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Immunohistochemical Expression of Caveolin-1 in Normal and Neoplastic Canine Mammary Tissue
J. Comp. Path. 2010, Vol. 143, 39e44
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www.elsevier.com/locate/jcpa
Immunohistochemical Expression of Caveolin-1 in Normal and Neoplastic Canine Mammary Tissue I. Amorim, C. C. Lopes, A. M. R. Faustino and P. Dias Pereira ICBAS-UP, Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal
Summary Caveolins are the major structural proteins of caveolae and play a role in tumour surveillance. The expression of caveolin-1 (Cav-1) was investigated immunohistochemically in samples of normal canine mammary tissue (n ¼ 5), benign (n ¼ 23) and malignant (n ¼ 45) mammary tumours, tumour emboli (n ¼ 10) and metastatic lesions (n ¼ 10). Cav-1 was not expressed by normal luminal epithelium, but was consistently expressed by normal myoepithelial cells. There was no epithelial expression of Cav-1 by the benign mammary lesions, but the molecule was detected in 35.6% of the malignant lesions, 80% of the tumour emboli and in 40% of the metastatic lesions. These findings link increased expression of Cav-1 to neoplastic transformation and suggest that Cav-1 expression is associated with more malignant canine mammary tumours and their vascular invasion and regional lymph node metastasis. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: caveolin-1; dog; immunohistochemistry; mammary gland
Introduction Caveolae (from the Latin ‘little caves’) are specialized plasmalemmal invaginations with a typically flaskshaped morphology, which are present in many types of mammalian cell (Shin and Abraham, 2001). The functional roles attributed to caveolae include involvement in cholesterol homeostasis, vesicular transport (transcytosis and endocytosis), tumorigenesis and regulation of signal transduction through immune and growth factor receptors (Razani et al., 2002). Caveolins are the major structural oligomeric proteins of caveolae. The caveolin (Cav) protein family is composed of three distinct proteins: Cav-1 (which includes the isoforms a and b; Okamoto et al., 1998), Cav-2 and Cav-3 (Glenney and Soppet, 1992; Tang et al., 1996). Cav-1 and Cav-2 are co-expressed in many cell types, with especially high levels in endothelial cells, adipocytes and type I pneumocytes. In contrast, Cav-3 is the only Cav expressed in skeletal
Correspondence to: P. Dias Pereira (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2009.12.019
and cardiac muscle cells, but all three caveolins are expressed in smooth muscle cells (Tang et al., 1996). Caveolins possess a 20 amino acid juxtamembrane domain, also termed the caveolin scaffolding domain (CSD), which mediates the functional binding and regulation of several caveolae-localized molecules, especially signalling proteins (Okamoto et al., 1998). It has been demonstrated that CSD serves a dual role, acting both as an anchor that holds various proteins within caveolae and as a regulator element capable of either inhibiting or enhancing protein signalling activity (Cohen et al., 2004). Cav-1, a 22e24 kDa protein of 178 amino acids, was initially identified as a major substrate for tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryonic fibroblasts, suggesting that it played an important role in the oncogenic process (Glenney, 1989). Cav-1 expression is down-regulated in sarcomas, lung carcinoma and ovarian carcinoma (Wiechen et al., 2001a,b; Be´langer et al., 2004); however, elevated expression of Cav-1 has been associated with metastasis of oesophageal squamous cell carcinoma and prostate cancer and negatively correlated with Ó 2010 Elsevier Ltd. All rights reserved.
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patient survival for these tumours (Yang et al., 1999; Kato et al., 2002). Therefore, it is reasonable to speculate that Cav-1 could function as both a negative and positive regulator of signalling and cell transformation (Lee et al., 2000) and that the role of this protein may vary considerably, depending on the tissue involved. Despite these controversial data, it is accepted that Cav-1 mediates the tumour surveillance process and therefore may constitute an important factor in cancer diagnosis and management (Williams and Lisanti, 2005). Cav-1 expression is observed in many different cell types in mammary tissue including the epithelium, myoepithelium, fibroblasts, adipocytes, endothelial cells and smooth muscle cells (Cohen et al., 2004; Savage et al., 2007). Immunohistochemical studies have shown increased expression of Cav-1 in primary and metastatic human breast cancer, when compared with normal epithelium (Yang et al., 1998; Pinilla et al., 2006). The role of caveolins in canine mammary tumours is still unknown. The aim of the present study was to evaluate the expression of Cav-1 in normal and neoplastic canine mammary tissue.
