**2.1 Biological functions of CS/DS chains**

CS/DS chains specifically interact with heparin binding proteins. The interaction of DS chains with fibroblast growth factor (FGF) activates FGF-2 to signal cell proliferation (Penc et al., 1998). DS also acts as a cofactor for FGF-7 (Trowbridge et al., 2002). In addition, DS has been shown to bind and activate hepatocyte growth factor/scatter factor (HGF/SF), a

On the Role of Cell Surface Chondroitin Sulfates and

potential CS-carrying PGs on the surface of breast tumor cells.

hematogenous metastasis (Monzavi-Karbassi et al., 2007).

**(Figure 2)** and is considered as a major CS-carrying PG.

present CS-GAGs in malignancy.

**3.1.1 CSPG4**

**3.1 Role of CS-carrying PGs in tumor progression and metastasis**

Their Core Proteins in Breast Cancer Metastasis 439

Many articles are now devoted to CD44 in cancer stem cells and its role in cancer progression and metastasis (Lesley et al., 1997; Naor et al., 1997; Lesley and Hyman, 1998; Kalish et al., 1999; Toole, 2009). Here we focus on SDC-1, SDC-4, CSPG4 and NRP-1 as

Alteration in the production and structure of GAG chains and the functional consequences of such alterations is dependent on the PG carrying the GAG chain. PGs isolated from carcinomas contained 32.2% more CS, 18% less DS, and 30% less HS than PGs of normal breast tissue (Vijayagopal et al., 1998). Chondroitin sulfate proteoglycans (CSPGs) were expressed significantly more often in metastases than in primary tumors of uveal melanoma (Kiewe et al., 2006). We have recently found that CSPGs on breast cancer cells also bind to Pselectin receptors, and interruption of this interaction leads to significant reduction in

Selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium may regulate their hematogenous spread in the microvasculature (Krause and Turner, 1999). Among selectin molecules, evidence strongly supports P-selectin involvement in tumor metastasis (Kim et al., 1998; Stevenson et al., 2005). Our data suggest that inhibition of Pselectin interaction with CS-GAGs significantly attenuates hematogenous lung metastasis (Monzavi-Karbassi et al., 2007)**.** We have demonstrated that P-selectin binding to the surface of the aggressive breast cancer cell line MDA-MB-231 and MDA-MET is also CS-dependent, suggesting a role for CSPGs in metastatic behavior of human cancer cells. Because of the role of some of these PGs in signaling and tumor phenotype, we speculate that P-selectin interaction with a particular PG may lead to an exclusive tumor cell activation, and consequently survival in circulation. Here, we review the role of the surface PGs able to

CSPG4 is a human homolog of Rat neuroglycan 2 (NG2), which is also known as High Molecular Weight Melanoma Associated Antigen and Melanoma Chondroitin Sulfate Proteoglycan (Stallcup, 1981; Bumol and Reisfeld, 1982; Pluschke et al., 1996) and exclusively carries CS chains (Bumol and Reisfeld, 1982; Nishiyama et al., 1991). This tumorassociated cell surface PG potentiates cell motility, promotes invasiveness and the metastatic potential of tumor cells in melanoma (Burg et al., 1998; Campoli et al., 2004; Iida et al., 2007; Wang et al., 2010), and modulates responses to growth factors (Grako and Stallcup, 1995; Yang et al., 2009), processes that are critical for the proliferation and migration of tumor cells. It is suggested that CSPG4 facilitates the invasion of aggressive primary tumors within the dermis by enhancing the local concentration and/or activation of specific matrix metalloproteinases (MMPs) at sites of contact between melanoma cells and the underlying ECM (Iida et al., 2001). The authors demonstrated that CSPG4 on WM1341D cells, interacts with membrane-type matrix metalloproteinase (MT3-MMP), facilitating invasion, and that the interaction is CS-dependent. Inhibiting CS presentation by treating cells with pnitrophenyl beta-D-xylopyranoside (beta-D-xyloside or DX), a compound that uncouples the CS chain from the PG, led to a decrease in melanoma cell invasion into type I collagen (Faassen et al., 1992). CSPG4 is highly expressed on aggressive breast cancer cell lines

paracrine growth factor whose receptor, c-met (previously characterized as a protooncogene), is also a transmembrane tyrosine kinase.

The CS/DS chains of the PG versican, which is expressed in many tissues including kidney, skin, aorta, and brain, bind the adhesion molecules L- and P-selectin (Kawashima et al., 2002), molecules that have been implicated in leukocyte trafficking, inflammatory disease, and tumor dissemination. Interestingly, these interactions are specifically inhibited by CS or DS containing the 'E' disaccharide unit GlcUA-GalNAc (4S, 6S) or the 'iE' unit IdoUA-GalNAc (4S, 6S), respectively.

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. This suggests that CS/DS-GAGs can mediate P-selectin binding and P-selectin-mediated adhesion of cancer cells to platelets and endothelial cells (Monzavi-Karbassi et al., 2007). In inhibition assays performed in vitro, we showed that among the CS types only CS-B (DS), and CS-E can efficiently block P-selectin binding to tumor cells (Monzavi-Karbassi et al., 2007). Other studies have also suggested important interactions mediated by CS-A and CS-E in tumor progression and metastasis (Iida et al., 2007; Li et al., 2008; Basappa et al., 2009). Therefore, enzymes involved in sulfation (sulfotransferases) or epimerization (DS epimerase) of CS chains may play a fundamental role in defining the malignant phenotype of breast tumors.

The expression of several sulfotransferases including CHST11 and CHST15 appears to be greater in human breast carcinoma compared to normal breast tissue (Potapenko et al., 2010). An increase in CHST11 expression is observed in malignant plasma cells from myeloma patients compared to normal bone-marrow plasma cells (Bret et al., 2009). In searches for genes involved in the transition of DCIS to IDC, Schuetz et al. (Schuetz et al., 2006) found a significant increase in DS epimerase (Maccarana et al., 2006).

Collectively, the evidence implicates CS/DS GAGs in a wide array of molecular and cellular interactions resulting in tumorigenesis and metastasis.
