| Abstract |
Osteosarcomas comprise approximately 1% of total malignant tumors diagnosed every
year and they have a mesenchymal origin. Osteosarcoma is the second most common
primary bone tumor, after myeloma and 20% of all sarcomas of the bone are
osteosarcomas. Its incidence is higher during the second decade and lower in the sixth
decade of the human life. An important characteristic of osteosarcoma is its
heterogeneity and ability to produce abundant non-mineralized ECM–osteoid, mainly
consisting of collagen type I, glycoproteins, and proteoglycans (PGs)/GAGs (Benayahu
et al., 2001; Nikitovic et al., 2013).
The small leucine-rich proteoglycans (SLRPs) were originally defined as proteoglycans
with a relatively small protein core (36–42 kDa) harboring tandem leucine-rich repeats
and undergoing post-translational modifications, including substitution with
glycosaminoglycan side chains of various types (Iozzo and Murdoch 1996; Iozzo
1997). Their ubiquitous tissue distribution and expression at strategic sites in
embryogenesis and tissue repair, coupled with their protein conservation, suggests that
SLRP functions are of no small consequence. Originally, the SLRPs were grouped into
three distinct classes based on nucleotide and protein sequence conservation, the
organization of disulfide bonds at their N and C termini, and their genomic organization
(Iozzo 1997; Danielson et al., 1993). More recently, the SLRP gene family has
expanded to encompass 18 genes classified into five distinct subfamilies (Schaefer and
Iozzo 2008; Iozzo et al., 2011), additionally based on N-terminal Cys-rich clusters of
the protein core and ear repeats (C-terminal repeats specific to SLRPs) (McEwan et al.,
2006), chromosomal organization (Schaefer and Iozzo 2008; Iozzo et al., 2011), and,
importantly, functional commonality in view of the fact that some SLRPs are not classical proteoglycans (Schaefer and Iozzo 2008). Thus, the canonical class I members
decorin and biglycan contain chondroitin or dermatan sulfate side chains, whereas the
more recently described asporin does not (Henry et al., 2001; Kisawa et al., 2005). On
the other hand, all class II members bear keratan sulfate chains or polylactosamine in
their leucine-rich repeats, whereas class III members carry keratan sulfate
(osteoglycin), chondroitin/dermatan sulfate (epiphycan), or no glycosaminoglycan
(opticin) chains (Schaefer and Iozzo 2008, Sanders et al., 2003). However, most noncanonical
class IV and V members (Neame et al., 1994; Pusch et al., 2000; Ohta et al.,
2006) unexpectedly lack any glycosaminoglycan chain, with the exception of
chondroadherin, which is substituted with keratan sulfate (Neame et al., 1994).
Therefore, the unique characteristics of their protein cores and the presence of
glycosaminoglycans, together with specific post-translational modifications, notably
changes in the degree of glycosaminoglycan epimerization or sulfation, characterize
this class of proteoglycans with high structural complexity.
The present PhD thesis investigated the role of two important SLRPs, lumican and
biglycan, in the regulation of osteosarcoma cell function.
Our research group has shown that human osteosarcoma cell lines express and secrete
lumican partly substituted with keratan sulfate glycosaminoglycans Nikitovic et al.,
2008. Lumican appears to have a role in osteosarcoma pathogenesis, as the growth of
Saos 2 osteosarcoma cells was inhibited by lumican, whereas their migration and
chemotactic response to fibronectin were found to be promoted (Nikitovic et al., 2008).
