Expression of LIN28A/B and Let-7 miRNAs in canine mammary carcinomas / Expressão de LIN28A/B e Let-7 miRNAs em carcinomas mamários caninos

LIN28 is a RNA-binding protein including two highly conserved homologous, LIN28A and LIN28B. Proto-oncogenes such as LIN28A and LIN28B are generally targeted by the let-7 miRNAs in different types of human cancers. Here, we determined the expression of LIN28A in canine mammary tumor samples and the LIN28/let-7 pathway in canine mammary cell lines. In those cell lines, we identified a functional LIN28/let-7 pathway which exhibited high expression of let-7 members and low expression of its targets, including LIN28A and LIN28B. However, the mammary carcinoma tissue samples showed a frequent expression of LIN28A being expressed mainly in the epithelial cells. No association was observed between LIN28A expression and histopathological classification and grade, TNM and survival time. Our results suggested a possible role of the LIN28A protein in the development of canine mammary carcinomas due to the high frequency observed in the tumor samples (28 of 32). The in vitro experiments suggested that the LIN28/let-7 pathway is active in the tumor cells evaluated. However, more studies are necessary to elucidate the exact role of LIN28/let-7 pathway in canine mammary carcinomas.

LIN28 é uma proteína de ligação ao RNA, com duas formas homólogas altamente conservadas, LIN28A e LIN28B. Os proto-oncogenes LIN28A e LIN28B são regulados pela família de miRNAs let-7 em diferentes tipos de cânceres em humanos. No presente trabalho, o objetivo foi determinar a expressão de LIN28A em amostras de tumor mamário de cadelas e a via LIN28/let-7 em linhagens celulares mamárias caninas. Nestas linhagens, através das técnicas de qPCR e RNAseq, foi identificado que a via LIN28/let-7 apresenta-se funcional, com alta expressão dos membros da família let-7 e baixa expressão de seus alvos, entre eles LIN28A e LIN28B. No entanto, as amostras de tecidos de carcinomas mamários caninos demonstraram expressão frequente de LIN28A, sendo observada principalmente em células epiteliais. Não foram observadas associações entre expressão de LIN28A com classificação e gradação histopatológicas, TNM e tempo de sobrevida. Nossos resultados sugerem uma possível relação da proteína LIN28A no desenvolvimento de carcinomas mamários caninos devido à alta frequência observada nas amostras tumorais (28 de 32). Os experimentos in vitro sugerem que a via LIN28/let-7 é ativa nas linhagens celulares caninas avaliadas. Entretanto, estudos funcionais ainda são necessários para elucidar a função exata da via LIN28/let-7 nos carcinomas mamários caninos.

Presença da mutação do gene BRCA1 em cadelas com tumores mamários malignos / Presence of BRCA1 gene mutation in bitches with malignant mammary tumors

Mammary tumors (MTs) in bitches are similar to breast cancers in women. Thus, they can be used as a model for human breast cancer and findings can be extrapolated for use in human medicine. BRCA1 is a tumor suppressor gene. When the gene has a mutation, it cannot repair damaged DNA, which causes genetic instability and tumorigenesis. Therefore, we aimed to study the frequency of single nucleotide polymorphisms (SNPs) in the BRCA1 gene that are associated with distinct histological types of malignant MT in bitches. The study population consisted of 91 bitches, including a control group of 6 animals with healthy mammary glands and 85 animals with MTs. All animals underwent a presurgery evaluation consisting of a questionnaire administered to the person responsible for the animal, a physical examination, collection of peripheral blood for hematological and serum biochemistry evaluations, an electrocardiogram, and a preanesthesia evaluation. In addition, distant metastasis was studied via chest radiography and abdominal ultrasound. After evaluations were complete, the animals that could undergo surgery were administered general anesthesia and underwent a mastectomy or mammary gland sample collection. Histopathological examination and molecular analysis were performed to identify mutations in the BRCA1 gene. Histopathological examinations found 10 different types of malignant tumors in 36 sick animals. Tumor samples plus samples from the 6 control animals were subjected to DNA extraction, polymerase chain reaction (PCR) analysis, and genetic sequencing. The tumor with the highest incidence (33.33%) was a complex carcinoma, followed by carcinoma in mixed tumor (13.88), tubular carcinoma (13.88) and carcinosarcoma (13.88). Molecular analysis revealed 3 different SNP points in 5 samples (4006G>A, 3619A>G, and 3761C>T). The allelic variant 4006G>A (1/36) resulted in the alteration of the amino acid valine by isoleucine (V1336 I). The mutation 3619A>G (2/36) inserted the amino acid alanine instead of threonine (T1207 A). The mutation 3761C>T (2/36) led to the alteration of the amino acid serine by phenylalanine (S1254 F), a mutation for which there are no published reports. The histological types that showed BRCA1 mutations were complex carcinoma (1/5), carcinoma in mixed tumor (1/5), papillary carcinoma (1/5) and tubular carcinoma (2/5). Software analysis identified the new SNP (nucleotide 3761) in BRCA1 and 2 point mutations in nucleotides 4006 and 3619 and responsible for genetic instability. The development of breast cancer is caused by many endogenous and exogenous factors. The results of our study show that these factors have a greater presence in female, mixed breed, uncastrated, and older dogs, confirming the data in the veterinary literature. In the present study, we found different histological types of malignant breast tumors with mutations in the BRCA1 gene, as other authors have reported. However, we also found the mutation 3761C>T, which, to the best of our knowledge, has not been reported in the literature. This shows the need for studies in veterinary medicine that assess mutations in the BRCA1 gene and the most common histological types. In conclusion, SNPs in the BRCA1 gene cause genetic instability, resulting in additional mutations that lead to the development of breast tumors. They are point mutations that affect transcription, resulting in truncated proteins. These proteins may have a loss of function, leading to carcinogenesis.(AU)

