Specific sulfation patterns in heparan sulfate promote a proinvasive phenotype of breast cancer cells via upregulation of Wnt and MAPK signaling

Abstract

Heparan sulfate proteoglycans (HSPGs) have been implicated in breast cancer metastasis partially due to their heterogeneous sulfation patterns, which allow for specific binding of a multitude of ligands relevant to tumor progression. HS3ST2, one of the enzymes involved in 3-O-sulfation of HS, is silenced by hypermethylation in breast cancer. Here, we elucidate the role of HS3ST2 in breast cancer using an ectopic overexpression approach. HS3ST2 was stably transfected into the MDA-MB-231 breast cancer cell line, and phenotypical changes were investigated in vitro using matrigel invasion chamber assays, cell proliferation assays, immunofluorescence microscopy, real-time PCR analysis, immunoblotting, and gelatin zymography. Compared to controls, HS3ST2 transfected MDA-MB-231 cells showed a highly significant increase in invasiveness and motility accompanied by significantly increased expression of several matrix metalloproteinases (MMPs), including MMP9 and MMP13. Treatment of cells with TIMP-1 hampered invasion, suggesting a role of MMPs in increased invasiveness. HS3ST2 overexpression lead to increased basal and FGF-specific signaling through the p44/42 MAPK pathway, which depended on the presence of HS. Increased MAPK activation was accompanied by a significantly increased expression of the transcription factor TCF4. MAPK inhibition with a MEK1/2 inhibitor also hampered invasion, suggesting that increased invasiveness might be also due to increased MAPK signaling. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion. Increased activation of the p44/42 MAPK signaling pathway and of TCF4 in the presence of HS3ST2-specific sulfation patterns emerge as novel mechanistic aspects leading to increased expression of proinvasive gene products. These results suggests that increased invasion in HS3ST2 overexpressing MDA-MB 231 cells is due to increased expression of proteases and increased MAPK signaling.