Abstract Integrin-linked Kinase (ILK) is an integrin-associated protein kinase, which regulates growth factor-signalling pathways and cell-ECM adhesion events. Abrogated ILK expression or activity has been implicated in contributing to oncogenic transformation. We examined the role played by ILK in growth factor-stimulated and integrin signalling events in five human oesophageal squamous cell carcinoma cell lines (HOSCCs), known to overexpress the EGF receptor. Western blot analysis revealed the presence of ILK (59kDa) in all the moderately differentiated HOSCC lines. ILK1 was confirmed as being the predominant isoform. Densitometrically analysed Western blots showed that, per unit of protein, ILK is expressed uniformly across the cell lines under standard culture conditions. Following EGF (10 ng/ml) and TGFβ1 (1 ng/ml) treatment, ILK expression increased in all five HOSCCs. Indirect immunofluorescence microscopy showed the majority of ILK to localise at a cytoplasmic/nuclear level, with a proportion of ILK localising at the membrane, which resembled the distribution pattern of the β3 integrin subunit. This membranal distribution most likely follows that of the adhesion plaques although lesser, and variable, amounts were also identified throughout the cytoplasm. The functionality of the ILK1 kinase domain was demonstrated using myelin basic protein (MBP)-based kinase assays. EGF and TGFβ1 treatment produced an increase in ILK activity in the WHCO3 cell line of 3.5 fold, but a decrease in activity in the other cell lines, which are suggested to involve the actions of PTEN. The identification of nuclear ILK was surprising, and the mechanism for nuclear ILK localisation was suggested to involve a caveolae-associated protein, caveolin-1. Cell adhesion assays revealed that KP-392-mediated inhibition of ILK resulted in a nonsignificant reduction in cell adhesion to collagen and fibronectin. These data provide distinctive evidence for the influence of growth factors on ILK expression, but a duality in the effect on ILK activity. This apparent dichotomy is noteworthy and may be of particular relevance in HOSCC. It is further suggested that KP-392-induced ILK inhibition destabilises the αβ integrin heterodimer and that PI3K acts upstream of ILK-mediated cell adhesion events in HOSCCs. This suggests that ILK mediates integrin associated processes in human oesophageal SCC cell lines.

Signaling networks have a crucial role in every aspect of plant communication with the environment. There is significant interest in identifying signal transduction pathways governing CW homeostasis in interactions with pathogens and symbionts. In previous work, our lab has demonstrated that the RAF-like Integrin-Linked Kinase 1 (ILK1) is a negative regulator of FLS2 (Flagellin Sensing2)-mediated signaling, required for defense against a low-virulence P. syringae T3SS-mutant, and modulating cellular ion homeostasis through functional interactions with Ca2+ sensor CML9 and HAK5 K+ transporter (Brauer et al., 2016). In this work we revealed that ILK1 homologs, ILK4 and ILK5, are required for plant response to PAMPs (elf18) and plant damage-associated molecules (pep1) but not flg22. Specifically, we found that ilk4 and ilk5 were unable to undergo priming for Pattern-Triggered Immunity (PTI) with elf18 and pep1 and showed increased susceptibility to virulent P.syringae pv.tomato DC3000. A global transcriptomic analysis revealed the role of ILK1 in modulating the temporal dynamics and range of Arabidopsis transcriptional re-programming postlg22 treatment. In the absence of the PAMP challenge, ilk1-1 showed derepression of a sector of PTI, osmotic and ionic stress, and iron starvation transcriptional programs. Postlg22 challenge, genes for innate immunity, microtubule (MT) structure and movement, CW biosynthesis, and plant growth were differentially regulated in ilk1-1 compared to wt. Phenotyping of ilk1-1 alongside ilk4, ilk5, and OE-ILK5 mutant lines revealed significant de-regulation of induction of defense genes, upregulation of auxin (SAURs) genes, and repression of tubulins and MT-motor protein genes. Moreover, the mutants showed abnormal insensitivity to MT-depolymerizing treatments and defective root growth and displayed CW-specific defects (i.e., ectopic lignin accumulation in the xylem and defects in the pectin-rich seed mucilage). We postulate that ILKs are components of a CW-integrity signaling pathway that suppresses PTI and facilitates CW biosynthesis during normal growth, whereas post-pathogen challenge this pathway is required for defensive re-modeling of the CW and MT cytoskeleton and resumption of plant growth.

No description available.

No description available.

