Epithelial-mesenchymal transition inhibition by metformin reduces melanoma lung metastasis in a murine model.

Melanoma is an aggressive cancer with fast metastatic spread and reduced survival time. One common event during the neoplastic progression is the epithelial-mesenchymal transition (EMT), which enhances invasiveness, cell migration, and metastasis. In this study, we investigated the effects of metformin at EMT in melanoma cell lines B16-F10 and A-375, in vitro, and the impact of EMT downregulation on melanoma progression in vivo. The metformin cells treatment reduces the migration potential in vitro and reduced the development of pulmonary metastases and the expressions of N-cadherin, vimentin, ZEB1, and ZEB2 at the metastases site, in vivo. These results indicate that metformin can promote EMT downregulation impairing the metastatic potential of melanoma cells.

COX-2 expression in mammary invasive micropapillary carcinoma is associated with prognostic factors and acts as a potential therapeutic target in comparative oncology.

Pure human and canine mammary invasive micropapillary carcinoma is a rare malignant epithelial tumor accounting for 0.9 to 2% of all invasive mammary carcinomas and present a high rate of lymphatic invasion and metastasis, with unfavorable prognosis. Surgery and chemotherapy are standard treatments for almost all mammary cancer in both species, as well as hormonal and target therapies available for human patients. However, depending on the patient's clinical staging, satisfactory therapeutic results for invasive micropapillary carcinoma are a challenge due to its high capacity of invasion and metastasis. Cyclooxygenase-2 (COX-2) isoform is an important enzyme stimulated by cytokines, growth factors and oncogenes activation to synthetizes prostaglandins in inflammatory process. COX-2 overexpression is associated with angiogenesis and invasion and contributes to cancer development, disease progression, tumor recurrence and regional lymph node metastasis in human and canine mammary carcinomas. This enzyme can be targeted by non-steroidal anti-inflammatory drugs and its inhibition can reduce tumor growth and metastasis in several cancer types. Given the similarity between both species, the present study aims to elucidate the involvement of COX-2 mRNA and protein expression in canine (cIMPC) and human (hIMPC) pure invasive mammary micropapillary carcinoma, with clinicopathological and survival data. Twenty-nine cases of cIMPC and 17 cases of hIMPC were analyzed regarding histologic type, grade, age, tumor size, lymph node condition, extracapsular extension, inflammatory infiltrate and immunophenotype. When available, information on adjuvant treatment, recurrence, metastasis and overall survival were collected. The present study demonstrated COX-2 protein expression in 65.5% of cIMPC and 92.3% of hIMPC, and an association with more advanced histological grades in bitches and higher Ki67 in women. COX-2 mRNA expression was significantly higher in cIMPC than in hIMPC, and its expression was not associated with COX-2 protein expression in both species. COX-2 mRNA expression was associated with negative-ER hIMPC as well as higher Ki67. cIMPC demonstrated proportional early development, more regional metastasis, and a prevalence of negative estrogen receptor, than hIMPC. This is the first time COX-2 expression is associated with negative prognostic factors in both cIMPC and hIMPC, besides the overexpression of COX-2 protein in such unfavorable histological type, which suggests that COX-2 can act as a potential target in IMPC.

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.

Versican and Tumor-Associated Macrophages Promotes Tumor Progression and Metastasis in Canine and Murine Models of Breast Carcinoma.

Versican and tumor-associated macrophages (TAMs) are involved in growth and metastases in several cancers. Here, we investigated the potential role of versican, a matrix proteoglycan, and its correlation with TAMs infiltrates in different stages of two different breast cancer models: spontaneous canine mammary gland carcinomas and the murine 4T1 breast cancer model. The stromal versican expression was correlated with TAMs accumulation in tumors with an advanced stage from spontaneous canine mammary carcinoma samples. Versican expression in mice, identified in late stages of tumor progression, was associated to a high number of peri-tumoral infiltrating TAMs. Indeed, TAMs were related to a pro-inflammatory and pro-angiogenic state in the primary tumor. Furthermore, TAMs accumulation was related to versican expression in the lungs and an increased number of pulmonary metastatic nodules with pulmonary mechanical dysfunction, which was due to leukocyte influx in the airways and elevated growth factor levels in the microenvironment. Thus, we suggest that versican and TAMs as attractive targets for breast cancer therapy.

Quantification of EGFR family in canine mammary ductal carcinomas in situ: implications on the histological graduation.

The epithelial growth factor receptors are transmembrane proteins with an important role in the neoplastic progression of tumors, and in this context, DCIS is an important phase in the progression of canine mammary tumors. Studies on the molecular profile and its relationship to a progression of canine mammary tumors are important to improve the treatment of patients and for a better understanding of canine mammary carcinogenesis. The aim of this study was to determine, by immunohistochemistry, the relation between the expression of EGFR, ErbB-2, ErbB-3, and ErbB-4 in 52 canine mammary gland DCIS with high and low histological grade. A positive correlation between histological grade and expression of membrane ErbB-2 and cytoplasmic ErbB-4 was observed. Increased ErbB-4 membrane expression was correlated with increased ErbB-3 expression in low and high-grade DCIS. Our data suggest that increased expression of ErbB-2 and ErbB-4 may be related to more aggressive DCIS and probabily involved with canine mammary neoplastic progression.

