Altered expression of versican and hyaluronan in melanocytic tumors of dogs

María-José Docampo Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

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Rosa M. Rabanal Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

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Laia Miquel-Serra Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

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Daniel Hernández Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

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Clelia Domenzain Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

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Anna Bassols Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

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Abstract

Objective—To analyze the expression of versican and hyaluronan in melanocytomas and malignant melanomas of dogs, to correlate their expression with expression of the hyaluronan receptor CD44, and to identify enzymes responsible for the synthesis and degradation of hyaluronan in canine dermal fibroblasts and canine melanoma cell lines.

Sample Population—35 biopsy specimens from melanocytic tumors of dogs, canine primary dermal fibroblasts, and 3 canine melanoma cell lines.

Procedures—Versican, hyaluronan, and CD44 were detected in tumor samples by use of histochemical or immunohistochemical methods. Expression of hyaluronan-metabolizing enzymes was analyzed with a reverse transcriptase–PCR assay.

Results—Versican was found only in some hair follicles and around some blood vessels in normal canine skin, whereas hyaluronan was primarily found within the dermis. Hyaluronan was found in connective tissue of the oral mucosa. Versican and, to a lesser extent, hyaluronan were significantly overexpressed in malignant melanomas, compared with expression in melanocytomas. No significant difference was found between malignant tumors from oral or cutaneous origin. The expression of both molecules was correlated, but hyaluronan had a more extensive distribution than versican. Versican and hyaluronan were mainly associated with tumor stroma. Canine fibroblasts and melanoma cell lines expressed hyaluronan synthase 2 and 3 (but not 1) and hyaluronidase 1 and 2.

Conclusions and Clinical Relevance—Versican may be useful as a diagnostic marker for melanocytic tumors in dogs. Knowledge of the enzymes involved in hyaluronan metabolism could reveal new potential therapeutic targets.

Abstract

Objective—To analyze the expression of versican and hyaluronan in melanocytomas and malignant melanomas of dogs, to correlate their expression with expression of the hyaluronan receptor CD44, and to identify enzymes responsible for the synthesis and degradation of hyaluronan in canine dermal fibroblasts and canine melanoma cell lines.

Sample Population—35 biopsy specimens from melanocytic tumors of dogs, canine primary dermal fibroblasts, and 3 canine melanoma cell lines.

Procedures—Versican, hyaluronan, and CD44 were detected in tumor samples by use of histochemical or immunohistochemical methods. Expression of hyaluronan-metabolizing enzymes was analyzed with a reverse transcriptase–PCR assay.

Results—Versican was found only in some hair follicles and around some blood vessels in normal canine skin, whereas hyaluronan was primarily found within the dermis. Hyaluronan was found in connective tissue of the oral mucosa. Versican and, to a lesser extent, hyaluronan were significantly overexpressed in malignant melanomas, compared with expression in melanocytomas. No significant difference was found between malignant tumors from oral or cutaneous origin. The expression of both molecules was correlated, but hyaluronan had a more extensive distribution than versican. Versican and hyaluronan were mainly associated with tumor stroma. Canine fibroblasts and melanoma cell lines expressed hyaluronan synthase 2 and 3 (but not 1) and hyaluronidase 1 and 2.

Conclusions and Clinical Relevance—Versican may be useful as a diagnostic marker for melanocytic tumors in dogs. Knowledge of the enzymes involved in hyaluronan metabolism could reveal new potential therapeutic targets.

Malignant melanomas are relatively common neoplasms in dogs. They account for 7% of all malignant tumors and 3% of all neoplasms. Oral melanomas in dogs have typically been considered malignant,1 although the existence of benign oral melanocytic tumors has been reported.2 Cutaneous melanocytic lesions are benign in most dogs.1,2 Despite the importance of these neoplasms, little is known about the composition of their ECM.

