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.

ZEB and Snail expression indicates epithelial-mesenchymal transition in canine melanoma.

Melanoma progression is associated with the epithelial-mesenchymal transition (EMT) when tumor cells reduce E-cadherin and increase N-cadherin expression resulting in an escape from the microenvironment via loss of cellular adhesion and gain of motility. Transcription factor proteins Snail and ZEB trigger EMT by repression of epithelial markers and activation of mesenchymal properties. This study evaluated E-cadherin, N-cadherin, Snail, ZEB1 and ZEB2 expression by IHC and investigated their relationship with morphological characteristics in cutaneous and oral canine melanoma. Results from melanoma cases demonstrated E-cadherin expression in 45% (9/20) of oral and 58% (22/38) of cutaneous tumors, while N-cadherin expression was observed in 95% (18/19) of oral and 92% (34/37) of cutaneous melanoma. Cytoplasmic and nuclear N-cadherin expression was positively correlated with ZEB1 expression, while the cell membrane N-cadherin expression was positively correlated with ZEB2. In addition, an increase in nuclear N-cadherin expression was associated with reduced Snail expression in cutaneous melanoma and an increase in Snail expression in oral melanoma, indicating that the correlation between N-cadherin and Snail expression is coincident with tumor location. Our data suggest that ZEB family protein is associated with N-cadherin translocation from cell membrane to the cytoplasm and nuclei, and may act as important transcription factors of EMT regulation in canine melanoma.

Diverse roles of epidermal growth factors receptors in oral and cutaneous canine melanomas.

BACKGROUND: The epidermal growth factor receptors participate in the physiological processes such as regulation of morphogenesis, proliferation and cell migration, but when overexpressed or overactivated they may play an important role in neoplastic progression. Melanoma is the most aggressive skin neoplasm and is characterized by elevated invasion and low survival rates in both humans and dogs. In human melanomas the overexpression of EGFR, HER3 or HER4 is associated with poor prognosis. In canine melanomas the epidermal growth factor receptors expression has not been evaluated. Therefore, this study evaluated the expression of epidermal growth factor receptors by immunohistochemistry and investigated their relationship with morphological characteristics and proliferative indices in cutaneous and oral canine melanoma. RESULTS: In cutaneous melanoma an increased proliferative index was associated with increased cytoplasmic HER4 and reduced EGFR and HER3 protein expression. In oral melanomas, membranous HER2 protein expression correlated with occurrence of emboli, but ERBB2 gene amplification wasn't observed. CONCLUSION: Thus, our work evidenced the relationship between HER4 and the stimulus to cell proliferation in cutaneous melanomas, in addition to the relationship between HER2 and the occurrence of emboli in oral melanomas.

Frontline Science: Abnormalities in the gut mucosa of non-obese diabetic mice precede the onset of type 1 diabetes.

Alterations in the composition of the intestinal microbiota have been associated with development of type 1 diabetes (T1D), but little is known about changes in intestinal homeostasis that contribute to disease pathogenesis. Here, we analyzed oral tolerance induction, components of the intestinal barrier, fecal microbiota, and immune cell phenotypes in non-obese diabetic (NOD) mice during disease progression compared to non-obese diabetes resistant (NOR) mice. NOD mice failed to develop oral tolerance and had defective protective/regulatory mechanisms in the intestinal mucosa, including decreased numbers of goblet cells, diminished mucus production, and lower levels of total and bacteria-bound secretory IgA, as well as an altered IEL profile. These disturbances correlated with bacteria translocation to the pancreatic lymph node possibly contributing to T1D onset. The composition of the fecal microbiota was altered in pre-diabetic NOD mice, and cross-fostering of NOD mice by NOR mothers corrected their defect in mucus production, indicating a role for NOD microbiota in gut barrier dysfunction. NOD mice had a reduction of CD103+ dendritic cells (DCs) in the MLNs, together with an increase of effector Th17 cells and ILC3, as well as a decrease of Th2 cells, ILC2, and Treg cells in the small intestine. Importantly, most of these gut alterations precede the onset of insulitis. Disorders in the intestinal mucosa of NOD mice can potentially interfere with the development of T1D due the close relationship between the gut and the pancreas. Understanding these early alterations is important for the design of novel therapeutic strategies for T1D prevention.