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC₅₀ of < 0.1 μM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Background: Pancreatic ductal adenocarcinoma (PDAC), one of the fourth most lethal malig-nancies in the world, has a less than 5% five-year relative overall survival rate. Thus, there is a great need for novel therapies. PDAC is characterized as a stroma rich malignancy, composed of a large amount of extracellular matrix and pancreatic stellate cells. Accordingly, cell-matrix adhesion is crucial for cancer cell survival, invasion, and therapy resistance. We embarked on a high-throughput assay to identify the function of 117 focal adhesion proteins (FAP) in PDAC cell radiochemoresistance. Material and methods: We generated and performed a 3D tumoroid high-throughput esiRNA-based screening assay (3DHT-esiRNAs) in PDAC cell cultures (established and PDC) grown in laminin-rich extracellular matrix (IrECM). In addition to characterizing the β8 integrin expres-sion, distribution, and co-localization with other cellular organelles, such as Golgi apparatus, we also performed 3D tumoroid formation assay, sphere formation assay, type I collagen-based 3D invasion assay, and 2D clonogenic survival assay following esiRNA-mediated knockdown, 6 Gy x-ray irradiation and gemcitabine treatment. Image analysis was performed to determine Pearson's correlation coefficient, vesicle distribution and expression patterns upon irradiation or gemcitabine treatment by Fiji software (NIH). Immunoprecipitation-mass spectrometry (IP-MS) was performed to investigate the interactome of β8 integrin in the normal versus 6 Gy x-ray irradiation group. Inhibitor screen was conducted following 6 Gy x-ray irradi-ation or gemcitabine treatment to identify pathways involved in changes of β8 integrin localiza-tion upon treatment Autophagy flux was detected by monitoring LC3B puncta. Results: We identified a series of novel targets, including β8 integrin and PINCH1. Intriguingly, depletion of either β8 integrin or PINCH1 both showed radiosensitizing effect, with β8 integrin knockdown exerting a more profound radiosensitizing effect in a panel of PDAC cell lines. Without cytotoxicity, β8 integrin depletion evoked radiochemosensitization in PDAC, PDCs cell lines, and reduced sphere formation and 3D invasion into collagen-I. Intriguingly, β8 integrin was found to be located in the perinuclear region where it co-localized with the cis-Golgi matrix protein, GM130. Upon irradiation and gemcitabine treatment, β8 integrin was translocated from the perinuclear region to the cytosol, showing a slightly increased compart-mentalization in the exosome; a process that was abrogated by treatment with cytoskeletal inhibitors (paclitaxel, latrunculin B, and colchicine) and ATP synthase inhibitors (antimycin A and oligomycin). Depletion of β8 integrin influenced the autophagy via decreasing the LC3B puncta in a microtubule independent manner. Conclusion: Our results generated in 3D lrECM PDAC cell cultures, propose that β8 integrin, but not PINCH1, is a novel determinant of PDAC radiochemoresistance. Moreover, β8 integrin, although not localized to the cell membrane to facilitate cell adhesion, has a critical role in regulating intracellular vesicle trafficking under stress conditions and autophagy flux.

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC50 of < 0.1 µM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation. / Public Authority for Applied Education and Training (PAAET)

No description available.

Hypercholesterolemia is a significant risk factor in the development of cardiovascular disease and is associated with chronic leukocyte adhesion in the microvasculature. While the underlying mechanisms behind this have yet to be determined, it may be possible that hypercholesterolemia impairs the fluid shear stress (FSS) inactivation of neutrophils through the rigidifying effect of cholesterol on membrane fluidity. FSS restricts surface expression of CD18 integrins through cathepsin B (ctsB) proteolysis, which minimizes neutrophil adhesivity. If hypercholesterolemia blocks FSS mechanotransduction, then the inhibition of CD18 cleavage may link pathologic blood cholesterol elevations with dysregulated neutrophil adhesion. We hypothesized that elevated cholesterol contributes to dysregulated neutrophil adhesion by impairing ctsB FSS-induced CD18 cleavage through membrane fluidity changes. In the first part of this study, we demonstrated that FSS-induced CD18 cleavage is a robust response of neutrophils and involves selective cleavage of macrophage 1-antigen (Mac1) through ctsB proteolysis. The second part of this study confirmed that ctsB regulates neutrophil adhesion through its proteolytic actions on Mac1, an important integrin involved in adhesion and chemotaxis. Specifically, ctsB accelerated neutrophil motility through an effect on Mac1 integrins during pseudopod retraction. Furthermore, by using a flow-based assay to quantify the mechanoregulation of neutrophil adhesivity, we demonstrated that FSS-induced ctsB release promoted neutrophil detachment from platelet-coated substrates and unstimulated endothelium. For the third part of this study, we linked cholesterol-related membrane fluidity changes with the ability of FSS to restrict neutrophil adhesion through Mac1. We also determined that pathologic cholesterol elevations associated with hypercholesterolemia could block FSS-induced Mac1 cleavage and were linked to disrupted tissue blood flow. This was accomplished using low-density lipoprotein receptor deficient (LDLR-/-) mice fed a high-fat diet. Ultimately, the results provided in the present study confirmed that cholesterol-related changes in membrane fluidity blocked the ability of ctsB to regulate neutrophil adhesion through FSS-induced Mac1 cleavage. This implicates an impaired neutrophil FSS mechanotransduction response in the dysregulation of neutrophil adhesion associated with hypercholesterolemia. Since dysregulated adhesion may be one of the earliest upstream features of cardiovascular disease associated with hypercholesterolemia, the present study provides a foundation for identifying a new mechanobiological factor in the pathobiology of microcirculatory dysfunction.