The proteoglycan versican is one of the most studied components of the extracellular matrix associated with human breast cancer . Versican is synthesized mainly by stromal cells and possesses anti-adhesive properties that interfere with cell motility . This property appears to be related to the association of versican with hyaluronic acid, which exhibits altered expression in neoplastic conditions .
The most important criteria for the diagnosis of invasion in mammary carcinomas is the absence of myoepithelial cells surrounding neoplastic proliferations associated with basal layer rupture . Nevertheless, the recognition of these areas based solely on HE staining evaluation can be quite difficult. Therefore, staining for p63, α-SMA and PAS was implemented to aid in the evaluation of invasive regions.
In this study, myoepithelial cells revealed a progressive loss of immunoreactivity for both p63 and α-SMA from in situ to invasive areas. In in situ areas, the aberrant expression of these molecules suggests that myoepithelial cells have suffered dedifferentiation and no longer express important functional and characterization molecules [12, 27, 28]. In sites suspected of invasion, the absence of p63 and α-SMA staining denotes a flaw in the myoepithelial layer, which in association with the discontinuity of the basal membrane, confirms the lesion. In such sites, double-staining immunohistochemistry is useful for evaluating these areas.
Once areas with infiltration of neoplastic cells in the adjacent stroma were confirmed, the presence of versican was evaluated. Versican expression differed significantly between in situ and invasive areas (P < 0.0001), both in cases of low and of high versican expression. There was also statistically significant difference (P = 0.0002) in the versican immunoreactivity in the in situ areas between the two groups (1 and 2). In addition, a significant difference was observed when comparing versican immunoreactivity in invasive and in situ areas (P < 0.0001), suggesting a direct relationship between versican and invasion.
Researchers have previously analysed versican expression in areas adjacent to in situ and invasive carcinomatous areas in human breast cancer . The mechanisms involved in cell and stromal interaction are believed to lead to the development of invasive lesions from in situ mammary lesions. Furthermore, elements typically expressed in the invasive stroma, such as versican, suggest that pre-invasive lesions can acquire some characteristics commonly attributed to invasive areas, most likely contributing to the progression of neoplastic processes.
Many authors have also shown that extracellular matrix components, including the proteoglycan versican, play a critical role in facilitating the progression and dissemination of malignant neoplastic cells [14, 18, 19, 26, 29–31]. Mukaratirwa and colleagues (2004) observed a significant correlation between stromal immunoreactivity intensity for versican and invasion in colorectal carcinomas in dogs, suggesting that this proteoglycan supports tumour progression . The carcinomatous cells that invade the stroma are believed to be capable of stimulating fibroblasts to produce versican, which in turn plays a crucial role in tumour progression.
In this study, the possible association between versican expression and clinico-pathological factors was evaluated to verify the biological significance and prognostic value of versican. However, no statistically significant relationship was observed between the evaluated prognostic factors and high or low expression of versican or between the prognostic factors and versican staining in in situ and invasive areas. These results may be explained by the fact that the studied cases mostly represented low grade, nonaggressive tumours associated with a good prognosis, suggesting that our samples were somewhat homogenous. Furthermore, no association was found between the evaluated biological factors and the immunohistochemical expression of versican in human pharyngeal squamous cell carcinomas . In non-small cell lung cancer, versican has been shown to be associated with an unfavourable prognosis but is not considered to be an independent indicator of patient survival . Finally, in ovarian cancer, versican expression does not appear to have prognostic significance, despite the finding that high stromal staining is related to poor disease-free survival .
Versican has also been linked to neoplastic cell adhesion and proliferation [15, 16, 21, 34–37]. A potential significant role for the G3 versican domain, with its EGF-like motif, in influencing tumour cell viability, proliferation and local tumour growth has been suggested . Some studies also suggest that versican expression is involved in an increase in cell proliferation, apoptosis-resistance and the regulation of the cadherin family protein expression, inducing mesenchymal-epithelial transitions [38, 39]. Versican isoforms v1 and v2 seem to play distinct roles in cell proliferation functions that are mediated by the GAG-α and GAG-β domains, respectively [34, 38]. It has been shown that the GAGβ domain is responsible for activation, whereas the GAGα domain plays a role in the suppression of epidermal growth factor receptor (EGFR) expression and its downstream signaling pathway . The extracellular environment might become favourable for cell proliferation and survival when v1 expression is increased, as in the case of tissue development and tumour formation .
In canine mammary tumours, the mechanisms that regulate the signal pathways and the role of the different isoforms remain poorly elucidated. In this study, no difference regarding the proliferation index was observed between low and high versican expression groups. Thus, Ki-67 expression did not indicate a relationship between proteoglycan overexpression and tumour proliferation in CBMTs.
Studies indicating a relationship between versican and E-cadherin expression in epithelial tumours are scarce. Therefore, one goal of this study was to investigate the association between versican and E-cadherin. However, despite the existence of statistically significant differences between both E-cadherin and versican expression in in situ and invasive areas, no relationship between the two molecules was observed in CBMTs. This finding most likely indicates that versican expression interferes in carcinomatous cell adhesion through other mechanisms that are not yet understood.