown mice (cKDILK) challenged with a high-fat diet (HFD) or control diet (STD) for 2 weeks. Body and WAT weight gains were compared. The expression of ILK and other markers was determined in the visceral epididymal (epi) and inguinal subcutaneous (sc) WAT.ResultsTF reduced TG content and the expression of adipogenesis markers and transporters in adipocytes, while UCP-1 expression was increased and the expression of lipases, cytokines or PCNA was not affected. Mechanistically, TF rapidly increased and faded the intracellular phosphorylation of INTB1 but not AKT or GSK3β. F-actin levels were rapidly decreased, and INTB1 blockade avoided the TF effect. After 24 h, ILK expression and phosphorylation rates of AKT and GSK3β were upregulated, while ILK silencing increased TG content. INTB1 blockade and ILK silencing avoided TF effects on the TG content and the transcriptional expression of PPARγ and UCP1. In HFD-challenged mice, the systemic administration of TF for several days reduced the weight gain on WAT depots. TF reduced adipogenesis and pro-inflammatory biomarkers and increased lipolysis markers HSL and FAT in epiWAT from HFD, while increased UCP1 in scWAT. In both WATs, TF upregulated ILK expression and activity, while no changes were observed in other tissues. In HFD-fed cKDILK, the blunted ILK in epiWAT worsened weight gain and avoided the anti-obesogenic effect of in vivo TF administration.ConclusionsILK downregulation in WAT can be considered a biomarker of obesity establishment. Via an INTB1-ILK axis, TF restores malfunctioning hypertrophied WAT by changing the expression of adipocyte-related genes, increasing ILK expression and activity, and reducing TG storage. TF prevents obesity, a property to be added to its anticoagulant and cardiovascular protective advantages.

Downregulation of PARVA promotes metastasis by modulating integrin-linked kinase activity and regulating MAPK/ERK and MLC2 signaling in prostate cancer.

BackgroundMetastasis is the predominant cause of mortality in prostate cancer (PCa); however, the underlying mechanisms are largely uncharted. Here, we found that Parvin alpha (PARVA) is downregulated in PCa and its loss is associated with clinical metastasis. We further explored the mechanistic basis of this finding.MethodsThe mRNA expression of PARVA was identified by analysis of the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) data sets. Immunohistochemistry (IHC) analysis was performed to evaluate the PARVA expression pattern in 198 PCa tissues, and 36 metastatic lymph node tissues. The function and molecular mechanism by which PARVA affects PCa were investigated in vitro using knockdown and overexpression cell lines. The effect of PARVA in cell proliferation, migration, and invasion in PCa cells was detected by MTS assay and Transwell assay. Real-time polymerase chain reaction (PCR) and Western blot analysis were used to assess the gene expression in mRNA and protein level.ResultsThe microarray data analysis indicated that PARVA was drastically downregulated in primary and metastatic PCa compared with normal and primary samples, respectively (all P<0.001). Multivariate Cox regression analysis suggested that downregulation of PARVA in PCa was an independent prognostic factor for poor biochemical recurrence (BCR)-free survival (P<0.01). IHC analysis confirmed that PARVA was frequently downregulated in metastatic and primary PCa tissues (All P<0.001). Furthermore, PARVA expression was found to be associated with Gleason score, pathological stage, extracapsular extension, and lymph node invasion (All P<0.05). Knockdown of PARVA triggered cell migration and invasion in vitro, whereas overexpression of PARVA reverted the invasive phenotypes. Mechanistic investigations identified that overexpression of PARVA repressed the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) phosphorylation via inhibiting the integrin-linked kinase (ILK) biological function. With knockdown of ILK, the downregulated MAPK/ERK phosphorylation and Myosin Light Chain 2 (MLC2) expression by PARVA overexpression were abolished, indicating that the PARVA effect on PCa is ILK/MAPK/ERK pathway dependent.ConclusionsOur study revealed that loss of PARVA expression in PCa promotes metastasis by releasing the inhibition of ILK activity, followed by the activation of MAPK/ERK and MLC2 signaling.

Cardiomyocyte Proteome Remodeling due to Isoproterenol-Induced Cardiac Hypertrophy during the Compensated Phase.