Materials and Methods The study was conducted with samples obtained either during surgery or necropsy examination. These included samples of five normal mammary glands, 23 benign and 45 malignant tumours, 10 tumours with neoplastic emboli and 10 metastatic lesions (nine regional lymph nodes and one pulmonary metastasis). Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin wax. Serial sections (3 mm) were stained with haematoxylin and eosin (HE) or used for the immunohistochemical study. Table 1 Histological classification of the canine mammary tissues examined in this study Histological classification Normal mammary gland Benign tumours Simple adenoma Complex adenoma Benign mixed tumour Malignant tumours Tubulopapillary carcinoma Solid carcinoma Anaplastic carcinoma Complex carcinoma Carcinosarcoma Neoplastic emboli Metastatic lesions
Number of samples 5 23 7 7 9 45 9 10 10 12 4 10 10
Mammary tumours were classified (Table 1) according to the criteria proposed by the World Health Organization (Misdorp et al., 1999). Sections were independently examined by three pathologists and when there was a divergence of opinion, an agreed diagnosis was reached by using a multiheaded microscope. For immunohistochemistry (IHC), antigen retrieval was performed on dewaxed sections by boiling in a steamer in 10 mmol/l sodium citrate buffer (pH 6.0) for 2 min. Slides were cooled for 10 min at room temperature and rinsed twice in triphosphate buffered saline (TBS) for 5 min. After blocking endogenous peroxidase activity with hydrogen peroxide 3% in methanol for 10 min, sections were subjected to immunohistochemical labelling with a polyclonal antiserum specific for Cav-1 (C13630; 1 in 400 dilution; Transduction Laboratories, Erembodegem, Belgium) for 1.5 h. Secondary detection was with the NovolinkÔ Max-Polymer detection system (Novocastra, Newcastle, UK). Sections were rinsed with TBS between each step of the procedure. Labelling was ‘visualized’ by incubation of the sections with a freshly prepared solution of 3, 30 -diamino-benzidine (DAB) for up to 7 min at room temperature. Finally, sections were lightly counterstained with haematoxylin, dehydrated and mounted. Normal mesenchymal tissue at the periphery of each tumour and endothelial cells of capillaries within the tumours were used as internal positive controls, while in negative controls the primary antibody was omitted and replaced by TBS. The distribution, intensity and proportion of labelling were assessed. Distribution was defined in epithelial cells (E), myoepithelial cells (M) and mesenchymal cells (Me). The myoepithelial population was further subdivided into the single layer of myoepithelial cells between the basement membrane and the luminal epithelial cells of the mammary gland (M1) and the myoepithelial cells arranged in whorls or in small aggregates, independent of the basement membrane and luminal epithelial cells and characteristically found in complex tumours (M2). The intensity of labelling was recorded as: 0, absent; 1, weakly positive; 2, moderately positive; 3, intense labelling equivalent to that seen with normal adipocytes or endothelial cells. The proportion of tumour cells labelled was graded as: 0, <5% of the total neoplastic population labelled; 1, 5e25% of tumour cells labelled; 2, 26e50% of tumour cells labelled; 3, 51e75% of tumour cells labelled; 4, 76e100% of tumour cells labelled. Finally, a semiquantitative estimation of Cav-1 expression was made, using a composite score obtained by multiplying the values of the intensity and grade of the labelling for each location, according to the
Caveolin-1 Expression in Canine Mammary Gland
41
method described by Park et al. (2005). A mean score of $6 was considered positive and a score of <6 as negative.
Results In normal canine mammary gland tissue, Cav-1 was expressed consistently by M1 cells arranged as a continuous layer around ducts and lobular units, whereas epithelial cells were devoid of any labelling (Fig. 1). None of the 23 benign tumours had epithelial expression of Cav-1, but M1 cells were positively labelled in all cases. In 14 benign tumours an M2 component was identified and in nine of these cases these cells expressed Cav-1 (three complex adenomas and six benign mixed tumours; Fig. 2). Five benign tumours did not have Cav-1 expression of M2 cells (three complex adenomas and two benign mixed tumours). All benign tumours had Me cells that expressed Cav-1 (Fig. 3). Cav-1 was expressed by epithelial cells in 16 of 45 malignant lesions (one tubulopapillary carcinoma, two complex carcinomas, four solid carcinomas, seven anaplastic carcinomas and two carcinosarcomas; Fig. 4). M1 cells were characteristically difficult to identify in malignant canine tumours and were observed in only 23 cases. In 22 of these 23 cases these M1 cells expressed Cav-1 (Fig. 5), but M1 cells in one tubulopapillary carcinoma were not labelled. M2 cells were identified in 16 malignant tumours and in 15 of these cases these cells expressed Cav-1 (11 complex carcinomas and four carcinosarcomas); one complex carcinoma was negative for such expression. Me cells, represented by cartilaginous or osseous neoplastic foci present in the four carcinosarcomas, expressed Cav-1 in three of these samples.
