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Ponnusamy et al. Cancer Drug Resist 2019;2:297-312 I http://dx.doi.org/10.20517/cdr.2018.11 Page 307

breast cancer cells through epigenetic aberrations. During acquired resistance development to doxorubicin,
significant morphological changes with acquisition of EMT phenotype and/or CSC-like growth properties
are reported only in intermittently treated breast cancer cells (MCF-7 and MDA-MB-231 cells) . In this
[37]
study, MCF-7 cells that are typically polarized epithelial cells with cuboidal to columnar shape acquired
elongated spindle shaped mesenchymal morphology upon intermittent exposure to doxorubicin. More
interestingly, the degree of acquired EMT phenotype was increased with treatment and correlated with
level of acquired resistance to doxorubicin. With similar treatment, MDA-MB-231 cells which are originally
mesenchymal in origin, acquired further enhanced mesenchymal phenotype. These changes accompanied
with down-regulation of epithelial markers, elevated mesenchymal promoting transcription factor FoxC2,
repressor transcription factor of E-cadherin Snail 1, and mesenchymal markers (N-cadherin, vimentin and
fibronectin).

Besides, accompanied the EMT phenotype were cells with CSC-like characteristics with formation of free-
floating colonies (tumorospheres) upon intermittent doxorubicin treatment. These features uniquely noted in
MCF-7 cells and corresponded with relatively early and higher level of resistance developed. Interestingly, in
this published study, acquisition of EMT and CSC phenotypes were observed only in intermittently exposed
cells and not in continuously exposed cells. In a continuation study , when the treatment continued for
[78]
18 months, the acquired resistant cells were enriched for CSC and metastasis markers (ALDH1A1, CD44,
Nanog, OCT4, MMP2 and VEGFA) and acquired increased tumorigenicity and resistance to therapy .
[78]
This study further tracked the transcriptional level epigenetic changes in these cells after 3 months (when
cells acquired pronounced EMT and CSC-like phenotype) and after 18 months (when 30-fold increase in
resistance observed with enrichment for CSC-markers) and found temporal increase in epigenetic markers
expression including DNMT1, DNMT3a, DNMT3b, and HMT1 . DNA demethylating agent 5-Aza-2’-
[78]
deoxycytidine significantly inhibited the tumorigenicity, tumorospheres formation and sensitized resistant
cells to doxorubicin. These recent reports along with other evidences further support the role of cellular
reprogramming through epigenetic changes involves in acquired chemoresistance.

Epigenetic therapy focused on cellular reprogramming associated with acquired chemoresistance
Reversible and dynamic nature of the epigenetic aberrations create potential opportunities in terms of
therapy. Epigenetic modifying drugs are potential therapy approach mainly because they target epigenetic
enzymes that regulate cell’s genetic programming rather than targeting cancer cells as such. Given
the fact that cellular programming initiates the transient drug tolerance that further lead to resistance
development, addressing cellular programming is crucial. Targeting epigenetic landscape of cancer stem
cells as potential therapeutic intervention have been explored for different cancer subtypes . Since
[79]
CSC cells enter quiescent state, inducing cell differentiation from their quiescent states seem promising
approach to resensitize the resistant cells to chemotherapy. This can be achieved using DNA demethylating
agents, histone deacetylase (HDAC) inhibitors, histone methyltransferase (HMT) inhibitors and histone
demethylase (HDM) inhibitors.

Currently, the epigenetic treatment approaches addressing the EMT-CSC pathway are experimental, and
clinical investigations thus far are scarce. Potential epigenetic reprogramming target to address EMT-
CSC include the Twist-Snail-E-Cadherin-ZEB axis, Wnt-TGF-β-BMI axis, and Wnt-TGF-β-SOX axis.
Accumulating evidence suggests that the effect of DNA demethylating agents and HDAC inhibitors either
alone or in combination with chemotherapeutic drugs to resensitize chemoresistance works through cellular
reprogramming [6,78,80] . Besides reactivating silenced tumor suppressor genes and inducing differentiation of
CSCs, the low dose DNA methylating agents and HDAC/ HMT/ HDM inhibitors reduce tumorigenicity and
prevent CSC invasiveness and tumor metastasis .
[79]