Integrin-linked Kinase Research Articles

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.

PSVIII-B-5 Dynamic of Accessible Chromatin Regions in Cattle Rumen Epithelial Tissue During Weaning

Abstract Weaning in ruminants is characterized by the transition from a milk-based diet to a solid diet which drives a critical gastrointestinal tract transformation. Identifying regulatory control of this transformation during weaning can help identify strategies to improve rumen health. This study aimed to identify regions of accessible chromatin in rumen epithelial tissue in pre- and post-weaning calves and investigate differentially accessible regions (DARs) to uncover regulatory elements in cattle rumen development using the ATAC-seq approach. A total of 126,071 peaks were identified, covering 1.15% of the cattle genome. From these accessible regions, 2,766 DARs were discovered. Gene ontology enrichment resulted in GO terms related to cell adhesion, anchoring junction, growth, cell migration, motility, and morphogenesis. In addition, putative regulatory canonical pathways were identified (TGFβ, Integrin-linked kinase, Integrin signaling, and regulation of the epithelial-mesenchymal transition). Canonical pathways integrated with co-expression results showed that TGFβ and ILK signaling pathways play essential roles in rumen development through the regulation of cellular adhesions. In this study, DARs during weaning have been identified, revealing enhancers, transcription factors, and candidate target genes that represent potential biomarkers for the bovine rumen development which will serve as a molecular tool for rumen development studies.

Laminin-α4 Negatively Regulates Adipocyte Beiging Through the Suppression of AMPKα in Male Mice.

Laminin-α4 (LAMA4) is an extracellular matrix protein implicated in the regulation of adipocyte differentiation and function. Prior research describes a role for LAMA4 in modulating adipocyte thermogenesis and uncoupling protein-1 (UCP1) expression in white adipose; however, the mechanisms involved are poorly understood. Here, we describe that Lama4 knockout mice (Lama4-/-) exhibit heightened mitochondrial biogenesis and peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) expression in subcutaneous white adipose tissue (sWAT). Furthermore, the acute silencing of LAMA4 with small interfering RNA in primary murine adipocytes was sufficient to upregulate the expression of thermogenic markers UCP1 and PR domain containing 16 (PRDM16). Silencing also resulted in an upregulation of PGC1-α and adenosine 5'-monophosphate-activated protein kinase (AMPK)-α expression. Subsequently, we show that integrin-linked kinase (ILK) is downregulated in the sWAT of Lama4-/- mice, and its silencing in adipocytes similarly resulted in elevated expression of UCP1 and AMPKα. Last, we demonstrate that treatment of human induced pluripotent stem cell-derived thermogenic adipocytes with LAMA4 (LN411) inhibited the expression of thermogenic markers and AMPKα. Overall, our results indicate that LAMA4 negatively regulates a thermogenic phenotype and pathways involving mitochondrial biogenesis in adipocytes through the suppression of AMPKα.

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.

Extracellular matrix stiffness regulates degradation of MST2 via SCF βTrCP

The Hippo pathway plays central roles in relaying mechanical signals during development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we found that chemical inhibition of proteasomal proteolysis resulted in increased levels of MST2 in human breast epithelial cells. MST2 binds SCFβTrCP E3 ubiquitin ligase and silencing βTrCP resulted in MST2 accumulation. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that βTrCP binds MST2 via a non-canonical degradation motif. Additionally, stiffer extracellular matrix, as well as hyperactivation of integrins resulted in enhanced MST2 degradation mediated by integrin-linked kinase (ILK) and actomyosin stress fibers. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and underscores how alterations in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.

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.

Differentially CTCF-Binding Sites in Cattle Rumen Tissue during Weaning.