Fig. 1. Normal mammary gland. Note Cav-1 expression by M1 and lack of immunoreactivity in luminal epithelium. IHC. 400.
Fig. 2. Complex adenoma. Note strong positive labelling of M2 population. IHC. 100.
Eight of 10 epithelial tumour emboli expressed Cav-1 (Fig. 6). Four of 10 metastatic tumour foci showed Cav-1 expression by epithelial cells (Fig. 7). In two metastatic foci an M1 cell component was identified and in both of these foci the M1 cells expressed Cav-1. A single metastatic focus had evidence of M2 and Me cells and both of these cell types were also positively labelled for Cav-1 expression.
Discussion In recent years there have been numerous investigations of the role of Cav-1 in human breast cancer (Yang et al., 1998; Park et al., 2005; Liedtke et al., 2007). Immunohistochemical studies have demonstrated overexpression of Cav-1 in breast cancer (Yang et al., 1998; Park et al., 2005) and suggested its importance as a prognostic marker, associating
Fig. 3. Benign mixed tumour. Cartilaginous foci exhibit strong Cav-1 expression. IHC. 400.
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Fig. 4. Solid carcinoma. Neoplastic epithelial cells show strong Cav-1 expression. IHC. 200.
Fig. 6. Neoplastic tumour emboli showing strong Cav-1 expression by neoplastic epithelial cells. IHC. 100.
Cav-1 overexpression with metastasis, shorter diseasefree interval and overall survival in women (Savage et al., 2007). The present study represents the first immunohistochemical investigation of Cav-1 expression in canine mammary tissue. This molecule is strongly expressed by myoepithelial cells, endothelial cells and adipocytes of normal canine mammary gland; however, there is no expression of Cav-1 by luminal epithelial cells. There remains controversy regarding Cav-1 expression in normal human breast tissue. Yang et al. (1998), Park et al. (2005) and Savage et al. (2007) reported Cav-1 expression by normal human mammary epithelium, while Hurlstone et al. (1999) reported no such expression. Liedtke et al. (2007) concluded that some of these discrepancies may reflect the choice of primary antibody (monoclonal or polyclonal) and to the nature
of the scoring system adopted to evaluate the immunoreactivity. None of the benign canine mammary tumours showed epithelial expression of Cav-1; however, 35.6% of the malignant canine mammary tumours had positive Cav-1 labelling of the epithelial cell population. Curiously, this pattern was mostly observed (68.8% of cases) in solid and anaplastic carcinomas, tumours that are characteristically more aggressive and usually associated with a worse prognosis. This finding is concordant with data previously reported on human breast cancer, documenting Cav-1 overexpression in tumours with unfavourable behaviour, namely invasive breast cancer (Park et al., 2005) and inflammatory breast cancer cell lines and specimens (Van den Eynden et al., 2006). The recent discovery that Cav-1 is preferentially expressed in human basal cell-like breast carcinomas, characterized by high histological grade and high proliferation
Fig. 5. Tubulopapillary carcinoma. Note strong positive labelling of the M1 population. IHC. 200.
Fig. 7. Lymph node metastasis of a solid carcinoma, showing Cav-1 expression by neoplastic cells. IHC. 100.
