Loss of p27 expression in canine mammary tumors and their metastases

The p27 gene is a member of the cyclin-dependent kinase inhibitors, which arrest G1- to S-phase transition of the cell cycle. We have previously shown a significant reduction of p27 mRNA expression level in laser-microdissected mammary carcinomas and their lymph node metastases when compared to non-neoplastic mammary gland of the same dog. Here, p27 expression was analyzed on the protein level in non-neoplastic mammary gland, primary mammary carcinomas, their lymph node metastases and intravascular tumor cells of 49 dogs, adenomas of 49 dogs and non-neoplastic mammary gland of 98 dogs by immunohistochemistry. A significantly (p ? 0.05) decreased percentage of p27 positive tissue samples was found when normal gland was compared with adenomas, carcinomas and lymph node metastases. Specifically, 91% of normal gland epithelium displayed nuclear p27 expression. In contrast, only 22% of the adenomas, 20% of carcinomas, 12% of lymph node metastases and 32% of intravascular tumor cells had p27 reactivity. Cell cycle control by p27 is therefore lost in the majority of canine mammary tumors. The lack of significant differences between benign and malignant mammary tumors indicates that decreased p27 expression is an early step in carcinogenesis of canine mammary tumors and hinders the use of p27 as a marker of malignancy for this tumor type.     

Growth hormone receptor (GHR) RNAi decreases proliferation and enhances apoptosis in CMT-U27 canine mammary carcinoma cell line

Canine mammary gland has been identified as a major site of the extrapituitary growth hormone (GH) production. This finding is linked to its role in tumourigenesis of the mammary gland. Our previous studies indicated the role of GH and GH receptor (GHR) in regulation of proliferation and apoptosis. Thus, we have optimized the ghr RNA interference method in canine mammary carcinoma cell line CMT-U27. We have analysed the effect of GHR reduction on the intracellular signalling and the cell cycle and apoptosis. The results showed that GHR reduction decreased the p-ERK1/2 expression and caused increase of apoptosis and decrease in number of cells at S and G2M phases. This study indicates that GHR besides proliferative effect promotes growth by increasing cell survival. It can tilt the balance between proliferation and death in cancer cells.     

COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

Differential expression of cell cycle regulators p21, p27 and p53 in metastasizing canine mammary adenocarcinomas versus normal mammary glands

The cyclin dependent kinase inhibitors p21 and p27 are important regulators of cell cycle progression. To analyze their role in the malignant progression of canine mammary tumors expression levels of p27 and p21 and its major regulator p53 were compared in simple adenomas, adenocarcinomas of the mammary gland and lymph node metastases with normal mammary gland. Laser microdissection of tissue samples and real-time PCR were used for quantification of mRNA expression levels. p21 was overexpressed in adenocarcinomas, whereas adenomas and metastases expressed p21 more heterogeneously. Comparison of p21 expression in adenocarcinomas and their metastases revealed a significant decrease in expression in metastases. In contrast, p27 expression was reduced in the adenocarcinomas but heterogeneously expressed in adenomas and metastases. Taken together the results suggest that loss of p21 overexpression is associated with tumor metastasis while reduced cell cycle inhibition by p27 is associated with malignant progression.     

Expression and significance of PTEN in canine mammary gland tumours

To explore the expression and clinical importance of the anti-oncogene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in canine mammary gland tumours, PTEN expression was compared in 50 cases of canine mammary tumour and four examples of normal mammary tissue using real-time quantitative PCR. PTEN expression was similar in benign mammary tumours and normal mammary tissues (P>0.05), but was lower in malignant tumours than in normal mammary tissues or benign mammary tumours (P<0.001). PTEN expression was also low in the lymph node metastases of malignant mammary tumours. The expression profile of PTEN in malignant mammary tumours compared to those without lymph node metastasis varied significantly. Low-level PETN expression might play an important role in carcinogenesis and the progression of canine mammary tumours, and PTEN protein detection might be useful in evaluating tumour development and prognosis.     

Fractal dimension of canine mammary gland epithelial tumors on cytologic smears

BACKGROUND: Fractal geometry is a tool that can be used for describing, modeling, analyzing, and processing irregular and complex figures. Past investigations in medicine have revealed that fractal analysis could also be applied in tumor pathology to characterize irregular boundaries of the nuclei of tumor cells. OBJECTIVE: The aim of this study was to define whether the fractal dimension parameter could be used on cytologic specimens to differentiate benign from malignant canine mammary gland epithelial tumors. METHODS: The fractal dimension of nuclear surface was determined by computer-assisted morphometry on Hemacolor-stained cytologic smears obtained by fine needle aspiration of normal canine mammary gland epithelial cells, and cells in mammary adenomas, tubulopapillary carcinomas, solid carcinomas, and anaplastic carcinomas. Data were analyzed by Mann-Whitney U test. RESULTS: Significant differences (P <.001) were observed in mean fractal dimension among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for the fractal dimension between solid carcinomas and anaplastic carcinomas). CONCLUSION: The morphometric parameter, fractal dimension, could help in the diagnostic discrimination between benign and malignant canine mammary gland epithelial tumors on cytologic specimens.     

