Abstract
IntroductionWe have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread.MethodsQuantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group).ResultsThe CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002).ConclusionsCell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.
Highlights
We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands
In previous studies we found that Chondroitin Sulfate/Dermatan Sulfate (CS/Dermatan Sulfate (DS))-GAGs are expressed on the cell surface of murine and human breast cancer cell lines with high metastatic capacity and that they play a major role in P-selectin binding and P-selectin-mediated adhesion of cancer cells to platelets and endothelial cells [24]
Carbohydrate Sulfotransferase 11 (CHST11) is overexpressed in aggressive human breast cancer cell lines and its expression correlates with Pselectin binding We have shown that CS/DS-GAGs expressed on the cell surface of MDA-MB-231 and MDA-MET human breast cancer cells function as P-selectin ligand and that exogenous Chondroitin Sulfate E unit (CS-E) efficiently inhibits P-selectin binding to cells [24]
Summary
We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread. Among glycans that play a critical role in stromal tumor cell interactions are glycosaminoglycans (GAGs) attached to proteoglycans (PGs). In previous studies we found that CS/DS-GAGs are expressed on the cell surface of murine and human breast cancer cell lines with high metastatic capacity and that they play a major role in P-selectin binding and P-selectin-mediated adhesion of cancer cells to platelets and endothelial cells [24]. It is likely that P-selectin binding to tumor cells and the functional consequences of such binding are dependent on which sulfotransferases define the relevant CS/DS and which core proteins carry the CS polysaccharide
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