Caveolin-1 Expression in Canine Mammary Gland
rate, calls into question the tumour-suppressor functions of Cav-1 in breast cancer proposed by some investigators, particularly for this subgroup of lesions (Pinilla et al., 2006; Savage et al., 2007). Moreover, 80% of tumour emboli also showed overexpression of Cav-1 by epithelial cells and in 40% of metastatic lesions overexpression of Cav-1 by epithelial cells was also noted. Yang et al. (1998) also described Cav-1 expression of lymph node metastasis of human breast cancer. Cav-1 expression in M1 cells was found in normal mammary glands and benign mammary tumours, as well as in 95.7% of malignant lesions and in all cases of metastatic lesions where this population was identified. When present, M2 cells expressed Cav-1 in 64.3% of the benign lesions and in 94.4% of the malignant lesions. Myoepithelial cells are attached to the basement membrane and to adjacent luminal epithelial cells by desmosomes. The location of myoepithelia, between stroma and luminal epithelial cells, places them in an ideal position to control many aspects of mammary structure and function such as polarity, electrolyte and fluid flow. They may also act as an intermediary in signalling processes, passing information both to and from epithelial cells in a paracrine fashion or via intra-epithelial gap junctions (Locke et al., 2000). This model is consistent with the involvement of Cav-1 in diverse cellular processes including cholesterol homeostasis, vesicular transport, cell migration, cell cycle, cell polarity and signal transduction (Williams and Lisanti, 2005). The Me cell component was clearly labelled by Cav-1 antibody in all benign tumours and in 75% of malignant lesions. Mixed tumours are unusual in the human breast (Kumar et al., 2004), but they are frequently found in both human salivary glands and in the canine mammary gland (Misdorp et al., 1999). Expression of Cav-1 in normal salivary gland and in mucoepidermoid carcinoma of the human salivary gland has been recently documented (Shi et al., 2007). Strong expression of Cav-1 in ductal epithelial cells of normal salivary glands and down-regulation of Cav-1 expression in neoplastic epithelial cells was observed. However, that study does not describe Cav-1 expression by the myoepithelial or mesenchymal cell population. Many studies have documented an interaction between the CSD of Cav-1 and various signalling molecules that can stimulate or suppress their activity. It appears that Cav-1 plays a complex role that might be dependent on the type of molecules in question, the different regions of the CSD to which it binds, the type of tissue and the microenvironmental conditions involved and the stage of disease (Williams and Lisanti, 2005). Extensive evidences suggest that
43
Cav-1 normally regulates the growth and transformation of mammary luminal epithelial cells, representing a key factor in the neoplastic transformation process, and that overexpression of this molecule may represent an acquired feature that contributes to a malignant phenotype. This can explain why luminal epithelial cells of the normal mammary gland and benign mammary lesions are Cav-1 negative, while a significant number of cases of malignant lesions, neoplastic emboli and metastatic lesions are Cav-1 positive. To the best of the authors’ knowledge this represents the first report of Cav-1 expression in canine mammary tissue. The study has documented absence of Cav-1 expression by normal luminal cells and its consistent expression by myoepithelial cells of the normal canine mammary gland. There is a clear association between increased expression of Cav-1 and neoplastic transformation of the epithelium and the data suggest that Cav-1 expression is associated with a more malignant phenotype of canine mammary neoplasia and with vascular invasion and regional lymph node metastasis. Additional studies comprising a larger group of animals are warranted in order to confirm these findings and to further investigate the usefulness of Cav-1 immunoreactivity as a prognostic factor in canine mammary tumours.
References Be´langer M, Roussel E, Couet J (2004) Caveolin-1 is down-regulated in human lung carcinoma and acts as a candidate tumor suppressor gene. Chest, 125, 106S. Cohen A, Hnasko R, Schubert W, Lisanti M (2004) Role of caveolae and caveolins in health and disease. Physiological Reviews, 84, 1341e1379. Glenney J (1989) Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus. Journal of Biological Chemistry, 264, 20163e20166. Glenney J, Soppet D (1992) Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in Rous sarcoma virus-transformed fibroblasts. Proceedings of the National Academy of Sciences of the United States of America, 89, 10517e10521. Hurlstone A, Reid G, Reeves J, Fraser J, Strathdee G et al. (1999) Analysis of the caveolin-1 gene at human chromosome 7q31.1 in primary tumours and tumour derived cell lines. Oncogene, 18, 1881e1890. Kato K, Hida Y, Miyamoto M, Hashida H, Shinohara T et al. (2002) Overexpression of caveolin-1 in esophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Cancer, 94, 929e933.
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Kumar P, Sobhani S, Monabati A, Talei A, Shirazi B (2004) Cytologic findings of a pleomorphic adenoma of the breast: a case report. Acta Cytologica, 48, 849e852. Lee H, Volonte D, Galbiati F, Iyengar P, Lublin D et al. (2000) Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: identification of a c-Src/Cav-1/Grb7 signaling cassette. Molecular Endocrinology, 14, 1750e1775. Liedtke C, Kersting C, Bu¨rger H, Kiesel L, Wu¨lfing P (2007) Caveolin-1 expression in benign and malignant lesions of the breast. World Journal of Surgical Oncology, 5, 110e119. Locke D, Perusinghe N, Newman T, Jayatilake H, Evans W et al. (2000) Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland. Journal of Cell Physiology, 183, 228e237. Misdorp W, Else R, Hellmen E, Lipscomb T (1999) Histological Classification of Mammary Tumours of the Dog and Cat, 2nd Edit., Armed Forces Institute of Pathology, Washington DC, pp 1e59. Okamoto T, Schlegel A, Scherer P, Lisanti M (1998) Caveolins, a family of scaffolding proteins for organizing ‘preassembled signaling complexes’ at the plasma membrane. Journal of Biology and Chemistry, 273, 5419e5422. Park S, Kim J, Kim Y, Kim Y, Kim S (2005) Caveolin-1 is down-regulated and inversely correlated with HER2 and EGFR expression status in invasive ductal carcinoma of the breast. Histopathology, 47, 625e630. Pinilla S, Honrado E, Hardisson D, Benı´ tez J, Palacios J (2006) Caveolin-1 expression is associated with a basal-like phenotype in sporadic and hereditary breast cancer. Breast Cancer Research and Treatment, 99, 85e90. Razani B, Woodman S, Lisanti M (2002) Caveolae: from cell biology to animal physiology. Pharmacological Reviews, 54, 431e467. Savage K, Lambros M, Robertson D, Jones C, Mackay A et al. (2007) Caveolin 1 is overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a morphological, ultrastructural, immunohistochemical, and in situ hybridization analysis. Clinical Cancer Research, 13, 90e101.