The weaning transition in calves is characterized by major structural changes such as an increase in the rumen capacity and surface area due to diet changes. Studies evaluating rumen development in calves are vital to identify genetic mechanisms affected by weaning. This study aimed to provide a genome-wide characterization of CTCF-binding sites and differentially CTCF-binding sites (DCBS) in rumen tissue during the weaning transition of four Holstein calves to uncover regulatory elements in rumen epithelial tissue using ChIP-seq. Our study generated 67,280 CTCF peaks for the before weaning (BW) and 39,891 for after weaning (AW). Then, 7401 DCBS were identified for the AW vs. BW comparison representing 0.15% of the cattle genome, comprising ~54% of induced DCBS and ~46% of repressed DCBS. Most of the induced and repressed DCBS were in distal intergenic regions, showing a potential role as insulators. Gene ontology enrichment revealed many shared GO terms for the induced and the repressed DCBS, mainly related to cellular migration, proliferation, growth, differentiation, cellular adhesion, digestive tract morphogenesis, and response to TGFβ. In addition, shared KEGG pathways were obtained for adherens junction and focal adhesion. Interestingly, other relevant KEGG pathways were observed for the induced DCBS like gastric acid secretion, salivary secretion, bacterial invasion of epithelial cells, apelin signaling, and mucin-type O-glycan biosynthesis. IPA analysis further revealed pathways with potential roles in rumen development during weaning, including TGFβ, Integrin-linked kinase, and Integrin signaling. When DCBS were further integrated with RNA-seq data, 36 putative target genes were identified for the repressed DCBS, including KRT84, COL9A2, MATN3, TSPAN1, and AJM1. This study successfully identified DCBS in cattle rumen tissue after weaning on a genome-wide scale and revealed several candidate target genes that may have a role in rumen development, such as TGFβ, integrins, keratins, and SMADs. The information generated in this preliminary study provides new insights into bovine genome regulation and chromatin landscape.

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

Discovery of small molecule therapies targeting the Kinase pathway in ovarian cancer (310)

<b>Objectives:</b> Refining a novel drug compound to optimize the molecular inhibition of Integrin-Linked Kinase in ovarian cancer. <b>Methods:</b> Tissue from treatment-naïve ovarian cancer patients (<i>n</i>=24) was compared to normal adjacent ovarian tissue from the same patient using whole-genome microarray. When comparing the normal tissue to cancer tissue from the same patient, we identified the integrin-linked kinase pathway (ILK) to be upregulated (1.5-fold and FDR <0.05 threshold). Given this finding, we validated the role of ILK by knocking this gene out using CRISPR/Cas-9 in SKOV3 cells. Using SRB assay, we compared cell proliferation in parental SKOV3 to the SKOV3-ILK-Knockout cell line, which revealed decreased proliferation in ILK knockout cells. Both cell lines were injected subcutaneously into NOD SCID gamma immunocompromised mice. Tumor growth was assessed by measuring tumor volume thrice weekly and final tumor weight. <b>Results:</b> Tumor growth was significantly less (p <0.0001) in the SKOV3-ILK-Knockout injected mice compared with parental control. Given this finding, we utilized a library of 100 molecules in which several of them have structural similarity to published kinase inhibitors in an attempt to identify an inhibitor of ovarian cancer cell proliferation and tumor growth and kinase inhibition. We identified a small molecule that decreased the proliferation of SKOV3 cells and OV90 cells (Figure 1). In order to investigate whether this drug had antiproliferative effects on non-cancerous cells, we treated COS7 non-cancerous cells with the small molecule and found no antiproliferative effect. Fig. 1 <b>Conclusions:</b> This suggests that the effects of the small molecule are cancer-specific and will have minimal off-target toxicity if further investigated in xenograft models. Once validated in xenograft models, this molecule will be further investigated for potential use in ovarian cancer patients.

Role of the integrin-linked kinase/TGF-Β/SMAD pathway in sitagliptin mediated cardioprotective effects in a rat model of diabetic cardiomyopathy

Background and Aims : In this study, we investigate whether sitagliptin mediates cardioprotection via interference with transforming growth factor-beta (TGF-(β signaling through the SMAD and integrin-linked kinase (ILK) pathways in rats with streptozotocin-induced type 2 diabetes.

Insight into the mechanism of molecular recognition between human Integrin-Linked Kinase and Cpd22 and its implication at atomic level.

