An epigenetic screening determines BET proteins as targets to suppress self-renewal and tumorigenicity in canine mammary cancer cells

Pedro L P Xavier,Susanne Müller,Edson R Silva,Heidge Fukumasu,Bruno H Saranholi,Pâmela A Alexandre,Yonara G Cordeiro,Pedro R L Pires

doi:10.1038/s41598-019-53915-7

Pedro L P Xavier, Susanne Müller + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-53915-7

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Abstract

Targeting self-renewal and tumorigenicity has been proposed as a potential strategy against cancer stem cells (CSCs). Epigenetic proteins are key modulators of gene expression and cancer development contributing to regulation and maintenance of self-renewal and tumorigenicity. Here, we have screened a small-molecule epigenetic inhibitor library using 3D in vitro models in order to determine potential epigenetic targets associated with self-renewal and tumorigenicity in Canine Mammary Cancer (CMC) cells. We identified inhibition of BET proteins as a promising strategy to inhibit CMC colonies and tumorspheres formation. Low doses of (+)-JQ1 were able to downregulate important genes associated to self-renewal pathways such as WNT, NOTCH, Hedgehog, PI3K/AKT/mTOR, EGF receptor and FGF receptor in CMC tumorspheres. In addition, we observed downregulation of ZEB2, a transcription factor important for the maintenance of self-renewal in canine mammary cancer cells. Furthermore, low doses of (+)-JQ1 were not cytotoxic in CMC cells cultured in 2D in vitro models but induced G2/M cell cycle arrest accompanied by upregulation of G2/M checkpoint-associated genes including BTG2 and CCNG2. Our work indicates the BET inhibition as a new strategy for canine mammary cancers by modulating the self-renewal phenotype in tumorigenic cells such as CSCs.

Highlights

Mammary cancer in humans (HMC) and canines (CMC) share similar biological patterns, including high incidence, spontaneous development, associated risk factors, response to treatment and expression of molecular targets[1,2]
An initial screening was performed in order to determine the cytotoxic potential of a small library of 27 epigenetic inhibitors in the CF41.Mg cell line, considered the most malignant canine mammary cancer cell line of our cell bank, with higher tumorigenicity and self-renewal potential compared to the other cell lines[11]
We found important G2/M checkpoint-associated genes upregulated by (+)-JQ1 including B-cell Translocation Gene 2 (BTG2), Cyclin-G2 (CCNG2) and Epidermal Growth Factor Receptor (EGFR). (+)-JQ1 increased the gene expression of Bone Morphogenetic Protein 7 (BMP7) and BCL-2-like protein 11 (BCL2L11), commonly called Bim, both associated with programmed cell death

Summary

Introduction

Mammary cancer in humans (HMC) and canines (CMC) share similar biological patterns, including high incidence, spontaneous development, associated risk factors, response to treatment and expression of molecular targets[1,2]. These features make dogs valuable models for comparative oncology and the development of new targets and therapies. Inhibitors targeting epigenetic modulators (typically referred to as writers, erasers and readers) have recently gained interest as potential and innovative therapeutic approaches in cancer therapy[14,15]. More than 50 specific inhibitors are available, covering well the Bromodomain reader domains and epigenetic writers, histone lysine and arginine methyltransferases[16,17]

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