Integrin-linked Kinase Expression Research Articles

An ILK/STAT3 pathway controls glioblastoma stem cell plasticity.

Glioblastoma (GBM) is driven by malignant neural stem-like cells that display extensive heterogeneity and phenotypic plasticity, which drive tumor progression and therapeutic resistance. Here, we show that the extracellular matrix-cell adhesion protein integrin-linked kinase (ILK) stimulates phenotypic plasticity and mesenchymal-like, invasive behavior in a murine GBM stem cell model. ILK is required for the interconversion of GBM stem cells between malignancy-associated glial-like states, and its loss produces cells that are unresponsive to multiple cell state transition cues. We further show that an ILK/STAT3 signaling pathway controls the plasticity that enables transition of GBM stem cells to an astrocyte-like state invitro and invivo. Finally, we find that ILK expression correlates with expression of STAT3-regulated proteins and protein signatures describing astrocyte-like and mesenchymal states in patient tumors. This work identifies ILK as a pivotal regulator of multiple malignancy-associated GBM phenotypes, including phenotypic plasticity and mesenchymal state.

Breast Cancer Cells Exhibit Mesenchymal-Epithelial Plasticity Following Dynamic Modulation of Matrix Stiffness.

Mesenchymal-epithelial transition (MET) is essential for tissue and organ development and is thought to contribute to cancer by enabling the establishment of metastatic lesions. Despite its importance in both health and disease, there is a lack of in vitro platforms to study MET and little is known about the regulation of MET by mechanical cues. Here, hyaluronic acid-based hydrogels with dynamic and tunable stiffnesses mimicking that of normal and tumorigenic mammary tissue are synthesized. The platform is then utilized to examine the response of mammary epithelial cells and breast cancer cells to dynamic modulation of matrix stiffness. Gradual softening of the hydrogels reduces proliferation and increases apoptosis of breast cancer cells. Moreover, breast cancer cells exhibit temporal changes in cell morphology, cytoskeletal organization, and gene expression that are consistent with mesenchymal-epithelial plasticity as the stiffness of the matrix is reduced. A reduction in matrix stiffness attenuates the expression of integrin-linked kinase, and inhibition of integrin-linked kinase impacts proliferation, apoptosis, and gene expression in cells cultured on stiff and dynamic hydrogels. Overall, these findings reveal intermediate epithelial/mesenchymal states as cells move along a matrix stiffness-mediated MET trajectory and suggest an important role for matrix mechanics in regulating mesenchymal-epithelial plasticity.

The Receptor Tyrosine Kinase EphA2 and Integrin-linked Kinase Expression in Colorectal Cancer in Relation to the Severity of the Tumor

Abstract Background: Although colorectal carcinoma is still the second cancer-related death worldwide, the current knowledge of the prognostic profile of routine markers is still humble. This study aimed to study the receptor tyrosine kinase EphA2 and integrin-linked kinase expression in relation to the severity of the disease represented by the grade, extent of invasion, and lymph node involvement. Materials and Methods: This cross-sectional study included 87 colorectal cancer cases diagnosed between 2019 and 2022. The immunohistochemical expression and scoring of integrin-linked kinase and EphA2 was carried out and correlated to the histological variables of severity. Results: The mean age of the studied cases was 57.85 years, and approximately 56.3% of them were females. The majority of the cases were moderately differentiated carcinoma (81.61%). More than half of the patients presented in T3. No lymph-node involvement was detected in 65.52 cases. The majority of the studied cases showed negative integrin-linked kinase expression in tumor cells in the colon and lymph nodes. Even positive cases showed low scores. EphA2 marker showed higher positivity in tumor cells of the colon and lymph nodes with higher scores, but no significant correlation to the histopathological variables. Conclusion: The EphA2 marker is of higher benefit than the integrin-linked kinase and the last one is of limited prognostic role in colorectal cancer due to the very high negative and low score results.

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.

