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Epigenetics in breast cancer therapies - New strategies and future nanomedicine perspectives

Authors:

Abstract

Epigenetic dysregulation has been recognized as a critical factor contributing to the development of resistance against standard chemotherapy and to breast cancer progression via epithelial-to-mesenchymal transition. Although the efficacy of the first-generation epigenetic drugs (epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence showing that epigenome modulation, in synergy with other therapeutic approaches, could play an important role in cancer treatment, reversing acquired therapy resistance. However, the epigenetic therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving their stability and solubility, and minimizing off-target effects. Furthermore, the omics technologies, as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic biomarker identification, allowing for patient stratification and tailored management. In combination with new-generation epi-drugs, nanomedicine can help to overcome low therapeutic efficacy in treatment-resistant tumors. This review provides an overview of ongoing clinical trials focusing on combination therapies employing epi-drugs for breast cancer treatment and summarizes the latest nano-based targeted delivery approaches for epi-drugs. Moreover, it highlights the current limitations and obstacles associated with applying these experimental strategies in the clinics.
cancers
Review
Epigenetics in Breast Cancer Therapy—New
Strategies and Future Nanomedicine Perspectives
Verona Buocikova 1, Ivan Rios-Mondragon 2, Eleftherios Pilalis 3,4 ,
Aristotelis Chatziioannou 3,4 , Svetlana Miklikova 1, Michal Mego 5, Karlis Pajuste 6,
Martins Rucins 6, Naouale El Yamani 7, Eleonora Marta Longhin 7, Arkadij Sobolev 6,
Muriel Freixanet 8, Victor Puntes 8,9 ,10 , Aiva Plotniece 6, Maria Dusinska 7,
Mihaela Roxana Cimpan 2, Alena Gabelova 1, and Bozena Smolkova 1, *,
1Cancer Research Institute, Biomedical Research Center of the Slovak Academy of Sciences,
Dubravska Cesta 9, 845 05 Bratislava, Slovakia; verona.buocikova@savba.sk (V.B.);
svetlana.miklikova@savba.sk (S.M.); alena.gabelova@savba.sk (A.G.)
2Department of Clinical Dentistry, University of Bergen, Aarstadveien 19, 5009 Bergen, Norway;
ivan.rios-mondragon@uib.no (I.R.-M.); Mihaela.Cimpan@uib.no (M.R.C.)
3e-NIOS Applications Private Company, Alexandrou Pantou 25, 17671 Kallithea, Greece;
epilalis@e-nios.com (E.P.); achatzi@e-nios.com (A.C.)
4
Center of Systems Biology, Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece
52nd Department of Oncology, Faculty of Medicine, Comenius University and National Cancer Institute,
Klenova 1, 833 10 Bratislava, Slovakia; michal.mego@nou.sk
6Latvian Institute of Organic Synthesis, Aizkraukles str. 21, LV-1006 Riga, Latvia; kpajuste@osi.lv (K.P.);
rucins@osi.lv (M.R.); arkady@osi.lv (A.S.); aiva@osi.lv (A.P.)
7Health Eects Laboratory, NILU-Norwegian Institute for Air Research, 2007 Kjeller, Norway;
ney@nilu.no (N.E.Y.); eml@nilu.no (E.M.L.); mdu@nilu.no (M.D.)
8Vall d Hebron, Institut de Recerca (VHIR), 08035 Barcelona, Spain; muriel.freixanet@vhir.org (M.F.);
victor.puntes@vhir.org (V.P.)
9Institut Catalàde Nanosciència i Nanotecnologia (ICN2), Bellaterra, 08193 Barcelona, Spain
10 InstitucióCatalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain
*Correspondence: bozena.smolkova@savba.sk
Alena Gabelova and Bozena Smolkova share the senior authorship.
Received: 31 October 2020; Accepted: 30 November 2020; Published: 3 December 2020
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Simple Summary:
Despite advances in cancer treatment, dicult-to-treat tumor subtypes remain
a challenge. New multidisciplinary approaches can help overcome current obstacles posed by
tumor heterogeneity, activation and enrichment of cancer stem cells, and acquired drug resistance
development. Epigenome modulation, currently unsuccessful in solid tumors due to epigenetic drug
instability, toxicity, and o-target eects, might be enabled by implementing nano-based delivery
strategies aiming to improve breast cancer patient outcomes.
Abstract:
Epigenetic dysregulation has been recognized as a critical factor contributing to
the development of resistance against standard chemotherapy and to breast cancer progression via
epithelial-to-mesenchymal transition. Although the ecacy of the first-generation epigenetic drugs
(epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence
showing that epigenome modulation, in synergy with other therapeutic approaches, could play
an important role in cancer treatment, reversing acquired therapy resistance.
However, the epigenetic
therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to
medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving
their stability and solubility, and minimizing o-target eects. Furthermore, the omics technologies,
as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic
biomarker identification, allowing for patient stratification and tailored management. In combination
with new-generation epi-drugs, nanomedicine can help to overcome low therapeutic ecacy in
Cancers 2020,12, 3622; doi:10.3390/cancers12123622 www.mdpi.com/journal/cancers
Cancers 2020,12, 3622 2 of 32
treatment-resistant tumors. This review provides an overview of ongoing clinical trials focusing on
combination therapies employing epi-drugs for breast cancer treatment and summarizes the latest
nano-based targeted delivery approaches for epi-drugs. Moreover, it highlights the current limitations
and obstacles associated with applying these experimental strategies in the clinics.
Keywords: epigenetics; breast cancer; nanomedicine; epi-drugs; targeted delivery; drug resistance
1. Introduction
The most common cancer diagnosed among women is breast cancer (BC), the second leading cause of
cancer deaths [
1
]. Besides well-studied genetic changes, epigenetic alterations, resulting in aberrant gene
expression, are among the key contributors to breast carcinogenesis. Different mechanisms introduce and
maintain epigenetic modifications, including DNA methylation, post-translational histone modifications,
and non-coding RNA-mediated regulation [
2
]. Epithelial-mesenchymal transition (EMT) is a complex
developmental program, which plays a crucial role in the hematogenous and lymphatic dissemination
of tumors. EMT facilitates phenotypic metamorphosis of epithelial tumor cells into highly motile and
more aggressive mesenchymal cells that can colonize distant organs. Moreover, this multistep process
enables the generation of tumors with stem cell properties, which play a significant role in developing
therapeutic resistance [
3
]. The reversibility of EMT, allowing circulating tumor cells (CTCs) to remain
epithelial in their origin, endowing them with a potential to seed metastasis, supports the hypothesis
about its epigenetic regulation [
4
]. EMT is triggered by extracellular signals, including extracellular matrix
proteins and soluble growth factors, or by intracellular cues. It is mediated by a group of pleiotropic
transcription factors (TFs), which control a heterogeneous network of epigenetic effectors, thus allowing
potent gene expression changes [
4
]. This epigenetic plasticity not only permits dynamic regulation of
expression but also offers numerous therapeutic opportunities.
Currently, BC treatment involves a multidisciplinary approach. Although the effectiveness of various
therapeutic regimens has increased, resulting in reduced mortality [
5
], there are still many obstacles to
overcome. These include serious side effects, hard-to-treat tumor subtypes, intratumoral heterogeneity,
and at present incurable metastatic disease. The success achieved so far in treating hematological
malignancies using epigenetic inhibitors has stimulated interest in their use to treat solid tumors.
Promising preclinical results suggest that epigenetic drugs (epi-drugs) can sensitize resistant cancer cells
to traditional approaches. Unfortunately, these results have not yet been confirmed by clinical studies,
as the early-generation
epi-drugs were basically broad-spectrum reprogrammers, causing large-scale
gene expression changes. This “one size fits all” approach has mostly failed due to off-target effects,
significant toxicities, the risk of large-scale epigenomic repatterning, and the lack of appropriate biomarkers
for patient selection. However, this failure has led to the development of selective new-generation epi-drugs,
which, together with precision medicine design, provide a new chance for epigenetic therapy of solid
tumors [6,7].
Among the options that could contribute to successful clinical applications of epi-drugs are
new technologies for safer and more ecient cancer cell epigenome modulation. Advances in
nanotechnology and material science have provided a broad variety of more precise and safer
nanoscale organic and inorganic nanomaterials for drug delivery (e.g., dendrimers, micelles,
liposomes, gels, metal- and carbon-based nanomaterials). To date, several nanomaterials have
been successfully studied and introduced in cancer treatment, and many others are undergoing clinical
trials. Encapsulation by intelligent nanocarriers of antitumor drugs, conventional chemotherapeutics,
epi-drugs, or both, can improve their solubility and stability by protecting the drugs from fast clearance
and degradation, thus prolonging their half-life in the systemic circulation [
8
]. Nanocarriers can
also be tuned to ensure targeted, controlled, and sustained release, thereby reducing toxicity [
8
10
].
Nanoscale size and unique physicochemical properties (e.g., shape, surface area, and charge) allow
Cancers 2020,12, 3622 3 of 32
the accumulation of nanocarriers in the tumor mass due to the enhanced permeability and retention
(EPR) eect, which is the basis of passive targeting [
11
]. The spatial and temporal heterogeneity of
tumors is one of the limitations of therapeutic ecacy in passive targeting [
12
]. Functional surface
modifications of nanocarriers by specific ligands (antibodies, aptamers, proteins, etc.) with a high
anity for particular receptors overexpressed on the tumor cells allows active targeting ofdrug delivery
to the tumor mass, thereby increasing treatment ecacy and reducing side eects [
13
]. The biggest
challenge to combat BC is to eliminate cancer stem cells (CSCs) that play a crucial role in metastasis and
the development of multidrug resistance to therapy. Nanoscale delivery systems represent a promising
tool for their eradication [14].
In this review, we provide an outline of current achievements in epigenetic therapy of BC,
focusing on the ability of epi-drugs to sensitize resistant cancer cells to standard therapeutic approaches.
Furthermore, we highlight the promise of nanomedicine with regard to overcoming obstacles associated
with the successful use of epi-drugs for the treatment of solid tumors.
