J Vet Sci.  2017 Sep;18(3):359-367. 10.4142/jvs.2017.18.3.359.

Evaluation of expression of the Wnt signaling components in canine mammary tumors via RT² Profiler PCR Array and immunochemistry assays

• Yu F
• Rasotto R
• Zhang H
• Pei S
• Zhou B
• Yang X
• Jin Y
• Zhang D
• Lin D

• KMID: 2412454
• DOI: http://doi.org/10.4142/jvs.2017.18.3.359

Abstract

The Wnt signaling pathway and its key component β-catenin have critical roles in the development of diseases such as tumors in mammals. However, little has been reported about involvement of the Wnt/β-catenin signaling pathway in canine mammary tumors (CMTs). The present study detected expression of 30 Wnt signaling pathway-related genes in CMTs; the results are potentially useful for molecular-based diagnosis of CMTs and the development of new targeted therapies. Significant upregulations of dickkopf-1 protein, secreted frizzled-related sequence protein 1 (SFRP1), frizzled 3, β-catenin, and lymphoid enhancer-binding factor 1 (LEF1) were detected in highly malignant CMTs compared to levels in normal mammary gland tissues; moreover, highly significant upregulation of WNT5A was observed in low malignancy CMTs. Downregulation was only detected for SFRP4 in malignant CMT samples. The subcellular location of β-catenin and cyclin D1 in 100 CMT samples was investigated via immunohistochemical analysis, and significantly increased expressions of β-catenin in cytoplasm and cyclin D1 in nuclei were revealed. Western blotting analysis revealed that the expression of β-catenin and LEF1 increased in in the majority of CMT samples. Taken together, the results provide important evidence of the activation status of the Wnt pathway in CMTs and valuable clues to identifying biomarkers for molecular-based diagnosis of CMT.

Keyword

RT² Profiler PCR Array; Wnt signaling pathway; beta-catenin; canine mammary tumor

MeSH Terms

Animals
Blotting, Western/veterinary
Cyclin D1/metabolism
Dog Diseases/*metabolism/pathology
Dogs
Female
Gene Expression Regulation, Neoplastic
Mammary Neoplasms, Animal/*metabolism/pathology
Polymerase Chain Reaction/veterinary
Retrospective Studies
*Wnt Signaling Pathway
Wnt-5a Protein/metabolism
beta Catenin/metabolism
Wnt-5a Protein
beta Catenin
Cyclin D1

Figure

• 

  Fig. 1 Transcriptional expression profiles of Wnt signaling-related genes in canine mammary tumors (CMTs). Total RNA extracted from CMT samples or normal mammary gland tissues were reverse transcribed into cDNA for quantitative PCR analysis with an RT2 Profiler PCR Array system. The transcriptional level of these genes are expressed as fold changes. (A and B) Wnt and Wnt antagonist genes. (C) Frizzled receptors and co-receptor. (D) β-catenin and destruction complex-related factors. (E) Transcription factors. (F) Target genes. (G) Upregulated genes in 4 groups of different CMT subtypes. Benign, benign tumor; LM, low malignant tumor; HM, high malignant tumor; CC, complex carcinoma; CMM, carcinoma and malignant myoepithelioma; EC, epithelial carcinoma (malignant tumors with generally only one component (epithelial)). Significant differences expressed as *p < 0.05 while highly significant differences are expressed as **p < 0.01. Asterisk(s) directly above the error bar of each column indicates a comparison between the indicated group and the benign group; while the Asterisk(s) above the drawn lines means the comparison between the tumor groups indicated by the lines.

• 

  Fig. 2 The expression of β-catenin and cyclin D1 in CMTs detected via immunohistochemical staining. Tissue sections were probed with either anti-β-catenin antibodies (A–H) or anti-cyclin D1 antibodies (I–L). (A) Reduced staining of both membrane and cytoplasm; weak (D), moderate (B, E and F) and strong (C and G) staining of the cytoplasm; distinct (D), partially disappeared (C and G) or mostly disappeared (B, E and F) staining of membrane; and (H) weakly staining in cytoplasm and more than 70% of membrane staining in normal tissue section. (I–K), nuclear staining of cyclin D1. Negative control staining using PBS instead of primary antibodies was performed with normal mammary tissue (M). The red arrows indicate membrane staining of β-catenin, while the red arrowhead indicates cytoplasmic staining of β-catenin. The black arrows indicate nuclear staining of cyclin D1. The subtype and grading of the CMT samples are shown below each image. Scale bar = 20 µm.

• 

  Fig. 3 Analysis of subcellular localization of β-catenin in normal mammary tissues or CMTs. The 100 CMT samples included 37 benign CMTs, 30 low malignant (LM) CMTs and 33 highly malignant (HM) CMTs, as well as 8 normal mammary tissue samples were subjected to IHS analysis. Scores of membrane-stained (A) or cytoplasm-stained (B) samples ranged from 0 (non-staining) to 3 (strongly positive stain) according to the criteria of Khramtsov et al. [11].

• 

  Fig. 4 Expression of β-catenin, TCF7, LEF1 and cyclin D1 in CMTs analyzed by immunoblotting. The samples include 1 normal canine mammary tissue (N), 2 complex adenomas (33 and 35), 2 simple adenomas (4 and 2), 1 complex carcinoma (43), 2 simple tubulo-papillary carcinomas (78, Grade I; 80, Grade II), 2 intraductal papillary carcinomas (86, Grade I; 84, Grade II) and 2 solid carcinomas (93, Grade II; 92, Grade III). Primary antibodies against β-catenin, TCF7, LEF1 and cyclin D1 were used for detection. β-actin was used as the reference protein. The malignant grades of the CMTs are marked as I, II, and III on the top of the image.

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