Development Of Canine Mammary Tumors Research Articles

Machine learning determines stemness associated with simple and basal-like canine mammary carcinomas

Simple and complex carcinomas are the most common type of malignant Canine Mammary Tumors (CMTs), with simple carcinomas exhibiting aggressive behavior and poorer prognostic. Stemness is an ability associated with cancer initiation, malignancy, and therapeutic resistance, but is still few elucidated in canine mammary tumor subtypes. Here, we first validated, using CMT samples, a previously published canine one-class logistic regression machine learning algorithm (OCLR) to predict stemness (mRNAsi) in canine cancer cells. Then, using the canine mRNAsi, we observed that simple carcinomas exhibit higher stemness than complex carcinomas and other histological subtypes. Also, we confirmed that stemness is higher and associated with basal-like CMTs and with NMF2 metagene signature, a tumor-specific DNA-repair metagene signature. Using correlation analysis, we selected the top 50 genes correlated with higher stemness, and the top 50 genes correlated with lower stemness and further performed a gene set enrichment analysis to observe the biological processes enriched for these genes. Finally, we suggested two promise stemness-associated targets in CMTs, POLA2 and APEX1, especially in simple carcinomas. Thus, our work elucidates stemness as a potential mechanism behind the aggressiveness and development of canine mammary tumors, especially in simple carcinomas, describing evidence of a promising strategy to target this disease.

LncRNA34977 promotes the proliferation, migration, and invasion and inhibits the apoptosis of canine mammary tumors by regulating the expression of miR-8881/ELAVL4

Long-stranded noncoding RNAs (lncRNAs) play different roles in various diseases. lncRNA34977 has been shown to play a relevant role the development of canine mammary tumors (CMTs). However, the mechanism of lncRNA34977 in canine mammary tumors has not been fully investigated. The aim of this study was to investigate the effects of lncRNA34977 on the proliferation, migration, invasion, and apoptosis of canine mammary tumor (CMT) cells through the regulation of miR-8881/ELAVL4 expression. The apoptosis was detected by an in situ fluorescence assay and flow cytometry. The expression levels were analyzed by RT-qPCR. CCK-8, colony formation, wound healing, and Transwell assays were used to assess the proliferation, migration, and invasion. The expression of protein was detected by western blot. The siRNA-induced silencing of lncRNA34977 promoted the apoptosis of CHMp cells, and in overexpression of lncRNA34977, the result is the opposite. LncRNA34977 has a direct targeting relationship with miR-8881 and that miR-8881 is correlated with ELAVL4. Transfection of miR-8881 mimics inhibited the proliferation, migration, invasion, and promoted the apoptosis of CHMp cells of CHMp cells. In the transfection with miR-8881 inhibitors, the result is the opposite. Co-transfected with lncRNA34977, miR-8881, or ELAVL4, we found that lncRNA34977 could regulate the expression of miR-8881 or ELAVL4. Our study shows that lncRNA34977 promotes the proliferation, migration, and invasion and suppresses the apoptosis of CMT cells by regulating the expression of miR-8881/ELAVL4.

Expression of Arginine Vasopressin Type 2 Receptor in Canine Mammary Tumours: Preliminary Results

The aims of this study were to investigate the potential association of arginine vasopressin type 2 receptor (AVPR2) in canine mammary tumours with expression of oestrogen receptors α (ORα) and β (ORβ) and clinicopathological features of the neoplasms. Twenty-six canine mammary tumour samples (11 benign, 15 malignant) were immunolabelled for AVPR2, ORα and ORβ antigens. Moderate to intense immunolabelling of AVPR2 antigen, found in all neoplasms, was not significantly associated with expression of ORα or ORβ antigens or with clinicopathological features. These findings indicate a potential role for AVPR2 in the development of canine mammary tumours and the use of AVPR2-selective vasopressin analogues as therapeutic options.

A first immunohistochemistry study of transketolase and transketolase-like 1 expression in canine hyperplastic and neoplastic mammary lesions.