Cyclooxygenase-2 expression in canine mammary tumors

Mammary tumors are the most common neoplasms in female dogs. Induction of cyclooxygenase-2 (COX-2) has been implicated in various cancers in humans. However, expression of COX-2 has not been investigated in canine mammary tumors. Normal mammary gland (n = 4), simple or complex adenomas (n = 63), and simple or complex adenocarcinomas (n = 84) were studied by immunohistochemistry. Results showed that COX-2 was not expressed in the normal gland but was detected in 24% of adenomas and in 56% of adenocarcinomas (P < 0.001). The incidence of COX-2 expression and the intensity of the COX-2 signal were higher in adenocarcinomas than in adenomas (P < 0.001). These results demonstrate for the first time that COX-2 is induced in a proportion of canine mammary tumors and that COX-2 expression is more frequent and more intense in malignant than in benign tumors, suggesting a potential role for COX-2 in canine mammary tumorigenesis.     

Acute phase proteins and biomarkers of oxidative status in feline spontaneous malignant mammary tumours

Acute phase proteins (APP) and biomarkers of oxidative status change in human and canine mammary tumours, however, they have not been studied in feline mammary tumours. The aims of this study were to investigate the APP and antioxidant responses in feline malignant mammary tumours, to evaluate their relation with tumour features, and to assess their prognostic value. Serum amyloid A (SAA), haptoglobin (Hp), albumin, butyrylcholinesterase (BChE), insulin‐like growth factor1 (IGF1), paraoxonase1 (PON1), total serum thiols (Thiol), glutathione peroxidase (GPox) and total antioxidant capacity determined by different assays, including trolox equivalent antioxidant capacity assessed by two different methodologies (TEAC1/2), ferric reducing ability of plasma (FRAP), and cupric reducing antioxidant capacity (CUPRAC), were determined in serum of 50 queens with spontaneous mammary carcinomas and of 12 healthy female cats. At diagnosis, diseased queens presented significantly higher SAA and Hp, and lower albumin, BChE, GPox, TEAC1, TEAC2 and CUPRAC than controls. Different tumour features influenced concentrations of APP and antioxidants. Increases in serum Hp, and decreases in albumin, Thiol and FRAP were significantly associated with neoplastic vascular emboli, metastasis in regional lymph nodes and/or in distant organs. Distant metastasis development during the course of the disease was associated with increases in SAA and TEAC1. At diagnosis, decreased albumin was associated with a longer survival, and BChE <1.15 μmoL/mL.minute was associated with a shorter survival time on multivariate analysis. Feline malignant mammary tumours are associated with an APP response and oxidative stress, and different tumour features influence the inflammatory response and the oxidative damage. Furthermore, some of these analytes proved to have prognostic value.     

Immunohistochemical evaluation of expression of heat shock proteins HSP70 and HSP90 in mammary gland neoplasms in bitches

Heat shock proteins have essential roles in a number of pathophysiologic conditions including carcinogenesis and represent a group of novel molecular markers in cancer management. The aim of this study was to investigate heat shock protein expression in correlation with other neoplasm traits such as: histological type, differentiation grade, proliferative activity, estrogenic receptor expression, and cyclooxygenase-2 and p53 proteins. Material for the investigation comprised 133 tumors of the mammary gland collected from bitches. In total 14 adenomas, 66 complex carcinomas, 47 simple carcinomas and 6 solid carcinomas were collected. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Expression of heat shock protein 70 was observed in all types of evaluated neoplasms. A higher average number of cells undergoing expression of heat shock protein 70, which was statistically insignificant, was established in complex and simple cancers and in cancers with the 1st and the 2nd degree of histological malignancy. Expression of heat shock protein 90 was observed in all studied neoplasms; it was very insignificant in adenomas, compared to cancers, and the highest expression was established in the solid cancers, as well as in cancers with the 2nd degree of histological malignancy. This high expression of heat shock protein 90 was correlated with proliferative activity. The results suggest that heat shock protein 90 is involved in canine mammary gland carcinogenesis. The results also suggest that heat shock protein 90 may be a prognostic factor, but this requires detailed clinical confirmation.     