Molecular changes in the ECM and in the interaction between tumor cells and stroma have been implicated in the tumorigenic process. During tumor development, the ECM undergoes structural changes that contribute to alterations in proliferation, migration, and differentiation of tumor cells that contribute to tumor progression.3,4 The ECM contains a complex formed by hyaluronan, versican, and tenascin.5,6 Hyaluronan is a polysaccharide produced by numerous cells and tissues.7–9 Versican is an ECM proteoglycan that has the ability to bind hyaluronan.10–12 Both versican and hyaluronan are overexpressed in a number of tumors in humans. These versican-hyaluronan aggregates supposedly provide an expanded and water-enriched environment for tumor cells and modify the normal architecture of the ECM, which facilitates cell movement and growth.13

Versican has been found in human melanoma cell lines and biopsy specimens obtained from melanomas in humans. Versican has been proposed14 as a useful diagnostic marker that is involved in biological processes of tumors on the basis that purified versican is able to decrease cell adhesion in melanomas of humans and increase cell proliferation in vitro, which may contribute to the malignant properties of tumor cells.15 The production of extracellular proteoglycans, including versican and its regulation by growth factors, in canine melanoma cell lines has been described,16 but detailed knowledge of the expression of versican in naturally developing melanocytic tumors of dogs is lacking. Furthermore, it has been proposed that versican is an important ECM component in other types of tumors in dogs, such as mammary gland tumors (for which it has been found to be fundamentally expressed in the invasive areas of malignant tumors17,18) and colonic tumors (for which it has been found to be correlated with the depth of invasion19).

In contrast, hyaluronan is enriched in virtually all epithelial tumors of humans. In general, it is a strong, independent, and negative predictor of survival in humans patients.8,9,20 The situation is controversial regarding melanocytic tumors of humans in which reduced concentrations of hyaluronan and the hyaluronan receptor CD44 have been associated with poor prognosis in clinical stage I cutaneous melanomas.21

Tumor-promoting properties of versican and hyaluronan are largely dependent on the self-interaction abilities and attachment to the cell surface through the hyaluronan receptor CD44. Hyaluronan receptor CD44 is a transmembrane protein with an extracellular domain that binds hyaluronan, whereas the cytoplasmic domain selectively interacts with cytoskeletal proteins, such as ankyrin and proteins in the ezrin-radixin-moesin family. Through these interactions, CD44-mediated extracellular signals regulate specific pathways that lead to the concomitant onset of multiple functions, such as changes in cell proliferation, adhesion, and migration.22–24

Fibroblasts are important cells that contribute to tumor progression. It is accepted that they are primarily responsible for the synthesis, deposition, and remodeling of the ECM as well as for the production of many soluble paracrine growth factors that regulate cellular proliferation, morphology, survival, and death.3 Dermal fibroblasts produce hyaluronan25 and versican.26 In another study16 conducted by our laboratory group, we determined that canine melanoma cells are also capable of producing versican and that this production may be enhanced by treatment of cells with growth factors produced in an autocrine or paracrine manner. Melanoma cells also produce variable amounts of hyaluronan when grown in culture.27 Hyaluronan is synthesized by HAS, which exists in 3 isoforms (ie, HAS1, HAS2, and HAS3). Hyaluronan is degraded by Hyals (eg, Hyal1, Hyal2, or Hyal3),28,29 which differ in their catalytic properties.30 However, the actual role and distribution of the HAS and Hyal enzymes are largely unknown.

The primary objective of the study reported here was to investigate the expression of versican and hyaluronan in melanocytic tumors of dogs and to relate findings to the expression of CD44 and the degree of malignancy. A secondary objective was to identify the enzyme isoforms responsible for metabolism of hyaluronan in canine primary dermal fibroblasts and canine melanoma cell lines.

Materials and Methods

Sample population—Thirty-five melanocytic tumors (14 melanocytomas and 21 malignant melanomas) of dogs were used in the study. All melanocytomas were of dermal origin. Fourteen of 21 malignant melanomas were within the oral cavity, and the other 7 were cutaneous. Biopsy specimens that included skin and oral mucosa were obtained from dogs with no history of melanocytic tumors and used as normal tissues. Tumors were on slides and in paraffin blocks of the archives of the Pathology Department at the Facultat de Veterinària of the Universitat Autònoma de Barcelona. Sections (4 μm) were cut from paraffin blocks, mounted on slides, and stained with H&E. Tumors were classified as malignant or benign on the basis of cytologic features, mitotic activity, ulcers and necrosis, melanin production, junctional activity, and infiltration into deeper tissues in accordance with the World Health Organization classification.31,32

