| Abstract |
Melanoma is an aggressive cancer, deriving from malignant transformation of
melanocytes. Melanocytes are attached on to the basal membrane-containing
keratinocytes and produce melanin. Extracellular matrix and melanoma cell
interactions are important regulators of melanoma progression and their study
becomes essential because of the increased frequency and poor prognosis at the early
stages of melanoma stages.
Proteoglycans (PGs) are complex macromolecules consisting of
glycosaminoglycans (GAGs) - covalently attached on to the protein core. They are
divided into different isoforms according to their location, in extracellular, cell
membrane and intracellular PGs. Heparan sulplhate proteoglycans (HSPGs) act as
coreceptors on the cell surface. These receptors may affect many cellular events,
including cell proliferation, cell adhesion and differentiation, by inducing intracellular
signals. Melanoma cells interact with their surrounding environment through cell -cell
contact, producing numerous molecules. Autocrine growth factors such as FGF-2
produced by melanoma cells stimulate cell proliferation, whereas paracrine growth
factors such as PDGF and VEGF, regulate tumour cell growth and cell invasion by
inducing changes in the environment. Growth factors expressed by melanoma cells
including PDGF, FGF-2, VEGF, and TGFb, stimulate radial (RGP) or vertical growth
phase (VGP) formation, indicating a key role in cancer progression. FGF-2, TGFb
and PDGF are overexpressed either in VGP or in metastatic melanoma cells.
Fibroblast growth factor-2 (FGF-2), the most abundant growth factor produced
by melanoma cells but not by normal melanocytes, is an important regulator of cell
proliferation, migration and differentiation. In this study we show that M5 human
metastatic melanoma cells’ ability to migrate is significantly enhanced by
exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their
adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the
synthesis of individual (GAGs/PGs) subclasses, changing both their amounts and
distribution in M5 cells. Here, treatment with FGF-2 strongly reduces the expression
levels of the heparan sulphate containing proteoglycan, syndecan-4. Syndecan-4 is a
focal adhesion component in a range of cell types, adherent to several different matrix
molecules, including fibronectin (FN). The reduction in syndecan- 4 expression by
Abstract
II
utilizing specific siRNA discriminately increased melanoma cell motility and
decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on
these cell functions. Syndecan-4 has previously been demonstrated to regulate focal
adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to
downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion,
promoting their migration. The observed decrease in FAK Y397 activation was
correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression
perpetrated by FGF-2 may be required for optimal M5 cell migration.
Heparin and its various derivatives affect cancer progression in humans. In
this study, we show that heparin uptaken intracellularly by melanoma cells activated a
signaling cascade, which in turn inhibited melanoma cell adhesion and migration. The
reduced ability of M5 cells to adhere onto the FN substrate was directly correlated to
a decrease in the expression of focal adhesion kinase (FAK), which is a key regulator
of melanoma motility. Cell treatment with heparin caused a marked downregulation in
FAK expression (P <0.01). This is followed by an analogous inhibition of both
constitutive and FN-induced FAK Y397-phosphorylation (P< 0.01). Moreover,
heparin stimulated the P53 expression (P <0.001) of M5 cells and its increased
accumulation in the nucleus. This favours a decrease in FAK promoter activation and
explains the reduced FAK transcript and protein levels. In conclusion, the results of
this study clearly demonstrate that the action of heparin in the regulation of melanoma
cell adhesion and migration involves a P53/FAK/signaling pathway.
Low molecular weight heparin (LMWH) has significant antimetastatic
capabilities and affects cancer progression in humans. However, the mechanism by
which LMWH affects cancer cell behavior is not known. Here we evaluated its
activity at the intracellular level and how it is correlated with melanoma cell adhesion
and migration. Treatment of M5 melanoma cells with LMWH caused a marked down
regulation of constitutive phosphorylation as well as the FN-induced phosphorylation
(p<0,01) in protein kinase alpha (PKCa), as demonstrated by confocal microscopy
and western blotting, LMWH treatment altered the distribution of pPKCa between the
cytoplasm and nuclear region with a profound decrease in the cytoplasmic pPKCa (p≥
0,05) and a simultaneous enhancement of nuclear pPKCa localization (p< 0,01). A
significant decrease in the levels of pJNK (p<0,01), which is a downstream effector of
PKCa, was also demonstrated in the LMWH-treated cells. The lineage activation of
PKCa-JNK / p38 and their correlation to M5 cell adhesion was confirmed with the
Abstract
III
utilization of specific inhibitors. The reduced ability of the LMWH-treated M5
melanoma cells to adhere onto the fibronectin (FN) substrate (p≤0,01) was directly
correlated to a decrease in the activation of PKCa which is an important regulator of
cell motility. LMWH-treated cells had disorganized actin stress fibers with a diffuse
distribution of non-polymerized actin correlated to a strong decrease in cell –
substratum interface area (p<0,05) and altered morphology. It is postulated that
LMWH through the downregulation of pPKCa and redistribution to nuclear region
attenuates JNK activation, which in turn induces cytoskeleton changes correlated to
M5 cell decreased adhesion / migration. Conclusively, we show that the action of
LMWH in the regulation of melanoma cell adhesion and migration involves a PKCa/
JNK/ actin signalling pathway, which may be of paramount importance in the
pharmacological targeting of melanoma.
In conclusion we described important information regarding the mechanisms
involved in the regulation of HSPGs and their biological behavior in human
melanoma cells. Melanoma is one of the most aggressive skin cancers, and there is no
prognosis available in the early stages of cancer. Thus continuous studies of the
mechanisms involved in the development of melanoma, may offer candidate
molecules and markers to aid melanoma prognosis and treatment.
|
Search
