MicroRNA expression patterns in canine mammary cancer show significant differences between metastatic and non-metastatic tumours.

BACKGROUND: MicroRNAs may act as oncogenes or tumour suppressor genes, which make these small molecules potential diagnostic/prognostic factors and targets for anticancer therapies. Several common oncogenic microRNAs have been found for canine mammary cancer and human breast cancer. On account of this, large-scale profiling of microRNA expression in canine mammary cancer seems to be important for both dogs and humans. METHODS: Expression profiles of 317 microRNAs in 146 canine mammary tumours of different histological type, malignancy grade and clinical history (presence/absence of metastases) and in 25 control samples were evaluated. The profiling was performed using microarrays. Significance Analysis of Microarrays test was applied in the analysis of microarray data (both unsupervised and supervised data analyses were performed). Validation of the obtained results was performed using real-time qPCR. Subsequently, predicted targets for the microRNAs were searched for in miRBase. RESULTS: Results of the unsupervised analysis indicate that the primary factor separating the samples is the metastasis status. Predicted targets for microRNAs differentially expressed in the metastatic vs. non-metastatic group are mostly engaged in cell cycle regulation, cell differentiation and DNA-damage repair. On the other hand, the supervised analysis reveals clusters of differentially expressed microRNAs unique for the tumour type, malignancy grade and metastasis factor. CONCLUSIONS: The most significant difference in microRNA expression was observed between the metastatic and non-metastatic group, which suggests a more important role of microRNAs in the metastasis process than in the malignant transformation. Moreover, the differentially expressed microRNAs constitute potential metastasis markers. However, validation of cfa-miR-144, cfa-miR-32 and cfa-miR-374a levels in blood samples did not follow changes observed in the non-metastatic and metastatic tumours.

In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma.

Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.

Extensive expression of craniofacial related homeobox genes in canine mammary sarcomas.

The global gene expression in three types of canine mammary tumors: carcinoma, fibrosarcoma and osteosarcoma were investigated by Affymetrix gene array technology. Unsupervised clustering analysis revealed a close clustering of the respective tumor types, with fibrosarcomas clustering close to the osteosarcomas and the carcinomas clustering closer to non-malignant mammary tissues (NMTs). A number of epithelial markers were expressed in both carcinomas and NMTs, whereas the sarcomas expressed genes related to mesenchymal differentiation. A comparison of the gene expression profile of the sarcomas versus carcinoma/NMTs revealed that the sarcomas, in particular the osteosarcomas, showed a striking upregulation of a panel of homeobox genes previously linked to craniofacial bone formation. In line with this finding, osteosarcomas showed an upregulation of bone morphogenetic proteins (BMPs) and of genes associated with retinoic acid signaling. Increased homeobox gene expression in sarcomas was also confirmed at the protein level by immunohistochemical analysis of tumor tissue, and in an osteosarcoma cell line after stimulation by BMP-2. These findings suggest that the development of mammary sarcomas specifically involves triggering of a set of homeobox genes related to neural crest and craniofacial bone development.

Evaluation of Angiogenesis Process after Metformin and LY294002 Treatment in Mammary Tumor.

BACKGROUND: The angiogenesis process is regulated by many factors, such as Hypoxia-Inducible Factor-1 (HIF-1) and Vascular Endothelial Growth Factor (VEGF). Metformin has demonstrated its ability to inhibit cell growth and the LY294002 is the major inhibitor of PI3K/AKT/mTOR pathway that has antiangiogenic properties. METHODS: Canine mammary tumor cell lines CMT-U229 and CF41 were treated with metformin and LY294002. Cell viability, protein and gene expression of VEGF and HIF-1 were determined in vitro. For the in vivo study, CF41 cells were inoculated in female athymic nude mice treated with either metformin or LY294002. The microvessel density by immunohistochemistry for CD31 as well as the gene and protein expression of HIF-1 and VEGF were evaluated. RESULTS: The treatment with metformin and LY294002 was able to reduce the cellular viability after 24 hours. The protein and gene expression of HIF-1 and VEGF decreased after treatment with metformin and LY294002. In the in vivo study, there was a decrease in tumor size, protein and gene expression of HIF-1 and VEGFA, in addition to the decreasing of CD31 expression after all treatments. CONCLUSION: Our results demonstrate the effectiveness of metformin and LY294002 in controlling the angiogenesis process in mammary tumors by VEGF and HIF-1, the most important angiogenic markers.