Consumption of Diet Containing Free Amino Acids Exacerbates Colitis in Mice.

Dietary proteins can influence the maturation of the immune system, particularly the gut-associated lymphoid tissue, when consumed from weaning to adulthood. Moreover, replacement of dietary proteins by amino acids at weaning has been shown to impair the generation of regulatory T cells in the gut as well as immune activities such as protective response to infection, induction of oral and nasal tolerance as well as allergic responses. Polymeric and elemental diets are used in the clinical practice, but the specific role of intact proteins and free amino acids during the intestinal inflammation are not known. It is plausible that these two dietary nitrogen sources would yield distinct immunological outcomes since proteins are recognized by the immune system as antigens and amino acids do not bind to antigen-recognition receptors but instead to intracellular receptors such as mammalian target of rapamycin (mTOR). In this study, our aim was to evaluate the effects of consumption of an amino acid-containing diet (AA diet) versus a control protein-containing diet in adult mice at steady state and during colitis development. We showed that consumption of a AA diet by adult mature mice lead to various immunological changes including decrease in the production of serum IgG as well as increase in the levels of IL-6, IL-17A, TGF-ß, and IL-10 in the small and large intestines. It also led to changes in the intestinal morphology, to increase in intestinal permeability, in the number of total and activated CD4+ T cells in the small intestine as well as in the frequency of proliferating cells in the colon. Moreover, consumption of AA diet during and prior to development of dextran sodium sulfate-induced colitis exacerbated gut inflammation. Administration of rapamycin during AA diet consumption prevented colitis exacerbation suggesting that mTOR activation was involved in the effects triggered by the AA diet. Therefore, our study suggests that different outcomes can result from the use of diets containing either intact proteins or free amino acids such as elemental, semielemental, and polymeric diets during intestinal inflammation. These results may contribute to the design of nutritional therapeutic intervention for inflammatory bowel diseases.

Qa-2 expression levels is related with tumor-infiltrating lymphocytes profile during solid Ehrlich tumor development.

The Qa-2 has been described as Human Leucocyte Antigen G (HLA-G) murine homolog. This homology is well accepted to gene and protein structure, in different pathology process and embryos implantation. However, in some neoplasm, this homology is questioned, where Qa-2 has been proposed as an immunogenic molecule, associated to tumor rejection. In this way, the aim of this study was to describe the pattern of Qa-2 expression and its relationship with the profile of tumor-infiltrating lymphocytes in solid Ehrlich tumor. The Ehrlich tumor growth was evaluated in Balb/c female mice in different tumor stages. The inflammatory infiltration features were determined by histopathology and, both lymphocyte type and tissue Qa-2 expression by immunohistochemistry. ELISA kit was used to determine soluble Qa-2 in the serum from the animals. We observed that Qa-2 in neoplastic cells increases in intermediate tumor development stages, while, serum Qa-2 increases in the late stage. Qa-2 increasing is correlated with CD3+ increase. Our results suggest that Qa-2 has a role opposite to HLA-G in Ehrlich solid carcinoma, and may be modulating the immune response by attracting the inflammatory infiltrate, especially T CD8+ Lymphocytes.

Evaluation of Antitumor Activity of Long-Circulating and pH-Sensitive Liposomes Containing Ursolic Acid in Animal Models of Breast Tumor and Gliosarcoma.

Background Ursolic acid (UA) is a triterpene found in different plant species, possessing antitumor activity, which may be a result of its antiangiogenic effect. However, UA has low water solubility, which limits its use because the bioavailability is impaired. To overcome this inconvenience, we developed long-circulating and pH-sensitive liposomes containing ursolic acid (SpHL-UA). We investigated the antiangiogenic effect of free UA and SpHL-UA in murine brain cancer and human breast tumor models by means of determination of the relative tumor volume, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and histopathological analysis. Methods The animals were treated with dimethyl sulfoxide in 0.9% (w/v) NaCl, free UA, long-circulating and pH-sensitive liposomes without drug (SpHL), or SpHL-UA. The animals were submitted to each treatment by intraperitoneal injection for 5 days. The dose of free UA or SpHL-UA was equal to 23 mg/kg. Results Tumor growth inhibition was not observed in human breast tumor-bearing animals. For murine gliosarcoma-bearing animals, a slight tumor growth inhibition was observed in the groups treated with free UA or SpHL-UA (9% and 15%, respectively). No significant change in any of the parameters evaluated by DCE-MRI for both experimental models could be observed. Nevertheless, the evaluation of the mean values of magnetic resonance parameters of human breast tumor-bearing animals showed evidence of a possible antiangiogenic effect induced by SpHL-UA. Histopathological analysis did not present significant change for any treatment. Conclusion SpHL-UA did not show antiangiogenic activity in a gliosarcoma model and seemed to induce an antiangiogenic effect in the human breast tumor model.