Integrin-linked kinase mRNA expression in circulating mononuclear cells as a biomarker of kidney and vascular damage in experimental chronic kidney disease

BackgroundTraditional biomarkers of chronic kidney disease (CKD) detect the disease in its late stages and hardly predict associated vascular damage. Integrin-linked kinase (ILK) is a scaffolding protein and a serine/threonine protein kinase that plays multiple roles in several pathophysiological processes during renal damage. However, the involvement of ILK as a biomarker of CKD and its associated vascular problems remains to be fully elucidated.MethodsCKD was induced by an adenine-rich diet for 6 weeks in mice. We used an inducible ILK knockdown mice (cKD-ILK) model to decrease ILK expression. ILK content in mice's peripheral blood mononuclear cells (PBMCs) was determined and correlated with renal function parameters and with the expression of ILK and fibrosis and inflammation markers in renal and aortic tissues. Also, the expression of five miRNAs that target ILK was analyzed in whole blood of mice.ResultsThe adenine diet increased ILK expression in PBMCs, renal cortex, and aortas, and creatinine and urea nitrogen concentrations in the plasma of WT mice, while these increases were not observed in cKD-ILK mice. Furthermore, ILK content in PBMCs directly correlated with renal function parameters and with the expression of renal and vascular ILK and fibrosis and inflammation markers. Finally, the expression of the five miRNAs increased in the whole blood of adenine-fed mice, although only four correlated with plasma urea nitrogen, and of those, three were downregulated in cKD-ILK mice.ConclusionsILK, in circulating mononuclear cells, could be a potential biomarker of CKD and CKD-associated renal and vascular damage.Graphical ILK content in circulating mononuclear cells reflects renal and vascular damage in a CKD experimental model.

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.

Glutaredoxin 2 protects lens epithelial cells from epithelial-mesenchymal transition by suppressing mitochondrial oxidative stress-related upregulation of integrin-linked kinase

Glutaredoxin 2 (Grx2), a mitochondrial glutathione-dependent oxidoreductase, is crucial for maintaining redox homeostasis and cellular functions in the lens. The oxidative stress-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is related to posterior capsule opacification. In this study, we investigated the effects of Grx2 on oxidative stress-induced EMT in LECs during posterior capsule opacification. We found that Grx2 expression was substantially decreased during the EMT of LECs and in a mouse model of cataract surgery. Deletion of Grx2 aggravated the generation of reactive oxygen species, including those that are mitochondria-derived, and promoted the proliferation and EMT of the LECs. This was reversed by Grx2 overexpression. In vivo, proteomic liquid chromatography-mass spectrometry analysis showed that integrin-linked kinase (ILK) was significantly upregulated in the lens posterior capsule of a Grx2 knockout (KO) mouse model. Compared with that of the wild-type group, the expression of ILK and EMT markers was increased in the Grx2 KO group which was reversed in the Grx2 knock-in group. Inhibition of ILK partially blocked Grx2 knockdown-induced EMT and prevented the increased phosphorylation of Akt and GSK-3β and the nuclear translocation of β-catenin in the Grx2 KO group. Finally, inhibition of the Wnt/β-catenin pathway partially blocked the Grx2 knockdown-induced EMT. In conclusion, we demonstrated that Grx2 protects LECs from oxidative stress-related EMT by regulating the ILK/Akt/GSK-3β axis.

Upregulation of integrin‑linked kinase enhances tumor progression in gemcitabine‑resistant pancreatic cancer.

Pancreatic cancer (PaCa) tends to be resistant to chemotherapy and is associated with a very poor prognosis. It has been previously reported by the authors that integrin‑linked kinase (ILK) is a prognostic factor in PaCa. ILK expression was examined in a newly established gemcitabine (Gem)‑resistant (Gem‑R) PaCa cell line and it was demonstrated that ILK expression was upregulated compared with that in Gem‑sensitive (Gem‑S) cells. In the present study, the effects of increased ILK expression in Gem‑R PaCa cells were evaluated and it was examined whether compound22 (Cpd22), an ILK inhibitor, exerted antitumor effects not only in Gem‑S cells but also in Gem‑R cells. Reverse transcription‑quantitative polymerase chain reaction and western blotting revealed that ILK expression was higher in Gem‑R PaCa cells than in Gem‑S PaCa cells. Cpd22 inhibited the growth of PaCa cells in a concentration‑dependent manner. Cpd22 also inhibited the growth of Gem‑R PaCa cells. The invasive and angiogenic potential of Gem‑R PaCa cells was enhanced compared with that in Gem‑S cells; however, ILK small interfering RNA and Cpd22 treatment suppressed this enhancement of invasive potential compared with that in Gem‑S cells. The addition of Cpd22 to Gem also improved the sensitivity of Gem‑R cell lines to Gem. Furthermore, enhanced Akt signaling was associated with increased malignancy in Gem‑R cell lines. In conclusion, ILK was upregulated with resistance and may be involved in tumor angiogenesis, invasive potential, and chemotherapy resistance, which were all suppressed by Cpd22 treatment. Thus, Cpd22 may be a novel therapeutic agent for the treatment of PaCa.

