Pharmacological Research 163 (2021) 105320

Contents lists available at ScienceDirect

Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs

Breast cancer stem cells, heterogeneity, targeting therapies and

therapeutic implications
Xiaobin Zeng a, b, 1, Chengxiao Liu a, 1, Jie Yao a, Haoqiang Wan a, b, c, Guoqing Wan d,
Yingpeng Li c, **, Nianhong Chen a, e, f, *
a
Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People’s Hospital, The Second Clinical Medical College, Jinan University, The First
Affiliated Hospital, Southern University of Science and Technology, Shenzhen, Guangdong, 518020, PR China
b
Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, Medicine School of Shenzhen University, Shenzhen, Guangdong Province, 518037, PR China
c
Department of Gastroenterology, (Longhua Branch), Shenzhen People’s Hospital, 2nd Clinical Medical College of Jinan University, Shenzhen, Guangdong Province,
518120, PR China
d
Shanghai University of Medicine and Health Sciences Affiliated Zhoupu Hospital, Shanghai, 201318, PR China
e
Department of Cell Biology & University of Pittsburgh Cancer Institute, School of Medicine, University of Pittsburgh, PA, 15261, USA
f
Laboratory of Signal Transduction, Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, 10065, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Both hereditary and sporadic breast cancer are suggested to develop from a stem cell subcomponent retaining
Breast cancer stem cell most key stem cell properties but with dysregulation of self-renewal pathways, which drives tumorigenic dif­
Breast cancer therapy ferentiation and cellular heterogeneity. Cancer stem cells (CSCs), characterized by their self-renewal and dif­
Cancer heterogeneity
ferentiation potential, have been reported to contribute to chemo-/radio-resistance and tumor initiation and to
Ubiquitination
be the main reason for the failure of current therapies in breast cancer and other CSC-bearing cancers. Thus, CSC-
targeted therapies, such as those inducing CSC apoptosis and differentiation, inhibiting CSC self-renewal and
division, and targeting the CSC niche to combat CSC activity, are needed and may become an important
component of multimodal treatment. To date, the understanding of breast cancer has been extended by advances
in CSC biology, providing more accurate prognostic and predictive information upon diagnosis. Recent im­
provements have enhanced the prospect of targeting breast cancer stem cells (BCSCs), which has shown promise
for increasing the breast cancer remission rate. However, targeted therapy for breast cancer remains challenging
due to tumor heterogeneity. One major challenge is determining the CSC properties that can be exploited as
therapeutic targets. Another challenge is identifying suitable BCSC biomarkers to assess the efficacy of novel
BCSC-targeted therapies. This review focuses mainly on the characteristics of BCSCs and the roles of BCSCs in the
formation, maintenance and recurrence of breast cancer; self-renewal signaling pathways in BCSCs; the BCSC
microenvironment; potential therapeutic targets related to BCSCs; and current therapies and clinical trials tar­
geting BCSCs.

1. Introduction
WHO statistics indicate that in 2018, breast cancer caused 627,000
deaths and 2.1 million new cases were diagnosed. Among the causes of
death, many patients die of metastasis, recurrence and drug resistance
due to failure of current therapies. Over the past decades, mortality from
breast cancer has decreased significantly in the United States and else­
where, largely due to improvement in early detection and the develop­
ment of more effective adjuvant therapies [1]. However, cancer
mortality is still high, and there is no curative treatment.
Accumulating evidence indicates that normal breast cancer cells can
acquire cancer stem cell (CSC) properties, resulting in an increase in

* Lead corresponding author at: Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People’s Hospital, The Second Clinical Medical
College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology, Shenzhen, Guangdong, 518020, PR China.
** Corresponding author at：Department of Gastroenterology, (Longhua Branch), Shenzhen People’s Hospital, 2nd Clinical Medical College of Jinan University,
Shenzhen, Guangdong Province, 518120, PR China.
E-mail addresses: pqr9hs@163.com (Y. Li), nhchen2004@hotmail.com (N. Chen).
1
These authors have contributed equally to this work.