2. Molecular Pathology of Breast Cancer
The BC incidence rate varies from 27.9 per 100,000 people in Middle Africa to 92.6 per 100,000 in
Western Europe [
1
]. The dierences in incidence are attributed to dierent risk factors and the availability
of improved imaging techniques for screening and diagnosis [
15
]. The five-year relative survival rate
for women diagnosed with the regional disease was recently estimated at 86%, whereas it was 27% for
those with metastatic disease [
16
]. Therapeutic resistance and metastatic potential are influenced by
the heterogeneity of phenotypic and molecular characteristics [
17
20
]. While the luminal A subtype is
considered a low-grade disease with a good prognosis and likely to benefit from endocrine therapy alone,
luminal B tumors have a higher proliferation rate, worse prognosis, and patients require additional
chemotherapy treatment. The human epidermal receptor 2 (HER2)-overexpressing tumors tend to
grow faster and can have higher histological grade than luminal-like tumors, but generally, they are
successfully treated by targeted anti-HER2 therapies. Triple-negative/basal-like BC is a histologically
high-grade disease associated with a poor prognosis. Patients with this subtype do not benefit from
targeted therapies, and the standard chemotherapy regimen is the only suitable therapeutic approach
at present.
2.1. Role of Epigenetics in BC Pathogenesis
Approximately 10% of BCs are considered to be hereditary. Most of them are associated with
mutations in tumor suppressor genes BRCA1 and BRCA2 or other high or moderate-penetrance
genes, such as CHEK2, ATM, PALB2, PTEN, STK11, and TP53 [
21
]. In general, BC initiation,
followed by histological progression from premalignant stages to invasive carcinoma, develops from
the accumulation of genetic and epigenetic changes. These aberrations involve the inactivation of
tumor suppressor genes and/or oncogene activation, enabling the continuous malignant transformation
of cells [
22
]. The main genetic changes comprise point mutations (single nucleotide substitutions,
small insertions, and deletions), structural rearrangements, and large scale copy number changes [
23
].
The most frequently altered genes reported in early BCs are TP53,PIK3CA,MYC,PTEN, CCDN1,
ERBB2,FGFR, and GATA3 [24].
Apart from genomic alterations, cancer initiation and progression are driven by the combined
action of multiple epigenetic changes [
25
]. The term epigenetics describes heritable DNA modifications
that do not change the DNA sequence but can aect gene expression. Epigenetic regulation is involved
in many normal cellular processes, including cell growth and dierentiation [
26
]. The major epigenetic
modifications encompass changes in DNA methylation, post-translational histone modifications,
and non-coding RNA expression. These modifications are thought to participate in early BC
carcinogenesis events and can be useful as biomarkers for early detection and the determination of
prognosis and response to treatment [27].
Cancers 2020,12, 3622 4 of 32
2.1.1. DNA Methylation
DNA methylation is the chemical modification caused by covalent attachment of a methyl group at
cytosine within CpG dinucleotides, resulting in the formation of 5-methyl-cytosine (5mC). The methyl
group transfer from the S-adenosyl-L-methionine donor is catalyzed by three DNA methyltransferases
(DNMTs), DNMT1, DNMT3A, and DNMT3B. DNMT1 maintains existing methylation patterns
during replication, while DNMT3A and DNMT3B are responsible for de novo methylation [
28
].
DNA methylation occurring in the promoter regions inhibits gene expression by preventing
the binding of the transcriptional machinery to their recognition sequences or by binding proteins
with a methyl-binding domain (MBD) that have a higher anity to promoters [
29
]. MBD proteins
interlink DNA methylation and histone modifications by engaging histone deacetylase complexes
and chromatin remodeling factors, leading to chromatin condensation and thus to transcriptional
repression [30].
Aberrant DNA methylation is a hallmark of cancer. The methylation of normally unmethylated
promoter CpGs, known as hypermethylation, can lead to tumor suppressor genes’ inactivation, thus acting as
a potential biomarker for early cancer detection and prognosis [
31
].
Furthermore, DNA hypomethylation
on
a genome-wide scale, reported in many cancers, can induce genomic instability [
32
].
De Almeida et al.
identified 368 dierentially methylated individual CpG sites in BC tumors compared to healthy breast
tissues [
33
]. Hypermethylated CpG sites were mostly present in upstream promoter regions (56%),
while hypomethylated CpG sites were localized mostly in the gene bodies (66%).
Nevertheless, in many
cancers, CpG island shores, the regions of low CpG density flanking traditional CpG islands (up to 2 kb
distant) show distinct subtype-specific methylation signatures [
34
]. Lately, more than 100 genes
have been found to be hypermethylated in BC [
35
]. They play essential roles in various cell
mechanisms, including DNA repair (e.g., MGMT,BRCA1,MLH1), cell-cycle regulation (e.g., CCND2,
AK5,FOXA2), apoptosis (e.g., BCL2,APC) [
36
], cell adhesion (e.g., CDH1), tissue invasion and
metastasis (e.g., RASSF1A,RAR
β
,TWIST,HIN1), and hormone-mediated cell signaling (ESR1,ESR2,
and THRB) [37].
For a long time, DNA methylation was referred to as an irreversible epigenetic event, which could
only be passively depleted through DNA replication. However, this consideration changed with
the discovery of ten-eleven translocation (TET) proteins [
38
]. The TET family, comprising TET1, TET2,
and TET3 proteins, oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine
and accordingly facilitates active DNA demethylation [
32
]. TET proteins bind preferentially to
unmethylated CpGs within CpG islands, thus maintaining CpG islands in a hypomethylated
state, associated with active transcription [
39
]. Besides, decreased 5-hydroxymethylcytosine and
loss-of-function mutations of TET proteins can potentially aect malignant transformation [40].
2.1.2. Histone Modifications
Chromatin is a dynamic structure composed of nucleosomes. The main components of the nucleosomal
subunit are the histones. These alkaline proteins form an octamer consisting of two identical subunits,
each containing four histones: H2A, H2B, H3, and H4. The nucleosome core particle includes 147 bp
of DNA sequence, wrapped in nearly two superhelical turns around a histone octamer [
41
]. In general,
histones are highly conserved proteins that can be post-translationally modified at the amino acid
residues located on their C- and N-terminal tails [
42
]. Post-translational histone modifications (PTMs)
do not aect the DNA sequence but can influence gene expression by changing chromatin structure
from the non-condensed transcriptionally active state (euchromatin) to the condensed inactive state
(heterochromatin). Histone tail residues can undergo covalent PTMs, including methylation, acetylation,
phosphorylation, sumoylation, glycosylation, ubiquitination, ADP-ribosylation,
and carbonylation [43,44].
For instance, acetylated histone H3 and di- or tri-methylated histone H3 lysine 4 (H3K4me2, H3K4me3)
in the promoter region result in the activation of gene expression. Conversely, repression of
the promoters is usually caused by histone deacetylation and by tri-methylated histone H3 lysine 27
(H3K27me3) and tri-methylated histone H3 lysine 9 (H3K9me3) [
45
,
46
]. It is not surprising that many
Cancers 2020,12, 3622 5 of 32
aberrations in histone modifications are found in cancer due to the essential roles of these modifications
in DNA-mediated cellular processes. Furthermore, it has been shown that global loss of acetylation on
lysine K16 and tri-methylation on lysine K20 of histone H4 is a hallmark of various cancers [
47
]. In BC,
reduced levels of lysine acetylation (H3K9ac, H3K18ac, and H4K12ac), lysine methylation (H3K4me2,
H4K20me3), and arginine methylation (H4R3me2) have been observed in poor prognostic tumors.
Additionally, down-regulation of H4K16ac and the corresponding enzyme histone acetyltransferase
(HAT) hMOF, in most primary breast tumors, represent an early sign of BC [48,49].
Dierent enzymes catalyze histone PTMs, including histone deacetylases (HDACs), which remove
acetyl groups, histone methyltransferases (HMTs), demethylases (HDMs), HATs and others. At least
18 HDACs have been identified in humans and classified into four groups [
50
]. Histone acetylation
marks are read by bromodomain protein modules, which are evolutionarily conserved domains of
110 amino acids within many chromatin-associated proteins (including HATs) and other eector
enzymes [
51
]. HATs are categorized into five families: GNAT, MYST, p300/CBP, SRC, and TAFII250.
Members of the bromodomain family, bromodomain and extra-terminal domain (BET) proteins can
increase proliferation and may potentiate the overexpression of several oncogenes such as MYC [
52
].
HMTs, along with HDMs, introduce or remove methyl groups and modify other proteins in addition
to histones [53].
Molecular changes that aect the expression of histone-modifying enzymes could also
contribute to cancer development and progression. In BC, various alterations have been identified,
including overexpression of p300, HBO1, HDAC1, HDAC2, HDAC3, HDAC6 [
54
], amplification and
overexpression of enhancer of zeste homolog 2 (EZH2) [
55
], depletion of H3K9 trimethyl-demethylase
(JMJD2B) [
56
], downregulation of lysine-specific histone demethylase 1A (LSD1) [
57
], and others.
Due to more demanding methodological approaches, the role of histone modification in cancer is less
studied than DNA methylation or changes in non-coding RNA (ncRNAs).
2.1.3. Epigenetic Regulation by Non-Coding RNA
The ncRNAs are molecules of RNA that are not translated into proteins. However, they play
an important role in epigenetic modifications. RNAs with regulatory functions are divided into two
major classes based on their size: short-chain ncRNAs (including siRNAs, miRNAs, and piRNAs) less
than 200 nucleotides in length, and long ncRNAs (lncRNAs) [
58
]. The ncRNAs can directly influence
gene expression or be epigenetically regulated themselves [
59
]. Alterations in their expression
contribute to the pathogenesis of most human cancers [6062].
MicroRNAs (miRNAs) are endogenous, short, single-stranded RNAs, 19–25 nucleotides in
length, which regulate gene expression by post-transcriptional silencing or the degradation of target
mRNAs [
61
]. In general, they can be categorized into tumor-promoting (oncomiRs) and tumor-suppressing
miRNAs. During carcinogenesis, oncomiRs are usually amplified, and tumor-suppressing miRNAs are
down-regulated [
63
]. miRNAs can be readily identified in tumor samples (non-circulating miRNAs) and
different body fluids (circulating miRNAs). Deregulation of miRNAs is involved in each cancer hallmark,
such as proliferation, invasion and metastasis, apoptosis, etc. [64,65].
In BC, the expression profiles of miRNAs have been determined as promising biomarkers for
diagnosis, more precise subtyping, prognosis, and therapy response [
66
]. It was observed that luminal
A tumors have higher expression of the miR-99a/let-7c/miR-125b-2 miRNA cluster compared to luminal
B tissues [
67
]. The miR-1, miR-92a, miR-133a, and miR-133b were determined as essential diagnostic
markers by analyzing miRNA profiles between tumor and serum samples [
68
]. Furthermore, it was
shown that patients with up-regulated expression of miR-1307-3p, miR-940, and miR-340-3p had
a worse overall survival [
69
], while miR-21 and miR-205 were associated with disease-free survival
interval and miR-205 with overall survival [70]. In addition, miRNA-449a overexpression was found
to have propitious prognostic significance in BC patients [71].