Extracellular vesicle-packaged ILK from mesothelial cells promotes fibroblast activation in peritoneal fibrosis.

Progressive peritoneal fibrosis and the loss of peritoneal function often emerged in patients undergoing long-term peritoneal dialysis (PD), resulting in PD therapy failure. Varieties of cell-cell communications among peritoneal cells play a significant role in peritoneal fibrogenesis. Extracellular vesicles (EVs) have been confirmed to involve in intercellular communication by transmitting proteins, nucleic acids or lipids. However, their roles and functional mechanisms in peritoneal fibrosis remain to be determined. Using integrative analysis of EV proteomics and single-cell RNA sequencing, we characterized the EVs isolated from PD patient's effluent and revealed that mesothelial cells are the main source of EVs in PD effluent. We demonstrated that transforming growth factor-β1 (TGF-β1) can substitute for PD fluid to stimulate mesothelial cells releasing EVs, which in turn promoted fibroblast activation and peritoneal fibrogenesis. Blockade of EVs secretion by GW4869 or Rab27a knockdown markedly suppressed PD-induced fibroblast activation and peritoneal fibrosis. Mechanistically, injured mesothelial cells produced EVs containing high level of integrin-linked kinase (ILK), which was delivered to fibroblast and activated them via p38 MAPK signalling pathway. Clinically, the expression of ILK was up-regulated in fibrotic peritoneum of patients undergoing long-term PD. The percentage of ILK positive EVs in PD effluent correlated with peritoneal dysfunction and the degree of peritoneal damage. Our study highlights that peritoneal EVs mediate communications between mesothelial cells and fibroblasts to initiate peritoneal fibrogenesis. Targeting EVs or ILK could provide a novel therapeutic strategy to combat peritoneal fibrosis.

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.

Plantamajoside alleviates hypoxia-reoxygenation injury through integrin-linked kinase/c-Src/Akt and the mitochondrial apoptosis signaling pathways in H9c2 myocardial cells

Myocardial ischemia–reperfusion injury(MIRI) is one of the common complications after myocardial infarction surgery, Oxidative stress is among the main mechanisms of myocardial ischemia–reperfusion injury. Plantamajoside (PMS), the main effective ingredient in the genus Plantain, has been reported to possess an antioxidation, anti-inflammatory and anti-apoptosis role. However, whether PMS can attenuate myocardial ischemia–reperfusion injury is not yet known. Herein, we explored the effects of PMS on hypoxia-reoxygenation (H/R) injury in H9c2 cardiomyocytes and the underling molecular mechanisms of the treatment. Network pharmacological analysis screened the top 31 key genes in the treatment of MIRI disease treated with PMS, and the result of molecular docking further illustrated the roles that the PMS play in the treatment of MIRI through its interference with integrin-linked kinase (ILK) target protein. PMS was not cytotoxic in the concentration range of 5–40 μM and increased cell survival after H/R injury in a concentration-dependent manner without affecting proliferation or growth. PMS significantly reduced the levels of lactate dehydrogenase, malonic dialdehyde, reactive oxygen species and cell apoptosis, and increased soperoxide dismutase activity compared with those of the H/R injury group. PMS promoted the protein and mRNA expression of ILK and Bcl-2, the protein expression of p-Akt, and reduced the protein and mRNA expression of Bax, Caspase-3, and Cytochrome c, the protein expression of p–c-Src. PMS has protective effects against H/R injury in H9c2 cells, and its protective mechanism may be related to reactive oxygen species clearance, activation of the ILK/c-Src/Akt pathway and inhibition of the mitochondrial apoptosis.

Investigating the sarcolemma-sarcomere connection and force transmission in the heart.