https://doi.org/10.1016/j.phrs.2020.105320
Received 17 October 2020; Received in revised form 27 November 2020; Accepted 27 November 2020
Available online 1 December 2020
1043-6618/© 2020 Elsevier Ltd. All rights reserved.
X. Zeng et al.
their chemo-/radio-resistance ability. In addition, the proportion of
breast cancer stem cells (BCSCs) in tumors is increased after chemo-/
radio-therapy, which further increases tumor heterogeneity and the
presence of BCSCs, subsequently leading to therapeutic failure or cancer
recurrence. Eradicating CSCs, which are proposed to be the root cause of
cancer initiation and recurrence, has been thought to be a promising
approach to improve cancer survival or even to cure cancer. In research
on killing CSCs, many promising methods have been developed,
including molecular targeted therapy, microRNA (miRNA)-targeted
therapy, differentiation therapy, tumor microenvironment-targeted
therapy, and immunotherapy.
In this article, we review the characteristics of BCSCs, discuss the
regulatory pathways of these cells and potential targets for therapy, and
consider the implications of CSC models for clinical trial design.
2. BCSCs
As early as 1937, CSCs were confirmed to be present in human acute
myeloid leukemia. In 2003, the first solid CSCs were identified in breast
tumors. Subsequently, other CSCs were isolated and identified from
many tumors, including brain, melanoma, prostate, ovarian, colon,
pancreatic, and other types of tumors. CSCs can be defined by three
functional characteristics: the capacity for tumor initiation and forma­
tion, long-term self-renewal, and differentiation into non-self-renewing
cells [2,3]. Self-renewal can occur via either symmetric division or
asymmetric cell division. Two daughter stem cells are produced via
symmetric self-renewal. Alternatively, via asymmetric self-renewal, a
daughter stem cell and a nonstem differentiated cell are produced [4,5].
The serial tumor transplantation assay, which can demonstrate
self-renewal and differentiation potential, has been used as a gold
standard to ascertain whether a specific population of cells comprises
CSCs [6]. CSCs may originate from normal adult stem cells, progenitor
cells, differentiated cells, fused cells from stem cells, and cancer cells or
cells that acquire DNA from other cells [7,8]. CSCs are more likely to be
transformed from normal stem cells than from other cells due to accu­
mulated mutations during their relatively long lives [9]. Progenitors and
other differentiated cells are also thought to give rise to CSCs via mu­
tations, especially mutations in self-renewal genes. However, these cells
must survive for a relatively shorter time and harbor more mutations
than stem cells.
One hypothesis proposed that mutations in mammary stem cells can
primarily trigger the formation of breast cancer and that mutations in
progenitor cells contribute to transforming events in the mammary gland.
Mouse BCSCs, characterized as having an EpCAM+/CD44+/CD24− /low
phenotype, were first identified in a tumorigenesis experiment in xeno­
transplanted immune-deficient mice [10]. Later, human cells with a
CD44+/CD24–/low/Lin– expression phenotype were isolated and identi­
fied as BCSCs. Tumors formed when 100 CD44+/CD24–/low/Lin– cells
were injected into nonobese diabetic/severe combined immunodefi­
ciency (NOD/SCID) mice. Conversely, no tumors formed when 20,000
cells without this phenotype were injected. This experiment demonstrated
that CD44+/CD24–/low/Lin– subtype cells have key characteristics,
including self-renewal and differentiation abilities, and that tumors can
originate from a rare population of BCSCs with specific markers [10].
Molecular classification of breast cancer has been successfully
applied for the design of targeted therapies, resulting in significant ef­
fects and an increase in the survival rate [11,12]. Breast cancers of
different subtypes and histological stages have been reported to express
unique markers, including the stem cell markers CD44 and CD24, as well
as other markers, such as vimentin, osteonectin, connexin 43, MUC1,
and CK18 [13]. Generally, highly heterogeneous expression of these
markers in different tumor subtypes and histological stages have been
demonstrated in previous studies. CD44 and CD24 are most commonly
expressed in basal-like tumors. Another stem cell marker, ALDH1, has
the highest expression in HER2+ and basal-like tumors. In contrast,
CK18, GATA3 and MUC1 are more commonly expressed in luminal-type