Immunohistochemical labeling of versican—Paraffin was removed from the slides and the sections rehydrated by use of a series of xylol and graded alcohol solutions. For versican retrieval, 2 initial enzymatic treatments were used. Sections were incubated with 0.1% trypsina in 50mM Tris-HCl buffer (pH, 7.8) for 10 minutes at 37°C. Sections then were digested with 0.5 U of chondroitinase ABC/mLb in a solution consisting of 50mM Tris and 60mM sodium acetate (pH, 8.0) for 2 hours at 37°C to remove chondroitin sulfate chains from the versican core protein. Activity of endogenous peroxidase was blocked by immersing slides in 0.3% H2O2 in methanol for 30 minutes. After nonspecific sites were blocked by incubation with 20% normal goat serum in PBS solution for 30 minutes at 25°C, sections were incubated overnight at 4°C with primary antibody (ie, murine anti-human 2B1 antibodyc diluted 1 in 1,000 in PBS solution). The murine anti-human large chondroitin sulfate monoclonal antibody 2B1 (IgG1 subclass [affinity purified]) recognizes the core protein of versican. Slides were then incubated with biotinylated goat anti-mouse immunoglobulind (1 in 200 dilution in PBS solution) at 25°C for 45 minutes, which was followed by incubation with the avidin-biotin-peroxidase complex.e The reaction was developed by use of a commercial staining kit,f and sections were counterstained with Mayer's hematoxylin. Sections incubated with an irrelevant isotype-matched monoclonal antibody were used as negative control samples.

Histochemical labeling of hyaluronan—After removal of paraffin, rehydration, and inhibition of endogenous peroxidase activity, melanin was bleached by treating sections with 0.25% potassium permanganate for 30 minutes, which was followed by treatment with 1% oxalic acid for 5 minutes. Negative control samples were digested with 40 U of hyaluronate lyaseg/mL in a solution of 50mM sodium acetate and 0.15M NaCl (pH, 6.0) for 2 hours at 37°C. Sections were incubated in parallel with the same solution without enzyme. After being washed with PBS solution, all samples were blocked for 1 hour at 25°C by incubation with 20% normal goat serum in PBS solution. Specimens then were incubated overnight at 4°C with 5 μg of biotinylated hyaluronan-binding proteinh/mL in PBS solution with 0.1% bovine serum albumin, which was followed by incubation with the avidin-biotin complex. The reaction was developed by use of 0.05% 3,3′diaminobenzidine tetrahydrochloride with 0.015% H2O2 in Tris buffer (pH, 7.6) with 3.1mM NiCl2 6•H2O, and sections were counterstained with Mayer's hematoxylin.

Immunohistochemical labeling of CD44—After removal of paraffin, rehydration, inhibition of endogenous peroxidase activity, and blocking of nonspecific sites with 20% rabbit serum in TBS solution for 30 minutes, sections were incubated overnight at 4°C with anti-CD44 antibodyi diluted 1 in 100 in TBS solution. The rat monoclonal antibody 2D10 (IgG2a subclass [hybridoma supernatant]) recognized canine CD44.33 Slides were then incubated with biotinylated rabbit anti-rat immunoglobulinj (1 in 200 dilution in TBS solution) for 1 hour at 25°C, which was followed by incubation with the avidin-biotin complex. The reaction was developed by use of 0.04% 3-amino-9-ethylcarbazole with 0.015% H2O2 in 50mM acetate buffer (pH, 5.0) containing 0.05% dimethylformamide. Sections were counterstained with Mayer's hematoxylin. Sections incubated with rat preimmune serum were used as negative control samples.

Evaluation of expression—Sections were examined independently by 2 investigators (MJD and RMR). Slides were examined by use of a microscopek and photographed with an integrated digital camera system.l Intensity of labeling for versican and CD44 was categorized as negative, weak, moderate, or intense. Expression of hyaluronan was evaluated as described elsewhere.34 Hyaluronan staining in stroma was classified as weak, moderate (< 50% of stroma expressed hyaluronan intensely), or extensive (> 50% of stroma expressed hyaluronan intensely).

Statistical analysis—Expression was classified by use of a scale from 0 to 3 (0, negative; 1, weak; 2, moderate; and 3, intense or extensive expression). Statistical analysis was performed by use of commercial software.m The χ2 test was used to examine the correlation between versican or hyaluronan expression and tumor grade. Significance was set at values of P < 0.05.

Cell culture—Canine melanoma cell lines CML-1, CML-10c2, and CML-6Mn were originally derived from melanoma tumors of dogs.16,35 Canine primary dermal fibroblasts were obtained from skin biopsy specimens.o Cells were grown in a humidified atmosphere at 37°C with 5% carbon dioxide in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum, 100 U of penicillin/mL, and 100 μg of streptomycinp/mL.

