PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

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.     

Immunohistochemical analysis of cyclin A, cyclin D1 and P53 in mammary tumors, squamous cell carcinomas and basal cell tumors of dogs and cats

The involvement of cyclin A, cyclin D1 and p53 proteins in canine and feline tumorigenesis was analyzed immunohistochemically. In the present study, a total of 176 cases were examined, among which there were 108 canine cases (75 mammary lesions, 16 squamous cell carcinomas and 17 basal cell tumors) and 68 feline cases (43 mammary lesions, 20 squamous cell carcinomas and 5 basal cell tumors). Speckled nuclear staining for cyclin A was observed in 19/38 (50%) canine malignant mammary tumors and 18/37 (48.6%) feline mammary carcinomas, while this was not seen in benign mammary tumors of either dogs or cats. Marked intense nuclear cyclin A staining was seen in 7/16 (43.8%) canine squamous cell carcinomas and 18/20 (90.0%) feline squamous cell carcinomas. Only 3/17 (17.6%) canine basal cell tumors showed slight and scattered staining for cyclin A. Expression of cyclin D1 was very rare in both canine and feline tumors. Nuclear staining of p53 was found in 7/37 (18.9%) feline mammary carcinomas. Intense immunoreactivity for p53 was found in 6/16 (37.5%) canine squamous cell carcinomas and 8/20 (40%) feline squamous cell carcinomas. These results suggest that cyclin A may have a role in the proliferation of canine malignant mammary tumors, feline mammary carcinomas and squamous cell carcinomas of dogs and cats, and p53 may associate with the tumorigenesis of feline mammary carcinomas and squamous cell carcinomas of dogs and cats.     

Cellular proliferative and telomerase activity in canine mammary gland tumors

In canine mammary tumors, we examined the telomerase activity, proliferative activity by proliferative cell nuclear antigen (PCNA) immunohistochemistry, and percentage of apoptotic cells by the deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The relationship between these measures and histopathologic malignancy was also investigated. PCNA index was highest in malignant tumors (adenocarcinoma: 27.0%; malignant mixed tumor: 15.7%), followed by benign tumors (adenoma: 4.4%; benign mixed tumor: 5.3%), hyperplasia (2.1%), and normal mammary gland (0.9%). In adenoma and adenocarcinoma, papillary and solid types showing higher cellularity tended to have higher PCNA indices than did cystic and tubular types. Although the TUNEL index was <1% in all cases, the relationship between this measure and histopathologic diagnosis showed the same tendency as observed in PCNA immunostaining. Telomerase activity was detectable in all adenomas, benign mixed tumors, and adenocarcinomas examined. In contrast, all normal mammary glands, hyperplasias, and malignant mixed tumors were negative for telomerase. Relative telomerase activity (RTA) of adenocarcinoma (56.5) was significantly higher than that of adenoma (27.8) and benign mixed tumor (33.9), and a significant positive correlation (P < 0.001) was noted between RTA and PCNA index. No significant correlations were noted between either PCNA or TUNEL index and clinical features such as metastasis and tumor diameter. PCNA index and telomerase activity may be useful markers for judging malignancy of canine mammary tumors.     

Role of CXCR4 and SDF-1 in mammary tumor metastasis in the cat

It has recently been suggested that the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) promote metastasis of various cancers in humans. Since feline mammary tumors also metastasize to distant organs frequently, we used real-time quantitative PCR to examine the expression of feline CXCR4 (fCXCR4) in ten feline mammary tumor cell lines and seven feline mammary tumor tissues, and also the expression of feline SDF-1 (fSDF-1) in various organs. Cell lines derived from metastatic regions expressed more fCXCR4 than those derived from primary tumors. Mammary tumor tissues overexpressed more fCXCR4 than normal mammary tissues. Organs with high levels of fSDF-1 expression represent common sites of metastasis. Migration assays using the feline mammary tumor cell line NAC were also performed to test the activity of TN14003 and TC14012, antagonists of human CXCR4, to antagonize fCXCR4 expressed on NAC cells. TN14003 and TC14012 inhibited migration of NAC cells. We conclude that fCXCR4 may be a therapeutic target for feline mammary tumors.     

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.     