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Pharmacological Research 163 (2021) 105320
tumors. In addition to these findings based on preclinical and experi­
mental data, a competing hypothesis by Campbell et al. suggests that
CD44+/CD24− and CD44− /CD24+ cells may be competitive sub­
populations of cancer cells, and this hypothesis is supported by evidence
indicating that CD44+/CD24− cells are not associated with the pro­
gression or prognosis of breast cancer, however, CD44− /CD24+ cells are
negatively correlated with prognosis [14–18]. In addition, the
CD44+/CD24– profile is more closely related to basal breast cancer.
Expression of CD44 may indicate that any feature of CSCs enhances their
implantation [19]. Notably, luminal-type breast cancer-specific CSCs
have not been identified. However, the above stem cell markers are not
exclusively expressed on BCSCs but are also expressed on other cells.
Recently, a study reported the identification of a BCSC-like population
among 3 populations in a breast cancer cell line based on differential
gene transcription detected using single-cell RNA sequencing [20].
Therefore, DNA/RNA sequencing may reveal more information about
genes and allow more accurate identification of BCSCs in the future.
3. The roles of BCSCs in models of heterogeneity in breast cancer
and the case for the development of BCSC-targeted therapy
Variability among tumors arising in the same organ or patient is
often referred to as intertumor heterogeneity. Because of intertumor
heterogeneity, tumors can be classified into different types according to
their morphology, molecular expression profiles or genomic copy
number patterns [21,22]. Two hypothetical models have been proposed
to explain the intertumor heterogeneity: 1) different genetic and/or
epigenetic events occur within the same target cell, leading to different
tumor phenotypes, and 2) different tumor subtypes are derived from
different cell of origin in tissues [22] (Fig. 1). Breast cancer can be
classified into three subtypes based on the intertumor heterogeneity in
cell surface molecule expression: 1) hormone receptor-positive (HR+)
breast cancer, with expression of the estrogen receptor (ER) and/or
progesterone receptor (PR); 2) human epidermal receptor 2 (HER2)-­
amplified (also called ERBB2-amplified) breast cancer; and 3)
triple-negative breast cancer (TNBC), lacking expression of ER, PR, and
HER2 [23].
The cancer cells within a given primary tumor show amazing clonal,
genetic and morphological diversity (i.e., intratumor heterogeneity).
Tumor cells often have diverse functional properties. Different pop­
ulations of cancer cells endow the given tumor with multiple charac­
teristics supporting tumorigenesis, such as angiogenesis, invasion and
metastasis [24]. The diverse expression of markers in cells within a
tumor also reflects intratumor heterogeneity. For example, the per­
centage of ER-expressing cells can vary widely and range from 1 to 100
% within a single breast tumor.
The ‘clonal evolution’ and ‘CSC’ hypotheses constitute the two major
hypothetical models of tumorigenesis [14, 25]. These two models were
developed to explain intratumoral heterogeneity and intrinsic differ­
ences in the breast tumor regeneration capacity [26] (Fig. 2A and B).
The clonal evolution model is based largely on the hypothesis that any
undifferentiated or differentiated cell can accumulate mutations that
lead to tumor formation and to the generation of clonal populations of
cells within a tumor. In contrast, the CSC model proposes that exclu­
sively normal stem cells or their early progeny can be transformed into
CSCs by the accumulation of mutations. These proliferating stem cells
then drive tumor formation and recurrence by undergoing a carcino­
genic differentiation process [25,27]. In addition, a third hypothesis for
tumor generation denies the existence of clear subpopulations of cancer
cells within tumors (Fig. 2C), stating instead that cancer cells have
substantial diversity resulting from the accumulation of random and
gradual mutations [28,29]. The CSC model can explain breast cancer
tumorigenesis [10]. Some evidence supports the CSC model in tumori­
genesis: 1) the results from the breast tumorigenesis experiment of CSCs
in xenotransplanted immune-deficient mice, 2) markers for distant BCSC
clones, 3) the model showing intratumor heterogeneity of breast cancer
X. Zeng et al. Pharmacological Research 163 (2021) 105320