Although the pathophysiological response of cardiac tissue to pro-hypertrophic stimulus is well characterized, a comprehensive characterization of the molecular events underlying the pathological hypertrophy in cardiomyocytes during the early compensated cardiac hypertrophy is currently lacking. A quantitative label-free proteomic analysis of cardiomyocytes isolated was conducted from mice treated subcutaneously with isoproterenol (ISO) during 7 days in comparison with cardiomyocytes from control animals (CT). Canonical pathway analysis of dysregulated proteins indicated that ISO-hypertrophy drives the activation of actin cytoskeleton and integrin-linked kinase (ILK) signaling, and inhibition of the sirtuin signaling. Alteration in cardiac contractile function and calcium signaling are predicted as downstream effects of ISO-hypertrophy probably due to the upregulation of key elements such as myosin-7 (MYH7). Confocal microscopy corroborated that indeed ISO-treatment led to increased abundance of MYH7. Potential early markers for cardiac hypertrophy as APBB1, GOLGA4, HOOK1, KATNA1, KIFBP, MAN2B2, and SLC16A1 are also reported. The data consist in a complete molecular mapping of ISO-induced compensated cardiac hypertrophy model at cardiomyocyte level. Marker candidates reported may assist early diagnosis of cardiac hypertrophy and ultimately heart failure.

P0992PHARMACOLOGICAL UPREGULATION OF INTEGRIN-LINKED KINASE (ILK) REDUCES VISCERAL ADIPOSITY IN A METABOLIC SYNDROME MICE MODEL

Abstract Background and Aims Important mechanism associated to the development of Insulin resistance (IR) are dysregulation of lipid adjustment and excessive deposition of extracellular matrix (ECM) in the visceral white adipose tissue (WAT). The transgenic depletion of the ECM-to-cell scaffold intracellular protein Integrin-linked kinase (ILK) modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017] and body weight gain and adipose malfunction correlates with early downregulation of ILK expression in WAT during a short-term high fat diet (HFD) mice model [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Thus, we demonstrated that transcriptional regulation of ILK might govern the expansion of adipocytes. Between several transcriptional factors implicated in lipid metabolism, the activation of the transcription factor peroxisome proliferator activated receptor (PPAR) family member PPARβ/δ ameliorates IR. Taking in consideration that it modulates the expression of ILK in other contexts aside metabolism [Di-Poï N et al. J Steroid Biochem Mol Biol. 2003, Zhu B et al. Oncogene. 2014], here we activated PPARβ/δ in vivo and in vitro to upregulate adipose ILK expression and prevent WAT expansion. Method Adult mice were fed with either standard (STD) or HFD diets during 4 weeks and divided into groups treated with PPARβ/δ activator L-165041 (i.p. 2 mg/kg b.w per day) or vehicle for 2 additional weeks with maintained diet challenges [Lim HJ et al. Eur J Pharmacol. 2009]. Body and epidydimal WAT depot (eWAT) weights changes were compared and ILK expression was determined by RT-qPCR. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). After cells were treated with L-165041 (1 microM, 24h), we determined the intracellular triglyceride content (by adipo-red dye), the expression of ILK and its downstream substrate AKT (total and active p-AKT in ser473) by RT-qPCR or Western blot. Results HFD increased progressively eWAT and whole body weight gains, which coincide with increase in IR. PPARβ/δ-activated mice under STD or HFD have preventive reduction of weight gains and increased expression of ILK in eWAT. Downregulated ILK expression in culture adipocytes coincide with increased triglyceride content, whereas the activation of PPARβ/δ reduced it at the same time that increased ILK expression and activity. Conclusion We suggest ILK modulation as a novel therapeutic strategy during the obesity establishment. The pharmacological activation of PPARβ/δ increases ILK expression and activity, which correlates with a weight loss in IR-based models in vivo. Although the increased presence of ILK induces the phosphorylation of AKT, in turn one of the main mediators during insulin signalling, we cannot conclude the role of ILK-mediated AKT modulation during the reduction of the adipocyte expansion observed

P0675LEUCOCYTE PODOSOME FORMATION AND ADHESION TO ENDOTHELIUM IS MEDIATED BY INTEGRIN LINKED KINASE IN CHRONIC KIDNEY DISEASE (CKD)