External mechanical signals are communicated through cardiac muscle cells by converting them to internal biochemical signals during mechanotransduction. In the heart, altered transmission of these signals can affect myocardial structure and function. This study examines the impact of sarcospan deletion, a sarcolemma protein, on cardiac muscle mechanics. Sarcospan is an integral member of the dystrophin-glycoprotein complex (DGC) and we are interested in investigating how it influences sarcomere contractile function. Measurements of cardiac contractile parameters were conducted using permeabilized left ventricular papillary muscle bundles obtained from wild-type and SSPN-deficient (SSPN-/-) mouse hearts. Myofilament Ca2+-sensitivity and cooperativity of thin filament activation was similar between WT and SSPN-/- cardiac muscle preparations. However, SSPN-/- muscle bundles exhibited a significantly faster rate of tension redevelopment (kTR) at submaximal activation conditions (pCa 5.8) but no significant changes in maximal kTR (recorded at maximal Ca2+ activation, pCa 4.5). Maximal steady-state isometric force and sinusoidal stiffness were proportionally increased in SSPN-/- cardiac muscle preparations during Ca2+ activation. These intriguing observations suggest that SSPN may play an inherent role in balancing pathways involved in force transmission and its deletion may cause compensatory remodeling of sarcomeric proteins (e.g. isoform switching or protein modification) changing mechanical properties of cardiac muscle. Mouse hearts lacking SSPN-/- exhibit increased sarcolemmal expression of integrin-linked kinase (ILK) and vinculin. ILK is an important biomechanical sensor shown to regulate cardiac contractility and ventricular hypertrophy whereas increasing vinculin expression has been shown to improve heart function and impact myofilament organization. Further examination of the WT and SSPN-/- myofibrils will allow us to assess sarcomere protein alterations that contribute to increased force at submax and max levels of Ca2+ activation. Therefore, the concentric enlargement observed in SSPN-/- mouse hearts could be explained by remodeling of the sarcomere itself.

Integrin-linked kinase expression in myeloid cells promotes colon tumorigenesis.

Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and treatment options for advanced CRC, which has a low 5-year survival rate, remain limited. Integrin-linked kinase (ILK), a multifunctional, scaffolding, pseudo-kinase regulating many integrin-mediated cellular processes, is highly expressed in many cancers. However, the role of ILK in cancer progression is yet to be fully understood. We have previously uncovered a pro-inflammatory role for myeloid-specific ILK in dextran sodium sulfate (DSS)-induced colitis. To establish a correlation between chronic intestinal inflammation and colorectal cancer (CRC), we investigated the role of myeloid-ILK in mouse models of CRC. When myeloid-ILK deficient mice along with the WT control mice were subjected to colitis-associated and APCmin/+-driven CRC, tumour burden was reduced by myeloid-ILK deficiency in both models. The tumour-promoting phenotype of macrophages, M2 polarization, in vitro was impaired by the ILK deficiency and the number of M2-specific marker CD206-expressing tumour-associated macrophages (TAMs) in vivo were significantly diminished in myeloid-ILK deficient mice. Myeloid-ILK deficient mice showed enhanced tumour infiltration of CD8+ T cells and reduced tumour infiltration of FOXP3+ T cells in colitis-associated and APCmin/+-driven CRC, respectively, with an overall elevated CD8+/FOXP3+ ratio suggesting an anti-tumour immune phenotypes. In patient CRC tissue microarrays we observed elevated ILK+ myeloid (ILK+ CD11b+) cells in tumour sections compared to adjacent normal tissues, suggesting a conserved role for myeloid-ILK in CRC development in both human and animal models. This study identifies myeloid-specific ILK expression as novel driver of CRC, which could be targeted as a potential therapeutic option for advanced disease.

The Role of Hippo Signaling Pathway and ILK in the Pathophysiology of Human Hypertrophic Scars and Keloids: An Immunohistochemical Investigation.

Keloids and hypertrophic scars are characterized by abnormal fibroblast activation and proliferation. While their molecular pathogenesis remains unclear, myofibroblasts have been associated with their development. Hippo pathway effectors YAP/TAZ promote cell proliferation and matrix stiffening. Integrin-linked kinase (ILK), a central component of focal adhesions that mediates cell-matrix interactions, has been linked to tissue repair and fibrosis. The aim of this study was to investigate the expression of key Hippo pathway molecules and ILK in hypertrophic scars and keloids. YAP/TAZ, TEAD4, ILK and a-SMA expression were evaluated by immunohistochemistry in keloids (n = 55), hypertrophic scars (n = 38) and normal skin (n = 14). The expression of YAP/TAZ, TEAD4, ILK and a-SMA was higher in fibroblasts of keloids compared to hypertrophic scars while negative in normal skin. There was a significant positive correlation between the expression of ILK and Hippo pathway effectors. Our results suggest that the deregulation of Hippo signaling and ILK are implicated in keloid and hypertrophic scar formation.

