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Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI3K-AKT and p38 signaling

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Abstract

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.

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Abbreviations

CM:

Conditioned medium

ECM:

Extracellular matrix

bHABC:

Biotinylated hyaluronan binding complex

ELSA:

Enzyme-linked sorbent assay

HAS:

Hyaluronan synthase

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Acknowledgments

We acknowledge expert technical help from Eija Kettunen, Arja Venäläinen, Eija Rahunen and Tuula Venäläinen. Academy of Finland (S.P-S.), Paavo Koistinen Foundation (S.P-S.), The North Savo Cancer Fund (S.P-S), Cancer Center of Eastern Finland (M.T. and R.T.) and Sigrid Juselius Foundation (R.T. and M.T.) supported this work financially.

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Authors and Affiliations

  1. School of Medicine, Institute of Biomedicine/Anatomy, University of Eastern Finland, Kuopio, Finland

    Sanna Pasonen-Seppänen, Piia Takabe, Leena Rauhala, Kirsi Rilla, Markku Tammi & Raija Tammi

  2. Section of Dermatology, School of Medicine, University of Glasgow, Glasgow, G12 8QQ, UK

    Michael Edward

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  1. Sanna Pasonen-Seppänen
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  2. Piia Takabe
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Correspondence to Sanna Pasonen-Seppänen.

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418_2012_1000_MOESM1_ESM.eps

Supplementary Figure 1. The effect of PDGFRβ siRNA transfection on PDGFRβ protein expression. Cell lysates were collected 24 h after siRNA transfection and analyzed by Western blotting. (EPS 522 kb)

418_2012_1000_MOESM2_ESM.eps

Supplementary Figure 2 . The effect of melanoma cell CM and PDGFR inhibitor on MMP1, 2, 9 and 14 mRNA expression. Fibroblast cultures were treated with melanoma cell CM with or without the PDGFR inhibitor for 24 h and mRNA levels of different MMPs were analyzed with qRT-PCR. The data represent means ± SEM of four independent experiments. (EPS 421 kb)

Supplementary material 3 (DOCX 45 kb)

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Pasonen-Seppänen, S., Takabe, P., Edward, M. et al. Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI3K-AKT and p38 signaling. Histochem Cell Biol 138, 895–911 (2012). https://doi.org/10.1007/s00418-012-1000-x

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