Comparison of B-mode and Doppler ultrasonographic findings with histologic features of benign and malignant mammary tumors in dogs.

OBJECTIVE: To compare and correlate B-mode and color Doppler ultrasonographic characteristics with the histologic findings of benign and malignant mammary tumors in dogs. STUDY POPULATION: 49 mammary tumors in 26 dogs. PROCEDURES: Before excision, tumors were evaluated via B-mode and color Doppler ultrasonography to assess size, echogenicity, echopattern, acoustic transmission, invasiveness, and vascularity. Paraffin-embedded microsections of the tumors were stained with H&E and examined for presence of necrosis, cysts, cartilage, bone, mineralization, invasion of surrounding tissue, and tissue heterogeneity. To assess vascularity, the number and distribution of vessels that were stained by the Verhoeff van Gieson technique were recorded. RESULTS: Tumor echogenicity and echopattern on ultrasonographic images correlated with tissue heterogeneity detected histologically. Acoustic enhancement was correlated with the presence of necrotic or cystic areas. Tumor invasion into surrounding tissues as determined ultrasonographically did not correlate with the histologic findings. There was a significant correlation between the number of detected vessels and distribution of flow within the tumors determined via ultrasonographic and histologic examinations. CONCLUSIONS AND CLINICAL RELEVANCE: In canine mammary tumors, ultrasonographic characteristics appear to be correlated with histopathologic changes. Data suggest that ultrasonography may have an important role in the evaluation of mammary tumors in dogs, particularly in the evaluation of tissue composition and tumor vascularity.

Unclassified sarcomas: a study to improve classification in a cohort of Golden Retriever dogs.

Morphologically, canine soft-tissue sarcomas (STSs) resemble human STSs. In humans, proper classification of STSs is considered essential to improve insight in the biology of these tumors, and to optimize diagnosis and therapy. To date, there is a paucity of data published on the significance of detailed classification of STSs in the dog. We revised a cohort (n = 110) of proliferative lesions obtained from a study in Golden Retrievers that were considered "soft tissue sarcoma, not otherwise specified or of uncertain subtype" in order to optimize the diagnoses of these lesions. The criteria according to the veterinary WHO classification, recent veterinary literature, and the WHO classification for humans were applied. Revision was initially based on morphologic characteristics of hematoxylin and eosin-stained histologic sections of the neoplasms. If considered necessary (n = 76), additional immunohistochemistry was applied to aid characterization. The diagnosis of STS was confirmed in 75 neoplasms (68%). Of this group, diagnosis of a specific subtype of the STSs was possible in 58 neoplasms. Seven neoplasms had morphologic characteristics that were suggestive for sarcoma subtypes only described in the WHO classification for humans. Seventeen neoplasms remained "unclassified STSs." Thirty-one lesions (28%) were diagnosed "neoplasm, not being STS." Four lesions (4%) were considered nonneoplastic. Because incorrect classification of a tumor could lead to inappropriate therapeutic intervention and prognostication, the results of our study clearly illustrate the importance of revision and further diagnosis of "unclassified STSs" in dogs.

Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines.

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44(+)/CD24(low/-) marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44(+)/CD24(low/-) positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.

MDSCs mediate angiogenesis and predispose canine mammary tumor cells for metastasis via IL-28/IL-28RA (IFN-λ) signaling.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients. METHODS: Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway. RESULTS: In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration. CONCLUSIONS: We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.

The antiprogestins mifepristone and onapristone reduce cell proliferation in the canine mammary carcinoma cell line CMT-U27.

Canine mammary tumours (CMTs) represent nearly half of all tumours in female dogs and some 50% have malignant behaviour. Simple epithelial carcinomas have shorter disease free periods after surgery and a higher reduction of the proliferation index reduction after antiprogestin aglepristone treatment in vivo related to the expression of progesterone receptors (PR). These findings make simple carcinomas good candidates for endocrine therapy. To further explore this possibility, the effects of the antiprogestins mifepristone (RU486) and onapristone (ZK299) on cell viability and PR expression of the canine mammary carcinoma cell line isolated from a simple epithelial carcinoma CMT-U27 were studied. Twenty five percent of CMT-U27 control cells expressed PR. RU486 (p<0.05) and ZK299 (p<0.05) reduced the number of viable cells (WST-8 test) at 24h but only the latter treatment reduced significantly PR expression in viable tumour cells at 24h of incubation. The results suggest that both RU486 and ZK299 induce a decrease in the number of viable CMT-U27 tumour cells with different effects on PR expression. The canine mammary carcinoma cell line CMT-U27 is sensitive to the effects of antiprogestins and may serve to further explore the role of these drugs in canine mammary carcinomas.