Immunohistochemical expression of Epidermal Growth Factor Receptor (EGFR) in canine mammary tissues

Epidermal Growth Factor Receptor (EGFR) has been extensively studied in human breast cancer; however, systematic studies of EGFR protein expression in canine mammary gland tumours are lacking. Therefore, we evaluated its immunohistochemical expression in a series of 136 canine mammary tumours and representative areas of adjacent normal and hyperplastic mammary tissue and investigated a possible correlation between EGFR overexpression and several clinicopathological parameters and survival. In normal and hyperplastic canine mammary glands, EGFR expression was consistently observed in myoepithelial cells, with luminal cells usually negative. In tumour tissues, EGFR overexpression was found in 9 benign (19.6%) and 38 malignant (42.2%) lesions, with EGFR positivity significantly related with malignancy. Besides animal age and tumour size, there were no significant associations between other clinicopathological parameters and EGFR overexpression. On survival analysis, tumours with EGFR overexpression showed a reduced disease-free and overall survival; however these associations failed to reach statistically significant levels.     

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.     

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.     

The expression of sialyl Lewis X in canine and feline mammary gland tumors

The expression of sialyl Lewis X (sLe(x)) in 93 canine and 15 feline mammary gland tumors (MGT) obtained by surgical resection at Veterinary Medical Center, the University of Tokyo was examined by immunohistochemistry. Their clinicopathological features and prognosis were also reviewed. Approximately 60% of MGT tissues showed sLe(x) positive expressions, while all normal mammary gland tissues were negative. However, its expression was not correlated with clinicopathological features and prognosis significantly. This study suggests that sLe(x) may be a tumor-associated antigen in canine and feline MGTs.     

p63: a novel myoepithelial cell marker in canine mammary tissues

Several immunohistochemical markers have been used to demonstrate the presence of myoepithelial cells in order to determine their role in the histogenesis of mammary tumors. p63, a recently characterized p53 homologue, is consistently expressed in myoepithelial cells of the human breast; however, no assessment of its immunoreactivity has been reported so far in canine mammary tissues. We investigated p63 immunohistochemical expression, as a novel myoepithelial cell nuclear marker, in 81 samples of normal (n = 2), hyperplastic (n = 11), and neoplastic (n = 68) canine mammary tissues. Myoepithelial phenotype was confirmed by using complementary monoclonal antibodies: alpha-smooth muscle actin, cytokeratin 14, cytokeratin AE1/AE3, and vimentin. p63 expression was observed in 91.4% (74/81) of the samples evaluated. Normal mammary glands, mammary hyperplasias, and benign tumors showed 100% immunoreactivity, with p63 expression restricted to myoepithelial cell nuclei. In general, benign mixed tumors showed a basal cell compartment immunoreactive to p63, with a gradual decrease of its expression during myoepithelial transformation. p63 expression was found in 72% of malignant tumors, allowing myoepithelial or basal cell identification in spindle-cell carcinomas (2/2), tubulopapillary carcinomas (8/9), solid carcinomas (7/10), and carcinosarcomas (1/3). The osteosarcoma analyzed was p63 negative. In our series, stromal components were consistently nonreactive to p63. In conclusion, the present study reveals p63 as a sensitive and highly specific marker of myoepithelial cells in canine mammary tissues, and the authors suggest p63 as an additional marker for defining myoepithelial histogenesis.     

miR-502在犬乳腺癌中的表达及意义

旨在研究微小miR-502（cfa-miRNA-502）在犬乳腺癌组织中的表达情况及其临床意义。采用实时定量PCR方法检测30例犬乳腺癌组织及癌旁正常组织中miR-502的表达水平,分析miR-502表达与犬乳腺癌组织临床病理特征（年龄、肿瘤大小、组织学分级和转移）的关系。结果显示：与癌旁组织相比,犬乳腺癌组织中miR-502的表达水平显著升高,差异具有统计学意义（P<0.01）;miR-502表达与犬乳腺癌的组织学分级与转移有关（P<0.05）,但与年龄和肿瘤大小无关（P>0.05）。本研究提示miR-502在犬乳腺癌组织中表达上调,其表达与犬乳腺肿瘤的发生、发展有一定关系,miR-502可能作为犬乳腺癌诊断的肿瘤标记物。     

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.     

p53 gene mutations occurring in spontaneous benign and malignant mammary tumors of the dog

Sixty-three cases of benign and malignant canine mammary tumors were analyzed to define the alteration of exons 5-8 for the p53 tumor suppressor gene using polymerase chain reaction direct sequence analysis with paraffin-embedded tissues. Four missense mutations were found in 38 benign mammary tumors (11%), and five missense (one tumor had two missense mutations) and one nonsense mutations were found in 25 mammary carcinomas (20%). These data suggest that the p53 gene alterations might be initiated at an early stage of canine mammary carcinogenesis and p53 mutations might be associated with malignancy. However, there was no evidence of any relationship between the p53 alterations and the histologic types of tumors or breeds of dogs.     

