In the tumour environment, versican is secreted mainly by the fibroblasts in the adjacent stroma. This secretion is elevated by paracrine signalling from the cancer, as shown in studies where conditioned media from cancer cell lines induced an increase in release of versican in mammary fibroblasts (Ricciardelli et al., 2009; Sakko et al., 2001). Specifically TGF-β1 induces versican secretion in human smooth muscle cells, gingival fibroblasts amongst other normal cell types (Haase et al., 1998; Kähäri, Larjava & Uitto, 1991). The presence of a TGF-β1 antibody was able to block induced versican expression in response to both exogenous TGF-β1 and conditioned media in prostate fibroblasts (Sakko et al., 2001), suggesting that TGF-β1 signalling is one of the primary ways that cancer cells are able to regulate versican levels. In addition to expression by stromal cells, versican mRNA has been detected in the cytosol of epithelial tumour and melanoma cell lines (Gulyás & Hjerpe, 2003; Touab et al., 2002).
High stromal versican levels are correlated with poor prognostic outcome and a more aggressive phenotype in many cancers, including breast (Ricciardelli et al., 2002), pharyngeal (Pukkila et al., 2004) oral squamous cell carcinoma (Pukkila et al., 2007), and bladder (Said et al., 2012). In addition, versican expression has been detected to be greater in metastatic sites (Pukkila et al., 2004). The same study’s data suggested that versican expression is greater in the earlier stages of pharyngeal tumours, however similar immunohistological studies have not seen any correlation with versican expression and the staging (Pukkila et al., 2007; Suwiwat et al., 2004). The presence of versican also increases the chance of relapse in prostate and breast malignancies. One study showed that high peritumoural versican levels were 6 fold more likely to relapse (Ricciardelli et al., 2002). On the other hand, versican levels found in epithelial cells have been indicative of a longer disease free survival in colon cancer (de Wit et al., 2012). Versican can cause proliferation and inhibit apoptosis in NIH3T3 fibroblasts (Sheng et al., 2005). Versican is an anti-adhesive molecule due to its negatively charged chondroitin sulphate side
59 chains (Ricciardelli et al., 2009), therefore another method by which it could promote cancer progression and relapse is by allowing cancer cell invasion through a non-cohesive extracellular matrix, allowing dissemination of cancer cells. It also destabilises focal adhesions between cells which is believed to permit cellular proliferation and hence tumour growth.
Versican has been implicated in muscle invasive bladder carcinomas, where it is expressed highly (Said et al., 2012). Rho-GTP dissociation inhibitor 2 (Rho-GDI2), a molecule preventing invasion which is lost in these tumours, works through the action of versican. When Rho-GDI2 was introduced into a bladder urothelial cell line, versican expression was decreased by more than 8-fold. Specifically, GDI was able to downregulate V1 and V3 isoforms. When Rho-GDI2 was introduced into a cancer urothelium and macrophage co-culture system there was a decrease in versican expression and a reduction in the release of pro- inflammatory mediators MCP-1 (also known as CCL2) and IL-6, suggesting that versican induces an inflammatory response resulting in the recruitment of macrophages. The presence of macrophages was shown to be crucial for the GDI2-mediated upregulation of versican to promote invasion and metastasis, as the depletion of macrophages in mice inhibits versican’s ability to stimulate lung-metastasis. This study eloquently revealed versican’s pro-metastisic role through the regulation of inflammation with the tumour microenvironment.
Versican has been found to act as an agonist for TLR-2 (Toll-like receptor-2) present on macrophages resulting in activation and subsequent secretion of chemokines TNF-α (tumour necrosis factor-alpha) and IL-6 (interleukin-6) (Kim et al., 2009). These metastatic mediators can then recruit more inflammatory mediators, increase permeability of blood vessels and activate the NF-KB pathway which is anti-apoptotic and pro-proliferative; all of which will aid metastasis. This in vivo study went on to show that both recombinant knock out of TLR-2 and the silencing of versican, in the cancer cells used to inoculate the mice, significantly attenuated metastatic spread and improved survival. It was clear from this study that versican is able to strongly affect the tumour microenvironment by behaving as a ligand, however it is not clear whether this effect was through its association with hyaluronan.
Extracellular versican levels are responsible for change in cellular phenotype. Hattori et al., (2011) reported that pericellular, the extracellular compartment surrounding the cell, accumulation of versican, via the loss of ADAMTS-5 mediated cleavage, was responsible for the acquisition of myofibroblast phenotype within dermal fibroblasts. ADAMTS-5 -/- knockout mice dermal fibroblasts had an attenuation of versican cleavage shown by a reduction in detection,
60 epitope) compared to the wild type. Moreover there was an increase in the unprocessed versican levels shown by an increase in detection of anti-GAGβ which is present in both V0 and V1. These ADAMTS-5-/- fibroblast had an extensive pericellular matrix and characteristics that resemble myofibroblasts: altered cell shape, increased αSMA expression, and greater contractility (Hattori et al., 2011). This phenotype could be rescued by the addition of ADAMTS- 5 and by crossing ADAMTS-5 -/- mice with haploinsufficient VCAN, suggesting that the phenotype was due to accumulation of versican. Again, heterologous expression of full-length versican induced a myofibroblast phenotype within ADAMTS-5 -/-, VCAN hdf/+ fibroblasts; suggesting that it was in fact accumulation of pericellular versican inducing dermal myofibroblast differentiation.
Hattori et al., (2011) also showed evidence to suggest that the myofibroblast phenotype was related to increased TGF-β signalling and HA. ADAMTS-5 -/- fibroblasts had an increase in psmad 2/3 levels, and when an inhibitor to TGF-β R was added there was a reduction in αSMA expression and contractility. Likewise, inhibiting the synthesis of HA, which had previously been shown to be involved with the myofibroblast transition (Meran et al., 2007), had the same ameliorating effect on the myofibroblast phenotype. This study further exposes the links between HA, versican, and TGF-β signalling, and indeed the action of ADAMTS family members, which appear to contribute towards myofibrotic phenotype (Hattori et al., 2011).
ADAMTS-1 and -4 are able to proteolytically cleave versican at the Glu441-Ala442 bond resulting in a truncated form of versican (Ricciardelli et al., 2009). This cleaved version of versican co- stained with both ADAMTS-1 and ADAMTS-4 in malignant prostate cancer sections. A separate study has suggested that higher levels of this cleaved form in cancers contributes to invasion, as an antibody to the neoepiptope formed by ADAMTS cleavage inhibited cancer cell migration (Arslan et al., 2007). How these cleaved products of versican contribute to cancer and metastasis needs to be explored further.