Cancers 2020,12, 3622 6 of 32
2.2. Epigenetics in BC Progression
EMT is a fundamental process having a crucial role during embryogenesis and tissue regeneration.
EMT,together with the mesenchymal-epithelial transition (MET), is required to form organs and cellular
structures [
72
]. Moreover, they are involved in malignant progression, where EMT is responsible for
the loss of cellular adhesion, cytoskeleton remodeling, acquisition of cancer cells’ migratory capacity
as well as their ability to intravasate and survive in circulation [
73
]. Typical phenotypic EMT stages
range from fully dierentiated epithelial to dedierentiated mesenchymal cells. EMT is triggered
by extracellular agents such as growth factors (e.g., TGF-
β
[
74
]) and by non-growth factor stimuli
(e.g., hypoxia, oxalate, Galectin-8 [
72
]). Stromal constituents of the tumor microenvironment can
activate EMT via secretion of various cytokines and chemokines acting in a paracrine fashion on nearby
carcinoma cells. Tumor-associated stromal cells include cancer-associated fibroblasts,
regulatory T cells
,
CD4+helper T and CD8+cytotoxic T cells, tumor-associated macrophages, and myeloid-derived
suppressor cells. These cells can promote tumor progression and metastasis. Moreover, activation of
EMT can contribute to the immunosuppressive tumor microenvironment, which has been observed in
BC [
75
,
76
]. In mammary epithelial cells, the up-regulation of TWIST1 or SNAI1-induced formation of
neoplastic cells with CSC properties, which are associated with a more aggressive basal-like BC subtype.
During EMT, epithelial cells down-regulate the expression of several cell adhesion molecules,
among them E-cadherin and
β
-catenin. EMT-inducing TFs play a critical role in this process.
While several of them are well known, notably SNAI1/2, TWIST1/2, ZEB1/2, FOXC1/2, and TCF3,
others may yet be discovered [
77
]. Until recently, the down-regulation of E-cadherin has been
considered a hallmark of EMT; however, this assumption seems to be too simple in light of recent
discoveries. For instance, Padmanaban et al. showed that E-cadherin is required for invasive ductal
carcinoma cell survival and metastasis [
78
]. Although E-cadherin loss was associated with increased
invasion into local tissue, in parallel, it was connected with a reduction in cell growth and survival,
as well as a decreased amount of CTCs in the peripheral blood of patients. Notably, cell clusters
rather than individual invading cells are involved in the initiating steps of malignant progression.
E-cadherin is expressed in the cells that form these clusters [
79
]. Thus, the notion that E-cadherin
is repressed during EMT contradicts recent findings, which have helped to explain why E-cadherin
expression is often observed in the BC tumors and metastases. Although several reports describe
the occurrence of metastases formed by cells that never underwent EMT [
80
], these studies have been
criticized for insucient experimental evidence [81].
While EMT is driving the dissemination of cancer cells, MET is crucial for metastatic colonization [
82
].
MET can occur as a passive consequence of the down-regulation of EMT-TFs or their active repression [
83
].
In BC, MET can be promoted by TFs such as Prrx1 or ZEB1/2 or the expression of ID1 and ID3
genes [
84
].
In the past
few years, evidence has accumulated to demonstrate that the phenotypic plasticity
of the metastatic process is mediated via epigenetic modifications driven by these TFs. After their
binding to promoters and enhancers, they interact with epigenetic regulators and chromatin remodeling
machinery to up-regulate the expression of pro-mesenchymal genes and down-regulate epithelial
genes [
85
]. These DNA-binding proteins act as intermediaries between cellular signaling and chromatin
remodeling, thus guiding the epigenetic machinery to their target sites.
It has been demonstrated that during EMT, HMT G9a, which can also methylate non-histone
proteins, interacts with Snail to recruit it to the CDH1 promoter [
86
,
87
]. A number of these TFs
contain methyl-lysine binding motifs, crucial for correct chromatin recognition, supporting their role
in EMT
´
s epigenetic regulation. It was shown that Zeb2 inhibits E-cadherin via DNA methylation [
88
].
Hypoxia-induced EMT is regulated via HDACs, namely HDAC3, coupled with H3K4Ac, present
at promoter regions of traditional EMT genes such as CDH1 and VIM. Chromatin changes are also
responsible for TGF-
β
mediated EMT, where the new chromatin modifiers were identified, e.g., UTX,
PRMT5, Rad21, or RbBP5 [
89
]. Cells that have undergone EMT can also acquire drug resistance and
stem cell-like properties [
90
]. Traditional EMT-associated TFs were shown to confer resistance to
Cancers 2020,12, 3622 7 of 32
oxaliplatin-based and cisplatin-based chemotherapeutics, while the loss of E-cadherin was associated
with resistance to several growth factors and kinase inhibitors [91,92].
Given their mode of action, epigenetic inhibitors can restore aberrant DNA methylation
or histone deacetylation or impede the recognition of acetylated lysine residues and, therefore,
potentially re-establish epithelial phenotypes during EMT. Comprehensive bioinformatic analysis
has identified and validated various drug combinations together with HDAC inhibition that hamper
EMT [
93
,
94
]. Thus, EMT, the crucial step in cancer progression and metastasis, can be potentially
modulated by epi-drugs.
3. Breast Cancer Therapeutic Opportunities
3.1. The Biomarker-Directed Approach in BC Treatment
To overcome the limitations incurred by resistance mechanisms in the clinical management of
advanced cancers, an increasing effort is being made towards biomarker-driven cancer treatments.
This aims to identify important biomarkers capable of addressing tumor heterogeneity and effectively
predicting a favorable clinical outcome as a response to a particular treatment. BC, in particular,
is considered a family of distinct diseases with a varying molecular basis. Initial gene expression
studies using cDNA microarrays have resulted in a classification of BC into five subtypes, establishing
as major BC biomarkers estrogen receptor (ER), progesterone receptor (PR), and HER2 [
95
,
96
].
Therefore, currently, there is
a mandatory need to define their expression status, lymph node
involvement, and tumor size for all patients with invasive BC for therapy decision making.
These markers are established in international guidelines as essential factors for the clinical management
of primary BC patients [
15
]. However, the stratification of patients based solely on ER, PR, and HER2
expression has proven inecient, unable to capture the substantial phenotypic complexity and
heterogeneity of BC, thus stressing the need to integrate additional biomarkers for a more refined
characterization.
Ki-67, a non-histone
nuclear protein, is used as a marker of cell proliferation.
Ki-67 expression
is significantly higher in malignant tissues with poorly dierentiated tumor cells than
in normal tissue and is thus used to assess tumor aggressiveness [97].
Traditional therapy of non-metastatic BC involves multidisciplinary strategies combining surgery,
radiotherapy, neo-/adjuvant, endocrine, and targeted therapy [
98
]. For non-metastatic BC, the primary
therapy approach consists of eradicating tumor and regional lymph nodes and preventing metastatic
relapse. The first two goals are usually achieved by locoregional therapy that involves surgery
and radiotherapy and/or neoadjuvant therapy in case of locally advanced disease. Prevention of
metastatic relapse is achieved with systemic therapies that comprise anthracycline and taxane-based
chemotherapy, anti-estrogen hormonal therapy, and anti-HER treatment, depending on receptor
status [99].
In contrast to early BC, metastatic disease (stage IV), with common sites of spread in bones, brain,
lung, and liver, is considered incurable, and the therapy aims to prolong life while minimizing symptoms
or side eects. The combinations of endocrine, targeted therapy, chemotherapy, and immunotherapy
can be administered to the metastatic patients, taking into account the tumor subtype, extent,
and localization of the disease and the presence of specific molecular alterations. Beyond HER2 and
ER/PR, new predictive biomarkers for targeted therapy in metastatic BC include BRCA1/2and PI3KCA
mutations for PARP and PI3KCA inhibitors, respectively, and PD-L1 expression and/or MSI status
for immunotherapy. The eective new biological therapies like CDK4/6 or mTOR inhibitors are now
emerging. However, we still lack predictive biomarkers for these treatments [100].
3.2. Precision Medicine Concept
The maturation of omic technologies as powerful molecular epidemiological screening tools
has empowered the emergence of manifold predictive biomarker signatures. The integration of
genomic and transcriptomic profiles of 2000 breast tumors from the METABRIC (Molecular Taxonomy
Cancers 2020,12, 3622 8 of 32
of Breast Cancer International Consortium) cohort revealed ten BC subtypes, termed integrative
clusters (IntClust/s) and characterized by distinct genomic drivers [
101
]. Currently, there are five main
standardized genetic prognostic platforms for BC, aiming to assist decision on therapeutic options,
mainly including hormone therapy, chemotherapy, and anti-HER2 treatment:
Oncotype DX provides prognostic information in terms of 10-year distant recurrence. It predicts
the likelihood of adjuvant chemotherapy benefit in ER+BC patients, based on the expression of
a panel of 21 genes (16 cancer-related and five reference genes) [102].
Breast Cancer Index assesses the expression of 7 genes to predict the benefit from extended,
adjuvant, endocrine therapy (Tamoxifen) in HR+patients. It is a gene expression signature
comprising two functional biomarker panels, the molecular grade index (MGI) and the two-
gene ratio HOXB13/IL17BR (H/I), that evaluate tumor proliferation and estrogen signaling,
respectively [
103
]. MGI is a gene expression assay, measuring the expression of five genes
(BUB1B, CENPA, NEK2, RACGAP1, RRM2) related to histological grade and tumor progression,
which recapitulates tumor grade and can predict the clinical outcome with high performance [
104
].
EndoPredict (Myriad Genetics, Inc., Salt Lake City, UT, USA) is a genomic test for people newly
diagnosed with early-stage, ER+, HER2-negative BC (node-negative). It assesses the expression
of 12 genes (8 target genes, 3 normalization genes, and 1 control gene) to predict response to
chemotherapy [105].
MammaPrint (Agendia, Irvine, CA, USA) is a 70-gene signature test that predicts the clinical
outcome/response to chemotherapy in ER+early-stage BC [106].
Prosigna Breast Cancer Prognostic Gene Signature Assay (Nanostring, Seattle, WA, USA),
formerly PAM50, assesses Tamoxifen response for HR+BC patients based on the expression of
58 genes after 5 years of hormonal therapy treatment in postmenopausal women [107].
