**3.1.1 CSPG4**

438 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

paracrine growth factor whose receptor, c-met (previously characterized as a proto-

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-

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

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.,

Collectively, the evidence implicates CS/DS GAGs in a wide array of molecular and cellular

Malignant neoplasms exhibit changes in production of PGs (Bumol and Reisfeld, 1982; Iozzo, 1985; Iozzo, 1988; Stylianou et al., 2008). The variation, abundance and function of CS/DS-GAGs are also affected by the expression of the PG core protein presenting them. Therefore, it is imperative to study these polysaccharides in the context of their carrying PG. PG are involved in signaling and tumorigenicity and their attached GAG contributes to their functions. There is a growing list of PGs that have been implicated as possessing CS/DS side chains (Esko et al., 1999; Taylor and Gallo, 2006). PGs that may be modified by CS/DS chains include aggrecan, neurocan, brevican, bamacan, a CD44 isoform, chondroitin sulfate proteoglycan 4 (CSPG4), syndecans, betaglycan, serglycin, versican, decorin, biglycan, and endocan, most of which are extracellular matrix PGs. Our focus is on the cell membrane PGs that are able to bind to P-selectin (Monzavi-Karbassi et al., 2007). CD44 variants (CD44v), CSPG4, syndecan-1 (SDC-1) and syndecan-4 (SDC-4) are among the cell surface candidates (Faassen et al., 1992; Jackson et al., 1995; Barbareschi et al., 2003; Burbach et al., 2003; Baba et al., 2006; Gotte et al., 2007; Wang et al., 2010). Recently, It has been demonstrated that substantial fraction of neuropilin-1 (NRP-1), a membrane glycoprotein, is a PG modified

2006) found a significant increase in DS epimerase (Maccarana et al., 2006).

**3. Potential cell membrane CS/DS-carrying PGs of breast carcinoma** 

interactions resulting in tumorigenesis and metastasis.

with either HS or CS-GAG chains (Shintani et al., 2006).

oncogene), is also a transmembrane tyrosine kinase.

GalNAc (4S, 6S), respectively.

phenotype of breast tumors.

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

On the Role of Cell Surface Chondroitin Sulfates and

**3.1.3 SDC-1 and SDC-4** 

a potential CS-carrying PG that can present CS-GAGs to P-selectin.

expression, in metastatic breast cancer cells (**Figure 3**).

Their Core Proteins in Breast Cancer Metastasis 441

formation, and defects in heart development (Kawasaki et al., 1999). In contrast, overexpression of NRP-1 leads to over-stimulation of blood vessel formation (Kitsukawa et al., 1995). Studies have shown that NRP-1interacts with a subset of heparin binding proteins like FGF-1, FGF-2, FGF-4, FGF-7, FGF receptor-1, and HGF/SF (West et al., 2005). Investigation of the role of NRP-1 in human glioma progression, Hu et al. (Hu et al., 2007) have shown that NRP-1 expression correlates with tumor progression in clinical setting, and that NRP-1 expression promotes tumor growth and survival through an autocrine HGF/SF/c-met signaling pathway. We observed an overexpression of NRP-1 in aggressive human breast cancer cell line MDA-MB-231 compare to MCF-7 cells (**Figure 2**). This PG is also considered as

SDC-1 is mainly expressed by epithelia and plasma cells. Although there are inconsistent reports (Barbareschi et al., 2003; Tsanou et al., 2004), the expression of SDC-1 is generally down-regulated in malignant tumors, and lower levels of expression have been associated with high metastatic/aggressive potential in many tumors (Nackaerts et al., 1997; Kumar-Singh et al., 1998; Mikami et al., 2001; Numa et al., 2002). SDC-1 has also been shown to act as a tumor suppressor molecule by inhibiting cell growth and inducing apoptosis (Mali et al., 1994; Dhodapkar et al., 1998). Therefore, during tumor development the decrease of SDC-1 expression may be an important step from tumorigenesis to a metastatic phenotype. However, there are conflicting data on the role of SDC-1; both its loss and over-expression in

carcinoma cells have been associated with malignant progression (Baba et al., 2006).

Fig. 3. Relative expression of SDC-1 and SDC-4 in human breast cancer cells using quantitative real-time PCR. Means of three independent experiments (±SD) are shown.

SDC-4 is more ubiquitously expressed by most cell types, and little is known about its role in malignancy. Among the four members of the syndecan family, SDC-4 is the only one involved in the formation of fibronectin-induced focal adhesions, in cooperation with β1 integrin receptors (Woods and Couchman, 1994; Woods et al., 2000). SDC-4 has been implicated in cytoskeletal organization and regulation of cell adhesiveness. The migratory capacity of lymphocytes and dendritic cells has been reported to be mediated by SDC-4 (Kaneider et al., 2002; Greene et al., 2003; Feistritzer et al., 2004; Averbeck et al., 2007). Our data suggest a role for relative expression of SDC-1 and SDC-4, low SDC-1 and high SDC-4

Fig. 2. Expression of NRP-1, SDC-4 and CSPG4 in breast cancer cells. Cells were grown in standard medium, harvested and then stained with monoclonal antibodies against the indicated targets. Stained cells were then analyzed by flow cytometry.

### **3.1.2 NRP-1**

NRP-1 is a 120-130 kDa transmembrane glycoprotein, initially characterized as a neuronal receptor for specific secreted members of the semaphorin family involved in exon repulsation (Kolodkin et al., 1997). A substantial fraction of NRP-1 is a PG with a GAG chain attached (Shintani et al., 2006). In addition to being a receptor for a number of class 3 semaphorins, NRP-1 also serves as a receptor for some members of vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) (Migdal et al., 1998; Soker et al., 1998; Makinen et al., 1999; Wise et al., 1999; Klagsbrun et al., 2002).

Considerable data support a functional role for NRP-1 in regulating VEGF activities in endothelium. It has been shown that semaphorin-3A competes with VEGF165 binding to NRP-1 and inhibits angiogenesis *in vitro* (Miao et al., 1999). NRP-1 knock-out mice, in addition to neural defects, exhibit transposition of large vessels, disorganized and insufficient capillary formation, and defects in heart development (Kawasaki et al., 1999). In contrast, overexpression of NRP-1 leads to over-stimulation of blood vessel formation (Kitsukawa et al., 1995). Studies have shown that NRP-1interacts with a subset of heparin binding proteins like FGF-1, FGF-2, FGF-4, FGF-7, FGF receptor-1, and HGF/SF (West et al., 2005). Investigation of the role of NRP-1 in human glioma progression, Hu et al. (Hu et al., 2007) have shown that NRP-1 expression correlates with tumor progression in clinical setting, and that NRP-1 expression promotes tumor growth and survival through an autocrine HGF/SF/c-met signaling pathway. We observed an overexpression of NRP-1 in aggressive human breast cancer cell line MDA-MB-231 compare to MCF-7 cells (**Figure 2**). This PG is also considered as a potential CS-carrying PG that can present CS-GAGs to P-selectin.