BackgroundCanine mammary tumors represent the most common neoplasm in female dogs, and the discovery of cancer biomarkers and their translation to clinical relevant assays is a key requirement in the war on cancer. Since the description of the ‘Warburg effect’, the reprogramming of metabolic pathways is considered a hallmark of pathological changes in cancer cells. In this study, we investigate the expression of two cancer-related metabolic enzymes, transketolase (TKT) and transketolase-like 1 (TKTL1), involved in the pentose phosphate pathway (PPP), an alternative metabolic pathway for glucose breakdown that could promote cancer by providing the precursors and energy required for rapidly growing cells.ResultsTKT and TKTL1 protein expression was investigated by immunohistochemistry in canine normal (N = 6) and hyperplastic glands (N = 3), as well as in benign (N = 11) and malignant mammary tumors (N = 17). TKT expression was higher in hyperplastic lesions and in both benign and malignant tumors compared to the normal mammary gland, while TKTL1 levels were remarkably higher in hyperplastic lesions, simple adenomas and simple carcinomas than in the normal mammary glands (P < 0.05).ConclusionsThis study reveals that the expression of a key PPP enzyme varies along the evolution of canine mammary neoplastic lesions, and supports a role of metabolic changes in the development of canine mammary tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-017-0961-3) contains supplementary material, which is available to authorized users.

Immunohistochemical Detection of Estrogen Receptors in Canine Mammary Tumors

Abstract Mammary tumors are among the most common neoplasms in intact female dogs. They have a complex morphology, usually affecting middle age and older bitches. Almost 50% of the mammary tumors in dogs are malignant neoplasms. Prognosis is based on several factors: stage, age, tumor size, metastasis, histopathology, ovariectomy status and hormone-receptor activity. Immunohistochemical (IHC) measurement has become increasingly an important diagnostic and prognostic parameter, with the development of monoclonal antibodies against nuclear estrogen and progestin receptors. The aim of this study was to detect the presence of ER receptors in malignant canine mammary tumors and to identify their association with the clinical course of the tumor. Mammary tumor samples have been obtained by mastectomy from dogs presented at our clinic. Detailed clinical examination, CBC and basic serum biochemical profile were performed in all patients. Surgery was the only treatment. Histopathological examination and immunohistochemical detection of estrogen α receptors (ERα) was performed on 8 formalin-fixed, paraffin-embedded tissue samples, using the PT LINK immunoperoxidase technique. Histopathological examination of the mammary tumor samples (n=11) revealed tubular adenocarcinoma (n=6,54.5%) and ductal adenocarcinoma (n=3, 27.3%), one patient with benign adenoma and one with mastitis. Patients with positive ER tumors are alive, without remission, while 3 of the patients that were ER negative died due to lung metastases. According to our results, it can be concluded that the appearance and development of canine mammary tumors is highly connected with ovarian steroid hormones and that immunostaining of the tumors may be used as a good prognostic parameter in these patients.

Relationship between three novel SNPs of BRCA1 and canine mammary tumors.

The BRCA1 gene plays an important role in the development of human breast cancer, and recent research indicated that genetic variations of BRCA1 are also related to canine mammary tumors (CMTs). Here, using rapid amplification of cDNA ends (RACE), we cloned the 5′- and 3′-UTRs of BRCA1. By direct sequencing of the flanking sequences of the 5′- and 3′-UTRs of BRCA1, three previously unreported single-nucleotide polymorphisms (SNPs) were identified, two (−1228T >C, −1173C >T) in the putative promoter regions and one non-synonymous SNP (63449G >A) in exon 23. Compared with 16 normal samples, the sequences from 34 CMTs suggested that SNP (−1173C >T) was associated with the development of CMTs (odds ratio (OR)=2.57, 95% confidence interval (CI): 1.07–6.15).

Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma.

BackgroundProgesterone receptors play a key role in the development of canine mammary tumours, and recent research has focussed on their possible value as therapeutic targets using antiprogestins. Cloning and sequencing of the progesterone receptor gene has shown that the receptor has two isoforms, A and B, transcribed from a single gene. Experimental studies in human breast cancer suggest that the differential expression of progesterone receptor isoforms has implications for hormone therapy responsiveness. This study examined the effects of the antiprogestin aglepristone on cell proliferation and mRNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n = 22) or vehicle (n = 5) twice before surgery.ResultsFormalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total progesterone receptor and both isoforms by RT-qPCR and Ki67 antigen labelling. Both total progesterone receptor and isoform A mRNA expression levels decreased after treatment with aglepristone. Furthermore, a significant decrease in the proliferation index (percentage of Ki67-labelled cells) was observed in progesterone-receptor positive and isoform-A positive tumours in aglepristone-treated dogs.ConclusionsThese findings suggest that the antiproliferative effects of aglepristone in canine mammary carcinomas are mediated by progesterone receptor isoform A.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-014-0296-2) contains supplementary material, which is available to authorized users.