Besides Prosigna, Nanostring has developed a more extensive assay, the human nCounter Breast
Cancer 360 panel, which comprises 776 genes across 23 key BC pathways and processes. Results are
grouped in 48 signatures across 13 categories, measuring biological variables crucial to BC tumor
biology. This panel has been developed for the evaluation of diverse BC aspects, including BC
subtyping (luminal A/B, HER2-enriched, basal-like, triple-negative), expression of BC receptors and
signaling (ESR1, PGR, ERBB2, AR, PTEN, CDK4, CDK6), mutational content (HRD, BRCA, P53),
markers for tumor proliferation, apoptosis and dierentiation (FOXA1, SOX2), cell adhesion (claudin),
and immunity (chemokines, TGF-
β
, PD-1). Although a detailed description of the nCounterBC
360 panel exceeds this review’s scope, it contains a vast number of gene expression markers and
indicates the intense molecular heterogeneity characterizing the diversity of BC phenotypes. Therefore,
investigating the epigenetic landscape of BC may provide an additional layer of information that could
improve our fundamental understanding of BC
´
s molecular complexity and the putative rational
development of more eective and precise treatments.
Finally, various studies have identified and suggested epigenetic modifications and regulators
as prognostic biomarkers for BC [
108
110
]. In this context, miRNAs have been considered a pool
of highly potent biomarkers, as they have been linked to the identification of distinct molecular
subtypes and tumor-related processes. miRNA expression profiling was successfully employed to
classify the breast tumors as luminal A, luminal B, basal-like, HER2+, and normal-like BC [
111
].
Aberrant expression of miRNAs has also been correlated with clinical features, such as angiogenesis,
metastasis, and EMT [
112
]. Besides, several lncRNAs have been reported as promising biomarkers
for prognosis, diagnosis, and therapy [
113
]. Overall, there is cumulative evidence focusing on
the systematic screening of epigenetic signals as a promising area for the discovery of novel molecular
BC biomarkers, combining sensitivity, specificity, and robustness, with a potentially decisive impact on
improving the quality of BC treatments in the context of precision medicine.
Cancers 2020,12, 3622 9 of 32
3.3. Potential of Epigenetic Therapy
The role of epigenetics in cancer initiation and progression, including its contribution to
the development of innate and acquired resistance to several therapeutic regimens, has led to
the scientific eort to reverse the aberrant cancer epigenome [
32
]. The lack of knowledge about
subtype-specific epigenome signaling pathways, and missing patient-specific epigenetic biomarker
profiles, are currently the main challenges hampering the wider clinical application of epigenetic agents
in the treatment of solid cancers [
114
]. Over the last decade, several epi-drugs have received US Food
and Drug Administration (FDA) approval for the treatment of blood-borne cancers: 5-azacytidine (AZA,
Vidaza
®
), 5-aza-2
´
-deoxycytidine (decitabine, DAC, Dacogen
®
), vorinostat (VOR, SAHA, Zolinza
®
),
romidepsin (FK228, Istodax
®
), belinostat (Beleodaq
®
), panobinostat (Farydak
®
), and chidamide
(Epidaza®) (Figure 1) [115117]. However, except for tazemetostat (Tazverik®), approved by FDA in
January 2020 for metastatic or locally advanced epithelioid sarcoma, there is no epigenetic therapy
approved for solid tumors, which are considered more epigenetically complex. Moreover, they exhibit
abnormal vascularization, a specific tumor microenvironment, and more dierentiated cells with
decreased epigenetic reprogramming [7,118].
Cancers 2020, 12, x FOR PEER REVIEW 9 of 33
scientific effort to reverse the aberrant cancer epigenome [32]. The lack of knowledge about subtype-
specific epigenome signaling pathways, and missing patient-specific epigenetic biomarker profiles,
are currently the main challenges hampering the wider clinical application of epigenetic agents in the
treatment of solid cancers [114]. Over the last decade, several epi-drugs have received US Food and
Drug Administration (FDA) approval for the treatment of blood-borne cancers: 5-azacytidine (AZA,
Vidaza®), 5-aza-2´-deoxycytidine (decitabine, DAC, Dacogen®), vorinostat (VOR, SAHA, Zolinza®),
romidepsin (FK228, Istodax®), belinostat (Beleodaq®), panobinostat (Farydak®), and chidamide
(Epidaza®) (Figure 1) [115–117]. However, except for tazemetostat (Tazverik®), approved by FDA in
January 2020 for metastatic or locally advanced epithelioid sarcoma, there is no epigenetic therapy
approved for solid tumors, which are considered more epigenetically complex. Moreover, they
exhibit abnormal vascularization, a specific tumor microenvironment, and more differentiated cells
with decreased epigenetic reprogramming [7,118].
Rational epi-drug discovery using validated targets is a recent phenomenon. During early
efforts, epi-drug development has been based on the demonstration of efficacy and phenotypic
observations rather than on knowledge of their molecular targets. The timeline and key events
influencing epi-drug development, including challenges and opportunities associated with their
implementation in clinics, have recently been reviewed in-depth by Ganesan and colleagues [119].
As a detailed characterization of epi-drugs is beyond this paper’s scope, we provide only their basic
characteristics and classification (Figure 1).
Figure 1. Different categories of epi-drugs, assessed in preclinical studies and clinical trials. Eight of
them (indicated by asterisks) were approved to treat several human malignancies (modified from
7,119,120). Abbreviations: DNMTIs-DNA methyltransferase inhibitors; HDACIs-histone deacetylase
inhibitors; HMTIs-histone methyltransferase inhibitors; HDMIs-histone demethylase inhibitors;
BETIs-bromodomain and extra-terminal domain inhibitors; HATIs-histone acetyltransferase
inhibitors; ncRNAs-non-coding RNAs; DOT1LIs-DOT1-like histone lysine methyltransferase
Figure 1.
Dierent categories of epi-drugs, assessed in preclinical studies and clinical trials.
Eight of them (indicated by asterisks) were approved to treat several human malignancies (modified
from 7,119,120). Abbreviations: DNMTIs-DNA methyltransferase inhibitors; HDACIs-histone
deacetylase inhibitors; HMTIs-histone methyltransferase inhibitors; HDMIs-histone demethylase
inhibitors; BETIs-bromodomain and extra-terminal domain inhibitors; HATIs-histone acetyltransferase
inhibitors; ncRNAs-non-coding RNAs; DOT1LIs-DOT1-like histone lysine methyltransferase inhibitors;
EZH2Is-enhancer of zeste homolog 2 inhibitors; PRMTIs-protein arginine methyltransferase inhibitor;
LSD1Is-lysine-specific histone demethylase 1A inhibitors.
Cancers 2020,12, 3622 10 of 32
Rational epi-drug discovery using validated targets is a recent phenomenon. During early eorts,
epi-drug development has been based on the demonstration of ecacy and phenotypic observations
rather than on knowledge of their molecular targets. The timeline and key events influencing epi-drug
development, including challenges and opportunities associated with their implementation in clinics,
have recently been reviewed in-depth by Ganesan and colleagues [
119
]. As a detailed characterization
of epi-drugs is beyond this paper’s scope, we provide only their basic characteristics and classification
(Figure 1).
Given that overexpression of DNMTs and HDACs are considered the critical factors in
carcinogenesis, demethylating agents and HDAC inhibitors (HDACIs) seem to be promising anticancer
drugs [
120
122
]. Cytidine analogs, AZA and DAC, were the first DNMT inhibitors (DNMTIs) approved
by the FDA in 2004 for the treatment of myelodysplastic syndrome and acute myeloid leukemia [
123
].
Although they were initially defined as cytotoxic agents, their therapeutic properties were achieved
at lower doses and with prolonged exposure [
124
]. In general, HDACIs block histone deacetylation,
causing reactivation of tumor suppressor genes that can inhibit cancer cell proliferation. Moreover,
they have been shown to induce cancer cell death at concentrations to which normal cells are relatively
resistant [
125
,
126
]. Chemically, HDACIs are classified into dierent subgroups: carboxylic acids,
benzamides, cyclic peptides, and hydroxamic acids [
127
]. Similar to DNMTs, the first-generation
HDACIs were characterized by poor bioavailability, low stability, and short half-life.
The development of second-generation epi-drugs has, therefore, been aimed to circumvent
these shortcomings. Guadecitabine, a second-generation DNMTI, has a novel molecular structure,
which prolongs its
in vivo
half-life, and increases efficacy [
128
]. As nucleoside analogs require active DNA
synthesis to incorporate them into the DNA, their use is limited in hypoproliferative cancers and could
be a major obstacle in the therapy of solid tumors [
129
]. Their common side effects could be avoided
using non-nucleoside analogs, such as hydralazine, procainamide, RG108, and MG98 [
130
]. Recently,
more efficient bi-substrate analogs have become potent DNMTIs [
119
].
Although the second-generation
HDACIs, such as hydroxamic acid, belinostat, panobinostat, chidamide, or valproic acid, possess improved
pharmacological properties, they achieved limited efficacy as single agents. However, their combination
with other therapeutic approaches has allowed new avenues of their clinical investigation [7].
The principle of precision medicine is now being applied to the development and the use
of third-generation epi-drugs, defined by a high degree of selectivity. This family includes HMT
inhibitors (e.g., EZH2, DOT1-like histone-lysine methyltransferase (DOT1L), G9a and PRMT inhibitors),
HDM inhibitors (e.g., LSD1 or Jumonji C domain inhibitors), BET inhibitors (BETIs) and HAT inhibitors
(HATIs) [
7
,
131
]. HMTIs are emerging therapies targeting specific modifications. For example, it has
been found that mutations in lymphomas activate the H3K27 histone methyltransferase EZH2,
leading to disease progression. Therefore, the EZH2I can selectively target and induce cell death
in cell lines with these mutations [
132
]. DOT1L is the only histone methyltransferase that targets
the histone H3 lysine 79 (H3K79) residue [
133
]. Aberrant H3K79 methylation is associated with
aggressive mixed-lineage leukemia and poor patient prognosis in lung, colorectal, and BCs [
134
].
This suggests that pharmacological inhibition of DOT1L can have therapeutic potential in several
cancer types. The H3K4 and H3K9 demethylase enzyme LSD1 has an essential regulatory role
in cell proliferation [
135
]. Its overexpression in several tumors has been correlated with a worse
prognosis [
136
]. LSD1 inhibition may slow down cell growth in LSD1-overexpressing tumor cells. BETIs,
JQ1, and I-BET762, are cell-permeable agents that reversibly and specifically bind the bromodomain
proteins, thus impeding their interaction with acetylated histone lysine residues. It has been shown that
they inhibit proliferation and induce apoptosis in various cancer cells [
137
]. HATIs include peptides,
small molecules derived from natural products (e.g., curcumin), and synthetic molecules [
138
].
Peptide-CoA bisubstrate inhibitors mimic the formation of the substrate and cofactor complex binding
to the HAT enzyme [
131
]. Well-conceived computational strategies and new screening platforms will
be needed to predict loci specific epi-drugs sensitivities.