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk‐3)

Reduced expression in immortalized cells (REIC/Dkk‐3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk‐3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk‐3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk‐3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk‐3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk‐3 represents a potential molecular target for the development of therapies against feline mammary cancers.     

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.     

血小板凝血酶蛋白1对犬乳腺肿瘤细胞体外生物学特性的影响分析

本研究旨在深入探讨血小板凝血酶蛋白1(THBS1)对犬乳腺肿瘤细胞CHMp的影响,并阐明其影响犬乳腺肿瘤发生发展的作用机制。通过构建THBS1慢病毒稳定过表达和THBS1沉默表达的犬乳腺肿瘤细胞CHMp细胞系,采用CCK-8试验、划痕试验、Transwell试验、原位荧光检测和流式细胞术检测THBS1对CHMp细胞增殖、迁移、侵袭、细胞凋亡和细胞周期情况的影响;通过qRT-PCR和Western blot检测THBS1对CHMp细胞凋亡相关因子(p53、Bcl-2、Bax)表达情况的影响,验证THBS1对CHMp细胞凋亡通路的影响。结果显示,过表达THBS1能有效增强犬乳腺肿瘤细胞CHMp的增殖、迁移和侵袭能力,并且减少CHMp细胞的凋亡数量,而沉默THBS1后结果与之相反;流式细胞术得出THBS1能够影响CHMp细胞的细胞周期分布;经qRT-PCR和Western blot检测发现,在THBS1过表达后,p53和Bcl-2的表达量均明显增高,Bax的表达量明显减少,在沉默THBS1后结果与之相反。结果表明,THBS1的异常表达能够影响犬乳腺肿瘤细胞CHMp的增殖、迁移、侵袭以及细胞周期;此外,THBS1能够通过影响细胞凋亡因子p53、Bcl-2和Bax的表达来影响CHMp细胞凋亡相关通路,进而影响犬乳腺肿瘤的发生发展。     

Effect of recombinant feline interferon-ω alone and in combination with chemotherapeutic agents on putative tumour-initiating cells and daughter cells derived from canine and feline mammary tumours

Interferons (IFNs) are naturally produced cytokines with multiple important biological functions. The activity of recombinant feline IFN-ω (rFeIFN-ω) alone and in combination with chemotherapeutic drugs was tested on canine and feline mammary carcinoma cell lines (REM134 and CAT-MT) and putative tumour-initiating cells (TIC) derived from these cell lines by sphere assay. Viability was measured by chemoluminescence and a one-way analysis of variance and Student's t -tests were used for statistical analysis. rFeIFN-ω showed in vitro antitumour activity on feline and canine mammary carcinoma cells and putative TICs with a dose-dependent and target cell-specific action. Putative TICs were more resistant to the action of rFeIFN-ω compared with daughter REM134 and CAT-MT cells. REM134 cells and TICs were more sensitive to rFeIFN-ω compared with the feline counterparts. An additive effect was noticed between rFeIFN-ω and conventional anticancer drugs, in particular following co-culture of cells with anthracycline drugs. The results suggest that rFeIFN-ω warrants further investigation as a therapeutic adjunct in feline and canine mammary tumours.     

Expression patterns of the BRCA1 splicing variants in canine normal tissues and mammary gland tumors

Human BRCA1 is familial breast cancer susceptibility gene. Recently, decreased BRCA1 mRNA and protein expression has been identified in sporadic breast tumors. In the reported human BRCA1 splicing variants, delta11b lacks the majority of exon11 and is suspected to have a distinct function in normal tissues. The splicing variants display a variety of expression pattern in breast cancer samples. Although mammary gland tumor is important disease in dog, there are few reports for BRCA1 in the canine tumors. In this study, we examined the relative amounts of BRCA1 splicing variants mRNA in canine normal and mammary tumor samples by RT-PCR to investigate whether there is the altered expression of variant mRNAs in the canine tumor as reported in human. The exon11b-defecting RT-PCR products were observed in all the normal tissues examined and the nucleotide sequence was quite similar to that of human BRCA1 delta11b. In some tumor samples, we did not detect the products targeted for exon10-13 and exon14-15, while these products were observed in all the normal samples examined. Especially, the relative amounts of the exon11-defecting products were remarkably decreased in most of the tumors (11/16).