J Vet Sci.  2017 Jun;18(2):149-158. 10.4142/jvs.2017.18.2.149.

Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer

• Kabir FM
• DeInnocentes P
• Agarwal P
• Mill CP
• Riese DJ
• Bird RC

• KMID: 2412567
• DOI: http://doi.org/10.4142/jvs.2017.18.2.149

Abstract

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer.

Keyword

breast cancer; canine; mammary epithelial cells; estrogen/progesterone receptor; c-erbB/HER-2 receptor

MeSH Terms

Animals
Cell Line, Tumor
Disease Models, Animal
Dog Diseases/*metabolism
Dogs
ErbB Receptors/*metabolism
Estrogen Receptor alpha/*metabolism
Female
Mammary Neoplasms, Animal/*metabolism
Phenotype
Receptors, Progesterone/*metabolism
Reverse Transcriptase Polymerase Chain Reaction/veterinary
Estrogen Receptor alpha
Receptors, Progesterone
ErbB Receptors

Figure

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  Fig. 1 Canine mammary epithelial cells (CMEC) and canine mammary tumor (CMT) cells. Phase contrast images of live CMT cells in culture were taken at 50–100% confluence in culture media by using a photomicroscope. The CMT primary cell image shows a representative colony of mammary carcinoma cells surrounded by a field of mammary fibroblasts prior to high-speed cell sorting on a MoFlo flow cytometer and cell sorter (Beckman Instruments). Magnifications for CMT12 and CMT27 cells were the same as CMT primary cell cultures. Representative images are shown. Scale bars = 1,000 µm (A and B), 400 µm (E and G), 200 µm (F and H).

• 

  Fig. 2 Expression of canine estrogen receptor α and progesterone receptor genes in canine mammary tumor (CMT) Cells. Quantitative reverse transcriptase polymerase chain reaction (QrtPCR) assays were developed to amplify discreetly each canine receptor mRNA in order to assess expression levels for each receptor in total cell RNA isolated from CMT cell lines CMT9, CMT12, CMT27, CMT28, CMT47, and CMT119. Agarose gel electrophoresis of semi-quantitative rtPCR for each amplicon is also shown to validate the QrtPCR values. (A) mRNA levels of estrogen receptor α (ER1) assessed by QrtPCR assay and normalized, for recovery and assay artifact, to expression levels of GAPDH and ribosomal protein L37 transcripts (L37). Normalized values were then expressed as a relative fold-function of the expression levels observed in normal canine mammary epithelial cells (CMEC), which were set to a value of 1 in the same assay. (B) mRNA levels of progesterone receptor (PR) were assessed by QrtPCR assay and calculated as described for ER1 mRNA expression. Agarose gels showing semi-quantitative levels of ERα and PR transcripts as well as GAPDH and L37 transcripts, which served as positive controls used for normalization, are also shown as positive controls to demonstrate the consistency of expression across samples. Significant differences, at the confidence levels (p values) noted, between CMEC and CMT expression levels are noted. An asterisk indicates significance at p≤0.05 or 0.01 as noted. FC, fold-change relative to control cells (CMEC); Neg, not expressed/detected in semi-quantitative or QrtPCR assays.

• 

  Fig. 3 Expression of canine c-erbB-1, c-erbB-2 (cHER2), c-erbB-3, and c-erbB-4 receptor genes in canine mammary tumor (CMT) cells. Quantitative reverse transcriptase polymerase chain reaction (QrtPCR) assays were developed to amplify discreetly each canine receptor gene to assess mRNA expression profiles of each receptor in total CMT cell RNA of cell lines CMT9, CMT12, CMT27, CMT28, CMT47, and CMT119. Agarose gel electrophoresis of semi-quantitative rtPCR for each amplicon is also shown to validate loading and as positive controls. The mRNA levels of (A) erbB-1/EGFr, (B) erbB-2/HER2, (C) erbB-3/HER3, and (D) erbB-4/HER4 are shown. Expression profiles were assayed by QrtPCR. Normalized levels calculated and significance determined as described for ER1 mRNA expression.

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  Fig. 4 ER/PR receptor family mRNA sequence analysis. mRNA sequences of ER1 and PR obtained from individual PCR products and derived amino acid sequences were compared. (A) Coding sequences (CDS) of ER1 from CMEC and canine mammary tumor (CMT)12 were aligned with published canine (GenBank No. XM_533454) and human (GenBank No. NM_000125) ER1 mRNA sequences. Inferred amino acid (AA) sequence in bold letters indicates differences between human and canine ER1 sequences. (B) Coding sequences of PR mRNAs from CMEC, CMT9, CMT27, CMT28, CMT47 and CMT119 were aligned with published canine (GenBank No. NM_001003074) and human (GenBank No. M15716) mRNA sequences and the inferred amino acid sequence is noted.

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  Fig. 5 c-erbB 1-4 receptor family mRNA sequence analysis. DNA sequencing of rtPCR amplicons of coding sequences of (A) c-erbB-1/EGFr, (B) c-erbB-2/HER2, (C) c-erbB-3/HER3, and (D) c-erbB-4/HER4 from canine mammary epithelial cells (CMEC) and canine mammary tumor (CMT) cell lines were aligned with corresponding canine and human sequences. Inferred amino acids (AA) in bold letters indicate differences between human and canine erbB sequences. GenBank accession Nos: canine c-erbB-1 No. XM_533073; human erbB-1 No. U48722; canine c-erbB-2 No. NM_001003217; human HER2 No. NM_004448; canine c-erbB-3 No. XM_538226; human erbB-3 No. NM_001982; canine c-erbB-4 No. XM_003640190; and human erbB-4 No. L07868.

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