Cancers 2020,12, 3622 11 of 32
The ncRNAs, like miRNAs or siRNAs, with their power to selectively “switch-o” specific
cancer genes, are attractive targets for the development of personalized cancer therapy. The main
hindrance to the implementation of ncRNA-based therapy in clinical practice is the absence of
eective delivery systems that can protect the RNA molecules from fast nuclease degradation before
delivering them into the target cells
´
cytoplasm [
139
]. There are two dierent possibilities to use
miRNAs as therapeutic agents; substitution of depleted miRNAs (MRX34, miR34a replacement)
and inhibition of overexpressed miRNAs by antagonistic oligonucleotides [
140
]. The inhibitor of
miR-155, MRG-106, has been successfully investigated in phase I clinical trials for the treatment of
hematological malignancies [
141
]. The miRNA-based therapeutic strategy also has great potential to
regulate lncRNAs. The siRNA-mediated silencing by oligonucleotide inhibitors results in the inhibition
of lncRNA-protein interactions and secondary structure changes, thus competing for their binding
partners [
142
]. Down-regulation of cancer-related genes by siRNAs, e.g., CALAA-01 (targeting RRM2),
Atu-027 (targeting PKN3), has been assessed in phase I/II clinical trials [139].
Although several ongoing clinical trials, including epi-drugs, exist on a wide range of diseases,
many obstacles remain to be resolved. Among them are enzyme isoform selectivity, dual substrates,
multimeric enzyme complexes involved in epigenetic regulations, high-order chromatin structure,
functional effects of inhibition, and off-target effects. Other challenges are the pharmacology of
the compounds, doses to be used, therapeutic regimens or duration of the treatment, and patient selection.
Epigenetic Therapy in BC
Several clinical studies have investigated the ecacy of DNMTIs (DAC, AZA) and HDACIs (VOR,
phenyl butyrate) administration as BC monotherapy. However, these epi-drugs have shown limited
antitumor ecacy at the maximum tolerated dose, suggesting the unsuitability of this approach [
143
].
In BC, the epi-drugs have been investigated in combination with cytotoxic agents, radiotherapy,
targeted and hormone therapy, immunotherapy, as well as the combination of epi-drugs themselves
(Table 1).
Table 1. Epigenetic drugs in clinical trials focused on breast cancer.
Epi-Drug Other Interventions Status Phase Trial No.
Decitabine
LBH589, Tamoxifen Terminated 1, 2 NCT01194908
Paclitaxel Unknown 1b NCT03282825
Carboplatin Recruiting 2 NCT03295552
Doxorubicin and 4 more Recruiting 2 NCT02957968
Azacitidine
Nab-paclitaxel Completed 1, 2 NCT00748553
Entinostat Active, not recruiting 2 NCT01349959
Definitive breast Withdrawn NA NCT01292083
Fulvestrant Terminated 2 NCT02374099
Durvalumab Active, not recruiting 2 NCT02811497
Valproic acid
FEC100 Terminated 2 NCT01010854
Hydralazine Terminated 2 NCT00395655
Bevacizumab, Cetuximab Recruiting 1 NCT01552434
Entinostat
Capecitabine Recruiting 1 NCT03473639
Exemestane Recruiting
Completed
Active, not recruiting
Active, not recruiting
3
2
3
2
NCT03538171,
NCT00676663,
NCT02115282,
NCT03291886
Fulvestrant Withdrawn 2 NCT02115594
Lapatinib Ditosylate, Trastuzumab Completed 1 NCT01434303
Anastrozole Terminated 2 NCT01234532
Nivolumab, Ipilimumab Active, not recruiting 1 NCT02453620
Atezolizumab and 6 more Recruiting 1, 2 NCT03280563
Romidepsin
Cisplatin, Nivolumab Suspended 1, 2 NCT02393794
Abraxane Terminated 1, 2 NCT01938833
Alone Completed 2 NCT00098397
Cancers 2020,12, 3622 12 of 32
Table 1. Cont.
Epi-Drug Other Interventions Status Phase Trial No.
Vorinostat
Olaparib Not yet recruiting 1 NCT03742245
Paclitaxel and 3 more Completed 1, 2 NCT00574587
Alone Completed
Terminated
Completed
Withdrawn
Completed
Terminated
1
2
2
NA
1
2
NCT00719875,
NCT00132002,
NCT00262834,
NCT01695057,
NCT00788112,
NCT00126451
Tamoxifen Completed
Terminated
2
2
NCT00365599,
NCT01194427
Tamoxifen, Pembrolizumab Terminated
Not yet recruiting
2
2
NCT02395627,
NCT04190056
Carboplatin, Nab-paclitaxel Active, not recruiting 2 NCT00616967
Ixabepilone Completed 1 NCT01084057
Lapatinib Terminated 1, 2 NCT01118975
Paclitaxel, Bevacizumab Completed 1, 2 NCT00368875
Anastrozole, Letrozole, Exemestane Completed
Completed
NA
NA
NCT01720602,
NCT01153672
Trastuzumab Completed 1, 2 NCT00258349
Belinostat Ribociclib Not yet recruiting 1 NCT04315233
Panobinostat
Alone Completed
Terminated
Withdrawn
2
2
1
NCT00777049,
NCT00777335,
NCT00993642
Trastuzumab Terminated 1, 2 NCT00567879
Letrozole Completed 1, 2 NCT01105312
Trastuzumab, Paclitaxel Completed 1 NCT00788931
Capecitabine, Lapatinib Completed 1 NCT00632489
The DAC-chemotherapy combination has been widely studied in BC preclinical studies,
and the results
suggest enhanced sensitivity compared to cytotoxic therapy alone [
144
146
].
Despite these findings, clinical trials have been disappointing due to systemic toxicities and limited
ecacy. Among the reasons leading to unsatisfactory results were the design of clinical trials using
unselected patient populations and high-dose administration. This has revealed a need to identify
appropriate epigenetic biomarkers for allowing a personalized approach and targeted delivery of
epi-drugs [7].
The preclinical data with HDACIs on HR+BC cell lines indicate an increase in antiproliferative
endocrine therapy action [
147
]. In ER- BCs, an AZA-entinostat combination, and HDACI therapy
(entinostat, valproic acid, TSA) alone, have shown a high ER re-expression and ecient restoration
of the sensitivity to antioestrogen treatment [
148
151
]. Furthermore, the BET inhibitor JQ1 alone
or in combination with the selective ER down-regulation by fulvestrant can eectively suppress
the growth of tamoxifen-resistant cells [
152
]. The synergy of combination therapy has been preclinically
demonstrated on triple-negative BC cells with mTOR-BETIs [
153
] and with PARP-HDACIs [
154
].
Based on promising preclinical results, the joint ecacy of HDACIs and anti-HER2 therapy with
trastuzumab has been assessed in a clinical study (NCT00258349). Unfortunately, the hypothesis
of reversing trastuzumab resistance by adding VOR in the therapeutic regimen has not been
confirmed [
155
].
However, with the appropriate
drug combination, it could be possible to target
several oncogenic pathways [156].
Cancers 2020,12, 3622 13 of 32
All in all, for the successful utilization of epi-drugs in clinics, there is an urgent need for
identification of new epigenetic therapeutic targets and mechanisms ensuring prolonged stability of
epi-drugs, decreasing undesirable side eects while allowing increased ecacy via targeted delivery.
4. Nanomedicine as a Tool to Overcome the Current Limitations of Epigenetic Therapy
Nanomedicine applies the knowledge from material science and nanobiotechnologies to healthcare
in order to improve the diagnosis, imaging, monitoring, prevention and regeneration, and to increase
the ecacy and safety of the treatment [
157
]. According to the European Commission definition,
a nanomaterial is a natural, incidental, or manufactured material with one or more external dimensions
in the nanoscale (size range 1 nm–100 nm), and having a volume-specific surface area larger than
60 m
2
/cm
3
[
158
]. The term nanomaterial in nanomedicine is broader and includes particles with
dimensions up to 1000 nm [
159
]. Besides, the International Organization for Standardization has defined
a nanoparticle (NP) as a nanomaterial, with the size in all three dimensions in the nanoscale [
160
].
However, these varied definitions and classifications allow a wide interpretation. Notably, the beneficial
or detrimental potential of nanomaterials for the environment and human health depends on their
physical and chemical properties, such as shape, composition, size, surface charge, and electron
transfer [161].
4.1. Smart Nanoformulations for Drug Delivery Applications
Nowadays, a central area for the use of nanomedical products, considering the number of scientific
publications and product approvals by regulatory agencies, is cancer treatment [
162
]. The conventional
cytotoxic anticancer therapy is often associated with low therapeutic ecacy and increased systemic
toxicity. The chemotherapeutic agents also aect rapidly dividing non-cancerous cells, causing common
(nausea, vomiting, fatigue, hair loss) and drug-specific(e.g., cardiotoxicity, neurotoxicity, nephrotoxicity)
side eects. Besides that, tumor cells have several defense mechanisms against the cytotoxic eect of
chemotherapy. The most significant one is the multidrug resistance resulting from the overexpression
of ATP-dependent eux pumps with broad drug specificity [163].
Innovative materials, including NPs, with their unique properties, have several advantages as
potential transport vehicles for standard and experimental anticancer agents. They allow encapsulation
of hydrophobic and lipophilic molecules, enhancing their pharmacokinetics and circulation time in
the body, leading to increased drug accumulation in the tumor site via passive targeting by the EPR
eect. Moreover, they can be conjugated with specific ligands to bypass biological barriers and deliver
a high drug concentration into the target tissue via active targeting. They also allow the drug
´
s
release in a stable and controlled manner, triggered by pH, temperature, redox potential, etc. [
164
,
165
].
However, the ecient
penetration of anticancer agents encapsulated in NPs remains an important
issue in solid tumor therapy [
166
]. Systemically administered NPs should range from 10 to 200 nm
in size to avoid early elimination by kidneys or subsequent entrapment by spleen and liver [
167
].
Biocompatibility, low toxicity, and low immunotoxicity are the prerequisite attributes of all biomedical
nanomaterials. Before being approved for clinical use by the FDA, they have to undergo complex
preclinical and clinical testing to confirm their biosafety. FDA-approved materials are, therefore,
preferentially employed for the development of innovative nanocarriers. There is an increasing body of
evidence from preclinical studies that the encapsulation of existing epi-drugs into dierent formulations
has resulted in their improved stability and enhanced targeted delivery while minimizing o-target
eects [168,169].
Nowadays, the range of nano-based delivery systems is growing [
170
]. Liposomes, solid lipid
NPs (SLNs), polymer NPs, polymer micelles, dendrimers, nanoemulsions, and polymer-lipid hybrid
NPs have been studied as promising nanocarriers for targeted drug delivery (Figure 2) [168].
