Involvement of Nestin in the Progression of Canine Mammary Carcinoma

Nestin, a class VI intermediate filament protein, is known to be expressed in various types of human neoplasms, including breast cancer, and is associated with their progression. However, its expression and role in canine mammary tumors remain unknown. We analyzed nestin expression in canine mammary tumors using in situ hybridization and immunohistochemistry. We also investigated its role in a canine mammary carcinoma cell line using RNA interference. Nestin expression was not observed in luminal epithelial cells of any of the 62 cases of benign mammary lesions examined, although myoepithelial cells showed its expression in most cases. In 16/50 (32%) primary mammary carcinomas and 6/15 (40%) metastases of mammary carcinomas, cytoplasmic nestin expression was detected in luminal epithelial cells. In luminal cells of primary mammary carcinomas, its expression was positively related to several pathological parameters that indicate high-grade malignancy, including histological grading ( P < .01), vascular/lymphatic invasion ( P < .01), Ki-67 index ( P < .01), and metastasis ( P < .05). Immunohistochemistry revealed that nestin expression was related to vimentin expression in mammary carcinomas ( P < .01). This relationship was confirmed using reverse transcription-quantitative polymerase chain reaction using 9 cell lines derived from canine mammary carcinoma ( P < .01). Finally, nestin knockdown in canine mammary carcinoma cells using small interfering RNA inhibited cell proliferation and migration based on WST-8, Boyden chamber, and cell-tracking assays. These findings suggest that nestin may at least partially mediate these behaviors of canine mammary carcinoma cells.

Canine Mammary Carcinoma With Vacuolated Cytoplasm: Glycogen-Rich Carcinoma, a Histological Type Distinct From Lipid-Rich Carcinoma

Lipid-rich carcinoma is a rare histotype of canine mammary tumors with cytoplasmic vacuolation. In humans, glycogen-rich carcinoma, secretory carcinoma, and myoepithelial neoplasms are included in the differential diagnosis for lipid-rich carcinoma. The aim of the study was to investigate the existence of histotypes other than lipid-rich in canine mammary carcinomas with vacuolated cytoplasm using a diagnostic algorithm based on histopathology, histochemistry, immunohistochemistry, and ultrastructure and to evaluate the molecular phenotype of these neoplasms. Ten mammary carcinomas were collected, histologically reviewed, and subjected to histochemistry (PAS, PAS with diastase, Alcian blue, Sudan III [1 case], and Congo red [1 case]); immunohistochemistry for CK19, CK5/6, CK14, p63, calponin, vimentin, ER, PR, and HER2; and transmission electron microscopy (TEM). Cytokeratin immunolabeling demonstrated the epithelial origin of all tumors. Sudan III and TEM confirmed the diagnosis of lipid-rich carcinoma in 8 tumors (one amyloid-producing). One tumor was reclassified as a glycogen-rich carcinoma based on PAS reactivity that was diastase-labile, and a second tumor was reclassified as a carcinoma-and-malignant myoepithelioma based on the differentiation markers. Lipid-rich carcinomas were basal-like (5/8), null-type (2/8), and luminal A phenotype (1/8). The glycogen-rich carcinoma was basal-like, while the carcinoma-and-malignant myoepithelioma was luminal A. Vacuolated morphology of neoplastic cells in canine mammary carcinoma can indicate either a neoplasm of luminal epithelial origin with cytoplasmic lipid or glycogen, or vacuolated neoplastic suprabasal myoepithelial cells. Glycogen-rich carcinoma is a novel histological type that should be considered in the differential diagnosis for canine mammary carcinomas with vacuolated cytoplasm.

Protease-Activated Receptor-2 Is Associated With Adverse Outcomes in Canine Mammary Carcinoma

Protease-activated receptor-2 (PAR2) is a G protein–coupled receptor that is activated by serine proteases. In humans, PAR2 is highly expressed in various cancers, including breast cancer, and is associated with cancer progression and metastasis. However, the expression and roles of PAR2 in canine mammary carcinoma remain unclear. The purpose of this study was to examine the expression of PAR2 in canine mammary carcinoma, the association between PAR2 expression and clinical characteristics, and the role of PAR2 in the metastatic phenotypes of tumor cells. Mammary carcinoma from 31 dogs and 10 normal mammary glands were included in this study, and used for immunohistochemical analysis of PAR2 expression. Normal mammary glands did not express PAR2. In contrast, mammary carcinomas showed PAR2 immunoreactivity in the cytoplasm, and its expression level varied between specimens from negative to strongly positive. The overall survival of dogs with high PAR2 expression was shorter than that of dogs with low PAR2 expression. Moreover, PAR2 expression level was associated with the presence of lymph node involvement, advanced clinical stage, and high histopathological grade. In vitro analyses revealed that a PAR2 agonist accelerated cell migration and invasion in a canine mammary carcinoma cell line. In addition, the PAR2 agonist induced epithelial-mesenchymal transition and actin polymerization. These results suggest that PAR2 expression plays a role in tumor progression and clinical outcomes in canine mammary carcinoma.