Study On Expression Profiles Of Molecules In Wnt Signaling Pathway In Canine Mammary Tumors With Different Subtypes And Grades

Mammary tumors are common neoplastic diseases in female dogs. Previous studies showed that the activation of Wnt signaling pathway plays an important role in cancer progression, such as proliferation, invasion and metastasis of the tumor cells. However, reports about Wnt signaling pathway in canine mammary tumors (CMTs) are very few and only focus on molecules such as β-catenin and cyclin D1. In this study, we analyzed the expression profiles of Wnt signaling pathway in CMTs.One hundred and six CMT tissue samples collected from the clinical were subjected to tissue section, HE staining and evaluation for classification and grading. Based on this,35 CMT samples with five normal mammary tissues (NMTs) were selected for transcriptional analysis by RT2 Profiler PCR Array. All 106 samples were used for the analysis of the subcellular location of β-catenin and cyclin D1 by immunohistochemical staining. Also, expression of β-catenin, cyclin Dl, LEF1, TCF7 and Axin2 in 15 selected CMT samples were detected by immunoblotting. Sequencing of the 3rd exon of β-catenin in 31 CMT samples was also carried out to find if mutations are existing within this region.Histopathological examination of the 106 CMT samples revealed 36 benign tumors (including 4 subtypes) and 70 malignant tumors (including 10 subtypes), the latter of which were further classified as grades I (n=32), II (n=27) and III (n=7), plus 4 sarcomas.Transcriptional expression of Wnt and Wnt antagonists (WNT5A, WNT3, DKK1 and SFRP1) was found increasing in different grades and subtype groups of CMTs when compared to those in NMTs. Similar trends were also found for Frizzled receptors (FZD3, FZD5 and FZD7), β-catenin, transcription factors (LEF1 and TCF7) and target genes (MMP7 and cyclin D1). A significant increase of expression was detected in high malignant tumors for DKK1, SFRP1, FZD3, β-catenin and LEF1, in low malignant tumors for WNT3, WNT5A and FZD5, and in benign tumors for LEF1 and MMP7, respectively, when compared to those of the NMTs. A significant increase of expression of WNT5A, DKK1, SFRP1, FZD3, β-catenin and LEF1 were detected in subtype CMM (carcinoma and malignant myoepithelioma) compared to NMTs; furthermore, expression levels of DKK1 and SFRP1 in CMM was much higher than those in other tumor subtypes.Membrane staining of β-catenin significantly decreased in all CMT samples when compared to that of the NMTs; while cytoplasm accumulation was found in 92.5% of CMTs. For cyclin D1, its expression was much higher in 80% of CMTs when compared to NMTs, as well as in malignant tumors when compared to benign tumors. In 15 CMT samples subjected to immunoblotting analysis, elevated expression of β-catenin, LEF1, TCF7, Axin2 and cyclin D1 was also detected in 5 high malignant tumor samples when compared to NMTs. Only a single nucleotide mutation was detected in 1 of 31 CMT samples.These results, taking together, demonstrate the existence of aberrant activation of genes related to Wnt signaling pathway in CMTs. In addition, DKK.1, as one of the genes with a significant change of expression in CMTs, is a valuable biomarker candidate for the molecular diagnosis and targeted therapy of canine mammary tumors. This work also provides solid data for using CMT as a model for the study of human breast cancers.