Abstract Background and Aims Patients with CKD have a higher risk of developing cardiovascular diseases. Protein-bound uremic toxins, such as p-cresol (pc) and indoxyl-sulfate (IS), are retention solutes, poorly removed during hemodialysis. They lead to endothelial dysfunction inducing the expression of adhesion molecules and leucocyte adhesion to endothelium. Monocyte extravasation can be carried out by dynamic adhesion structures called podosomes. Integrin-linked kinase (ILK), a kinase and an intracellular scaffold protein, links the cell-adhesion receptors, integrins and growth factors to the actin cytoskeleton and to a range of signalling pathways. We demonstrate the role of ILK in the accumulation of integrin-associated proteins inside the podosomes. The aim of this study was to investigate if ILK is involved in uremic toxin-induced leucocyte podosome formation and adhesion under uremic conditions that simulate CKD. Method In vitro experiments were carried out in human cell line of leukemic monocytes, THP-1. We tested the effect of uremic toxins on cell viability and ILK expression levels or activation by performing dose and time-response experiments. Cells were exposed to IS (25-100 µg ml-1) plus pc (10-100 µg ml-1) both combined, for different times. ILK expression levels were determined by Western Blot and RT-qPCR and its kinase activity was tested by its downstream effector GSK-3β phosphorylated levels. Cell adhesion of THP-1 cells stained with cell tracker to a monolayer of human endothelial cells (EA.hy926) and podosome formation and cell adhesion of THP-1 cells stained with phalloidin to a fibronectin extracellular matrix, was determined by fluorescence confocal microscopy. ILK co-localization with podosome specific protein cortactin was assessed by fluorescence confocal microscopy. For ex vivo experiments in ILK conditional-knockdown mice (cKD-ILK), male CRE-LOX mice were injected with tamoxifen (cKD-ILK) or vehicle (wild-type, WT), to induce ILK deletion. Peripheral blood mononuclear cells (PBMCs) were obtained and treated with uremic toxins. Cell adhesion to a fibronectin extracellular matrix was determined by cell count of the percentage of attached cells against the total cells collected. Results Our data suggests that uremic toxins did not induce toxicity in THP-1 cells. Furthermore, ILK was activated by uremic toxins both at low and high concentrations, without changes in the protein expression. In the cell adhesion assay to the endothelium, uremic toxins induces an increase of THP-1 cells adhesion, which is completely abolished when ILK is knocked down by ILK siRNA, both at low and high toxins concentrations. Similar results were obtained from the cell adhesion assay to the fibronectin extracellular matrix. Moreover, uremic toxins induced podosome formation in THP-1 cells, even at low concentrations, compared to the control, while ILK knockdown abrogated almost completely fibronectin adhesion and podosome formation in uremic toxin-treated cells. Interestingly, we tested that ILK co-localize with cortactin in podosomes, which confirmed the implication of ILK in podosome structures formation induced by uremic toxins. In cKD-ILK PBMC mice the transgenic ILK depletion significantly decrease non-excised ILK mRNA levels. These cKD-ILK PBMC mice exhibited a lower adhesion to the fibronectin extracellular matrix compared to WT. By last, uremic toxins induced a significant increase of mice PBMCs adhesion in WT animals compared to the control that was significantly lower in PBMCs of cKD-ILK animals. Conclusion These data suggest that ILK plays a critical role in the required processes for leukocyte extravasation. ILK deletion may prevent podosome formation and adhesion of leucocytes, decreasing the endothelial and vascular damage caused by the accumulation of uremic toxins in CKD. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.

Tumor Necrosis Factor‐α Activates Integrin Signaling in Vascular Smooth Muscle Cells via α5 Receptor