MUC1 expression in canine malignant mammary tumours and relationship to clinicopathological features

Mucin-1 (MUC1) is over-expressed in human breast carcinomas and is linked to a poorer prognosis. In this study, MUC1 expression in 32 spontaneous canine malignant mammary tumours was characterised in relation to histological type, mode of growth, grade, lymph node metastases and distant metastases. All tumours exhibited immunostaining for MUC1. In the normal canine mammary gland, MUC1 was expressed mainly in the apical cellular membrane, while in carcinomas MUC1 was detected in the cytoplasm only (56.3%) or in the cytoplasm with membrane accentuation (43.7%). There was a significant association between development of distant metastases and MUC1 over-expression (P=0.03), but no association with histological type, histological grade, mode of growth or lymph node metastasis.     

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERalpha and p53

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor α (ERα), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERα expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.     

Aglepristone decreases proliferation in progesterone receptor-positive canine mammary carcinomas

Background: Progesterone receptor (PR) antagonist aglepristone (RU534) has been used successfully for pregnancy termination and therapy of pyometra, vaginal tumors, and mammary hyperplasia in bitches and queens. All of these conditions share with canine mammary carcinomas the expression of PR. Objectives: To study the effect of RU534 on proliferation and apoptosis in canine mammary carcinomas in relation to PR expression. Animals: Twenty‐seven nonspayed bitches with mammary carcinomas were treated with either 2 doses of 20 mg/kg RU534 (n = 22, RU534‐treated group) or oil placebo (n = 5, control group) on days 1 and 8. Methods: Tumor samples were collected before (day 1) and after (day 15) treatment for immunohistochemistry. PR expression, proliferation index (PI), and apoptotic index (AI) were determined using antibodies against PR, Ki67, and cleaved lamin A/C antigens, respectively. The effect of treatment on these parameters was analyzed. Results: Differential expression of PR between day 1 (59.1% PR‐positive tumors) and day 15 (36.4% PR‐positive tumors) was observed in RU534‐treated tumors exclusively. After RU534 treatment, mean PI was significantly decreased in PR‐positive but unchanged in PR‐negative RU534‐treated tumors. A reduction of ≥20% in PI was found in 61.5% of RU534‐treated tumors with PR expression. Conversely, no effect on AI was observed after RU534 treatment. Conclusions and Clinical Importance: Neoadjuvant RU534 treatment had PR expression‐related inhibiting effects on proliferation of canine mammary carcinoma cells.     

Polymorphism in promoter region of growth hormone receptor is associated with potential production capacity of insulin‐like growth factor‐1 in pre‐pubertal Holstein heifers

Insulin‐like growth factor‐1 (IGF‐1) is one of the important factors for growth, milk production and reproductive functions and mainly released from the liver in response to growth hormone (GH) via GH receptor (GHR) in cattle. Recently, some single nucleotide polymorphisms (SNPs) were identified in the bovine GHR gene. Some GHR‐SNPs were shown to be related to plasma IGF‐1 concentration in cattle. Hence, the capacity to IGF‐1 production in the liver might be affected by GHR‐SNP and associated with performance in the future. This study examined whether GHR‐SNP is associated with IGF‐1 production in the liver of pre‐pubertal heifers. In 71 Holstein calves, blood samples for genomic DNA extraction were obtained immediately after birth. To genotype the GHR‐SNPs in the promoter region, polymerase chain reaction (PCR) products were digested with restriction enzyme NsiI (cutting sites: AA, AG and GG). All heifers at 4 months of age were intramuscularly injected with 0.4 mg oestradiol benzoate. Blood samples were obtained from the jugular vein just before (0 h) and 24 h after injection. The number of AA, AG and GG at the NsiI site was 0, 17 and 54 respectively. In AG and GG, plasma GH concentrations were higher pre‐injection than 24 h post‐injection (p < 0.01). Moreover, plasma GH concentrations in AG post‐injection were higher than in GG (p < 0.05). In contrast, the GG genotype exhibited higher plasma IGF‐1 concentrations in pre‐injection than post‐injection (p < 0.01), although oestradiol did not change IGF‐1 concentration in the AG genotype. We conclude that the GG polymorphism in the promoter region of GHR is associated with a higher potential capacity of IGF‐1 production in the liver of cattle.     