The ESR1 gene is associated with risk for canine mammary tumours

BackgroundThe limited within-breed genetic heterogeneity and an enrichment of disease-predisposing alleles have made the dog a very suitable model for the identification of genes associated with risk for specific diseases. Canine mammary cancer is an example of such a disease. However, the underlying inherited risk factors for canine mammary tumours (CMTs) are still largely unknown. In this study, 52 single nucleotide polymorphisms (SNPs) in ten human cancer-associated genes were genotyped in two different datasets in order to identify genes/alleles associated with the development of CMTs. The first dataset consisted of English Springer Spaniel (ESS) CMT cases and controls. ESS is a dog breed known to be at increased risk of developing CMTs. In the second dataset, dogs from breeds known to have a high frequency of CMTs were compared to dogs from breeds with a lower occurrence of these tumours.ResultsWe found significant associations to CMT for SNPs and haplotypes in the estrogen receptor 1 (ESR1) gene in the ESS material (best PBonf = 0.021). A large number of SNPs, among them several SNPs in ESR1, showed significantly different allele frequencies between the high and low risk breed groups (best PBonf = 8.8E-32, best PBPerm = 0.076).ConclusionsThe identification of CMT-associated SNPs in ESR1 in two independent datasets suggests that this gene might be involved in CMT development. These findings also support that CMT may serve as a good model for human breast cancer research.

Abstract 4651: Sorafenib and its derivatives induce apoptosis and autophagy in canine mammary tumor cells

Abstract Canine mammary tumors (CMTs) are the most common neoplasms in intact female dogs. Radiation treatment, chemotherapy and surgical removal are conventionally applied for treating CMTs; however, the efficacy is limited and a study showed around 48% of dogs died or were euthanized within one year due to recurrence or metastasis of the tumor after surgical removal. Recently, Sorafenib (Nexavar®), a multiple kinase inhibitor, has shown survival benefits in human patients with human hepatocellualr carcinoma (HCC) and is the first clinically approved drug for treating HCC. Except HCC, Sorafenib also showed certain effect on the treatment of human renal cell carcinoma, thyroid cancer, and, also, breast cancer. In this study, derivatives of sorafenib (SC-1 &amp; CY-13) made by depleting the kinase inhibitory activities were investigated for their therapeutic efficacy in canine mammary tumors. Three CMT cell lines including CMT-1, CF41.mg and MPG were treated with sorafenib, SC-1 and CY-13. The results showed SC-1 owned stronger potency than other two compounds in suppressing tumor cell growth. Lower LD50 and earlier onset of cell death were found in SC-1-treated groups. The SC-1-mediated CMT cell death was through both apoptosis and autophagy since DNA fragmentation and autophagosome were observed in treated cells. These sorafenib derivatives were previously reported as novel inhibitors for STAT3. Here we also found that SC-1 down-regulated phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested CMT cells. Expression of STAT3-driven genes, including cylcin D1 and survivin, was also repressed by SC-1. Although the in vivo anti-tumor efficacy of SC-1 will be soon evaluated, our preliminary results have provided a lead for drug development of canine mammary tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4651. doi:1538-7445.AM2012-4651

Is There a Malignant Progression Associated with a Linear Change in Protein Expression Levels from Normal Canine Mammary Gland to Metastatic Mammary Tumors?