RNA isolation and reverse transcriptase–PCR assay—Total RNA was extracted from subconfluent cells by use of an isolation kit.q For reverse transcriptase–PCR assay, 2 μg of total RNA was reverse transcribed in a 40-μL reaction volume by use of reverse transcriptase,r as described in a protocol provided by the manufacturer. An aliquot (2.5 μL) of the reaction mixture was subsequently used for PCR assays with primers for canine HAS1, HAS2, HAS3, Hyal1, and Hyal2. Primers used for extension were designed from the human sequences or from the predicted canine sequences when no amplification was achieved and significant differences between human and canine sequences were reported (Appendix). The assay used an initial denaturing step at 94°C for 2 minutes, which was followed by 30 to 33 cycles of reaction and a final extension step at 72°C for 5 minutes. Amplification of a fragment of GAPDH was used as an internal control sample. The PCR amplification products were resolved on 2% agarose gels (1% agarose gel for GAPDH), stained with ethidium bromide, and developed under UV light.

Results

Versican, hyaluronan, and CD44 expression in normal canine skin and oral mucosa—Normal canine skin and oral mucosa yielded negative results for versican, except for the dermal papilla and glassy membrane of some hair follicles, loose connective tissue around some blood vessels, and scarce foci at the dermal-epidermal junction (Figure 1). This was especially visible in skin adjacent to tumors. Other accessory structures (such as glands) had negative results for versican. In contrast, hyaluronan was abundant in the dermis and in connective tissue of oral mucosa. In addition, CD44 was highly expressed in the membrane of the basal layer of the epidermis and basal and suprabasal layers of the epithelium in the oral mucosa. Melanocytes, mast cells, and plasma cells also had positive results for CD44, similar to results described elsewhere for dogs.33

Figure 1—
Figure 1—

Photomicrographs of sections of normal canine skin (a, b, and c) and oral mucosa (d, e, and f) from lip biopsy specimens in which expression of versican (VS; a and d), hyaluronan (HA; ≤ and e), and CD44 (c and f) was analyzed histochemically or immunohistochemically. In panel A, notice that normal skin (a) and oral mucosa (d) had negative results for versican, whereas connective tissue of the dermis (b) and oral mucosa (e) had positive results for hyaluronan. The basal layer of the epidermis (c) and basal and suprabasal layers of the oral epithelium (f) had positive results for CD44; mast cells (black arrow) also had positive staining. In panel B, the dermal papilla and glassy membrane of a hair follicle (a) and loose connective tissue around a blood vessel (b) had positive results for versican. Avidin-biotin-peroxidase staining with Mayer's hematoxylin counterstain; panel A, bar = 100 μm (a) or 50 μm (b, c, d, e, and f); panel B, bar = 100 μm (a) or 50 μm (b).

Citation: American Journal of Veterinary Research 68, 12; 10.2460/ajvr.68.12.1376

Versican, hyaluronan, and CD44 expression in melanocytomas—Results of immunohistochemical analysis of versican expression in melanocytic tumors were summarized (Table 1). Most melanocytomas (11/14) had negative results for versican. Only 3 tumors had weak positive results. These included 2 dermal melanocytomas with junctional activity that had immunoreactivity at points of the dermal-epidermal junction (Figure 2). The third tumor had weak positive results in the peritumoral region.

Figure 2—
Figure 2—

Photomicrographs of sections of melanocytomas of dogs in which versican (a and c) and hyaluronan (b and d) expression were analyzed immunohistochemically or histochemically. Notice the negative results for versican (a) but weak positive results for hyaluronan (b). A melanocytoma with junctional activity had versican expression at sites of the dermal-epidermal junction (black arrows; c) and moderate expression of hyaluronan, especially surrounding nests of melanocytic cells (d). Melanin was bleached in slides stained for detection of hyaluronan. Avidinbiotin-peroxidase staining with Mayer's hematoxylin counterstain; bar = 100 μm (a and d), 500 μm (b), or 50 μm (c). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 68, 12; 10.2460/ajvr.68.12.1376

Table 1—

Versican expression in 35 melanocytic tumors of dogs.