Shi L, Chen X-M, Wang L, Zhang L, Chen Z (2007) Expression of caveolin-1 in mucoepidermoid carcinoma of the salivary glands: correlation with vascular endothelial growth factor, microvessel density, and clinical outcome. Cancer, 109, 1523e1531. Shin J-S, Abraham S (2001) Caveolae e not just craters in the cellular landscape. Science, 293, 1447e1448. Tang Z-L, Scherer P, Okamoto T, Song K, Chu C et al. (1996) Molecular cloning of caveolin-3, a novel member of the caveolin gene family expresses predominantly in muscle. Journal of Biological Chemistry, 271, 2255e2261. Van den Eynden G, Van Laere S, Van der Auwera I, Merajver S, Van Marck E et al. (2006) Overexpression of caveolin-1 and -2 in cell lines and in human samples of inflammatory breast cancer. Breast Cancer Research and Treatment, 95, 219e228. Wiechen K, Diatchenko L, Agoulnik A, Scharff K, Schober H et al. (2001a) Caveolin-1 is down regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene. American Journal of Pathology, 159, 1635e1643. Wiechen K, Sers C, Agoulnik A, Arlt K, Dietel M et al. (2001b) Down-regulation of caveolin-1, a candidate tumor suppressor gene, in sarcomas. American Journal of Pathology, 158, 833e839. Williams T, Lisanti M (2005) Caveolin-1 in oncogenic transformation, cancer and metastasis. American Journal of Physiology. Cell Physiology, 288, C494eC506. Yang G, Truong L, Timme T, Ren C, Wheeler T et al. (1998) Elevated expression of caveolin is associated with prostate and breast cancer. Clinical Cancer Research, 4, 1873e1880. Yang G, Truong L, Wheeler T, Thompson T (1999) Caveolin-1 expression in clinically confined human prostate cancer: a novel prognostic marker. Cancer Research, 59, 5719e5723.
March 14th, 2009 ½ Received, Accepted, December 29th, 2009
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
Immunohistochemical Expression of Caveolin-1 in Normal and Neoplastic Canine Mammary Tissue I. Amorim, C. C. Lopes, A. M. R. Faustino and P. Dias Pereira ICBAS-UP, Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal
Summary Caveolins are the major structural proteins of caveolae and play a role in tumour surveillance. The expression of caveolin-1 (Cav-1) was investigated immunohistochemically in samples of normal canine mammary tissue (n ¼ 5), benign (n ¼ 23) and malignant (n ¼ 45) mammary tumours, tumour emboli (n ¼ 10) and metastatic lesions (n ¼ 10). Cav-1 was not expressed by normal luminal epithelium, but was consistently expressed by normal myoepithelial cells. There was no epithelial expression of Cav-1 by the benign mammary lesions, but the molecule was detected in 35.6% of the malignant lesions, 80% of the tumour emboli and in 40% of the metastatic lesions. These findings link increased expression of Cav-1 to neoplastic transformation and suggest that Cav-1 expression is associated with more malignant canine mammary tumours and their vascular invasion and regional lymph node metastasis. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: caveolin-1; dog; immunohistochemistry; mammary gland
Introduction Caveolae (from the Latin ‘little caves’) are specialized plasmalemmal invaginations with a typically flaskshaped morphology, which are present in many types of mammalian cell (Shin and Abraham, 2001). The functional roles attributed to caveolae include involvement in cholesterol homeostasis, vesicular transport (transcytosis and endocytosis), tumorigenesis and regulation of signal transduction through immune and growth factor receptors (Razani et al., 2002). Caveolins are the major structural oligomeric proteins of caveolae. The caveolin (Cav) protein family is composed of three distinct proteins: Cav-1 (which includes the isoforms a and b; Okamoto et al., 1998), Cav-2 and Cav-3 (Glenney and Soppet, 1992; Tang et al., 1996). Cav-1 and Cav-2 are co-expressed in many cell types, with especially high levels in endothelial cells, adipocytes and type I pneumocytes. In contrast, Cav-3 is the only Cav expressed in skeletal
Correspondence to: P. Dias Pereira (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2009.12.019
and cardiac muscle cells, but all three caveolins are expressed in smooth muscle cells (Tang et al., 1996). Caveolins possess a 20 amino acid juxtamembrane domain, also termed the caveolin scaffolding domain (CSD), which mediates the functional binding and regulation of several caveolae-localized molecules, especially signalling proteins (Okamoto et al., 1998). It has been demonstrated that CSD serves a dual role, acting both as an anchor that holds various proteins within caveolae and as a regulator element capable of either inhibiting or enhancing protein signalling activity (Cohen et al., 2004). Cav-1, a 22e24 kDa protein of 178 amino acids, was initially identified as a major substrate for tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryonic fibroblasts, suggesting that it played an important role in the oncogenic process (Glenney, 1989). Cav-1 expression is down-regulated in sarcomas, lung carcinoma and ovarian carcinoma (Wiechen et al., 2001a,b; Be´langer et al., 2004); however, elevated expression of Cav-1 has been associated with metastasis of oesophageal squamous cell carcinoma and prostate cancer and negatively correlated with Ó 2010 Elsevier Ltd. All rights reserved.
