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Identifier 000416955
Title Μελέτη του ρόλου των συνδεκανών στην παθογένεια του ινοσαρκώματος και διερεύνηση της επίδρασης των αυξητικών παραγόντων
Alternative Title Study of the role of syndecans and growth factors on fibrosarcoma progression
Author Μυτιληναίου, Μαρία
Thesis advisor Τζανακάκης, Γεώργιος
Reviewer Κοντάκης, Γεώργιος
Κρασαγάκης, Κων/νος
Αγγελάκη, Σοφία
Debree, Elcon
Τζαρδή, Μαρία
Τσιαούσης, Ιωάννης
Abstract The focus of the present PhD thesis was to further understand the underlying mechanisms that are responsible for the progress of human adult fibrosarcoma. With the utilization of new, specialized techniques, which have resulted in new classification systems it is defined as a rare tumor with poor prognosis. This soft tissue sarcoma is derived from the malignant transformation of the fibroblasts, cells known to produce abundant extracellular matrix (ECM) which ultimately supports surrounding tissues. Fibrosarcoma cells are morphologically characterized as spindle cells, with enhanced motility. It is well established that fibrosarcoma cells strongly modulate the surrounding ECM. ECM is a complex extracellular network containing proteoglycans, fibrous proteins and hyaluronic acid, which supports and protects the cells within the tissues. Moreover, ECM is able to create and transfer multiple signals to the cells, thus affecting their basic functions and behaviour. Indeed, ECM is a pool of growth factors, such as TGF (transforming growth factor), EGF (epidermal growth factor), FGF (fibroblast growth factor), VEGF (vascular endothelial growth factor), IGF-Ι (insulin-like growth factor), enzymes like the MMPs (matrix metalloproteinases), synthases, transferases, sulfases and a whole group of other interesting macromolecules which can affect cell behaviour. Proteoglycans are molecules composed of glycosaminoglycan chains (GAG), covalently attached to their protein core. They are classified according to their GAG chains into separate groups: KSPGs (keratan sulfate proteoglycans), HSPGs (heparan sulfate proteoglycans), CSPGs (chondroitin sulfate proteoglycans) and DSPGs (dermatan sulfate proteoglycans). They possess the ability to affect cell functions and ultimately the evolution of a tumour with various mechanisms. Τheir complex functionality comes not only from their basic morphology, but also from their final stereotactic modification by specialized enzymes. These alterations can induce important changes like different localization, degradation and recycling, interplay with other factors, let alone different signalling, both outwards and inwards of the cell. One of the unique proteoglycan characteristics is the direct contact with the ECM and the consequent transport of environmental signals towards the inner part of the cells. Syndecan 2 (SDC2), a heparan sulfate proteoglycan, is known to be crucial for the proliferation and migration of human fibrosarcoma cells (HT1080). SDC2 is recognized to modify the actin cytoskeleton and consequently alter HT1080 cell migration through the FAK/PI3K/Rac signalling pathway. One of the well-studied growth factors, proven to affect the progress of fibrosarcoma, is TGFβ2. It has been reported that TGFβ2 can control the formation of ECM and subsequently, the invasiveness and migration of HT1080 cells. In this study, we show the ability of TGFβ2 to enhance the adhesion of HT1080 cells towards fibronectin (FN) (p ≤ 0.01). Adhesion of cells to other cells as well as the ECM is known to affect the invasive abilities of the cells. SDC2 seems to be essential when it comes to TGFβ2-induced HT1080 cell adhesion, as this ability is remarkably down-regulated in SDC2-deficient cells (p ≤ 0.01). We also show that SDC2 is important for Smad2 activation, an indispensable mediator of TGFβ signalling. It seems that TGFβ2-dependent Smad2 phosphorylation is significantly decreased in HT1080 cells that lack SDC2 (P ≤ 0.05). The co-localization and the regulation of the expression of TGFRIII, one of the TGFβ receptors, is another interesting quality of SDC2. In our study, we confirm the importance of SDC2 in the regulation of TGFRIII expression (p ≤ 0.01) and suggest that this proteoglycan participates in the presentation of TGFRIII on the cell membrane. We also showed that SDC2 modulates the TGF-dependent expression (p ≤ 0.05) and activation (p ≤ 0.01) of FAK, as well as of integrin β1 (p ≤ 0.01), two genes that are necessary for cell adhesion towards FN. Additionally, we studied the effect of HS chains on human fibrosarcoma cell functions. It is already known that HS chains can influence TGFβ-induced cell motility. In order to proceed with these experiments, we used heparitinase, to achieve the specialized digestion of HS chains. This resulted in the formation of HS stubs. Interestingly, the otherwise TGFβ2-enhanced HT1080 cell adhesion was completely reversed after the addition of heparitinase and the shedding of HS chains (p ≤ 0.001). This outcome highlights the important role of HS chains on TGFβ2- induced cell adhesion. Furthermore, HS shedding inhibited TGFβ2-dependent Smad2 activation (p ≤ 0.001). The consequent down-regulation of the TGFβ2 signalling pathway explains the inhibitory effect of HS chains on ΤGFβ2-promoted HT1080 cell adhesion. Finally, the present PhD thesis studied the potential role of IGF-I in fibrosarcoma progression. IGF-I is a known anabolic hormone, with proven oncogenic behaviour. Most of the downstream signalling is conducted through two main signalling pathways: PI3K/AKT and MEK/ERK. While examining the effect of IGF-I on HT1080 cells, we reported a remarkable increase in cell migration, when cells were treated with IGF-I (p ≤ 0.001). SDC2 is abundant in fibrosarcoma and, as previously mentioned, SDC2 can regulate cell motility by changing actin cytoskeleton. Taking into account its unique abilities, we examined its possible participation in IGF-I pathways. The silencing of SDC2 gene resulted interestingly in the inhibition of IGF-I induced HT1080 cell migration (p ≤ 0.001). Knowing that SDC2 can play the role of the co-receptor for several growth factors, such as FGF and GM-CSF (granulocyte-macrophage colony-stimulating factor), the next step was to examine whether SDC2 could be a co-receptor for IGF-I too. In the present study, we show for the first time that SDC2 is co-localized with IGF-IR, suggesting that SDC2 could be a co-receptor for IGF-I and subsequently affect its bioavailability and downstream signalling. Next, we examined the participation of SDC2 in IGF-I signalling and cell migration. As ERK1/2 is an important mediator of IGF-I signalling and affects cell motility in various types of cancer, we tested the effect of SDC2 in IGF-I induced ERK1/2 activation. We show that SDC2 is essential for ERK1/2 phosphorylation (p ≤ 0.001) and we also show that ERK1/2 is essential for the IGF-I dependent HT1080 cell migration (p ≤ 0.001). This could mean that SDC2 regulates IGF-Ι induced migration, at least partly through ERK1/2 activation and via the subsequent ERK1/2-dependent gene transcription towards specific cell behaviours. Finally, we examined the putative role of ezrin, a protein-linker between receptors of the cell membrane and actin cytoskeleton. It has been previously found to be co-localized with SDC2 in filopodia of fibroblast-like cells. Interestingly, we showed for the first time in our cells, that adding IGF-I to HT1080 cells leads to activation of ezrin (p ≤ 0.001) and we confirmed that ezrin is co-localized with SDC2 in ΗΤ1080 cells, making their lamellipodia more prominent. With these results, we present some of the mechanisms through which SDC2 participates in IGF-Idependent HT1080 cell migration.
Language Greek
Subject Cell adhesion
Cell migration
Κυτταρική μετανάστευση
Κυτταρική προσκόλληση
Issue date 2018-07-18
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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