| Abstract |
Backround: Osteosarcoma is the most common malignant bone tumor in children
and adolescents, with frequency 4,4 / 1000000. Despite the significant increase in the
survival rate of patients with osteosarcoma over the past decades, due to early
diagnosis and the use of chemotherapy, the tumor cell pathophysiology remains
largely unknown and the presence of metastases continues to be a major negative
prognostic factor.
Complex interactions between cancer cells, the extracellular matrix (ECM) and the
adjacent normal cells play an important role in development and tumor progression
.ECM composition affects important functions of normal and tumor cells, such as
proliferation, adhesion, migration and differentiation. One of the main components of
the extracellular matrix and cell membrane are proteoglycans (PGs), through which
their protein core or through their glycosaminoglycans (GAGs) chains are capable
to interact with macromolecules of the extracellular matrix such as collagen, growth
factors, growth factor receptors and adhesion molecules , participating in this way to
the regulation of various cellular functions.
Parathyroid hormone (PTH) as either an endogenous produced hormone or an
administered antiosteoporotic factor plays a key role in the physiology of
bone. However it remains largely unknown the importance of PTH action in primary
bone tumors such as osteosarcoma
Aim: To study the effect of different parathyroid hormone peptides in the migration of
human osteosarcoma cell lines and to identify possible changes of the extracellular
matrix induced by PTH peptides that affect the migratory capacity of these cells.
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Moreover, the investigation of potential extracellular and intracellular signaling
pathways that may regulate this specific cellular function
Results: we investigated whether the changes of HA metabolism induced by PTH (1–
34) and PTH (7–84) peptides in moderately MG-63 and well-differentiated Saos 2
osteosarcoma cell lines, are correlated to their migration capabilities. Our results
demonstrate that intermittent PTH (1–34) treatment significantly (P ≤ 0.01) supported
the migration of MG-63 cells, increased their HA-synthase-2 (HAS2) expression (P
≤0.001), and enhanced their high-molecular size HA deposition in the pericellular
matrix. Both increased endogenous HA production (P ≤ 0.01) and treatment with
exogenous high-molecular weight HA (P ≤ 0.05) correlated to a significant increase
of MG-63 cell migration capacity. Transfection with siHAS2 showed that PTH (1–
34), mainly through HAS2, enhanced HA and regulated MG-63 cell motility.
Interestingly, continuous PTH (1–34) treatment stimulated both Saos 2 cell HAS2 (P
≤0.001) and HAS1 (P ≤ 0.001) isoform expression inhibited their HYAL2 expression
(P ≤ 0.001) and modestly (P ≤ 0.05) enhanced their migration. Therefore, the PTH (1–
34) administration mode appears to distinctly modulate the migratory responses of the
MG-63 moderately and Saos 2 well-differentiated osteosarcoma cell lines.
Conclusively, the obtained data suggest that there is a regulatory effect of PTH (1–
34), in an administration mode-dependent manner, on HA metabolism that is essential
for osteosarcoma cell migration. Then we wanted to investigate the participation of
other potential pathways through which PTH(1-34) regulates the migratory capacity
of osteosarcoma cells, through regulation of the composition of the extracellular and
pericellular space. Thus, we investigated the possible participation of FGF-2 signaling
in PTH(1–34)-dependent osteosarcoma cell migration. FGF-2 treatment of
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osteosarcoma cells resulted in a significant increase (P ≤ 0.01) in MG63 cell
migration, similar to that caused by PTH(1–34). mRNA expression analysis of cells
treated with PTH(1–34) showed a strong increase in FGF-2 transcript levels (P =
0.0015). Interestingly, the addition of FGF-2 to MG63 cells led to significant
downregulation of small leucine-rich proteoglycan biglycan expression at both the
mRNA (P ≤ 0.0001) and protein (60%) levels. In order to examine the significance of
biglycan on MG63 cell migration, transfection with short interfering RNA specific for
biglycan was performed, resulting in a significant increase (P ≤ 0.01) in the migration
capacity of biglycan-deficient MG63 cells. In contrast, exogenous human
recombinant biglycan strongly inhibited the migration of these cells (P ≤ 0.01).
Finally, a direct correlation between PTH(1–34) action and biglycan expression was
established by the finding of a significant decrease (P ≤ 0.01) in biglycan transcript
levels in PTH(1–34)-treated cells. To summarize, the present study demonstrates a
novel cooperative mechanism of PTH(1–34) and FGF-2 action that results in specific
alteration of the biglycan extracellular matrix content to regulate osteosarcoma cell
migration.. Taking into consideration the importance of TGFb in bone biology and the
regulatory effect of the biglycan on TGFb action, in order to determine the necessity
of TGF b for cell migration we created MG 63 biglycan-deficient osteosarcoma cells.
We treated these cells with an antibody against TGFb and we found that while
biglycan-deficient cells increased their migration capacity (P <0.01), biglycandeficient
cells which were treated with the anti - TGFb1 antibody failed to increase
their migration activity. These results show the importance of TGFb1 in migration
capacity of MG63 cells .It has been shown that syndecan 4 plays an important role in
the biology of different tumor cells and that this role is achieved through complex
FGFR / syndecan 4 interactions. We sought to determine the importance of syndecan
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4 in MG63 osteosarcoma cell migration. We proceeded to create MG 63 cells
deficient in syndecan 4 (siSynd 4) in which we administered FGF 2 and found that
they did not show the expected increase in their ability to migrate. Thereby shown
that syndecan 4 is an essential molecule in the mechanism in which the FGF -2
achieves increased cell migration of MG 63 cells.
Conclusions: With this study we identified two possible pathways through which
PTH (1-34) achieves its action on migration ability of osteosarcoma cells.
1st Pathway: PTH (1-34) controlling the metabolism of hyaluronic acid (HA)
increases the migratory ability of osteosarcoma cells.
2nd Pathway: PTH (1-34) controlling the synthesis of biglycan through regulation of
FGF2 expression, manages to alter migration ability of osteosarcoma cells. Syndecan4
is an essential component for the action of FGF2 in this pathway.
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