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patient survival for these tumours (Yang et al., 1999; Kato et al., 2002). Therefore, it is reasonable to speculate that Cav-1 could function as both a negative and positive regulator of signalling and cell transformation (Lee et al., 2000) and that the role of this protein may vary considerably, depending on the tissue involved. Despite these controversial data, it is accepted that Cav-1 mediates the tumour surveillance process and therefore may constitute an important factor in cancer diagnosis and management (Williams and Lisanti, 2005). Cav-1 expression is observed in many different cell types in mammary tissue including the epithelium, myoepithelium, fibroblasts, adipocytes, endothelial cells and smooth muscle cells (Cohen et al., 2004; Savage et al., 2007). Immunohistochemical studies have shown increased expression of Cav-1 in primary and metastatic human breast cancer, when compared with normal epithelium (Yang et al., 1998; Pinilla et al., 2006). The role of caveolins in canine mammary tumours is still unknown. The aim of the present study was to evaluate the expression of Cav-1 in normal and neoplastic canine mammary tissue.
Materials and Methods The study was conducted with samples obtained either during surgery or necropsy examination. These included samples of five normal mammary glands, 23 benign and 45 malignant tumours, 10 tumours with neoplastic emboli and 10 metastatic lesions (nine regional lymph nodes and one pulmonary metastasis). Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin wax. Serial sections (3 mm) were stained with haematoxylin and eosin (HE) or used for the immunohistochemical study. Table 1 Histological classification of the canine mammary tissues examined in this study Histological classification Normal mammary gland Benign tumours Simple adenoma Complex adenoma Benign mixed tumour Malignant tumours Tubulopapillary carcinoma Solid carcinoma Anaplastic carcinoma Complex carcinoma Carcinosarcoma Neoplastic emboli Metastatic lesions
Number of samples 5 23 7 7 9 45 9 10 10 12 4 10 10
Mammary tumours were classified (Table 1) according to the criteria proposed by the World Health Organization (Misdorp et al., 1999). Sections were independently examined by three pathologists and when there was a divergence of opinion, an agreed diagnosis was reached by using a multiheaded microscope. For immunohistochemistry (IHC), antigen retrieval was performed on dewaxed sections by boiling in a steamer in 10 mmol/l sodium citrate buffer (pH 6.0) for 2 min. Slides were cooled for 10 min at room temperature and rinsed twice in triphosphate buffered saline (TBS) for 5 min. After blocking endogenous peroxidase activity with hydrogen peroxide 3% in methanol for 10 min, sections were subjected to immunohistochemical labelling with a polyclonal antiserum specific for Cav-1 (C13630; 1 in 400 dilution; Transduction Laboratories, Erembodegem, Belgium) for 1.5 h. Secondary detection was with the NovolinkÔ Max-Polymer detection system (Novocastra, Newcastle, UK). Sections were rinsed with TBS between each step of the procedure. Labelling was ‘visualized’ by incubation of the sections with a freshly prepared solution of 3, 30 -diamino-benzidine (DAB) for up to 7 min at room temperature. Finally, sections were lightly counterstained with haematoxylin, dehydrated and mounted. Normal mesenchymal tissue at the periphery of each tumour and endothelial cells of capillaries within the tumours were used as internal positive controls, while in negative controls the primary antibody was omitted and replaced by TBS. The distribution, intensity and proportion of labelling were assessed. Distribution was defined in epithelial cells (E), myoepithelial cells (M) and mesenchymal cells (Me). The myoepithelial population was further subdivided into the single layer of myoepithelial cells between the basement membrane and the luminal epithelial cells of the mammary gland (M1) and the myoepithelial cells arranged in whorls or in small aggregates, independent of the basement membrane and luminal epithelial cells and characteristically found in complex tumours (M2). The intensity of labelling was recorded as: 0, absent; 1, weakly positive; 2, moderately positive; 3, intense labelling equivalent to that seen with normal adipocytes or endothelial cells. The proportion of tumour cells labelled was graded as: 0, <5% of the total neoplastic population labelled; 1, 5e25% of tumour cells labelled; 2, 26e50% of tumour cells labelled; 3, 51e75% of tumour cells labelled; 4, 76e100% of tumour cells labelled. Finally, a semiquantitative estimation of Cav-1 expression was made, using a composite score obtained by multiplying the values of the intensity and grade of the labelling for each location, according to the
Caveolin-1 Expression in Canine Mammary Gland
41
method described by Park et al. (2005). A mean score of $6 was considered positive and a score of <6 as negative.
