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.

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLe x ). SLe x oligosaccharide on tumor cells can be recognized by E-and P-selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E-selectin reactivity, which was sLe x dependent, P-selectin reactivity with this cell line was sLe x -independent. The sLe x -Neg variant of the 4T1 cell line with markedly diminished expression of sLe x and lack of sLe a , provided a unique opportunity to characterize Pselectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E-selectin binding. We observed that P-selectin binding was Ca 21-independent and sulfation-dependent. We found that P-selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P-selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P-selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P-selectin-reactive ligands on the surface of human MDA-MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease. ' 2006 Wiley-Liss, Inc.Key words: P-selectin; chondroitin sulfate; breast cancer; cell-cell adhesion Breast cancer metastasis represents a terrible milestone associated with a poor prognosis. The multistep process of metastasis includes release of malignant cells from the primary neoplasm, migration of cancer cells into the blood circulation, interaction with platelets and leukocytes in circulation, adhesion to the vascular endothelium in distant organs and growth of the disseminated cancer cells within the vessels or within the tissue following extravasation.1-3 Each step in this process requires different types of interactions between cancer cells and the host microenvironment.A widely accepted hypothesis is that cancer cells exploit the adhesion molecules used by hematopoietic cells to migrate into distant organs. 4 In particular, the selectin family of adhesion molecules whose ligands, sialyl Lewis x (sLe x ) and sialyl Lewis a (sLe a ), are expressed at elevated levels on various cancer cells of human and murine origin 5-9 and play a significant role in cancer metastasis. The oligosaccharides sLe x and sLe a are the prime ligands for P-and E-selectin and many published works underscore the relative importance of interactions between sLe x/a and these vascular receptors in the hematogenous spread ...

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