Expression of insulin-like growth factor-1 by canine insulinomas and their metastases

The long-term prognosis after surgical resection of canine insulinoma is poor. Signs of hypoglycaemia often recur soon after surgery because tumour tissue has only been resected partially and/or functional (micro-)metastases were present. Using quantitative real-time PCR, the expression of 16 target genes was compared between primary canine insulinomas and their corresponding metastases. There was significantly higher expression of genes encoding for growth hormone (GH) and insulin-like growth factor-1 (IGF-1) in metastases compared to their primary tumours. Immunohistochemical examination of proteins of the GH-IGF-1 axis revealed expression of GH, IGF-1 and GH receptor (GHR) in both primary insulinomas and metastases. Immunohistochemical staining for IGF-1 was significantly higher in metastases compared to primary tumours.     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

Computerized morphometry of mean nuclear diameter and nuclear roundness in canine mammary gland tumors on cytologic smears

BACKGROUND: The use of computer-based image analysis systems in veterinary oncology has increased. Computerized morphometry is a part of image analysis that describes geometric figures of cellular structures in any dimension. Most investigators have performed morphometric analysis on histologic specimens. Computer-assisted nuclear cytomorphometry can provide important preoperative information on neoplastic lesions in animals. OBJECTIVES: The aim of this study was to define whether the morphometric parameters of mean nuclear diameter and nuclear roundness could be used to differentiate benign from malignant canine mammary gland tumors on cytologic specimens. METHODS: Mean nuclear diameter and nuclear roundness were determined by computer-assisted morphometry of epithelial cells in Hemacolor-stained cytologic smears from normal canine mammary gland (n = 7) and from canine mammary adenomas (n = 8), tubulopapillary carcinomas (n = 9), and solid carcinomas (n = 6). Data were analyzed by the Mann-Whitney U test. RESULTS: Significant differences (P <.001) were found in mean nuclear diameter and nuclear roundness among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for nuclear roundness between tubulopapillary carcinomas and solid carcinomas). CONCLUSIONS: The morphometric parameters of mean nuclear diameter and nuclear roundness can be used in the preoperative differentiation of benign from malignant canine mammary gland tumors.     

p62/SQSTM1 expression in canine mammary tumours: Evolutionary notes

Recent studies highlighted the role of autophagy as a cardinal regulatory system for homeostasis and cancer‐related signalling pathways. In this context, the deregulated expression of p62 – Sequestosome1 (p62/SQSTM1) – a protein acting both as an autophagy receptor and signalling hub, has been associated with tumour development and chronic inflammation. Multiple clinical studies test drugs targeting autophagy, and even more research is on the way to clinical trials. However, no comparative investigations have been carried out to identify adequate preclinical models to assess p62‐based medicine. In veterinary oncology the role of p62 in cancer‐related pathways has been largely ignored. We compared p62 sequences in multiple organisms and found that canine p62 significantly diverges from the humans and from other animals sequences. Then, we chart by immunohistochemistry the expression levels of p62 in canine mammary tumours. A total of 66 tumours and 10 non‐neoplastic mammary samples were examined. The expression of p62 was higher in normal tissue and adenomas than carcinomas, with lowest levels of p62 protein detected in high grade carcinomas. In all cases examined the tumour stroma appeared to be p62‐negative. Taken together our results would suggest that in dogs the association between p62 expression and cancer cells overturns that reported in human breast carcinoma, where p62 accumulates in malignant cells as compared to normal epithelium. Thus, at least in canine mammary tumours, p62 should be not considered a tumour‐rejection antigen for an anti‐cancer immunotherapy.     

Expression and significance of PTEN and VEGF in canine mammary gland tumours

To investigate the relationship between the expression of the PTEN (phosphatase and tensin homolog deleted on chromosometen) and VEGF (vascular endothelial growth factor) and the clinicopathological features in canine mammary gland tumours, the expression levels of PTEN and VEGF protein were assessed in 50 cases of canine mammary gland tumours tissues and 4 cases of normal mammary gland tissues with using immunohistochemical method. The over-expression rate of PTEN protein was 100% in normal and well-differentiated mammary gland tissues and 67% in breast cancer cases respectively with a significant difference between the two groups (P<0.01). Expression of PTEN was not related to age and tumour size, but closely correlated to lymph node metastasis (P<0.01). The over-expression rate of VEGF protein was 33.3% in normal mammary gland tissues, and 78% in canine mammary gland tumours with a significant difference between the two groups (P<0.01).Expression of VEGF was not related to age or tumour size, but closely correlated with lymph node metastasis and clinical stage (P<0.05).Therefore the combination detection of PTEN and VEGF could serve as an important index to estimate the biological behavior and prognosis of canine mammary gland tumours. Reduced expression of PTEN might be involved in carcinogenesis and progression of canine breast cancer by up-regulating the VEGF expression to enhance angiogenesis.