Integrin-Linked Kinase Expression in Human Valve Endothelial Cells Plays a Protective Role in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is highly prevalent during aging. CAVD initiates with endothelial dysfunction, leading to lipid accumulation, inflammation, and osteogenic transformation. Integrin-linked kinase (ILK) participates in the progression of cardiovascular diseases, such as endothelial dysfunction and atherosclerosis. However, ILK role in CAVD is unknown. First, we determined that ILK expression is downregulated in aortic valves from patients with CAVD compared to non-CAVD, especially at the valve endothelium, and negatively correlated with calcification markers. Silencing ILK expression in human valve endothelial cells (siILK-hVECs) induced endothelial-to-mesenchymal transition (EndMT) and promoted a switch to an osteoblastic phenotype; SiILK-hVECs expressed increased RUNX2 and developed calcified nodules. siILK-hVECs exhibited decreased NO production and increased nitrosative stress, suggesting valvular endothelial dysfunction. NO treatment of siILK-hVECs prevented VEC transdifferentiation, while treatment with an eNOS inhibitor mimicked ILK-silencing induction of EndMT. Accordingly, NO treatment inhibited VEC calcification. Mechanistically, siILK-hVECs showed increased Smad2 phosphorylation, suggesting a TGF-β-dependent mechanism, and NO treatment decreased Smad2 activation and RUNX2. Experiments performed in eNOS KO mice confirmed the involvement of the ILK-eNOS signaling pathway in valve calcification, since aortic valves from these animals showed decreased ILK expression, increased RUNX2, and calcification. Our study demonstrated that ILK endothelial expression participates in human CAVD development by preventing endothelial osteogenic transformation.

Niveles de quinasa ligada a la integrina endotelial y calcificación valvular: estudio molecular de la calcificación en la estenosis valvular aórtica

Introduction and objectivesThe endothelial integrin-linked kinase (ILK) controls the production of nitric oxide, modulating transduction signals of mechanical stress in the cell. Decreased endothelial ILK has been related to valve calcification. We analyzed the association of ILK expression with aortic valve calcification. MethodsObservational, single-center and prospective study. Aortic valve tissue samples were obtained from patients whit aortic stenosis, and controls for non-calcified aortic valves. Endothelial ILK levels were quantified using molecular analysis techniques, as well as bone growth markers (BMP2 and RUNX2). Stepwise multiple logistic regression was used to control for confounding factors, introducing clinical parameters related to atherosclerosis and valve calcification (age, DM, HBP, kidney failure). ResultsSamples from 75 patients whit aortic stenosis, and 28 controls were obtained. A percentage of 69.9 were men, mean age 67 years (SD 9.59 years). ILK levels were significantly lower in diseased valves than in controls (0.96 vs. 0.76; P<.001). There was a significant negative linear correlation between ILK and degree of valve calcification (Pearson linear correlation coefficient=−0.35; P=.002).In the multivariate model, ILK was significantly associated with valve calcification (OR adjusted 0.068; P<.001), as well as DM (OR 11.80; P=.03). In addition, an increase in bone growth markers was observed as calcification progressed, with a significant inverse correlation between ILK and BMP2 (R=−0.417; P<.001) and RUNX2 (R=−0.587; P<.001). ConclusionsThe decrease in the endothelial expression of ILK may influence the calcification of the aortic valve, marking the beginning of the process of transdifferentiation of the valvular endothelium to tissue with osteogenic capacity.

Abstract P1069: Integrin Linked Kinase Controls Human Valve Endotelial Cells Pro-osteogenic Activation In A TFG-b/NO Dependent Manner