Results In normal canine mammary gland tissue, Cav-1 was expressed consistently by M1 cells arranged as a continuous layer around ducts and lobular units, whereas epithelial cells were devoid of any labelling (Fig. 1). None of the 23 benign tumours had epithelial expression of Cav-1, but M1 cells were positively labelled in all cases. In 14 benign tumours an M2 component was identified and in nine of these cases these cells expressed Cav-1 (three complex adenomas and six benign mixed tumours; Fig. 2). Five benign tumours did not have Cav-1 expression of M2 cells (three complex adenomas and two benign mixed tumours). All benign tumours had Me cells that expressed Cav-1 (Fig. 3). Cav-1 was expressed by epithelial cells in 16 of 45 malignant lesions (one tubulopapillary carcinoma, two complex carcinomas, four solid carcinomas, seven anaplastic carcinomas and two carcinosarcomas; Fig. 4). M1 cells were characteristically difficult to identify in malignant canine tumours and were observed in only 23 cases. In 22 of these 23 cases these M1 cells expressed Cav-1 (Fig. 5), but M1 cells in one tubulopapillary carcinoma were not labelled. M2 cells were identified in 16 malignant tumours and in 15 of these cases these cells expressed Cav-1 (11 complex carcinomas and four carcinosarcomas); one complex carcinoma was negative for such expression. Me cells, represented by cartilaginous or osseous neoplastic foci present in the four carcinosarcomas, expressed Cav-1 in three of these samples.
Fig. 1. Normal mammary gland. Note Cav-1 expression by M1 and lack of immunoreactivity in luminal epithelium. IHC. 400.
Fig. 2. Complex adenoma. Note strong positive labelling of M2 population. IHC. 100.
Eight of 10 epithelial tumour emboli expressed Cav-1 (Fig. 6). Four of 10 metastatic tumour foci showed Cav-1 expression by epithelial cells (Fig. 7). In two metastatic foci an M1 cell component was identified and in both of these foci the M1 cells expressed Cav-1. A single metastatic focus had evidence of M2 and Me cells and both of these cell types were also positively labelled for Cav-1 expression.
Discussion In recent years there have been numerous investigations of the role of Cav-1 in human breast cancer (Yang et al., 1998; Park et al., 2005; Liedtke et al., 2007). Immunohistochemical studies have demonstrated overexpression of Cav-1 in breast cancer (Yang et al., 1998; Park et al., 2005) and suggested its importance as a prognostic marker, associating
Fig. 3. Benign mixed tumour. Cartilaginous foci exhibit strong Cav-1 expression. IHC. 400.
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Fig. 4. Solid carcinoma. Neoplastic epithelial cells show strong Cav-1 expression. IHC. 200.
Fig. 6. Neoplastic tumour emboli showing strong Cav-1 expression by neoplastic epithelial cells. IHC. 100.