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). Extracellular superoxide dismutase (ecSOD) converts O2•− to hydrogen peroxide (H2O2). It binds to the cell surface by association with either heparin‐sulfate or fibulin‐5, an extracellular matrix protein that interacts with integrin receptors. We previously found that ecSOD knockdown (siRNA) impaired TNFα signal transduction as reflected by MAPK and NF‐κB activation. Targeting of ecSOD also inhibited TNFα‐dependent activation of focal adhesion kinase (FAK) which reflects integrin receptor activation. Here we investigated the mechanism by which TNFα activates integrin signaling in VSMC. We analyzed NF‐κB activation in the presence of selective blocking antibodies (Ab) for integrin receptor subtypes previously shown to be expressed in VSMCs including α4, α5, αv, β1, and β3. TNFα‐induced NF‐κB activation was not affected by blockade of integrin receptors α4, αv, β1, and β3. However blocking of α5 reduced TNFα‐induced NF‐κB activation (control IgG + TNFα 7.36 ± 0.7, anti‐α5 Ab + TNFα 4.78 ± 0.3 fold change, p &lt; 0.05, n = 5). We next sought to determine if α5 integrin is associated with upstream steps of integrin signaling in VSMCs. FAK and integrin‐linked kinase (ILK) are downstream effectors of integrin receptor activation. After 10 min, TNFα induced phosphorylation of both FAK and Protein Kinase B (Akt) which was used as a surrogate for ILK activation. Blockade of α5 receptors decreased TNFα‐induced phosphorylation of both FAK and Akt. Thus, short term exposure of VSMCs to TNFα transactivates α5 integrins and subsequently FAK and ILK‐Akt. We speculate that ecSOD localizes TNFα‐induced H2O2 production to the vicinity of α5 integrins via physical association with fibulin‐5 and this redox environment affects activation of the receptor.

Integrin-Linked Kinase Activation Prevents Ventricular Arrhythmias Induced by Ischemia/Reperfusion Via Inhibition of Connexin 43 Remodeling

Ischemia reperfusion (I/R)-induced arrhythmia is a serious complication in patients with cardiac infarction. Remodeling of connexin (Cx) 43, manifested as phosphorylation, contributes significantly to arrhythmogenesis. Integrin-linked kinase (ILK) attenuated ventricular remodeling and improved cardiac function in rats after myocardial infarction. We hypothesized that ILK, through Cx43 phosphorylation, would be protective against I/R-induced ventricular arrhythmias. Our study showed that I/R-induced ventricular arrhythmias were attenuated by an ILK agonist LPTP and worsened by the ILK inhibitor Cpd22. I/R disrupted Cx43 distribution, but it was partially normalized in the presence of LPTP. Compared with I/R, the phosphorylation of Akt was increased significantly after pretreatment with LPTP. The increase in phosphorylated Akt was physiologically significant because, in the presence of the Akt inhibitor MK2206, the protective effects of LPTP were blocked. This indicated that ILK activation prevented I/R-induced-ventricular arrhythmia, an effect potentially related to inhibition of Cx43 remodeling via Akt activation.

Suppression of tensin 2 promotes intestinal tumorigenesis by liberating integrin-linked kinase-induced nuclear translocation of β-catenin

Tensin 2 (TNS2) is a focal adhesion-localized multidomain protein expressed in various tissues. TNS2 expression significantly decreases in many tumor cell lines, and low TNS2 expression is associated with a poorer relapse-free survival in some cancers, suggesting that the loss of TNS2 may be related to tumor progression. Deregulation of Wnt/β-catenin signaling is frequently observed in colorectal cancer. In the present study, we found that TNS2 negatively regulated Wnt signaling by suppressing the nuclear translocation of β-catenin by reducing integrin-linked kinase (ILK) activity in colon cancer cell lines. To investigate the role of TNS2 in intestinal tumorigenesis in vivo, we introduced Tns2 mutation into ApcMin/+ mouse, a model of human familial adenomatous polyposis. The compound mutant mice showed a significant increase in tumor number and size in the small intestine and colon. Thus, this study may contribute to the discovery of novel mechanisms underlying cancer malignancy, and pave the way for the development of treatment strategies for intestinal cancers.

SAT-197 Functional Cross-Talk Between MCP-1 and Integrin Linked Kinase Is Required for Endometriosis Disease Progression