Cancers 2020,12, 3622 14 of 32
Cancers 2020, 12, x FOR PEER REVIEW 14 of 33
Figure 2. Main types of soft nanocarriers for drug delivery. Schematic examples of surface
modifications and functionalization.
With the large body of evidence accumulated over the past decades, three important elements
in the development of controlled drug delivery can be defined: (1) utilization of the EPR effect for
passive targeting, (2) surface modification of NPs/nanocarriers to prolong their circulation time, (3)
their effective design [171]. Coating the surface of NPs with PEG (PEGylation), an FDA approved
polymer, is a commonly used approach for improving the efficiency of drug and gene delivery to
target cells and tissues. PEGylation protects the surface from aggregation, opsonization, and
phagocytosis and prolongs systemic circulation time [172]. PEG is a hydrophilic, biocompatible, and
biologically inert polymer, which is used to enhance the stability of NPs [173]. Moreover, the coating
allows loading of specific ligands, antibodies, peptides, drugs, folic acid, aptamers, tumor markers,
transferrin, vitamins, etc., on the surface of particles offering an exciting tool to make NPs target-
specific and increase their therapeutic benefit [174–176]. These ligands can be easily conjugated onto
PEG´s distal end via various chemical coupling strategies [177]. Cancer cells overexpress various
kinds of receptors and antigens that may be used as potential drug targets in cancer therapy [178].
Entrapment of the fluorescent compound in the NPs platform allows tracking the fate of NPs in vitro
and monitoring of the accumulation of the drug in the tumor and the treatment efficacy [179]. The
Quantum Dots (QDs) are fluorescent semiconductor nanocrystals of about 10 nm, composed of a core
coated with an envelope. QD-containing NPs have found applications in cancer medical imaging,
cancer cell tracking, cancer photodynamic therapy, and cancer diagnosis [180].
4.1.1. Liposomes
Liposomes (Figure 2A) are the most utilized nanomaterial for drug delivery, with many clinical
products currently available [181]. Liposomal vesicles consist of amphiphilic poly-molecular lipid
compounds that assemble into bi-layered self-closed spherical NPs [182]. Different structural
modifications of polar and non-polar parts of phospholipids have allowed the development of
various synthetic lipids with improved properties [183]. The advantage of liposomes is their ability
to deliver both hydrophilic and hydrophobic drugs. Surface functionalization of liposomes enhances
Figure 2.
Main types of soft nanocarriers for drug delivery. Schematic examples of surface modifications
and functionalization.
With the large body of evidence accumulated over the past decades, three important elements in
the development of controlled drug delivery can be defined: (1) utilization of the EPR eect for passive
targeting, (2) surface modification of NPs/nanocarriers to prolong their circulation time, (3) their
eective design [
171
]. Coating the surface of NPs with PEG (PEGylation), an FDA approved polymer,
is a commonly used approach for improving the eciency of drug and gene delivery to target cells
and tissues. PEGylation protects the surface from aggregation, opsonization, and phagocytosis and
prolongs systemic circulation time [
172
]. PEG is a hydrophilic, biocompatible, and biologically inert
polymer, which is used to enhance the stability of NPs [
173
]. Moreover, the coating allows loading of
specific ligands, antibodies, peptides, drugs, folic acid, aptamers, tumor markers, transferrin, vitamins,
etc., on the surface of particles oering an exciting tool to make NPs target-specific and increase
their therapeutic benefit [
174
176
]. These ligands can be easily conjugated onto PEG
´
s distal end
via various chemical coupling strategies [
177
]. Cancer cells overexpress various kinds of receptors
and antigens that may be used as potential drug targets in cancer therapy [
178
]. Entrapment of
the fluorescent compound in the NPs platform allows tracking the fate of NPs
in vitro
and monitoring
of the accumulation of the drug in the tumor and the treatment ecacy [
179
]. The Quantum Dots
(QDs) are fluorescent semiconductor nanocrystals of about 10 nm, composed of a core coated with
an envelope. QD-containing NPs have found applications in cancer medical imaging, cancer cell
tracking, cancer photodynamic therapy, and cancer diagnosis [180].
4.1.1. Liposomes
Liposomes (Figure 2A) are the most utilized nanomaterial for drug delivery, with many clinical
products currently available [
181
]. Liposomal vesicles consist of amphiphilic poly-molecular lipid
compounds that assemble into bi-layered self-closed spherical NPs [
182
]. Dierent structural
modifications of polar and non-polar parts of phospholipids have allowed the development of
various synthetic lipids with improved properties [
183
]. The advantage of liposomes is their ability to
Cancers 2020,12, 3622 15 of 32
deliver both hydrophilic and hydrophobic drugs. Surface functionalization of liposomes enhances
their stability and facilitates targeted drug, gene, or imaging agent delivery, even across the biological
and physiological barriers [
172
,
175
,
176
,
184
186
]. They are biocompatible, of low immunogenicity,
and they increase the solubility of a wide range of chemotherapeutics [
187
]. Several liposomal
formulations, namely Doxil
®
/Caelyx
(pegylated liposomal doxorubicin), Myocet
®
(non-pegylated
liposomal doxorubicin), DaunoXome
®
(liposomal daunorubicin), Marqibo
®
(liposomal vincristine),
Mepact®(liposomal mifamurtide)
, and Onivyde
®
(pegylated liposomal irinotecan) have already been
EMA and/or FDA-approved for the treatment of various cancer types, including BC [164,188].
To improve the ecacy of HDACI in BCs and other solid tumors, PEGylated liposomes were
prepared for encapsulation of trichostatin A (TSA), CG1521 (CG), and PXD101 (PXD) (Table 2) [
189
].
These liposome formulations are promising nanocarriers as they remained stable in size, charge,
and biological activity for one month when stored at 4 C. Wang et al. utilized the iron complexation
technique on VOR and LAQ824 before encapsulating them in PEGylated liposomes [
190
]. This strategy
resulted in an improved aqueous solubility of these drugs as well as liposomal encapsulation eciency.
Table 2. Nanoplatforms for epigenetic drug delivery.
Nanocarrier Loaded Drug Reference
PEGylated liposomes trichostatin A, CG1521, and PXD101 [189]
PEGylated liposomes with Fe complex VOR and LAQ824 [190]
Hybrid lipid-polymer NPs DAC [191,192]
Solid lipid NPs VOR [193]
Solid lipid NPs decorated with hyaluronic acid VOR [194]
Norbornene polyethylene oxide macromonomer CI-994 (tacedinaline) [195]
POEG blocks VOR [196]
PLGA NPs decorated with PGON belinostat and VOR [197,198]
PLGE-PEG nano-micelles AZA [199]
PEG-PLA di-block copolymer DAC [200]
Gelatinases-stimuli di-block copolymers (PEG, PCL) DAC [201,202]
LGE block copolymer VOR [203]
Lipid-polymer (DSPE-PEG-COOH-
PLGA-lecithin-PEG) core-shell NPs VOR and quisinostat [204]
Nanogels DAC [205]
4.1.2. Solid Lipid Nanoparticles
SLNs (Figure 2B) consist of a hydrophobic lipid core composed of lipids (e.g., fatty acids,
steroids, waxes, or triglycerides) that are in the solid state at room temperature. The lipid core is
stabilized by surfactants (emulsifiers) to prevent particle agglomeration [
206
]. The advantage of
SLNs is the easy entrapment of hydrophobic drugs in the lipidic matrix, while the outer hydrophilic
shell allows the conjugation of hydrophilic drugs to lipidic components. The solid state of the lipid
permits better-controlled drug release and improves the stability of the drug. Moreover, SLNs can be
administered by various routes, including systemic injection, oral, transdermal, pulmonary, and ocular
application [
168
]. SLNs can be modified to exhibit various advantages over liposomes and polymeric
NPs; they are non-toxic, biocompatible, biodegradable, and highly stable [207].
Lipid-based nanostructures as carriers of the potential oral delivery of DAC were designed and
synthesized by Neupane et al. [
191
] An ex vivo gut permeation study showed a nearly four-fold
increment in the drug
´
s permeation from the nanostructured lipid carrier compared with the plain
DAC solution and better accumulation of DAC encapsulated in lipid 4 h after oral administration
Cancers 2020,12, 3622 16 of 32
compared with i.v. injection of free DAC into the tumor. Based on these results, the authors suppose
that lipid-based nanocarrier systems represent a promising cancer cell treatment strategy through oral
delivery. Solid lipids also represent promising nanocarriers for HDACI delivery. Encapsulation of VOR
into solid lipid NPs enhanced its bioavailability and reduced the drug clearance rate compared to free
VOR
in vivo
, whether administered orally or i.v. and increased VOR’s toxicity in multidrug-resistant
BC cells
in vitro
[
193
,
194
,
208
,
209
]. Cyclin-dependent kinase 4 (CDK4) siRNA packaged in a lipid NP
(LNP)-based delivery system that consists of an ionizable cationic lipid, helper lipid, and polyethylene
glycol (PEG)-lipid showed a 16-fold increase in intracellular uptake of siRNA by BC cells and
a significant G1 cell cycle arrest due to ecient down-regulation of CDK4 at both mRNA and protein
levels [
210
]. The ncRNA nano-based drug patirisan (Onpattro
®
) has recently been approved by
the FDA (in 2018) to treat polyneuropathy. It is the first clinically approved example of an RNAi
therapy-delivering NP administered intravenously (i.v.) and the first FDA-approved RNAi therapeutic
in general [
211
]. ALN-18328, the active ingredient of patirisan, is formulated as a lipid nanoparticle to
targeted delivery to hepatocytes.
4.1.3. Polymeric Nanoparticles
Polymeric NPs (Figure 2C) are the simplest form of soft-materials for nanobiomedical
applications [
212
]. They can be composed of synthetic biodegradable polymers, some of which
were already approved by the FDA for tissue engineering scaolds and drug delivery vehicles [
213
].
Alternatively, they can be made of natural polymers such as chitosan, gelatin alginate, and albumin.
The advantages of polymeric NPs include the capacity to encapsulate both hydrophilic and hydrophobic
drugs and straightforward activation with specific molecules for targeted delivery to the tumor
mass [
214
]. While synthetic polymers allow sustained drug release, within a period of days to weeks,
natural polymers are more easily and rapidly degraded [
215
]. Polymeric NPs are stable, bioavailable,
have prolonged circulation time in peripheral blood, enable controlled drug release, and, compared to
liposomes, they possess superior drug loading capacities [
216
]. There are several FDA-approved
polymeric nano-drugs, such as Adagen
®
, Cimzia
®
, Macugen
®
, Neulasta
®
, Pegasys
®
, PegIntron
®
,
Renagel®, and Somavert®[212].
