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A Comparative Study on the In Vitro Effects of the DNA Methyltransferase Inhibitor 5-Azacytidine (5-AzaC) in Breast/Mammary Cancer of Different Mammalian Species

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An Erratum to this article was published on 26 April 2016

Abstract

Murine models are indispensible for the study of human breast cancer, but they have limitations: tumors arising spontaneously in humans must be induced in mice, and long-term follow up is limited by the short life span of rodents. In contrast, dogs and cats develop mammary tumors spontaneously and are relatively long-lived. This study examines the effects of the DNA methyltransferase (DNMT) inhibitor 5-Azacytidine (5-AzaC) on normal and tumoral mammary cell lines derived from dogs, cats and humans, as proof of concept that small companion animals are useful models of human breast cancer. Our findings show that treatment with 5-AzaC reduces in vitro tumorigenicity in all three species based on growth and invasion assays, mitochondrial activity and susceptibility to apoptosis. Interestingly, we found that the effects of 5-AzaC on gene expression varied not only between the different species but also between different tumoral cell lines within the same species, and confirmed the correlation between loss of methylation in a specific gene promotor region and increased expression of the associated gene using bisulfite sequencing. In addition, treatment with a high dose of 5-AzaC was toxic to tumoral, but not healthy, mammary cell lines from all species, indicating this drug has therapeutic potential. Importantly, we confirmed these results in primary malignant cells isolated from canine and feline adenocarcinomas. The similarities observed between the three species suggest dogs and cats can be useful models for the study of human breast cancer and the pre-clinical evaluation of novel therapeutics.

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Abbreviations

DNMT:

DNA methyltransferase

5-AzaC:

5-Azacytidine

HDAC:

Histone deacetylase

FDA:

Food and Drug administration

MCF10A:

Human normal breast epithelial cell line

MCF10CA1a:

Human malignant breast carcinoma cell line

DMEM:

Dulbecco’s modified Eagle medium

EGF:

Epidermal growth factor

ER:

Oestrogen receptor

MCF7:

Human breast adenocarcinoma cell line

FMEC:

Feline normal mammary epithelial cell line

K12-72.1:

Feline mammary adenocarcinoma cell line

CAT-MT:

Feline mammary carcinoma cell line

FBS:

Fetal bovine serum

CMEC:

Canine normal mammary epithelial cell line

REM134:

Canine mammary carcinoma cell line

CMT12:

Canine mammary carcinoma cell line

ECIS:

Electric Cell-substrate Impedance Sensing

BLMVEC:

Bovine microvessel lung endothelial cells

qRT-PCR:

Quantitative reverse-transcription PCR

DFNA5 :

Non-syndromic hearing impairment protein 5

SFRP1 :

Secreted frizzled-related protein 1

NTN4 :

Netrin 4

SYK:

Spleen tyrosine kinase

FKBP6 :

FK506 binding protein 6

LOXL4 :

Lysyl oxidase-like 4

PON1 :

Paraoxonase 1

TRIM50 :

Tripartite motif-containing 50

OSPBL3 :

Oxysterol-binding protein 3

DKK3 :

Dikkopf-related protein 3

PGP9.5 :

Ubiquitin carboxy-terminal hydrolase L1

HSPBC :

Heat shock protein 1

GAPDH :

Glyceraldehyde 3-phosphate dehydrogenase

HPRT :

Hypoxanthine guaine phosphoribosyl transferase

UBI :

Polyubiquitin

RPL30 :

Ribosomal Protein L30

YWHAZ :

14-3-3 protein zeta

ICC :

Immunocytochemistry

HRP :

Horseradish peroxidase

PBS:

Phosphate buffered saline

BSA:

Bovine serum albumin

TBS:

Tris buffered saline

PFA:

4 % paraformaldehyde

AEC :

3-amino-9-ethylcarbazole

CMADC:

Canine mammary adenocarcinoma-derived cells

FMADC:

Feline mammary adenocarcinoma-derived cells

ECM:

Extracellular matrix

NIH-3T3:

Murine fibroblast cell line

EC:

Endothelial cells

ING1:

Inhibitor of growth family member 1

PDX:

Patient-derived xenograft

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Acknowledgments

This work was supported by the Morris Animal Foundation (grant #D12MS-002). We are very grateful for the excellent technical assistance of Don Miller with the PCR and bisulfite sequencing analyses and Leen Bussche for the generation of the primary tumor cell cultures. We would like to thank Jen Olson and José Morales for sample collection and Katie Kelly for grading the tumor samples.

Author’s Contributions

RH carried out all laboratory procedures, was involved in conception and design, and manuscript writing; TC provided expertise and technical assistance with the Electric Cell-substrate Impedance (ECIS) assays; DA provided canine and feline mammary cancer cell lines and SC provided the human cell lines; DA, SC and GVdW were involved in conception and design; GVdW was involved in data analyses and manuscript writing. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14850, USA

    Rebecca M. Harman, Scott A. Coonrod & Gerlinde R. Van de Walle

  2. Department of Biological Sciences, State University of New York at Cortland, Cortland, NY, USA

    Theresa M. Curtis

  3. Royal (Dick) School of Veterinary Studies and Roslin Institute, The University of Edinburgh, Edinburgh, UK

    David J. Argyle

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  1. Rebecca M. Harman
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Correspondence to Gerlinde R. Van de Walle.

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Electronic Supplementary Material

Supplementary Figure 1

(A). Viability of canine, feline and human tumoral mammary cells lines/primary cells treated with 5 μM 5-AzaC as determined by MTT assays. Percent viable cells, compared to non-treated cells, set at 100 %, are shown. n = 3, *: P < 0.05. (B). Expression levels of the gene PGP9.5 in the feline cell line K12–72.1 treated with 5 and 10 μM 5-AzaC as determined by qRT-PCR. Fold changes from non-treated cells is shown. n = 3, *: P < 0.05. (GIF 71 kb)

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Harman, R.M., Curtis, T.M., Argyle, D.J. et al. A Comparative Study on the In Vitro Effects of the DNA Methyltransferase Inhibitor 5-Azacytidine (5-AzaC) in Breast/Mammary Cancer of Different Mammalian Species. J Mammary Gland Biol Neoplasia 21, 51–66 (2016). https://doi.org/10.1007/s10911-016-9350-y

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  • DOI: https://doi.org/10.1007/s10911-016-9350-y

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