Table 1—

Expression of hyaluronan was variable in distribution and intensity. In general, hyaluronan was associated with the stroma of tumors and surrounding nests of melanocytic cells (Table 2; Figure 2). Most (8/14) melanocytomas had weak positive results, whereas the remainder had moderate expression, except for 1 melanocytoma that had extensive labeling of the tumor stroma. All tumors with weak labeling for hyaluronan had negative results for versican. Tumors with moderate or extensive hyaluronan expression had negative or weak positive results for versican.

Table 2—

Hyaluronan expression in 35 melanocytic tumors of dogs.

Table 2—

Most melanocytomas expressed CD44, which was clearly located at the membrane of neoplastic cells. Staining was particularly intense in areas in which neoplastic melanocytes were organized in nests (data not shown). No relationship was found between CD44 and versican or hyaluronan expression or between expression and cytologic characteristics of the tumors.

Versican, hyaluronan, and CD44 expression in malignant melanomas—In contrast to melanocytomas, a higher proportion (16/21) of malignant tumors stained for versican, which ranged from weak to intense (Table 1; Figure 3). There was a significant (P = 0.002) correlation between versican expression and malignancy. Several tumors had a multifocal distribution, which was usually associated with the tumor stroma. In other tumors, versican had a remarkable peritumoral location or was evident in strands between nests of neoplastic cells. Versican expression or distribution did not differ significantly (P = 0.29) between oral and cutaneous malignant melanomas.

Figure 3—
Figure 3—

Photomicrographs of sections of malignant melanocytic tumors of dogs in which versican (a, b, d, e, g, and h) and hyaluronan (c, f, and i) expression were analyzed immunohistochemically or histochemically. Cutaneous malignant melanomas have mild multifocal expression of versican (a) and intense immunoreactivity of versican at the peritumoral stroma and inside the tumor (d and g). Notice the high number of mitotic figures in the tumor (g). Oral malignant melanomas have versican expression at the tumor-peritumoral stroma border and in strands between nests of tumor cells (b and e) and intense versican immunoreactivity (h). Notice the cutaneous malignant melanoma with moderate hyaluronan expression in the stroma (c), an oral malignant melanoma with moderate hyaluronan staining in the stroma between cords and nests of neoplastic cells (f), and an oral malignant melanoma with extensive intratumoral expression of hyaluronan (i). Melanin was bleached in slides stained for detection of hyaluronan. Avidin-biotin-peroxidase staining with Mayer's hematoxylin counterstain; bar = 100 μm (a, b, d, and f), 50 μm (c, e, h, and i), or 25 μm (g). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 68, 12; 10.2460/ajvr.68.12.1376

In both oral and cutaneous malignant melanomas, abundant expression of hyaluronan was found, although expression was highly variable (Table 2; Figure 3). Similar to the results for benign tumors, hyaluronan was associated with the stroma and surrounding cell nests. The subepidermal and oral subepithelial region of the tumors was usually intensely labeled, as well as the inflamed areas. Despite heterogeneity of the labeling, malignant tumors had a stronger and more extensive hyaluronan expression than melanocytomas, although the expression did not differ significantly (P = 0.054) between malignant tumors and melanocytomas. Hyaluronan expression was usually more extensive than that of versican, but there was good correlation between versican and hyaluronan, with hyaluronan always evident in versican-positive areas (Figure 4). Again, hyaluronan expression did not differ significantly (P = 0.63) between oral and cutaneous malignant melanomas.

Figure 4—
Figure 4—

Photomicrographs of sections of malignant melanocytic tumors of dogs in which versican (a, c, e, and g) and hyaluronan (b, d, f, and h) expression were analyzed immunohistochemically or histochemically. Notice that there is a similar distribution in the tumor for versican and hyaluronan staining. An oral malignant melanoma has intense, extensive reactivity for versican and hyaluronan (a and b), whereas a cutaneous malignant melanoma has focal expression of both molecules surrounding cell nests (c and d). Notice the oral malignant melanoma with reactivity at the tumor-peritumoral stroma border and some strains inside the tumor (e and f). A cutaneous malignant melanoma has peritumoral and focal intratumoral expression (g and h). Melanin was bleached in slides stained for detection of hyaluronan. Avidinbiotin-peroxidase staining with Mayer's hematoxylin counterstain; bar = 500 μm (a, b, e, f, g, and h) or 50 μm (c and d). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 68, 12; 10.2460/ajvr.68.12.1376

Most of the malignant tumors expressed CD44, and in some tumors, CD44 was more abundant at the periphery of the tumor. Similar to results for melanocytomas, CD44 was found at the membrane of the cells, and staining was particularly intense in areas in which cells were organized in nests (data not shown). Again, no relationship was found between labeling for CD44 and versican or hyaluronan expression and between expression and cytologic characteristics of the tumors.