An innovative aspect of the nano-based delivery platform is the conjugation of CI-994 (tacedinaline)
through a clickable acid-responsive linker to a macromonomer. Such a prodrug monomer could
be polymerized to create a formulation that releases the HDACI in a pH-dependent manner while
minimizing
in vivo
drug release during blood circulation [
195
]. A pendant SAHA derivatized polymer
able to form spherical micelles is a promising novel prodrug carrier. This nanoformulation retains
the pharmacological activity of SAHA, inhibits the proliferation of tumor cells, and induces histone
acetylation [
196
]. Belinostat-loaded polymer NPs, decorated with a novel cell-penetrating polymer,
poly(guanidinium oxanorbornene), significantly enhanced intracellular uptake,
decreased the tumor
volume, and increased the intratumoral acetylation of histone H4 [
197
]. The safety of orally administered
SAHA-loaded polymer NPs manifested low cytotoxicity in lung cancer cells and favorable
biodistribution patterns
in vivo
[
198
]. A VOR nanofiber-coated stent was shown to be a promising
candidate for cholangiocarcinoma treatment [
217
]. Using the emulsion-solvent diusion technique,
Alp et al
. have developed a biocompatible starch NP formulation for delivery of CG-1521 to
hard-to-treat BCs [
218
]. This formulation improves the bioavailability and half-life of this HDACI
without aecting the drug´s mechanism of action.
A stable nanosized ribophorin II (RPN2) siRNA–atelocollagen complex was prepared to improve
the
in vitro
and
in vivo
resistance of BC cells to DOX and taxanes. RPN2 silencing caused reduced
glycosylation of the P-glycoprotein in vitro and markedly reduced tumor growth in vivo [219].
4.1.4. Polymeric Micelles
Polymeric micelles (Figure 2D) are supramolecular delivery systems composed of multiple
amphiphilic block copolymers, self-assembling in aqueous environments at a defined concentration [
206
].
Cancers 2020,12, 3622 17 of 32
The micelles
´
core-shell structure presents a hydrophobic core and the outer hydrophilic shell. The core
can be tailored for the controlled delivery of hydrophobic drugs, while hydrophilic drugs can be adsorbed
or chemically linked to the shell [
9
]. Polymeric micelles have high stability and exhibit prolonged
circulation time in the blood [
168
]. Poly(ethylene glycol)–poly(lactic-co-glycolide) (PEG–PLGA) di-block
copolymer micelles represent one of the most promising biocompatible platforms for drug delivery [
220
].
Lipids can also be used to form the hydrophobic core of these hybrids for encapsulation of many types of
poorly water-soluble drugs [
221
]. FDA approved polymeric micelles, already used in clinical practice,
include Copaxone
®
, Eligard
®
, Estrasorb
, Oncaspar
®
, Krystexxa
®
, Plegridy
®
, and Adynovate
®
used
for the treatment of sclerosis, prostate cancer, and blood malignities [212].
The micelles are predominantly utilized as versatile nanocarriers for the delivery of DNMT
inhibitors. Encapsulation of AZA or DAC into micelles has increased their stability under physiological
conditions, markedly enhanced their therapeutic ecacy, provided controlled pH-dependent drug
release, significantly down-regulated DNMT1 and DNMT3b expression, and increased expression
of caspase-9 in murine xenograft models of BC [
199
,
200
]. Intelligent DAC-loaded micelles have
been prepared by inserting cancer-specific gelatinase-cleavable peptide between two polymers.
These micelles manifested superior cellular uptake in the tumor-bearing xenografts due to an active
(gelatinase-stimuli) targeting strategy and more ecient gene demethylation compared with their
counterparts lacking gelatinase features [
201
,
202
]. DAC-loaded hybrid lipid-polymer micelles were
eciently internalized by BC cells and rescued the expression of silenced tumor suppressor genes
in cancer cells [
192
]. Several micelle formulations have been proposed and tested as a promising
nanocarrier for HDAI delivery. Kwak et al. revealed higher ecacy of tumor growth inhibition,
enhanced drug accumulation, and higher inhibition rate of HDAC expression in a xenograft mice
model by VOR-encapsulated micelles compared with the free drug [
203
]. Similarly, higher therapeutic
ecacy was confirmed by VOR- or quisinostat-loaded hybrid lipid-polymer micelles compared with
free HDACIs [
204
]. These micelles suppressed DNA double-strand break repair in tumor cells and
showed a synergistic eect in mouse xenograft
in vivo
models. Micelles loaded with antisense-miR-21
and antisense-miR-10b were prepared to block the functions of endogenous miRNAs, regulating genes
involved in cell proliferation, dierentiation, and apoptosis. Treatment of mice bearing subcutaneous
tumor xenografts with this formulation resulted in a substantial tumor growth reduction, thus indicating
a potential new therapeutic approach for triple-negative BC [222].
4.1.5. Dendrimers
Dendrimers (Figure 2E) are three-dimensional, hyper-branched, or tree-like polymers having
a central core with repeated branches of interior layers and exterior terminal functionality. These structures
offer high availability and a wide range of molecular moieties for the chemical conjugations and
internal cavities to encapsulate drugs and nucleic acids [
223
]. Dendrimers can be used to deliver
various molecules (therapeutics, imaging, and targeting agents) in a single particle due to the presence
of different functional monomers [
220
]. Dendrimers are suitable drug-delivery systems because of
their nanometric size, ease of fabrication, monodispersity, lipophilicity, multidrug loading capacity,
controlled drug release, low toxicity, biocompatibility, and ability to easily penetrate cell membranes [
9
].
Among different commercially available dendrimers, diaminobutyric polypropylenimine (DAB),
polyamidoamine (PAMAM), poly (amidoamine-organosilicon) (PAMAMOS), poly (Lysine), and poly
(propylene imine) (PPI) are the most promising platforms for delivering cargos to target sites [223].
Finlay et al. used a modified poly (amidoamine) (PAMAM) dendrimer for delivery of siRNA,
targeting the TWIST1 transcription factor [
224
]. TWIST1 is often overexpressed in aggressive BCs and
is involved in regulating cell migration through EMT. These PAMAM-siRNA complexes significantly
down-regulated TWIST1 and EMT-related target genes
in vitro
as well as in xenograft orthotopic
tumors. PAMAM dendrimer was also used to deliver antisense oligonucleotides targeting the vascular
endothelial growth factor VEGF-ASODN to inhibit the tumor vascularization of breast tumor tissue
using a human breast tumor xenograft mice model [225].
Cancers 2020,12, 3622 18 of 32
4.1.6. Nanogels
Nanogels (Figure 2F) are three-dimensional polymeric networks with a high capacity for
water uptake. They are mostly made of synthetic polymers or biopolymers, which contain
hydrophilic and hydrophobic monomers, cross-linked by either physical or chemical bonds, which
influence many network properties, like swelling, elastic modulus, and transport properties [
226
,
227
].
In aqueous media, nanogels form semi-solid states (hydrogels). The porous network allows
high-drug entrapment eciency. Nanogels can carry various drugs with low or high molecular
weight and either hydrophilic or hydrophobic. The advantages of nanogels include high drug
encapsulation capacity, enhanced drug stability, minimal toxicity, biocompatibility, prolonged blood
circulation time, and stimulus responsiveness to drug release (pH, magnetic field, light, ionic content,
and temperature) [206].
Nanogels (NGs) have also been explored for treating solid tumors. DAC-loaded nanogels
decorated with PEG manifested sustained DNMT1 depletion, prolonged cancer cell arrest in the G2/M
cell-cycle phase, and significantly enhanced the antiproliferative eect of DAC [205].
4.2. Nanoplatforms for Combination Therapy
A promising strategy to achieve more significant therapeutic benefits appears to be the combination
therapy involving epi-drug-loaded NPs and either a chemotherapeutic-encapsulated nanocarrier or
free standard anticancer drugs, or co-packaging of epi-drugs and anticancer drugs in one nanoplatform.
Li et al. have recently shown that combination therapy with low-dose DAC-loaded NPs and
DOX-loaded NPs is more eective and opens up new possibilities for the management of BC [
200
].
In line with these results, Vijayaraghavalu et al. have revealed that a combination of epigenetic drugs
(DAC +SAHA) encapsulated in biodegradable NGs more eectively overcomes drug resistance than the
same drugs in solution [
228
]. Moreover, pretreatment with epigenetic drugs in nanogels, then with DOX
in nanogels, was most eective in overcoming resistance even at low doses of DOX. Co-packaging DAC
and arsenic trioxide (ATO) into alendronate-conjugated bone-targeting hybrid lipid-polymer micelles
should enhance the synergistic eect of DAC and ATO in the treatment of MDS and reduce systemic
toxicity of chemotherapeutics [
229
]. Using co-axial and multi-needle electrohydrodynamic atomization
(EHDA) technology, Parhizkaz et al. have co-encapsulated DAC and cisplatin in a single PLGA-based
nanocarrier [
230
]. The EHDA technique enables fine-tuning of individual drug release characteristics,
with rapid DAC and slower cisplatin release, thus achieving the maximum synergistic therapeutic eect
and overcoming the chemoresistance to cisplatin. Lysophosphatidic acid receptor 1 (LPAR1)-targeted
lipid nanoemulsions (nanoscale oil-in-water emulsions) were developed for encapsulation of two
drugs with dierent chemistries, DAC (hydrophilic) and panobinostat (PAN–HDACI, hydrophobic)
and targeted co-delivery in triple-negative BC tissues [231].
Pendant SAHA derivatized polymer (POEG-b-PSAHA) micelles were used as nanocarriers for
DOX delivery. A DOX/POEG-b-PSAHA formulation resulted in an improved therapeutic eect
in vivo
compared to free DOX, Doxil, or POEG-b-POM-loaded DOX micelles, indicating that SAHA-based
prodrug micelles may serve as a dual functional carrier for combination strategies in epigenetic-oriented
anticancer therapy [
196
]. Ruttala et al. developed a transferrin-anchored albumin nanoplatform
with PEGylated lipid bilayers (Tf-L-APVN) for the targeted co-delivery of paclitaxel and VOR in
solid tumors [
232
]. At
in vitro
conditions, Tf-L-APVN significantly enhanced the synergistic eects of
paclitaxel and VOR on the proliferation of breast and liver cancer cells. In HepG2 tumor-bearing mice,
the co-delivery of paclitaxel and VOR in one nano-based platform significantly inhibited the tumor
growth, thus oering great potential in the chemotherapy of solid tumors.
