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Shb gene knockdown increases the susceptibility of SVR endothelial tumor cells to apoptotic stimuli in vitro and in vivo
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
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2008 (English)In: Journal of Investigative Dermatology, ISSN 0022-202X, E-ISSN 1523-1747, Vol. 128, no 3, 710-716 p.Article in journal (Refereed) Published
Abstract [en]

The Shb adapter protein is an Src homology 2-domain containing signaling intermediate operating downstream of several tyrosine kinase receptors, including vascular endothelial growth factor receptor-2. Shb is multifunctional and apoptosis is one response that Shb regulates. Inhibition of angiogenesis can be used in cancer therapy, and one way to achieve this is by inducing endothelial cell apoptosis. The angiosarcoma cell line SVR is of endothelial origin and can be used as a tool for studying in vivo inhibition of angiogenesis, and we thus employed an Shb-knockdown strategy using an inducible lentiviral system to reduce Shb levels in SVR cells and to study their responses. Shb knockdown increases the susceptibility of SVR cells to the apoptotic agents, cisplatin and staurosporine. Simultaneously, Shb knockdown causes reduced focal adhesion kinase (FAK) activation, monitored as phosphorylation of the regulatory residues tyrosines 576/577. No detectable effects on Akt or extracellular signal-regulated kinase activity were noted. The altered FAK activity coincided with an elongated cell phenotype that was particularly noticeable in the presence of staurosporine. In order to relate the effects of Shb knockdown to in vivo tumorigenicity, cells were exposed to the angiogenesis inhibitor honokiol, and again the cells with reduced Shb content exhibited increased apoptosis. Tumor growth in vivo was strongly reduced in the Shb-knockdown cells upon honokiol treatment. It is concluded that Shb regulates apoptosis and cell shape in tumor endothelial cells via FAK, and that Shb is a potential target for inhibition of angiogenesis.

Place, publisher, year, edition, pages
2008. Vol. 128, no 3, 710-716 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-97231DOI: 10.1038/sj.jid.5701057ISI: 000253374300024OAI: oai:DiVA.org:uu-97231DiVA: diva2:172072
Available from: 2008-05-09 Created: 2008-05-09 Last updated: 2012-12-14
In thesis
1. The role of Shb in ES cell differentiation, angiogenesis and tumor growth
Open this publication in new window or tab >>The role of Shb in ES cell differentiation, angiogenesis and tumor growth
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors and intracellular signaling proteins. Previous studies have implied a wide spectrum of Shb-mediated cellular responses, which motivated me to further investigate the role of Shb in differentiation and angiogenesis. Embryonic stem (ES) cells differentiate into endoderm and mesoderm from a bipotent mesendodermal cell population. Interregulatory signals between these germlayers are required for further specification. ES cells overexpressing Shb with an inactive SH2 domain (R522K-Shb) altered the expression of endodermal genes as a consequence of upregulated FGF expression. This response was enhanced by addition of activin A, suggesting a synergistic mechanism operative between FGF and activin A signaling in endoderm specification. To investigate a role for Shb in mesodermal specification, Shb knockout ES cells were established. These cells showed a reduced ability to form blood vessels after VEGF stimulation and delayed downregulation of genes associated with mesendoderm, indicating a reduced capacity for these cells to enter later stages.

To assess a role for Shb in tumor cell apoptosis, Shb expression was silenced in angiosarcoma endothelial cells. FAK-phosphorylation was reduced in Shb knockdown cells and this made them more susceptible to apoptotic stimuli both in vitro and in vivo.

Shb knockout microvasculature in mouse kidney, liver, and heart showed irregular endothelial linings with cytoplasmic projections toward the lumen, a feature that was also related to increased vascular permeability. VEGF treatment failed to stimulate vascular permeability in Shb knockout mice.

In order to elucidate whether these features relate to reduced angiogenesis, tumor growth was examined. Tumors grown in knockout mice showed reduced growth capacity and lower vessel density. In conclusion, Shb is a multifunctional adaptor protein that may be involved in several cellular responses both during embryonic development and adult life.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 49 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 355
Cell biology, Shb, tyrosine kinase signaling, ES, endoderm, mesoderm, apoptosis, microvasculature, tumor growth, Cellbiologi
urn:nbn:se:uu:diva-8746 (URN)978-91-554-7205-4 (ISBN)
Public defence
2008-05-31, Auditorium minus, Gustavianum, Akademigatan 3, Uppsala, 13:00
Available from: 2008-05-09 Created: 2008-05-09Bibliographically approved

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Welsh, Michael
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