Increased circulating macrophage migration inhibitory factor levels are associated with coronary artery disease

BACKGROUND: To evaluate the macrophage migration inhibitory factor and E-selectin levels in patients with acute coronary syndrome. MATERIALS/METHODS: We examined the plasma migration inhibitory factor and E-selectin levels in 87 patients who presented with chest pain at our hospital. The patients were classified into two groups according to their cardiac status. Sixty-five patients had acute myocardial infarction, and 22 patients had non-cardiac chest pain (non-coronary disease). We designated the latter group of patients as the control group. The patients who presented with acute myocardial infarction were further divided into two subgroups: ST-elevated myocardial infarction (n = 30) and non-ST elevated myocardial infarction (n = 35). RESULTS: We found higher plasma migration inhibitory factor levels in both acute myocardial infarction subgroups than in the control group. However, the E-selectin levels were similar between the acute myocardial infarction and control patients. In addition, we did not find a significant difference in the plasma migration inhibitory factor levels between the ST elevated myocardial infarction and NST-elevated myocardial infarction subgroups. DISCUSSION: The circulating concentrations of migration inhibitory factor were significantly increased in acute myocardial infarction patients, whereas the soluble E-selectin levels were similar between acute myocardial infarction patients and control subjects. Our results suggest that migration inhibitory factor may play a role in the atherosclerotic process. .

The potential role of COX-2 in cancer stem cell-mediated canine mammary tumor initiation: an immunohistochemical study

Increasing evidence suggests that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. Additionally, it is becoming apparent that cyclooxygenase (COX) signaling is associated with canine mammary tumor development. The goals of the present study were to investigate COX-2 expression patterns and their effect on CSC-mediated tumor initiation in primary canine mammary tissues and tumorsphere models using immunohistochemistry. Patterns of COX-2, CD44, octamer-binding transcription factor (Oct)-3/4, and epidermal growth factor receptor (EGFR) expression were examined in malignant mammary tumor (MMT) samples and analyzed in terms of clinicopathological characteristics. COX-2 and Oct-3/4 expression was higher in MMTs compared to other histological samples with heterogeneous patterns. In MMTs, COX-2 expression correlated with tumor malignancy features. Significant associations between COX-2, CD44, and EGFR were observed in low-differentiated MMTs. Comparative analysis showed that the levels of COX-2, CD44, and Oct-3/4 expression varied significantly among TSs of three histological grades. Enhanced COX-2 staining was consistently observed in TSs. Similar levels of staining intensity were found for CD44 and Oct-3/4, but EGFR expression was weak. Our findings indicate the potential role of COX-2 in CSC-mediated tumor initiation, and suggest that COX-2 inhibition may help treat canine mammary tumors by targeting CSCs.

DNA flow cytometry of canine mammary tumors: comparative aspects with human breast tumors

Flow cytometric analysis of DNA content was performed on 28 samples of canine mammary tumors. Nine of them were benign and 19 were malignant. All benign tumors and 11 malignant tumors (57.9 percent) were diploid (P<0.05). Form the aneuploid tumors, five (26.3 percent) were hyperdiploid, one (5.3 percent) hypodiploid, one (5.3 percent) near triploid and one (5.3 percent) multiploid. The analysis of the expression of the markers PR and CD31 revealed a significant difference between diploid and aneuploid tumors (P<0.05). The immunoreactivity of PR was higher in diploid tumors, while the immunoreactivity of CD31 was stronger in aneuploid tumors. No difference between the markers MIB-1, c-erbB2, p53 and Cyclin D1 was observed (P>0.05). Using the flow cytometry analysis and immunohistochemistry, it was found a close relationship between aneuploidy and malignant character of neoplasias, progesterone receptor (PR) negative immunostaining and higher microvases density. No correlation between DNA content and S phase or immunoreactivity for the markers MIB-1, p53, c-erbB2 and Cyclin D1 was observed

