Integrins mediate adhesive interactions between cells and their environment, including neighboring cells and extracellular matrix (ECM). These heterodimeric transmembrane receptors bind... Show moreIntegrins mediate adhesive interactions between cells and their environment, including neighboring cells and extracellular matrix (ECM). These heterodimeric transmembrane receptors bind extracellular ligands with their globular head domains and connect to the cytoskeleton through multi-protein interactions at their cytoplasmic tails. Integrin containing cell-matrix adhesions are dynamic force-responsive protein complexes that allow bidirectional mechanical coupling of cells with their environment. This allows cells to sense and modulate tissue mechanics and regulates intracellular signaling impacting on cell faith, survival, proliferation, and differentiation programs. Dysregulation of these functions has been extensively reported in cancer and associated with tumor growth, invasion, angiogenesis, metastasis, and therapy resistance. This central role in multiple hallmarks of cancer and their localization on the cell surface makes integrins attractive targets for cancer therapy. However, despite a wealth of highly encouraging preclinical data, targeting integrin adhesion complexes in clinical trials has thus far failed to meet expectations. Contributing factors to therapeutic failure are 1) variable integrin expression, 2) redundancy in integrin function, 3) distinct roles of integrins at various disease stages, and 4) sequestering of therapeutics by integrin-containing tumor-derived extracellular vesicles. Despite disappointing clinical results, new promising approaches are being investigated that highlight the potential of integrins as targets or prognostic biomarkers. Improvement of therapeutic delivery at the tumor site via integrin binding ligands is emerging as another successful approach that may enhance both efficacy and safety of conventional therapeutics. In this review we provide an overview of recent encouraging preclinical findings, we discuss the apparent disagreement between preclinical and clinical results, and we consider new opportunities to exploit the potential of integrin adhesion complexes as targets for cancer therapy. Show less
Background: Approximately 15% of patients undergoing resection for presumed perihilar cholangiocarcinoma (PHC) have benign disease at final pathological assessment. Molecular imaging targeting... Show moreBackground: Approximately 15% of patients undergoing resection for presumed perihilar cholangiocarcinoma (PHC) have benign disease at final pathological assessment. Molecular imaging targeting tumor-specific biomarkers could serve as a novel diagnostic tool to reduce these futile surgeries. Imaging agents have been developed, selectively binding integrin alpha(nu)beta 6, a cell receptor upregulated in pancreatobiliary malignancies, for both (preoperative) PET and (intraoperative) fluorescent imaging. Here, expression of integrin alpha(nu)beta 6 is evaluated in PHC, intrahepatic cholangiocarcinoma (ICC), hepatocellular carcinoma (HCC) and benign disease mimicking PHC using immunohistochemistry.Materials & methods: Three tissue microarrays (TMA) including 103 PHC tumor cores and sixty tissue samples were selected from resection specimens of pathologically proven PHC (n = 20), ICC (n = 10), HCC (n = 10), metastatic PHC lymph nodes (n = 10) and benign disease (presumed PHC with benign disease at pathological assessment, n = 10). These samples were stained for integrin anb6 and quantified using the H-score.Results: Immunohistochemical staining for integrin alpha(nu)beta 6 showed membranous expression in all twenty PHC whole mount slides (100%) and 93 out of 103 (92%) PHC tumor cores. Mean H-score of PHC samples was 195 +/- 71, compared to a mean H-score of 126 +/- 57 in benign samples (p = 0.013). In both benign and PHC samples, inflammatory infiltrates and pre-existent peribiliary glands showed integrin anb6 expression. The mean H-score across ten ICC was 33 +/- 53, which was significantly lower compared to PHC (p < 0.001) but too weak to consistently discriminate ICC from HCC (H-score 0)(p = 0.062).Conclusion: Integrin anb6 is abundantly expressed in PHC and associated metastatic lymph nodes. Expression is significantly higher in PHC as compared to benign disease mimicking PHC, ICC and HCC, emphasizing its potential as a target for tumor-specific molecular imaging. (C) 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/ Show less
This thesis describes our search to identify and understand possible regulatory mechanisms of integrin α6β4 in cell-matrix adhesion and intracelular signaling.
Human embryonic stem cells (hESC) hold great potential as a model for human development, disease pathology, drug discovery and safety pharmacology. All these applications will depend on... Show moreHuman embryonic stem cells (hESC) hold great potential as a model for human development, disease pathology, drug discovery and safety pharmacology. All these applications will depend on comprehensive knowledge of their biology and control of their signaling mechanisms and fate choices. To begin to address this, we developed a standardized feeder-free hESC culture protocol. This system is optimized and tested for 12 independently derived stem cell lines, and optimal for clonal growth and efficient gene transfer without loss of pluripotency (Chapter 2,3). Using these protocols we created stem cells ubiquitously expressing EGFP, showed efficient SOX2 knockdown and created a fluorescent reporter stem cell line for the stem cell regulator OCT4. Next we used mass spectroscopy to investigate the plasma membrane of hESC (Chapter 4). We were able to show that these cells express a uniform epithelial plasma membrane profile and that VIMENTIN, normally associated with mesenchymal cells is also expressed. This expression turned out to be related to stress and associated with hardness of the tissue culture plastic substrate rather than differentiation. We continued to investigate the plasma membrane of hESC and decided to focus on integrins, the cell surface receptors that bind extracellular matrix proteins. Functional analyses of their function showed human embryonic stem cells have the capacity to bind to a wide range of extracellular matrix proteins via specific integrin receptors. We were able to show that recombinant vitronectin robustly supports the maintenance of hESC in an undifferentiated state in completely defined culture medium. Having validated a completely defined culture protocol we began to investigate differentiation mechanisms under defined conditions (Chapter 5). We used Stable Isotope Labeling in Cell Culture (SILAC) and quantitative phopspho-proteomics to investigate how human embryonic stem cells exit the pluripotent state. BMP4 was used to trigger differentiation and protein samples were analyzed at 30 min, 60 min and 240 min. We showed that approximately 50% of the 3067 identified phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships we reconstructed the hESC kinome, with CDK1/2 emerging as a key kinase in controlling self-renewal and lineage specification. Next we used gene targeting to create a fluorescent cardiac reporter cell line. EGFP was targeted into one allele of the NKX2-5 gene. EGFP fluorescence driven by the endogenous Nkx2-5 promoter faithfully reported cardiovascular lineage commitment of differentiating hESC under defined culture conditions. Using fluorescence activated cell sorting we showed that the early NKX2-5 positive cell population contained multipotent progenitor cells capable of directed differentiation to cardiomyocytes, endothelial and vascular smooth muscle cells (Chapter 7). Finally, we used the cardiomcyocytes from hESC to develop a system for cardiac safety pharmacology (Chapter 8). Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. In particular, blocking of the human Ether-a-go go Related Gene ion channel is associated with life-threatening arrhythmias, such as Torsade de Pointes. We demonstrated that, as predicted, patient serum levels of drugs and known responses on QT interval overlapped with field potential duration values derived from hESC-CM,. On this basis, we propose field potential duration prolongation as a directly applicable safety criterion for pre-clinical evaluation of new drugs in development. In conclusion, the availability of human cardiomyocytes from stem cell sources is now expected to accelerate cardiac drug discovery and safety pharmacology by offering more clinically relevant human culture models than presently available (Chapter 9,10). Show less
