COX-2 Silencing in Canine Malignant Melanoma Inhibits Malignant Behaviour.

Metastatic melanoma is a very aggressive form of cancer in both humans and dogs. Dogs primarily develop oral melanoma of mucosal origin. Although oral melanoma in humans is rare, both diseases are highly aggressive with frequent metastases. This disease represents a "One Health" opportunity to improve molecular and mechanistic understanding of melanoma progression. Accumulating evidence suggests that cyclooxygenase-2 (COX-2) may play a critical role in the malignant behaviour of melanoma. In this study we analysed 85 histologically confirmed melanomas from canine patients and showed that COX-2 is overexpressed in both oral and cutaneous melanomas and that COX-2 expression correlates with established markers of poor prognosis. To determine the role of COX-2 in melanoma we developed two melanoma cell lines with stable integration of an inducible doxycycline-regulated expression vector containing a COX-2 targeted micro-RNA (miRNA). Using this system, we showed that cellular proliferation, migration and invasion are COX-2 dependent, establishing a direct relationship between COX-2 expression and malignant behaviour in canine melanoma. We have also developed a powerful molecular tool to aid further dissection of the mechanisms by which COX-2 regulates melanoma progression.

Characterization of neoplastic cells outlining the cystic space of invasive micropapillary carcinoma of the canine mammary gland.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) is a rare malignant breast tumor and a variant form of invasive ductal carcinoma that is an aggressive neoplasm of the human breast and canine mammary gland. The importance of the tumor microenvironment in cancer development has gradually been recognized, but little is known about the cell types outlining the cystic space of canine IMPC. This study aimed to characterize the neoplastic cells outlining the cystic space of IMPC. RESULTS: Immunohistochemistry (IHC), immunofluorescence (IF), superresolution and transmission electron microscopy (TEM) were used to assess the cell types in the cystic areas of IMPCs. Cells expressing the mesenchymal markers alpha-smooth muscle actin (αSMA), Vimentin, and S100A4 outlined the cystic space of IMPC. Furthermore, loss of epithelial cell polarity in IMPC was shown by the localization of MUC1 at the stroma-facing surface. This protein modulates lumen formation and inhibits the cell-stroma interaction. Immunohistochemical and IF staining for the myoepithelial cell marker p63 were negative in IMPC samples. Furthermore, associated with peculiar morphology, such as thin cytoplasmic extensions outlining cystic spaces, was observed under TEM. These observations suggested cells with characteristics of myoepithelial-like cells. CONCLUSIONS: The cells outlining the cystic space of IMPC in the canine mammary gland were characterized using IHC, IF and TEM. The presence of cells expressing αSMA, Vimentin, and S100A4 in the IMPC stroma suggested a role for tumor-associated fibroblasts in the IMPC microenvironment. The reversal of cell polarity revealed by the limited basal localization of MUC1 may be an important factor contributing to the invasiveness of IMPC. For the first time, the cystic space of canine mammary gland IMPC was shown to be delimited by myoepithelial-like cells that had lost p63 expression. These findings may enhance our understanding of the cellular microenvironment of invasive tumors to improve cancer diagnosis and treatment.

Cyclooxygenase-2 expression is associated with infiltration of inflammatory cells in oral and skin canine melanomas.

Melanoma is a fast-growing tumour in dogs and represents 7% of the total malignant neoplasms from the skin and is the most common tumour found in the oral cavity. In these tumours, high expression of cyclooxygenase-2 (COX-2) is associated with a poor prognosis. The aim of this study was to verify if the overexpression of COX-2 is related to the modulation of lymphocytes and if it is associated with the angiogenic and proliferative capacity of the melanoma. Canine melanoma samples (n = 85) were analysed by immunohistochemistry to detect the expression of S-100, Melan-A, PNL-2, COX-2, Factor VIII, Ki-67 and immune cells markers (CD3, CD4, FOXP3 and MAC387); and expression levels of MAC387, NOS and CD206 were determined by immunofluorescence. Our study showed a concurrent difference between the expression of COX-2 and inflammatory cell infiltration: Oral melanomas showed positivity for COX-2 in 34% of the cases and this expression was associated with CD3 positivity in the inflammatory infiltrate and angiogenesis; whereas cutaneous melanomas presented positivity for COX-2 in 42% of the cases and this expression was associated with positive staining for CD3, CD4, FOXP3 and MAC387. These markers are associated with inflammatory cells, angiogenesis and proliferation. Interestingly, melanomas were highly infiltrated by FOXP3+ cells, this is related to angiogenesis, whereas CD3, CD4 and MAC387 expression was only associated with cutaneous melanomas. The macrophage profile analysis showed that both oral and cutaneous melanomas with low COX-2 expression have an M1 phenoptype, whereas the cases with high COX-2 expression demonstrate a hybrid M1/M2 profile pattern. We concluded that the COX-2 is overexpressed in 42% of cutaneous melanomas and in 34% of oral melanomas, with a direct association with angiogenesis, proliferation, and intratumoral lymphocyte infiltration. We propose that COX-2 is a key regulator of immune cell infiltration and may drive tumour associated macrophage activation.