Análise por citometria de fluxo de DNA foi realizada em 28 amostras de tumores mamários de cadela. Nove eram benignos e 19 malignos, sendo todos os benignos e 11 malignos (57,9 por cento) diplóides (P<0,05). Dos tumores aneuploides, cinco (26,3 por cento) eram hiperdiploides, um (5,3 por cento) hipodiploide, um (5,3 por cento) triploide e um (5,3 por cento) multiploide. A análise dos marcadores de expressão PR e CD31 revelaram significativa diferença entre tumores diploides e aneuploides (P<0,05). A imunorreatividade do PR foi maior em tumores diplóides e a imunoreatividade do CD 31 maior em tumores aneuploides. Para os marcadores MIB-1, c-erbB-2, p53 e Ciclina D1 não foi observada diferença significativa (P>0,05). Pela citometria de fluxo e pela imunoistoquímica verificaram-se uma relação entre aneuploidia e características malignas das neoplasias, receptor de progesterona imunoreação negativa e alta densidade de microvascular. Não foi observada correlação entre conteúdo de DNA e a fase S ou imunorreatividade para os marcadores MIB-1, c-erbB-2, p53 e Ciclina D1

c-erbB-2 expression and nuclear pleomorphism in canine mammary tumors

The objective of the present investigation was to study the expression of c-erbB-2 and MIB-1 and try to associate them with morphological features of the cell such as nuclear pleomorphism, mitotic count and histological grade in a series of 70 canine mammary gland tumors, 22 of them benign and 48 malignant. Tumors were collected at the Veterinary Hospital of UFMG (Brazil) and the Veterinary Faculty of Porto University (Portugal). c-erbB-2 expression was determined according to the guidelines provided by the manufacturer of the HercepTest system and nuclear pleomorphism, mitotic count and histological grade according the Elston and Ellis grading system. The HercepTest is the FDA-approved in vitro diagnostic test marketed by Dako. It is a semi-quantitative immunohistochemical assay used to determine overexpression of HER2 protein (human epidermal growth factor receptor) in breast cancer tissue. MIB-1 expression was also evaluated in 28 malignant tumors. Seventeen (35.4 percent) of the malignant tumors were positive for c-erbB-2 expression, which was positively associated with nuclear pleomorphism (P < 0.0001), histological grade (P = 0.0017) and mitotic count (P < 0.05). Nuclear pleomorphism also showed a positive association with MIB-1 index (P < 0.0001). These results suggest that some of the biological and morphological characteristics of the tumor are associated in canine mammary gland tumors, as also reported for human breast cancer. It was also possible to show that the immunoexpression of c-erbB-2 can be a factor in mammary carcinogenesis. This fact opens the possibility of using anti-c-erbB-2 antibodies in the treatment of canine mammary tumors.

Mutation and overexpression of p53 as a prognostic factor in canine mammary tumors

We concentrated ourselves to evaluate the prognostic significance of the p53 gene mutations, its protein expression and MIB-1 index as a proliferative marker in canine mammary tumors. In the present study, a total of 20 cases were examined, among which there were 5 malignant mixed tumors, 4 mammary gland adenocarcinomas, 1 papillary adenocarcinoma, 8 benign mixed tumors and 2 mammary gland adenomas. Positive immunostaining for p53 with PAb240 antibody was found in 2 benign (20%) and 3 malignant (30%) tumors. However, PAb421 antibody did not give positive result at all. In Western blot analysis, the p53 expression in benign and malignant tumors was detected in 4 and 3 cases, respectively. p53 mutations were found in 6 cases out of the cases with detected p53 protein expression. The MIB-1 index in benign and malignant tumors were 17.6+/-20.8% and 29.0+/-27.2%, respectively and there was no significant difference between tumor types. There was a significant correlation between p53 mutations and p53 overexpression (correlation coefficient = 0.5, p < 0.05). In Kaplan-Meier survival analysis, the p53 index was associated with significantly shortened survival time (p < 0.01). In multivariate analysis, p53 overexpression was only an independent factor for indicator of worse prognosis in canine mammary tumors (p = 0.01). These results demonstrated that p53 gene mutations and protein overexpression using the PAb240 anti-p53 antibody were useful predictors of increased malignant potential and poor prognosis in canine mammary tumors.

Mutations of p53 Tumor Suppressor Gene in Spontaneous Canine Mammary Tumors

Mutation of the p53 tumor suppressor gene has been related in the pathogenesis of numerous human and canine cancers, including breast cancers and mammary tumors. We have investigated exons 5-8 of the p53 gene for mutations in 20 spontaneous canine mammary tumors using polymerase chain reaction (PCR) with direct sequence analysis to evaluate the role of this gene in canine mammary tumorigenesis and analyzed to compare with other clinicopathological parameters including age, histology, stage, recurrence and death from tumor. Four missense (one case had two missense mutations) and one nonsense mutations were detected in 10 malignant lesions (40%), and two missense and one silent mutations were found in 10 benign mammary tumors (30%). Five of the missense mutations were located in highly conserved domains II, III, IV and V. After a follow-up period, four dogs showed a progression and three of these patients revealed death from mammary carcinoma with p53 mutation. These results demonstrated that the p53 gene mutations might be involved in the development of canine mammary tumors and contribute to the prognostic status in canine mammary carcinomas.