AbsractPseudokinases have received increasing attention over the past decade because of their role in different physiological phenomena. Although pseudokinases lack several active-site residues, thereby hindering their catalytic activity, recent discoveries have shown that these proteins can play a role in intracellular signaling thanks to their non-catalytic functions. Integrin-linked kinase (ILK) was discovered more than two decades ago and was subsequently validated as a promising target for neoplastic diseases. Since then, only a few small-molecule inhibitors have been described, with the V-shaped pyrazole Cpd22 being the most interesting and characterized. However, little is known about its detailed mechanism of action at atomic level. In this study, using a combination of computational chemistry methods including PELE calculations, docking, molecular dynamics and experimental surface plasmon resonance, we were able to prove the direct binding of this molecule to ILK, thus providing the basis of its molecular recognition by the protein and the effect over its architecture. Our breakthroughs show that Cpd22 binding stabilizes the ILK domain by binding to the pseudo-active site in a similar way to the ATP, possibly modulating its scaffolding properties as pseudokinase. Moreover, our results explain the experimental observations obtained during Cpd22 development, thus paving the way to the development of new chemical probes and potential drugs.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10822-022-00466-1.

Skeletal muscle fiber type-specific expressions of mechanosensors integrin-linked kinase, talin, and vinculin and their modulation by loading and environmental conditions in humans.

Mechanosensors control muscle integrity as demonstrated in mice. However, no information is available in human muscle about the distribution of mechanosensors and their adaptations to mechanical loading and environmental conditions (hypoxia). Here, we hypothesized that mechanosensors show fiber-type-specific distributions and that loading and environmental conditions specifically regulate mechanosensors. We randomly subjected 28 healthy males to one of the following groups (n=7 each) consisting of nine loading sessions within 3 weeks: normoxia moderate (NM), normoxia intensive (NI), hypoxia moderate (HM), and hypoxia intensive (HI). We took six biopsies: pre (T0), 4h (T1), and 24 h (T2) after the third as well as 4h (T3), 24 h (T4), and 72 h (T5) after the ninth training session. We analyzed subjects' maximal oxygen consumption (V̇O2 max), maximal power output (Pmax), muscle fiber types and cross-sectional areas (CSA), fiber-type-specific integrin-linked kinase (ILK) localizations as well as ILK, vinculin and talin protein and gene expressions in dependence on loading and environmental conditions. V̇O2 max increased upon NM and HM, Pmax upon all interventions. Fiber types did not change, whereas CSA increased upon NI and HI, but decreased upon HM. ILK showed a type 2-specific fiber type localization. ILK, vinculin, and talin protein and gene expressions differed depending on loading and environmental conditions. Our data demonstrate that mechanosensors show fiber type-specific distributions and that exercise intensities rather than environmental variables influence their profiles in human muscles. These data are the first of their kind in human muscle and indicate that mechanosensors manage the mechanosensing at a fiber-type-specific resolution and that the intensity of mechanical stimulation has a major impact.

OSU-T315 and doxorubicin synergistically induce apoptosis via mitochondrial pathway in bladder cancer cells.

Bladder cancer (BC) is a common urological malignancy that still lacks an effective treatment. Doxorubicin (Dox) has been widely used in the treatment of various cancers, including BC. However, chemoresistance often hampers the clinical application of Dox, therefore, it is necessary to develop effective strategies to improve its efficacy. By using high-throughput screening, we identified OSU-T315, an integrin-linked kinase (ILK) inhibitor, that can augment the cytotoxicity of Dox against BC cells. We found that OSU-T315 and Dox synergistically induce apoptosis of BC cells via mitochondrial pathway in a caspase-dependent. Mechanically, it was found that OSU-T315 and Dox synergistically induced activation of Bax which is critical for the induction of apoptosis. Moreover, it was also found that the downregulation of BCL-2 and MCL-1 is essential for the activation of BAX induced by OSU-T315 and Dox. OSU-T315 was found to downregulate MCL-1 via the GSK-3β/FBXW7 axis in BC cells. Our findings suggest that combined treatment with OSU-T315 and Dox may be a promising strategy to treat BC.

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.