Using a layer-by-layer approach, Deng et al. generated a siRNA-loaded film superimposed on
a simple DOX-loaded liposome [
233
]. They showed that combination therapy with siRNA targeting
multidrug resistance protein 1 (MRP1) significantly enhanced DOX ecacy
in vitro
, and
in vivo
resulted in a substantial reduction in the tumor of tumors, which otherwise are nonresponsive to
treatment with DOX.
Cancers 2020,12, 3622 19 of 32
5. Biosafety of Soft Nanocarriers
Despite the many advantages of using nano-based delivery systems, there are also significant
drawbacks that must be considered, as they might present a real challenge for clinical use [
186
].
Currently, there is very little regulatory guidance in the field of biomedical applications of NPs [
234
].
Although the EPR eect has been described as a basis for successful passive targeting of tumors,
the complexity of this process must also be highlighted. The EPR eect can be influenced by
the interactions of NPs with the biological systems, including proteins, blood flow, and the tumor
microenvironment. Nanoparticle properties, including size, shape, surface properties, porosity,
and structure, can aect the EPR eect [
235
]. For example, it has been reported that the high interstitial
fluid pressure in tumors can hinder the accumulation of nanocarriers since the high density of
the extracellular matrix can reduce the chances of NP penetration [236].
It is also dicult to predict the behavior and responses of nanocarrier interactions with biological
systems during the drug delivery process [
220
]. For instance, the interaction of NPs with the renal
system and its role in blood clearance is a crucial aspect to be taken into account. An ecient
bloodstream clearance is important to avoid NPs accumulating, which might lead to adverse long-term
eects. The eciency of kidney clearance has been reported to be aected by small changes in the size
of the NPs. At the same time, unspecific clearance, possibly leading to drug release far from the tumor
site, must also be avoided [237,238].
Thus, the nanocarriers
´
possible toxicity must be taken into account and thoroughly
investigated [
239
]. Nanomaterials pose particular challenges in the evaluation of their toxicity
in comparison with chemical substances. Besides the chemical composition, the particles physical
properties (e.g., their size, shape, agglomeration/aggregation, solubility/dispersibility, surface charge,
redox potential) play an important role in eliciting biological responses [
240
]. In this sense, a proper
hazard and risk assessment needs to be applied. This means that external and internal exposures, as
well as cellular uptake of the NPs, must be defined. In order to investigate all the possible pathways
through which toxicity might arise, a battery of relevant toxicity assays should be applied. As NPs
have been reported to de-regulate gene expression and aect epigenetic mechanisms involved in
several biological processes, including cancer development [
44
,
165
], these eects have to be taken into
consideration in addition to cytotoxicity and genotoxicity.
Attention must also be given to the choice of exposure models. Although the use of
in vivo
models cannot yet be completely excluded, alternative
in vitro
tests can be valid substitutes for initial
screenings of cyto- and genotoxicity and for investigating the underlying molecular mechanisms
of toxicity of nanomaterials. More advanced and relevant
in vitro
models to assess ecacy and
safety have been developed, moving beyond the traditional 2D monocultural models, towards 3D,
e.g., spherical tumor
models, and more complex multicellular models, which better resemble and
mimic real-life conditions [
241
]. Several nanomaterials have not yet had their potential adverse
biological eects fully assessed due to costs and time constraints associated with the experimental
assessment, frequently involving animals [242].
There are OECD test guidelines for
in vitro
assays adapted to the testing of NPs.
In vitro
assays
are usually robust, fast, and cost-eective compared to
in vivo
methods, and several high-throughput
screening methods for relevant cells from humans and other mammals are available to study the eects
of NPs [
243
,
244
]. Nowadays,
in vitro
assays adapted for testing nanomaterials are performed in
a controlled manner, taking physicochemical characterization and cellular uptake into consideration.
However, such tests need further validation because of putative NM-induced interferences [245].
To fully understand the advantages and disadvantages of NP-based therapeutics, more clinical
data are needed to identify the best applications for nano-chemotherapeutics [
13
]. In the field of cancer
nanomedicine research, it is important to take into account the complex heterogeneity within and
among human tumors and to focus on the design of precision nanomedicines to achieve personalized
cancer treatments [
246
]. In this sense, understanding which physicochemical properties are coupled
with adverse eects is critical for designing safer and more eective nanomaterials for cancer treatment.
Cancers 2020,12, 3622 20 of 32
This so-called “Safe(r)-by-Design” approach requires the implementation of safety evaluation early
in the development of nanomaterials [
247
], together with understanding their toxicokinetics and
modulation of the immune system [248250].
6. Conclusions
In BC, there are tumor subtypes, which are considered to be hard to cure, and do not respond to
standard therapeutic agents, and have a high recurrence probability. In recent decades, the intense
study of epigenetic deregulation in many cancers has uncovered the potential of new therapeutic
approaches, targeting reversible changes in the epigenome. The limited tolerability, low ecacy,
and o-target eects of most epi-drugs remain significant challenges in this experimental approach as
applied to solid tumors. The exploration of lower doses, sequential scheduling, and their targeted
delivery might considerably improve the therapeutic index. In this regard, nanotechnology has
revolutionized the field of drug delivery for epigenetic therapy of solid tumors. Dierent formulations
of soft nanocarriers have been employed to increase the stability, solubility, and specificity of DNMT
and histone inhibitors as well as non-coding RNAs. In addition, conventional chemotherapeutic agents
have been successfully co-packed with epi-drugs in nanocarriers for combination therapy. Data from
preclinical and clinical trials of nano-based epi-drugs for BC treatment demonstrate reduced systemic
toxicity and improved ecacy compared to conventional free-drug formulations.
Furthermore, nanomedicine offers the possibility to refine the detection, diagnosis, and conventional
therapy of many cancers by combining different agents in a single multi-component nano-drug.
Although promising, nano-based delivery systems should follow the Safe(r)-by-Design approach and be
rigorously tested at all phases of development to prevent adverse health effects and environmental hazards.
The use of nano-based delivery systems, together with the benefits of a new generation of epi-drugs
and the emergence of robust biomarker data, will help to advance personalized-targeted therapy and
the ecacy of treatment for breast and other cancers.
Author Contributions:
Conceptualization, B.S., A.G., and V.B.; Writing—original draft preparation and literature
search, V.B., B.S., A.G., M.M., E.P., A.C., K.P., M.R., A.S., A.P., M.F., E.M.L., N.E.Y., and S.M.; Writing—review and
editing, M.R.C., I.R.-M., V.P., and M.D.; Supervision, V.P.; Visualization, B.S. and V.B. All authors have read and
agreed to the published version of the manuscript.
Funding:
This research was funded by ERA-NET EuroNanoMed II INNOCENT, by the Slovak Research and
Development Agency (APVV), grant No. APVV-16-0010, APVV-16-0178, by the European Union’s Horizon 2020
research and innovation program under grant agreement No. 857381, project VISION (Strategies to strengthen
scientific excellence and innovation capacity for early diagnosis of gastrointestinal cancers), by ERA-NET
EuroNanoMed III CELLUX, H2020-NMBP-TO-IND-2019 SABYDOMA, contract No. 862296 and by Scientific
Grant Agency (VEGA), contracts No. 2/0271/17, 2/0138/20 and 2/0052/18.
Acknowledgments:
We are grateful to Andrew Collins for his critical reading and English corrections.
Graphical abstract and Figure 2were created with BioRender.com.
Conflicts of Interest: The authors declare no conflict of interest.
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... Breast cancer (BC) remains one of the most common cancer types and a leading cause of cancer-related deaths among women worldwide [1]. Although innovative medical modalities and multidisciplinary approaches have contributed significantly to improved effectiveness of modern therapeutic regimens, the gradual development of drug resistance remains a serious clinical problem [2,3]. HER2-positive BC is a hormone-receptor negative, HER2-enriched molecular subtype, with faster growth and worse prognosis than luminal cancer. ...
... These data, including the upregulation of MUC1 in JIMT-1 cells, indicate that the use of epigenetic regulators for treating solid tumors should be considered cautiously. Targeted delivery over systemic application might be a more efficient therapeutic mode to avoid possible off-target DAC effects [3]. ...
Article
Full-text available
Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.
... Luminal A is characterized by the presence of estrogenic receptor (ER+), presence or absence of progesterone receptor (PR±), and absence of epidermal growth factor receptor-2 (HER2-); luminal B is characterized by (ER+), (PR±), and (HER2+); whereas the TNBC lacks expression of the three receptors (ER-), (PR-) and (HER2-) [3,4]. Several BC subtypes are treated with different primary therapeutic protocols, but none of them use epigenetic drugs [5]. Interest in treating BC with epigenetic drugs is increasing, mostly because epigenetic alterations such as DNA methylation and acetylation status of histones have been identified as important factors that contribute to tumorigenesis and BC progression [6,7]. ...
Article
Full-text available
Breast cancer (BC) is the most frequently diagnosed cancer and is the second-most common cause of death in women worldwide. Because of this, the search for new drugs and targeted therapy to treat BC is an urgent and global need. Histone deacetylase 6 (HDAC6) is a promising anti-BC drug target associated with its development and progression. In the present work, the design and synthesis of a new family of dihydropyrazole-carbohydrazide derivatives (DPCH) derivatives focused on HDAC6 inhibitory activity is presented. Computational chemistry approaches were employed to rationalize the design and evaluate their physicochemical and toxic-biological properties. The new family of nine DPCH was synthesized and characterized. Compounds exhibited optimal physicochemical and toxicobiological properties for potential application as drugs to be used in humans. The in silico studies showed that compounds with –Br, –Cl, and –OH substituents had good affinity with the catalytic domain 2 of HDAC6 like the reference compounds. Nine DPCH derivatives were assayed on MCF-7 and MDA-MB-231 BC cell lines, showing antiproliferative activity with IC50 at μM range. Compound 2b showed, in vitro, an IC50 value of 12 ± 3 µM on human HDAC6. The antioxidant activity of DPCH derivatives showed that all the compounds exhibit antioxidant activity similar to that of ascorbic acid. In conclusion, the DPCH derivatives are promising drugs with therapeutic potential for the epigenetic treatment of BC, with low cytotoxicity towards healthy cells and important antioxidant activity.
... The reversibility of the modifications catalyzed by these epi-enzymes, and the discovery of several inhibitors of their activity, gave rise to investigations focusing on the use of epi-enzymes as molecular targets for BC treatment. Moreover, novel approaches based on combinatorial epigenetic therapy (epi-drug) with chemotherapies, ET, or immunotherapy may represent new therapeutic strategies to overcome tumor relapse exploitable for BC clinical management (Buocikova et al., 2020). ...