The molecular mechanisms of the development of canine mammary tumors are still incompletely understood. In the present study we hypothesized that there is a malignant progression from normal gland to malignant carcinomas that is associated with a linear change in protein expression. To this end, the proteome of canine normal mammary gland, adenomas, nonmetastatic carcinomas, and metastatic carcinomas was compared. Application of 2D-DIGE and MALDI-TOF-MS identified 48 proteins with significant changes (fold change >|1.5|; p < 0.05) in expression levels at the different stages of malignant progression. Forty-two of these followed three major stepwise but not linear expression patterns. Thirteen proteins showed the adenoma pattern characterized by a change in protein expression levels during progression from normal gland to adenomas which persisted on the same level at the subsequent stages of malignancy. Nine proteins followed the carcinoma pattern with an up- or down-regulation between adenomas and carcinomas. The majority of 20 proteins followed the metastasis pattern with a significant change of protein expression levels between nonmetastatic and metastatic carcinomas. The present study therefore shows that differences in malignancy are associated with a stepwise but not linear change in protein expression levels, which does not finally confirm or disapprove the existence of a malignant progression in canine mammary tumors. In addition, the acquisition of metastatic potential seems to be associated with the strongest changes in protein expression levels.

Increased Presence of Stromal Myofibroblasts and Tenascin-C With Malignant Progression in Canine Mammary Tumors

The aims of this study were to determine whether the appearance of stromal myofibroblasts and the expression of tenascin-C (Tn-C) correlate with the grade of malignancy in canine mammary tumors and to determine the main cellular source of Tn-C in these tumors. Single or double immunostaining using antibodies against α-smooth muscle actin (α-SMA) and Tn-C was performed on serial sections of normal canine mammary glands as well as those with lobular hyperplasia, simple adenoma, and simple carcinoma. Thirty-nine of 42 simple carcinomas (93%) exhibited stromal α-SMA-positive myofibroblasts and Tn-C expression. Only 6 of 11 cases of simple adenoma (55%) showed these changes, whereas no changes were observed in normal mammary gland tissue or cases of lobular hyperplasia. The distribution of stromal Tn-C correlated with the presence of myofibroblasts. However, Tn-C immunoreactivity was also occasionally observed in the basement membrane zone surrounding the myoepithelial layer in normal tissue, benign lesions, and tubulopapillary carcinomas. This pattern of staining was not related to the presence of myofibroblasts. The appearance of stromal myofibroblasts and expression of Tn-C were significantly correlated with higher histological grades of malignancy and vascular/lymphatic invasion in simple carcinomas. Stromal myofibroblasts appear to be a major cellular source of Tn-C and play an important role in the development of canine mammary tumors. The Tn-C expressed in the basement membrane zone of normal, hyperplastic, and neoplastic mammary tissue, which is likely produced by neighboring myoepithelial cells, may differ functionally from the Tn-C produced by myofibroblasts.

Immunohistochemical Analysis of Bcl-2 Protein in Canine Mammary Tumours

IMMUNOHISTOCHEMICAL ANALYSIS OF BCL-2 PROTEIN IN CANINE MAMMARY TUMOURS I. Dolka*, T. Motyl y and E. Malicka* *Department of Clinical Sciences, Warsaw University of Life Sciences, Warsaw and yDepartment of Physiological Sciences, Warsaw University of Life Sciences, Warsaw, Poland Introduction: Development of canine mammary tumours involves accumulation of mutant cells caused by excessive proliferation and insufficient apoptosis. Bcl-2 is an oncoprotein and plays a role in blocking programmed cell death. Moreover, p53 gene products have been linked to apoptosis pathways. Materials and Methods: Samples of 112 canine mammary tumours were fixed in 10% neutral buffered formalin. Histopathological diagnosis was based on the WHO classification system using sections stained with haematoxylin and eosin. Expression of Bcl-2, p53 protein, Ki67 antigen, oestrogen (ERa) and progesterone (PR) receptors was evaluated by immunohistochemistry. Results: Positive expression of Bcl-2 and p53, respectively, was noted in 73% and 38% of the tumours. While p53 had nuclear localization, Bcl-2 was found in the cytoplasm of tumour cells. The Bcl-2 protein was detected in 81% of benign tumours and in 40% of grade I carcinomas. Expression of Bcl-2 was significantly correlated with ERa, but not with PR or p53. Proliferative activity showed a significant positive correlation with histological type, tumour grade and p53. Conclusions: The positive correlation between Bcl-2 and ERa suggests that oestrogen may be a regulator of Bcl-2 in canine mammary tumours. Prospective determination of Bcl-2 and p53 status may be useful in tumour grading and prognosis.

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.