These key tumor cell functions appear to be modulated through, crucial for bone tumor
cells, Smad signaling pathway (Nikitovic et al., 2008). In this study, we examine the
effect of lumican on osteosarcoma cell adhesion and investigate the hypothesis that
lumican through the modulation of the pericellular availability of the transforming growth factor-beta (TGF-β) isoform/s regulates its downstream intracellular signaling
and thus affects cell functions. Human osteosarcoma cell lines were recently shown to
express and secrete the small leucine rich proteoglycan (SLRP) lumican, with the ability
to regulate the growth and motility of these cells. In this study, lumican-deficient Saos
2 cells were demonstrated to have increased adhesive capability onto fibronectin (FN)
(p≤0.01). Upon neutralization of endogenous transforming growth factor β2 (TGF-β2)
activity, no difference in the ability of lumican siRNA-transfected and scramble
siRNA-transfected Saos 2 cells to adhere onto FN was detected (p = NS). Exogenous
TGF-β2 was shown to stimulate Saos 2 cell adhesion to FN (p≤0.01). These results
therefore, suggest that the inverse correlation existing between lumican expression and
Saos 2 cell adhesion is dependent on active TGF-β signaling. Furthermore, the
significant increase in Smad 2 activation present in lumican-deficient cells (p≤0.01)
was annulled in the presence of the anti-TGF-β2 peptide, demonstrating that lumican is
an upstream regulator of the TGF-β2/Smad 2 signaling cascade. Crucial to FNdependent
adhesion, β1 integrin expression and pFAK activation were likewise
identified as downstream TGF-β2 effectors regulated by lumican expression. In
conclusion, this study demonstrates a novel out-in signaling circuit in human
osteosarcoma cells: secreted to extracellular matrix lumican is an endogenous inhibitor
of TGF-β2 activity, resulting in downstream effector modulation including pSmad 2,
integrin β1 and pFAK to regulate osteosarcoma adhesion.
Furthermore, this study investigated osteosarcoma cell function modulation of the
equally important SLRP, biglycan.
Biglycan, a small leucine rich proteoglycan (SLRP), is an important participant in bone
homeostasis and development as well as in bone pathology. In the present study
biglycan was identified as a positive regulator of MG63 osteosarcoma cell growth (p ≤ 0.001). IGF-I was shown to increase biglycan expression (p ≤ 0.01), whereas biglycandeficiency
attenuated significantly both basal and IGF-I induced cell proliferation of
MG63 cells (p ≤ 0.001; p ≤ 0.01, respectively). These effects were executed through
the IGF-IR receptor whose activation was strongly attenuated (p ≤ 0.01) in biglycandeficient
MG63 cells. Biglycan, previously shown to regulate Wnt/b-catenin pathway,
was demonstrated to induce a significant increase in b-catenin protein expression
evident at cytoplasmic (p ≤ 0.01), membrane (p ≤ 0.01), and nucleus fractions in MG63
cells (p ≤ 0.05). As demonstrated by immunofluorescence, increase in b-catenin
expression is attributed to co-localization of biglycan with the Wnt co-receptor lowdensity
lipoprotein receptor-related protein 6 (LRP6) resulting in attenuated b-catenin
degradation. Furthermore, applying anti-b-catenin and anti-pIGF-IR antibodies to MG-
63 cells demonstrated a cytoplasmic and to the membrane interaction between these
molecules that increased upon exogenous biglycan treatment. In parallel, the
downregulation of biglycan significantly inhibited both basal and IGF-I-dependent
ERK1/2 activation, (p ≤ 0.001). In summary, we report a novel mechanism where
biglycan through a LRP6/b-catenin/IGF-IR signaling axis enhances osteosarcoma cell
growth (Aggelidakis et al., 2018).
In summary, the results of this PhD thesis described two novel mechanisms with which
two important cancer cell functions were modulated, adhesion and proliferation by
lumican and biglycan respectively. These SLRPs are suggested to be used as target
molecules for the regulation of osteosarcoma progression and maybe part of therapy
treatments for tumors of mesenchymal origin.
|
Ο ρόλος των μικρών πλούσιων σε λευκίνη πρωτεογλυκανών (SLRPs) στη συμπεριφορά των καρκινικών κυττάρων ανθρώπινου οστεοσαρκώματος.