Cav-1 overexpression with metastasis, shorter diseasefree interval and overall survival in women (Savage et al., 2007). The present study represents the first immunohistochemical investigation of Cav-1 expression in canine mammary tissue. This molecule is strongly expressed by myoepithelial cells, endothelial cells and adipocytes of normal canine mammary gland; however, there is no expression of Cav-1 by luminal epithelial cells. There remains controversy regarding Cav-1 expression in normal human breast tissue. Yang et al. (1998), Park et al. (2005) and Savage et al. (2007) reported Cav-1 expression by normal human mammary epithelium, while Hurlstone et al. (1999) reported no such expression. Liedtke et al. (2007) concluded that some of these discrepancies may reflect the choice of primary antibody (monoclonal or polyclonal) and to the nature
of the scoring system adopted to evaluate the immunoreactivity. None of the benign canine mammary tumours showed epithelial expression of Cav-1; however, 35.6% of the malignant canine mammary tumours had positive Cav-1 labelling of the epithelial cell population. Curiously, this pattern was mostly observed (68.8% of cases) in solid and anaplastic carcinomas, tumours that are characteristically more aggressive and usually associated with a worse prognosis. This finding is concordant with data previously reported on human breast cancer, documenting Cav-1 overexpression in tumours with unfavourable behaviour, namely invasive breast cancer (Park et al., 2005) and inflammatory breast cancer cell lines and specimens (Van den Eynden et al., 2006). The recent discovery that Cav-1 is preferentially expressed in human basal cell-like breast carcinomas, characterized by high histological grade and high proliferation
Fig. 5. Tubulopapillary carcinoma. Note strong positive labelling of the M1 population. IHC. 200.
Fig. 7. Lymph node metastasis of a solid carcinoma, showing Cav-1 expression by neoplastic cells. IHC. 100.
Caveolin-1 Expression in Canine Mammary Gland
rate, calls into question the tumour-suppressor functions of Cav-1 in breast cancer proposed by some investigators, particularly for this subgroup of lesions (Pinilla et al., 2006; Savage et al., 2007). Moreover, 80% of tumour emboli also showed overexpression of Cav-1 by epithelial cells and in 40% of metastatic lesions overexpression of Cav-1 by epithelial cells was also noted. Yang et al. (1998) also described Cav-1 expression of lymph node metastasis of human breast cancer. Cav-1 expression in M1 cells was found in normal mammary glands and benign mammary tumours, as well as in 95.7% of malignant lesions and in all cases of metastatic lesions where this population was identified. When present, M2 cells expressed Cav-1 in 64.3% of the benign lesions and in 94.4% of the malignant lesions. Myoepithelial cells are attached to the basement membrane and to adjacent luminal epithelial cells by desmosomes. The location of myoepithelia, between stroma and luminal epithelial cells, places them in an ideal position to control many aspects of mammary structure and function such as polarity, electrolyte and fluid flow. They may also act as an intermediary in signalling processes, passing information both to and from epithelial cells in a paracrine fashion or via intra-epithelial gap junctions (Locke et al., 2000). This model is consistent with the involvement of Cav-1 in diverse cellular processes including cholesterol homeostasis, vesicular transport, cell migration, cell cycle, cell polarity and signal transduction (Williams and Lisanti, 2005). The Me cell component was clearly labelled by Cav-1 antibody in all benign tumours and in 75% of malignant lesions. Mixed tumours are unusual in the human breast (Kumar et al., 2004), but they are frequently found in both human salivary glands and in the canine mammary gland (Misdorp et al., 1999). Expression of Cav-1 in normal salivary gland and in mucoepidermoid carcinoma of the human salivary gland has been recently documented (Shi et al., 2007). Strong expression of Cav-1 in ductal epithelial cells of normal salivary glands and down-regulation of Cav-1 expression in neoplastic epithelial cells was observed. However, that study does not describe Cav-1 expression by the myoepithelial or mesenchymal cell population. Many studies have documented an interaction between the CSD of Cav-1 and various signalling molecules that can stimulate or suppress their activity. It appears that Cav-1 plays a complex role that might be dependent on the type of molecules in question, the different regions of the CSD to which it binds, the type of tissue and the microenvironmental conditions involved and the stage of disease (Williams and Lisanti, 2005). Extensive evidences suggest that
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Cav-1 normally regulates the growth and transformation of mammary luminal epithelial cells, representing a key factor in the neoplastic transformation process, and that overexpression of this molecule may represent an acquired feature that contributes to a malignant phenotype. This can explain why luminal epithelial cells of the normal mammary gland and benign mammary lesions are Cav-1 negative, while a significant number of cases of malignant lesions, neoplastic emboli and metastatic lesions are Cav-1 positive. To the best of the authors’ knowledge this represents the first report of Cav-1 expression in canine mammary tissue. The study has documented absence of Cav-1 expression by normal luminal cells and its consistent expression by myoepithelial cells of the normal canine mammary gland. There is a clear association between increased expression of Cav-1 and neoplastic transformation of the epithelium and the data suggest that Cav-1 expression is associated with a more malignant phenotype of canine mammary neoplasia and with vascular invasion and regional lymph node metastasis. Additional studies comprising a larger group of animals are warranted in order to confirm these findings and to further investigate the usefulness of Cav-1 immunoreactivity as a prognostic factor in canine mammary tumours.
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March 14th, 2009 ½ Received, Accepted, December 29th, 2009