Ligand-independent canonical Wnt activity in canine mammary tumor cell lines associated with aberrant LEF1 expression.

Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, ß-catenin, GSK3ß, CK1α and Axin1) and have a functional ß-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand-independent mechanisms.

Macrophages mediate a switch between canonical and non-canonical Wnt pathways in canine mammary tumors.

OBJECTIVE: According to the current hypothesis, tumor-associated macrophages (TAMs) are "corrupted" by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell-TAM interactions are complicated and controversial we aimed to better define this phenomenon. METHODS AND RESULTS: Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial-mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs. CONCLUSIONS: We demonstrated that TAMs mediate a "switch" between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis. Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being "corrupted" by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis. These data challenge the conventional understanding of TAM-cancer cell interactions.

Canine mammary tumors contain cancer stem-like cells and form spheroids with an embryonic stem cell signature.

We have investigated the presence of tentative stem-like cells in the canine mammary tumor cell line CMT-U229. This cell line is established from an atypical benign mixed mammary tumor, which has the property of forming duct-like structures in collagen gels. Stem cells in mammary glands are located in the epithelium; therefore we thought that the CMT-U229 cell line would be suitable for detection of tentative cancer stem-like cells. Side population (SP) analyses by flow cytometry were performed with cells that formed spheroids and with cells that did not. Flow cytometric, single sorted cells were expanded and re-cultured as spheroids. The spheroids were paraffin embedded and characterized by immunohistochemistry. SP analyses showed that spheroid forming cells (retenate) as well as single cells (filtrate) contained SP cells. Sca1 positive cells were single cell sorted and thereafter the SP population increased with repeated SP analyses. The SP cells were positively labeled with the cell surface-markers CD44 and CD49f (integrin alpha6); however the expression of CD24 was low or negative. The spheroids expressed the transcription factor and stem cell marker Sox2, as well as Oct4. Interestingly, only peripheral cells of the spheroids and single cells were positive for Oct4 expression. SP cells are suggested to correspond to stem cells and in this study, we have enriched for tentative tumor stem-like cells derived from a canine mammary tumor. All the used markers indicate that the studied CMT-U229 cell line contains SP cells, which in particular have cancer stem-like cell characteristics.

Diverse bone morphogenetic protein expression profiles and smad pathway activation in different phenotypes of experimental canine mammary tumors.

BACKGROUND: BMPs are currently receiving attention for their role in tumorigenesis and tumor progression. Currently, most BMP expression studies are performed on carcinomas, and not much is known about the situation in sarcomas. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the BMP expression profiles and Smad activation in clones from different spontaneous canine mammary tumors. Spindle cell tumor and osteosarcoma clones expressed high levels of BMPs, in particular BMP-2, -4 and -6. Clones from a scirrhous carcinoma expressed much lower BMP levels. The various clones formed different tumor types in nude mice but only clones that expressed high levels of BMP-6 gave bone formation. Phosphorylated Smad-1/5, located in the nucleus, was detected in tumors derived from clones expressing high levels of BMPs, indicating an active BMP signaling pathway and BMP-2 stimulation of mammary tumor cell clones in vitro resulted in activation of the Smad-1/5 pathway. In contrast BMP-2 stimulation did not induce phosphorylation of the non-Smad pathway p38 MAPK. Interestingly, an increased level of the BMP-antagonist chordin-like 1 was detected after BMP stimulation of non-bone forming clones. CONCLUSIONS/SIGNIFICANCE: We conclude that the specific BMP expression repertoire differs substantially between different types of mammary tumors and that BMP-6 expression most probably has a biological role in bone formation of canine mammary tumors.

Duplication of FGF3, FGF4, FGF19 and ORAOV1 causes hair ridge and predisposition to dermoid sinus in Ridgeback dogs.

The dorsal hair ridge in Rhodesian and Thai Ridgeback dogs is caused by a dominant mutation that also predisposes to the congenital developmental disorder dermoid sinus. Here we show that the causative mutation is a 133-kb duplication involving three fibroblast growth factor (FGF) genes. FGFs play a crucial role in development, suggesting that the ridge and dermoid sinus are caused by dysregulation of one or more of the three FGF genes during development.