Calcific aortic valve disease (CAVD) is expected in the aging population. CAVD physiopathology arises from endothelial dysfunction leading to lipid accumulation, inflammation, and osteogenic transformation. Many studies suggest that valve endothelial cells (VECs) undergoing EndMT may differentiate into osteoblastic interstitial cells. We previously showed that Integrin linked kinase (ILK) regulates vasomotor function by preventing endothelial nitric oxide synthase uncoupling. ILK expression in vascular endothelium plays an essential cardioprotective role. Moreover, we have found a significant negative correlation between human valve protein levels of ILK and the osteogenic marker Runx2 and BMP2 in 70 patients with calcific aortic stenosis, a higher decrease at the endothelial level. We hypothesized that Endothelial ILK plays a protective role in human valve endothelial cells osteogenic transition. Isolation of human valve endothelial cells from the aortic (aortic hVECs) and ventricular sides (ventricular hVECs) show a specific ILK decrease in the aortic hVECs from calcific compared to non-calcified valve (p&lt;0.05). Silencing ILK expression in hVECs decreased NO production and increased ROS (Reactive Oxygen Species) production as early as 72 hours. After five days of silencing ILK, hVEC express myofibroblast markers SM22a, α-SMA, MMP12, MMP9, and SNAI 1 while losing the endothelial markers (CD31, and vWF (p&lt;0.01). ILK silenced hVECs (siILK-hVECs) express the osteogenic marker RunX2 indicating osteogenic differentiation and developed calcified nodules. Cell treatment with Noggin, a BMP2 antagonist, did not reverse the increase of Runx2, thus excluding the implication of the BMP2/RUNX2 axis. However, silk-hVECs showed an increase in phosphorylated-SMAD 2, suggesting a TFG-β -dependent mechanism. Treatment of siILK-hVECs with the NO donor DETA-NO decreased Smad 2 activation and RunX2. Moreover, DETA-NO treated siILK hVECs cultured in pro-osteogenic media did not develop calcification. Collectively, our results point to a crucial role of ILK in keeping human valve endothelial cell phenotype preventing valve calcification in a Nitric Oxide-TFGβ1 dependent manner. Financed by the Regional European fund “a way to achieve Europe.” JCCM SBPLY/19/180501/000055S and ISCIII PI20/00930

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.

Integrin-Linked Kinase Expression Characterizes the Immunosuppressive Tumor Microenvironment in Colorectal Cancer and Regulates PD-L1 Expression and Immune Cell Cytotoxicity.

Integrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in both inflammation and tumorigenesis of the colon. However, a role for ILK in the tumor microenvironment (TME) and immune evasion has not been investigated. Here, we show a correlation of ILK expression with the immunosuppressive TME and cancer prognosis. We also uncover a role for ILK in the regulation of programmed death-ligand 1 (PD-L1) expression and immune cell cytotoxicity. Interrogation of web-based data-mining platforms, showed upregulation of ILK expression in tumors and adjacent-non tumor tissue of colorectal cancer (CRC) associated with poor survival and advanced stages. ILK expression was correlated with cancer-associated fibroblast (CAFs) and immunosuppressive cell infiltration including regulatory T cells (Treg) and M2 macrophages (M2) in addition to their gene markers. ILK expression was also significantly correlated with the expression of different cytokines and chemokines. ILK expression showed pronounced association with different important immune checkpoints including PD-L1. Deletion of the ILK gene in PD-L1 positive CRC cell lines using a doxycycline inducible-CRISPR/Cas9, resulted in suppression of both the basal and IFNγ-induced PD-L1 expression via downregulating NF-κB p65. This subsequently sensitized the CRC cells to NK92 immune cell cytotoxicity. These findings suggest that ILK can be used as a biomarker for prognosis and immune cell infiltration in colon cancer. Moreover, ILK could provide a therapeutic target to prevent immune evasion mediated by the expression of PD-L1.

Integrin-linked kinase affects the sensitivity of esophageal squamous cell carcinoma cells to chemotherapy with cisplatin via the Wnt/beta-catenin signaling pathway

ABSTRACT Recent studies have shown that the expression of integrin-linked kinase (ILK) was related to the occurrence, development, and malignant progression of esophageal squamous cell carcinoma (ESCC). However, research on the relationship between ILK and the chemosensitivity of ESCC has to date not been reported. The present study found that ILK was highly expressed in ESCC cell lines, and the overexpression of ILK in ESCC cells reduced the incidence of cell apoptosis and alleviated the cytotoxicity on cells induced by cisplatin (CDDP). Inversely, ILK knockdown increased CDDP-induced apoptosis and had an inhibitive effect on the malignant phenotype of ESCC, including cell proliferation, invasion, and migration. In addition, ILK knockdown in ESCC cells inhibited the expression of beta (β)-catenin and activated the wingless/integrated (Wnt) signaling pathway. Furthermore, cellular MYC (c-MYC) and Cylin D1 were the target genes of the Wnt signaling pathway. Rescue experiments showed that the overexpression of β-catenin reversed a tumor’s inhibition and apoptosis abilities induced by ILK knockdown. In conclusion, ILK potentially reduced the CDDP sensitivity of ESCC cells by influencing the activity of the Wnt/β-catenin signaling pathway.