In many
cancers hyaluronan content is increased, either by
tumor cells or the surrounding stromal cells and this increased
hyaluronan content correlates with unfavorable clinical prognosis. In the present work, we studied the effects of
melanoma cell (aggressive
melanoma cell line C8161)-derived factors on fibroblast
hyaluronan synthesis, intracellular signaling,
MMP expression and invasion. Treatment of the fibroblast cultures with
melanoma cell
conditioned medium (CM) caused accumulation of
hyaluronan in the culture medium and formation of thick pericellular
hyaluronan coat and
hyaluronan cables. The expression of Has2 was increased approximately 20-fold by the C8161
melanoma cell CM, while Has1 and Has3 were increased twofold. Knock-down of Has2 expression with
siRNA showed that Has2 was responsible for the increased
hyaluronan synthesis induced by the
melanoma cell CM. To find out the signaling routes, which led to Has2 upregulation, the phosphorylation profiles of 46
kinases were screened with phosphokinase array kit.
Melanoma cell CM treatment strongly induced a rapid phosphorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and cJUN. Treatment of the fibroblasts with specific inhibitors of PI3K, AKT and p38 reduced the
melanoma cell CM-induced
hyaluronan secretion, while the inhibitor of PDGFR totally blocked it. In addition,
siRNA for PDGFRα/β inhibited Has2 upregulation in
melanoma cell CM-treated fibroblasts. In parallel with the increased
hyaluronan synthesis the
melanoma cell CM-treated fibroblasts showed spindle shape, numerous long cell protrusions, enhanced
MMP expression and increased invasion into
collagen-Cultrex matrix.
siRNA blocking of Has2 or PDGFRα/β expression reversed the stimulatory effect of
melanoma cell CM on fibroblast invasion. PDGF secreted by
melanoma cells thus mediated fibroblasts activation, with HAS2 upregulation as a major factor in the fibroblast response. This effect on stromal matrix is suggested to favor
tumor growth.