Development of painful invasive endometriotic lesions is usually a combined function of the chronic inflammation and active sequential cellular events such as migration, cell adhesion, proliferation, colonization, and invasion in endometriosis. An elevated level of monocyte chemotactic protein-1 (MCP-1) in serum and peritoneal fluid has been reported in endometriosis. Further, Integrin-Linked Kinase (ILK) communicates with extracellular signals and regulates the sequential cellular events in uterine and ovarian cancer. The functional cross-talk of MCP-1 with ILK in the immune cells leads to the high MCP-1 production and increased ILK activity. However, the role of the elevated MCP-1 level and its functional interaction with ILK in the endometriosis development is not explored yet. In order to investigate this, we used the surgically induced heterologous mouse model of endometriosis and externally treated with MCP-1 intraperitoneally for two weeks. Our results demonstrated that MCP-1 boosted the growth of endometriotic lesions mediated through ILK, FAK, VAV and RAC1 signaling axis in endometriosis. In addition, in vivo pharmacological inhibition of ILK by CPD22 restricted the growth of endometriotic lesions in mice. Flow cytometric and inflammatory cytokines profiling in the peritoneal fluid demonstrated that ILK was involved in the MCP-1 mediated macrophageF4/80+ recruitment and inflammatory response in order to support the growth of endometriotic lesions. Inhibition of ILK induced the anti-inflammatory cytokine IL-10 and concomitantly suppressed the pro-inflammatory cytokines MCP-1, TNF-α and IL-10 levels in peritoneal fluid. In vitro studies on human ovarian cystic endometriosis cells (Hs832(C).T) and normal endometrial stromal cells (T-HESCs) revealed that MCP-1 accelerated the adhesion (on collagen and fibronectin substratum), colonization, migration and 3D-spheroids formation and invasion. As the basal level of CCR2, an MCP-1 receptor, expression was higher in ovarian endometriotic cells than the normal endometrial stromal cells giving the hint for the role of MCP-1 in endometriosis. Interestingly, the siRNA mediated knockdown of ILK renders the MCP-1 induced nuclear translocation of phosphorylated ILK and also reduced these accelerated cellular events. Taken together, our results provide compelling evidence that the functional cross-talk between MCP-1 and ILK drives endometriotic disease progression.

Extracellular Superoxide Dismutase (SOD3) Links Tumor Necrosis Factor‐alpha Receptor 1 to Integrin Signaling

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1). Extracellular superoxide dismutase (ecSOD) can convert this O2•− to hydrogen peroxide (H2O2). ecSOD is anchored to the cell surface by interaction with either heparin sulfate proteoglycan via a heparin binding domain, or by binding to fibulin‐5 (Fib5), an extracellular matrix protein which interacts with integrin receptors. Fib5 binding does not activate integrins, but blocks interaction with other components of the extracellular matrix. The physiologic significance of ecSOD‐Fib5‐integrin interaction has not been well defined. We hypothesized that ecSOD regulates extracellular redox signaling of TNFα/Nox1 by modulating secondary effects on integrin signaling. We analyzed TNFα‐induced interleukin‐6 (IL‐6) production and NF‐κB activation in cultured vascular smooth muscle cells (VSMC). Knockdown of ecSOD by siRNA blocked TNFα‐mediated IL‐6 (siControl+TNFα 2922±354, si ecSOD+TNFα 952.5±99 pg/mL, p&lt;0.05, n=8–9) and NF‐κB activation (siControl+TNFα 10.8±1.0, si ecSOD+TNFα 4.0±0.3 fold change, p&lt;0.05, n=7). Similar inhibitory effects were observed following knockdown of Fib5 (IL‐6; siControl+TNFα 2372±123, siFib5+TNFα 1686±124 pg/mL, p&lt;0.05, n=4. NF‐κB activation; siControl+TNFα 10.8±1.0, siFib5+TNFα 5.4±0.3 fold change, p&lt;0.05, n=7). We next sought to determine how ecSOD affected integrin signaling. Focal adhesion kinase (FAK) and integrin‐linked kinase (ILK) are critical downstream effectors of integrin activation. TNFα induced phosphorylation of both FAK and Akt, which was used as a surrogate for ILK activation. This approach was validated by the ability of ILK inhibition (Cpd22; 3 μM) to prevent Akt phosphorylation. Both FAK and Akt phosphorylation in response to TNFα were inhibited by an anti‐integrin β1 antibody. Knockdown of ecSOD significantly enhanced TNFα‐induced phosphorylation of both FAK and Akt. FAK inhibition (FAK inhibitor 14; 2 μM) blocked while ILK inhibition (Cpd22; 3 μM) markedly potentiated NF‐κB activation. These results suggest that TNFα transactivates β1 integrins and therefore FAK and ILK in VSMCs. We speculate that ecSOD localizes H2O2 production to the vicinity of integrin receptors via physical association with Fib5 and provides tonic suppression of β1integrin receptor activation. Knockdown of either ecSOD or Fib5 would promote ILK‐dependent inhibition of NF‐κB activation and IL‐6 secretion which occurs via direct TNFR1‐dependent signaling.Support or Funding InformationAmerican Heart Association (16SDG30610002)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.