Expression of HAS and Hyal in canine fibroblasts and melanoma cell lines—To analyze the enzymes responsible for hyaluronan metabolism in canine fibroblasts and melanoma cell lines, reverse transcriptase–PCR assay for mammalian HASs and the main Hyals was performed on RNA extracted from primary canine dermal fibroblasts and CML-1, CML-10c2, and CML-6M canine melanoma cell lines.

Primary dermal fibroblasts and melanoma cell lines expressed HAS2 and HAS3 (Figure 5). There was no amplification for HAS1, even in nonrestrictive conditions. Although this was a semiquantitative technique, expression of HAS2 appeared to be higher than expression of HAS3.

Figure 5—
Figure 5—

Expression pattern of HASs and Hyals in canine dermal fibroblasts and canine melanoma cell lines. Total RNA was extracted from subconfluent cultured cells by use of an isolation kit and subjected to reverse transcriptase–PCR assay in a 2-step reaction with primers for canine HAS1, HAS2, HAS3, Hyal1, and Hyal2. Amplification of a fragment of GAPDH was used as an internal control sample. The PCR amplification products were resolved on 2% agarose gels (1% agarose gel for GAPDH), stained with ethidium bromide, and developed under UV light. Numbers on the right side represent the number of base pairs.

Citation: American Journal of Veterinary Research 68, 12; 10.2460/ajvr.68.12.1376

All tested cells in culture expressed Hyal1 and Hyal2, although a higher expression was evident for Hyal1. Expression for canine dermal fibroblasts also appeared to be higher than expression for canine melanoma cell lines.

Discussion

The main objective of the study reported here was to determine whether versican or hyaluronan (or both) could be markers for malignancy in melanocytic lesions of dogs, similar to the situation described for lesions in humans.14 A second objective was to define whether there was a common distribution of the 3 members of the extracellular-pericellular complexes formed by versican, hyaluronan, and CD44.

In the study reported here, we found that versican was not evident in normal oral mucosa, whereas it was found only in some hair follicles and around some blood vessels but not in the dermis and epidermis of normal canine skin. This was a more restricted location than in skin of adult humans in which versican has been found in the basal layer of the epidermis as well as the papillary and reticular layers of the dermis, in addition to the hair follicles.36,37 In contrast, localization of versican in hair follicles of dogs is similar to the situation in humans in which it has been suggested38–40 that versican may play an essential role in mesenchymal condensation and hair induction. Our results are compatible with those reported in humans, rats, and mice in which versican has been found specifically in the dermal papilla in the anagen phase as well as the connective tissue sheath surrounding the bulge region, which leads to the suggestion that versican gene expression throughout the anagen phase is necessary for the maintenance of normal hair growth.40,41 It is interesting that versican expression in hair follicles was more frequent and intense in follicles located in tissues adjacent to tumors, whereas most of the hair follicles in normal skin samples had negative results. This would suggest that versican may be expressed more in tissues subjected to remodeling conditions.

Large amounts of hyaluronan were found in the dermis and corium of the oral mucosa. In the skin42 and oral mucosa43 of humans, hyaluronan has also been found in the basal and spinous layers of the epidermis and basal and middle layers of the oral epithelium. The more extensive expression of versican and hyaluronan in the epidermis of humans, compared with expression in the epidermis of dogs, may be related to the higher number of keratinocyte layers in human skin, compared with the number of layers in the skin of dogs. Nevertheless, some alteration of the protein via fixation or low cross-reactivity between human and canine protein for versican cannot be discarded.

Similarly, the hyaluronan receptor CD44 was found in the basal cell layer of the canine epidermis and basal and middle cell layers of the oral epithelium, which is again a more restricted localization than in human skin in which CD44 is found in the basal and spinous cell layers.44 Thus, CD44, the receptor of the hyaluronanversican complex, appears to be expressed only in the proliferative layers in the skin of humans and dogs. In canine epidermis, the early differentiation marker cytokeratine K10 is expressed in the suprabasal layer.45

Melanocytomas from dogs had negative or (rarely) slightly positive results for versican, with staining mostly at the dermal-epidermal junction in tumors with junctional activity, whereas most malignant tumors had positive results for versican with a variable degree of intensity. A significant (P = 0.002) correlation was detected between malignancy and the degree of versican expression. We found no significant (P = 0.29) difference in versican expression or distribution between oral and cutaneous malignant melanomas.