Descifrando el mecanismo de inhibición de una pseudoquinasa: la quinasa ligada a integrinas y el cpd22

According to World Health Organization (WHO) cancer is one of top non- infectious death causes worldwide. In this context, as such the searching of new potent and safe anticancer drugs is a hot point for pharmacy industry and academic investigation. Despite protein kinases have been one of the most explored anticancer targets, its evolutionary degenerated parents, pseudokinases, have attracted the attention of scientific community during the last decade. Integrin Linked Kinase (ILK) is a member of the human pseudokinome that has been claimed and validated as a prommissing target for neoplastic diseases. The only well-known ILK inhibitor, CPD22, has probed its activity in phenotypic assays, however very few knowledge regarding its mechanism of action at molecular level is available. Using chemoinformatic and molecular modelling techniques we were able to elucidate if this molecule is able to bind to ILK and how. Additionally, our model explains the SAR raised from the optimization campaign, and SPR experiments have probed, by first time, that this molecule binds to ILK, thus supporting the hypothesis of inhibition by direct binding and the computational model as well.

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.

Cardiac differentiation of human pluripotent stem cells using defined extracellular matrix proteins reveals essential role of fibronectin.

Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin (FN), laminin-111, and laminin-521 enabled hPSCs to attach and expand. However, only addition of FN promoted cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF in contrast to the inhibition produced by laminin-111 or laminin-521. hPSCs in culture produced endogenous FN which accumulated in the ECM to a critical level necessary for effective cardiac differentiation. Inducible shRNA knockdown of FN prevented Brachyury+ mesoderm formation and subsequent hPSC-CM generation. Antibodies blocking FN binding integrins α4β1 or αVβ1, but not α5β1, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase led to a decrease in phosphorylated AKT, which was associated with increased apoptosis and inhibition of cardiac differentiation. These results provide new insights into defined matrices for culture of hPSCs that enable production of FN-enriched ECM which is essential for mesoderm formation and efficient cardiac differentiation.

Abstract 161: Regulation of Abl at mitotic centrosomes and survival by ILK in glioblastoma cells

Abstract Many cancer cells possess supernumerary centrosomes, having more than one centrosome at each pole during mitosis which can lead to aberrant mitotic division and cell death. To prevent this fate, cancer cells cluster their centrosomes at two poles during division to divide in a pseudobipolar manner. As the presence of supernumerary centrosomes is rare in normal cells, targeting the declustering of supernumerary centrosomes in cancer cells is a promising strategy for cancer treatment. Integrin-linked kinase (ILK) is an oncogenic protein and chemotherapeutic target shown to be found at the centrosome where it has a role in facilitating centrosome clustering. Inhibition of ILK, via an anti-ILK chemotherapeutic drug QLT-0267, has been shown to increase centrosome declustering causing mitotic arrest and cell death. However, not all cancers cells are susceptible to anti-ILK treatment alone. Therefore, we investigate a combination treatment strategy targeting ILK and another oncogenic kinase, Abelson kinase (Abl). Both ILK and Abl are involved in cancer cell survival, regulated by the same signalling pathways and share common binding partners that occur at the centrosome and are critical for mitosis. The ILK inhibitor QLT-0267 induced a significant sub-G1 peak following propidium iodide FACS analysis and an increase in MTT labelling, indicating that QLT-0267 significantly decreased survival in glioblastoma cell lines. These effects were augmented in the presence of imatinib, an Abl inhibitor. Moreover, QLT-0267 and imatinib, in combination, significantly increased mitotically arrested cells over the ILK inhibitor alone. Increased levels of cytosolic Abl have been associated with its transformative abilities. The ILK inhibitor effects on survival correlated with its ability to decrease cytosolic Abl levels and inhibited Abl’s localization to mitotic centrosomes in dividing glioblastoma cells. These effects were reversed by the proteasomal inhibitor MG132 (a drug that inhibits the degradation of Abl). Together these results indicate that combination treatment with QLT-0267 and imatinib are more effective then QLT-0267 alone at decreasing successful mitoses and survival in glioblastoma cells. Future studies will unravel the relationship between these oncogenic kinases at centrosomes and its effect on cancer cell cycle progression and survival. Citation Format: Jonathan Dresselhuis, Maddisen Brown, Nicole Robinson, Jacqueline Tan, Michael E. Cox, Julia Mills. Regulation of Abl at mitotic centrosomes and survival by ILK in glioblastoma 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 161.

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.