Abstract |
This thesis focuses on studying the mechanisms which regulate the biological
functions of tumors with a mesenchymal origin. Sarcomas are types of tumor
which develop from differentiated cells of the connective tissues of the human
body, and therefore can rise from tissues, like bone, cartilage, blood or
vessels. Sarcomas are classified into two categories depending on the tissue
and the cell type of their origin. Thus, there are bone sarcomas, like
osteosarcoma, chondrosarcoma, and sarcomas of soft tissues, like
fibrosarcoma.
In this study, we aimed to identify the signaling pathways which participate in
sarcoma progression in three different human cell lines e.g., osteosarcoma
(MG63), chondrosarcoma (HTB94), and fibrosarcoma (HT1080) respectively.
Tumor cells produce a modified extracellular matrix compared to healthy
tissues, and interaction with the deregulated ECM modulates these cells's
biological functions. ECM consists of a complicated system of proteoglycans,
fibrous proteins and hyaluronic acid, and its role is supportive, but also
protective of each tissue's cells. Furthermore, ECM creates and transfers
many different signals between cells/tissues, affecting their behavior and
functions. ECM also acts as a reserve for growth factors like transforming
growth factor-beta (TFG-β) or insulin-like growth factor I (IGF-I) and other
biologically significant macromolecules able to change cellular behavior.
Proteoglycans (PGs) are molecules that consist of glycosaminoglycan chains
(GAGs) covalently bound into a protein core. These proteins undergo various
post-translational changes, and they can affect cellular function and cancer
progression through a plethora of signaling pathways. Many studies have
shown that PGs are localized to the cell surface but are also secreted into the
ECM, like small leucine rich proteoglycans (SLRPs). This direct interaction
with the extracellular environment gives them the ability to transmit cues from
the ECM to the cells and finally act as signaling molecules that control cellular
functions, like proliferation, migration, differentiation, and further ECM
composition. It is also shown that SLRPs interact with tyrosine kinase
receptors, which affects their downstream signaling and cellular behavior. More specifically, some SLRPs, like biglycan and lumican, participate in bone
formation and homeostasis regulation and have specific roles in the different
phases of these processes. Furthermore, transmembrane proteoglycans, like
syndecan 2, have essential roles in regulating cellular functions of
fibrosarcoma and other types of malignancy. Therefore, PGs act like
regulators of signaling molecules and pathways and could be used as target
molecules either for therapeutic purposes or as diagnostic molecules for
pathological conditions.
IGF-I is a well-established anabolic hormone with proven oncogenic
properties. Most of its functions are performed through two main signaling
pathways, PI3K / Akt (phosphoinositide 3-kinase/ serine/threonine kinase Akt)
and that of MAPK (mitogen-activated protein kinase). Biglycan, an SLRP, has
an active role in bone formation, development, and homeostasis. This study
shows how biglycan regulates the growth of MG63 human osteosarcoma cells
positively. IGF-I increases the expression of this SLRP, while biglycandeficient
cells have attenuated both basal and IGF-I induced proliferation.
These effects were mediated through the IGF-IR receptor whose activation
was strongly attenuated in biglycan-deficient MG63 cells. Previously, it has
been shown that biglycan regulates Wnt/b-catenin signaling pathway, and our
results indicate that biglycan increases b-catenin expression in all cellular
fractions due to its co-localization with Wnt co-receptor LRP6. This interaction
leads to reduced degradation of b-catenin in the cytoplasm and enhances its
translocation to the nucleus, where it activates the transcription of target
genes, responsible for controlling the cell cycle. Besides, we have shown an
interaction between b-catenin and the activated IGF-IR, which is increased
upon treating MG63 cells with exogenous biglycan. In parallel, the downregulation
of biglycan significantly inhibited both basal and IGF-I induced
ERK1/2 activation, a molecule that mediates IGF-I signaling, as we have
previously reported. In summary, biglycan, through a LRP6/IGF-IR/b-catenin
signaling axis, enhances osteosarcoma cell growth.
In the next part of the study, it was shown that lumican is the main expressed
and secreted SLRP in HTB94 human chondrosarcoma cells, while decorin
and biglycan are poorly expressed. Lumican positively affects the proliferation of HTB94 cells as lumican deficiency inhibited the basal and IGFI
induced HTB94 cell growth significantly. The oncogenic action of IGF-I is
mediated by its receptor, IGF-IR, whose phosphorylation levels are strongly
attenuated in lumican-deficient cells. Furthermore, lumican levels affect
ERK1/2 activation, which seems to be crucial to IGF-I dependent HTB94 cell
growth. Moreover, lumican-deficient cells exhibit increased mRNA levels of
p53 suggesting that lumican facilitates chondrosarcoma cell growth through
an IGF-IR/ ERK1/2/p53 signaling cascade. In summary, we show that
endogenous lumican is a novel regulator of HTB94 cell growth.
Finally, we studied the action of IGF-I on HT1080 fibrosarcoma cells, and our
results show that it enhances their migration ability. SDC2, a transmembrane
PG, appears to act as a co-receptor for IGF-I since it colocalizes with IGF-IR.
Suppression of this PG expression reduces IGF-I dependent cell migration. In
order to investigate how SDC2 affects this signaling pathway and
consequently, IGF-I induced cell migration, we studied its role in ERK1/2
activation. Our results indicate that SDC2 is essential for ERK1/2
phosphorylation and that it is necessary for IGF-I dependent HT1080
migration.
Additionally, we looked at ezrin, a protein that connects membrane receptors
to the cytoskeleton and is found to be colocalized with SDC2.The formation of
an immunoprecipitative complex revealed an association between SDC2 and
ezrin, which was enhanced through IGF-I action in HT1080 cells. Ezrin
activation is also affected by IGF-I action.To conclude, SDC2 mediates
directly IGF-I-induced ERK1/2 activation; it recruits ezrin, contributes to actin
polymerization, and ezrin/actin specific localization to cell membranes,
ultimately facilitating the progression of IGF-I dependent fibrosarcoma cell
migration.
The above-described mechanisms reveal PGs's involvement in mesenchymal
origin tumor progression and their interaction with the IGF-I signaling pathway
to regulate basal functions of these cancer cells.
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