To our knowledge, the study reported here is the first in which versican has been detected in malignant melanomas of dogs. Therefore, malignant melanomas should be added to other neoplasms of dogs in which overexpression of versican has been found. This is the situation for colonic tumors of dogs in which versican expression is increased in the peritumoral stroma of adenocarcinomas and reduced in adenomas, with a significant correlation between grade of tumor and degree of versican expression.46 Versican is also overexpressed in mammary gland tumors of dogs; it has been proposed that versican expression is associated with prechondrogenesis in these tissues.17,18 Thus, versican expression in melanocytic tumors of dogs appears to be similar to that for melanocytic tumors of humans in which versican has been proposed as a useful marker of malignancy because benign nevi had negative results for versican, whereas malignant and metastatic melanomas had strong positive results for versican.15 Furthermore, nevi with atypic dysplasia, a lesion that is considered to be a precursor of malignant melanoma in humans, had a correlation between the degree of positive results and the degree of atypia.14 Nevertheless, premalignant lesions are not clearly defined in veterinary medicine in which corporal location is an important factor in defining malignancy.

Hyaluronan was also overexpressed in melanocytic tumors of dogs, although the differences between normal and tumor tissues were not significant (P = 0.054) because of the extensive amount of hyaluronan in normal connective tissue. No significant (P = 0.63) difference was found for hyaluronan expression between oral and cutaneous malignant melanomas. Hyaluronan and versican are abundant in the stroma of malignant tumors, although versican appears to be more restricted than hyaluronan. Versican is always associated with hyaluronan, and both molecules are found in the same areas of a tumor, which would be expected on the basis of the hyaluronan-binding properties of versican.11 Hyaluronan-versican complexes interact with a cell through the hyaluronan receptor CD44, and detection of this receptor has been reported27 in melanocytic tumors of dogs. The CD44 molecule is highly abundant in these tumors, but no relationship was found between expression of CD44 and versican or hyaluronan or between location of CD44 and expression of versican or hyaluronan in the tumors of the study reported here. This finding suggested that CD44 may have roles other than as a receptor for extracellular hyaluronan; this hypothesis has been proposed by other authors.23

Our results supported the hypothesis expressed by other investigators47 for mammary gland tumors in which versican is predominantly incorporated and accumulated into the hyaluronan matrix surrounding stromal cells. Thus, hyaluronan produced endogenously by tumor and stromal cells may support compartmentalization of versican on the cell surface. Because of the coincidence of hyaluronan and versican expression, versican is believed to modulate hyaluronan function and vice versa. Thus, it would appear that hyaluronanversican–rich ECM allows cells to prepare for proliferation and migration. Versican may promote these cell properties by enhancing cell detachment from the ECM or by participating in the assembly of intracellular machinery and transmitting signals in concert with hyaluronan. Purified versican is able to increase cell proliferation and migration and to decrease cell adhesion of some substrates in vitro in malignant melanoma cells of humans.15 A similar situation may happen in malignant melanomas of dogs. Hyaluronan has an established role in cell proliferation and tumor progression in general8 and in melanomas in particular.48,49 Acting in combination and because of the unique physicochemical properties of hyaluronan, the increase in hyaluronan concentrations in local tissues may cause increased turgidity and hydration, which in turn facilitates cell migration. Furthermore, hyaluronan appears to promote cell motility by acting on intracellular signaling pathways through interaction with cell-surface receptors, and versican may influence this action. It has been proposed50–52 that hyaluronan, HASs, and Hyals are involved in tumor angiogenesis and invasion because the production of high–molecular-weight hyaluronan is believed to provide a hydrated microniche that facilitates the invasion of tumor cells into the ECM, whereas the Hyals degrade the larger polymers of hyaluronan into small angiogenesis-inducing oligomers that allow neovascularization of tumors.

An unresolved question concerns the cellular source of the ECM components in tumors. Results of the study reported here revealed that versican and hyaluronan were localized in the tumor stroma in vivo, but it has been reported elsewhere16,27 that canine malignant melanoma cell lines produce versican and hyaluronan. The possibility of neoplastic and stromal cells being involved in versican production should be considered; it is also possible that in vivo stromal cells are the main cells responsible for versican production because expression of versican or hyaluronan was not detected in the more cellular and compact regions of the tumors.

