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Page 302 Ponnusamy et al. Cancer Drug Resist 2019;2:297-312 I http://dx.doi.org/10.20517/cdr.2018.11

Currently, there is no refined method to differentiate tumor-derived CSCs from EMT-induced CSCs.
However, growing body of literatures and discovery of advanced and new selection markers direct toward
the cross talk between EMT and CSC in therapy resistance. Several in vitro, in vivo and clinical studies on
various solid tumors including breast carcinoma emphasized the co-existence of EMT and CSC markers
indicating the relationship between the activation of EMT leading to stemness [13,24,26] . Nevertheless, the order
of occurrence between EMT and CSC in acquired chemoresistance is still debatable, however, accumulating
evidences support that the epigenetic machinery regulating the EMT-CSC co-ordination [6-8,43] . Current
understanding of the reversible nature of the EMT and phenotypic plasticity of CSC cells and their co-
existence in resistance induction together asserts the notion of reversible epigenetic changes.

EPIGENETIC REGULATION OF ACQUIRED CHEMORESISTANCE
Tumorigenesis involves cellular reprogramming that are known to be modulated through epigenetic
deregulation, thus, it is more conceivable that epigenetic aberrations play an important role in acquired cancer
drug resistance [9,10] . This section briefly describes the common epigenetic mechanisms and their involvement
in acquired breast cancer chemoresistance followed by epigenetic regulation of cellular reprogramming
events (EMT and CSC phenotype) associated with chemoresistance. Briefly, DNA methylation, post-
translational histone modifications and associated chromatin remodeling regulate the epigenetic signaling
and the sequential heritable gene expressions.

DNA methylation, histone modifications and chromatin remodeling
DNA methylation, the most studied epigenetic mechanism in mammals, involves the covalent addition of
methyl group to the C5 of cytosine base present exclusively in CpG sites that forms 5 methyl cytosine (5-
mC). DNA methyl transferase (DNMTs) enzymes (writers of methylation), using methyl donor S-adenosyl-
methionine (SAM), catalyze the methylation of DNA molecules . DNA methylation stably silence gene
[44]
repression through direct inhibition of TFs as well as through recruitment of other group of repressive
proteins (methylation readers). These proteins include methyl-binding proteins such as MeCPs, MBDs, zinc-
finger domain proteins and UHRF (ubiquitin-like, containing PHD and RING finger domain) proteins, that
bind methylated cytosines .
[44]

Core nucleosome proteins (histones) and their covalent post-translational modifications together dynamically
regulate chromatin structure and constitutes the part of the epigenetic regulatory machinery that dictate
gene expression [44,45] . These epigenetic modifications are combinatorial and occur at histone H3 and H4
moieties through acetylation, methylation, phosphorylation, sumoylation and ubiquitination. Histone
acetylation class of enzymes include Histone acetyl transferases (HAT1, Gcn5/PCAF, MYST, p300/CBP, and
Rtt109) and Histone deacetylases (classic HDACs and sirtuins HDACs- SIRTs). Histone methylation class of
enzymes include Histone methyl transferases (lysine and arginine HMTs- EZHs, MLLs, SETs and PRMTs)
and Histone demethylases (HDMTs and KDM1s) . Both classes of enzymes dynamically co-ordinate to
[45]
relax (hyperacetylate) and condense (hypoacetylate) the chromatin and regulate global and/or promoter-
specific gene transcription involved in tumorigenesis and chemoresistance [45,46] . In addition, these histones
[45]
modifying enzymes act upon non-histone protein targets to regulate gene expression.

HATs and HMTs catalyze the addition of acetyl and methyl group, respectively, to either lysine or arginine,
and HDACs and HDMTs catalyze the removal of acetyl or methyl groups, respectively. For histone
methylation, depending upon the amino acid moiety and their site of methylation, gene function can be
activated or silenced. Typically, acetylation of H3/H4 in their lysine residue and methylation of H3 at its
lysine 4 (H3K4me and H3K4me3) residue mark for transcriptionally active chromatin while methylation of
H3 at its Lysine 9 and 27 (H3K9me2 and H3K27me) mark for repressive chromatin. In addition, methylated
histones serve as binding site for MBD proteins that co-orchestrate the downstream gene expression.