The other question addressed in the study reported here was the enzymatic origin and degradation of hyaluronan in melanomas of dogs. Canine dermal fibroblasts and melanoma cells expressed HAS2 and HAS3 but not HAS1. This expression pattern is similar to that described in human breast cancer cells50 and human prostate cancer cells,53 for which it has been suggested50 that the HAS3 isoform is primarily responsible for the synthesis of basal amounts of hyaluronan, whereas HAS2 is required for the synthesis of the large quantities of hyaluronan required for cancer invasion. In contrast, colon cancer biopsy specimens and malignant mesotheliomas of humans express the 3 types of HAS.54,55 Regarding the degradative enzymes, canine dermal fibroblasts and canine melanoma cells expressed both Hyal1 and Hyal2. These enzymes constitute the major Hyals of somatic tissues, and Hyal2 may be the more important enzyme because the Hyal2 null mutation is lethal in mice embryos.51 Both enzymes appear to be highly related to cancer progression because Hyal1 is a candidate tumor suppressor gene product, whereas Hyal2 appears to function as an oncogene or a tumor suppressor gene.51,52 Human dermal fibroblasts appear to express a constitutive Hyal2 and a growth factor–inducible Hyal1,25 whereas human breast cancer cell lines express primarily Hyal1 and Hyal2, with Hyal2 being more correlated with invasiveness.50 To our knowledge, the study reported here is the first in which analysis of HAS or Hyal expression in melanocytic tumor cells has been described in any species.

Analysis of results for the study reported here indicated that versican expression may be used as a diagnostic marker in melanocytic tumors of dogs and provided support to the hypothesis that versican-hyaluronan complexes may facilitate cell detachment and promote cell growth, thus contributing to the aggressive biological behavior of malignant melanomas of dogs. Furthermore, analysis of our results indicated that in canine fibroblasts and melanoma cells, the enzymes mainly responsible for hyaluronan synthesis were HAS2 and HAS3 and for hyaluronan degradation were Hyal1 and Hyal2. In view of the role of hyaluronan in the tumorigenic process, information on the enzymes involved in hyaluronan metabolism could indicate new potential therapeutic targets.

ABBREVIATIONS

ECM

Extracellular matrix

HAS

Hyaluronan synthase

Hyal

Hyaluronidase

TBS

Tris-buffered saline

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

a.

Sigma Chemical Co, St Louis, Mo.

b.

Chondroitinase ABC from Proteus vulgaris, Sigma Chemical Co, St Louis, Mo.

c.

Anti-human monoclonal large chondroitin sulphate 2B1, Seikagaku Ltd, Tokyo, Japan.

d.

Dako, Glostrup, Denmark.

e.

Immunopure ABC peroxidase staining kit, Pierce, Rockford, Ill.

f.

Vector NovaRED substrate kit, Vector Laboratories Inc, Burlingame, Calif.

g.

Hyaluronate lyase from Streptomyces hyalurolyticus, Sigma-Aldrich, St Louis, Mo.

h.

bHABP from bovine cartilage, Seikagaku Ltd, Tokyo, Japan.

i.

Monoclonal antibody 2D10 against canine CD44, courtesy of Dr. Susanne Alldinger, Institut für Veterinär-Pathologie, Justus-Liebig-Universitat Giessen, Giessen, Germany.

j.

Dako, Glostrup, Denmark.

k.

Microscope Olympus Vanox-S AH-2, Olympus, Hamburg, Germany.

l.

Leica DFC480 integrated digital camera system, Leica Microsystems, Wetzlar, Germany.

m.

SPSS for Windows, version 13.0, SPSS Inc, Chicago, Ill.

n.

Courtesy of Dr. E. Greg MacEwen, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison,Wis.

o.

Courtesy of Dr. Montserrat Serra, UNIVET, Universitat Autònoma de Barcelona, Bellaterra, Spain.

p.

Gibco BRL/Life Technologies, Rockville, Md.

q.

RNeasy isolation kit, Qiagen, Hilden, Germany.

r.

Expand reverse transcriptase, Roche, Mannheim, Germany.

s.

Expand high-fidelity PCR polymerase, Roche, Mannheim, Germany.

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Appendix

Oligonucleotide primers and PCR amplification conditions used for reverse transcriptase–PCR assay of melanocytic tumors of dogs.

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