Fig. 1. Two models describing the establishment of intertumor heterogeneity.

Fig. 2. Proposed models explaining intratumor heterogeneity in breast cancer. Different models of tumor progression can explain distinct types of intratumor
heterogeneity (A–C), exemplified here by the clonal evolution (A), cancer stem cell (B), and mutator phenotype (C) models. The different models can result in distinct
spatial distributions of subpopulations.

3
X. Zeng et al.
tumors, and 4) the model showing breast cancer tumor relapse and
metastasis. However, the CSC model is controversial in breast cancer
principally because the xenotransplanted immune-deficient mouse
model cannot mimic the normal mouse and human environment, and
the major difference in BCSCs make presence of BCSCs questionable. A
CSC dynamic concept that emphases evolution and adaptation might be
a supplement to explain tumorigenesis, tumor relapse and metastasis
[30]. In general, CSCs in one tumor are genetically unstable, resulting in
the development of multiple distinct CSC clones (intratumor CSCs clone
hierarchy) in advanced cancer. After environmental selection including
the niche and niche changes due to metastasis, targeted therapy and
chemotherapy, adapted CSCs survive from the CSC clone pool in the
intratumor and intertumor hierarchy and develop to evolved tumors.
Competition among the CSC clones is another factor in the evolution of
cancer leading to a reduction in the diversity of CSC clones and
inter-CSC clone dominance. Distinct BCSC clones can be found in one
tumor [31]. However, it should be noted that BCSCs can be classified
into clear subtypes by biomarkers so the BCSCs may be more stable
genetically. In breast cancer, the evolution and adaptation of tumors
promoted by intratumor heterogeneity lead to underestimation by
tumor genomic profiling via single tumor biopsy sampling analysis,
which poses a major challenge to personalized medicine and identifi­
cation of biomarkers, especially those of BCSCs. Intratumor and inter­
tumor heterogeneity may foster tumor adaptation and therapeutic
failure through Darwinian selection in breast cancer, even though some
therapies may increase the fraction of CSCs. Killing all types of BCSCs is
still a potential basic strategy for curing breast cancer. However, the
strategy only targeting BCSCs may be ineffective as mentioned above, as
new CSCs may re-emerge by dedifferentiation from progenitor cells,
differentiated cells, fused cells, and cancer cells or cells that acquire
stemness genes from other cells. Thus, eradicating the most types of
breast cancer cells may be the most effective basic strategy for curing
breast cancer.
4. BCSC-targeted therapies and their implication
Most currently used therapies aim to eradicate as many cancer cells
as possible to reduce the tumor size, but CSCs often survive under these
therapies, leading to treatment failure or recurrence. CSCs show rela­
tively high resistance to chemo-/radiotherapies [32,33]. As mentioned
above, BCSCs have high levels of heterogeneity and plasticity. Thus,
BCSCs express various proteins that can block drugs and promote the
transition between epithelial and mesenchymal states to contribute to
therapeutic resistance [34]. How BCSCs acquire the resistance from EMT
Fig. 3. Signal transduction networks in the BCSC microenvironment. Schematic illustration of important signal transduction networks, therapeutic targets, and
targeting agents.
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Pharmacological Research 163 (2021) 105320
is still unclear, but the resistance is related to antiapoptotic effects and
induction of quiescence [35]. Most CSCs are in the G0 phase of the cell
cycle and divide infrequently, which can lead to resistance to cell
cycle-specific chemotherapeutic agents. Some ATP-binding cassette
proteins (ABCG2, ABCB1 and ABCC1) highly expressed in the plasma
membrane of CSCs have been indicated to export chemotherapeutic
agents [36–38]. Furthermore, the BCSC marker ALDH plays important
roles in the reduction of oxidative stress; resistance to some chemo­
therapeutic agents, such as oxazolidine, taxanes, and platinum; and
removal of free radicals resulting from radiotherapy [39,40]. In addi­
tion, the active DNA damage response and its repair pathways in BCSCs
lead to a reduction in apoptosis or other forms of cell necrosis [19]. In
addition to the intrinsic genetic and epigenetic aberrations involved in
resistance, the extrinsic factor, the tumor microenvironment, also reg­
ulates BCSC heterogeneity and plasticity and further affects the thera­
peutic resistance.
Thus, several treatment options may be efficient for targeting BCSCs:
1) therapies targeting self-renewal signaling pathways, resulting in the
death of BCSCs; 2) therapies targeting the microenvironment of breast
CSCs; 3) therapies targeting drug transporters; 4) differentiation ther­
apy; and 5) immunotherapy. In addition, therapies targeting miRNAs
and the ubiquitin–proteasome system (UPS) may be options. Interest is
increasing in combining such treatments with conventional anticancer
treatments (chemo-/radiotherapy) targeting the renewal and differen­
tiation signaling pathways [such as the Notch and Hedgehog (Hh)
pathways] and/or with immunomodulatory components (such as PD-1
and PD-L1) exploited by CSCs [41].
5. BCSC self-renewal pathways and targeted therapies
The current understanding of the pathways that regulate the self-
renewal of breast stem cells indicates that dysregulation of these path­
ways may lead to carcinogenesis. In the mammary gland, the signaling
pathways regulating the self-renewal of breast stem cells, such as the Hh,
Wnt/β-catenin, and Notch pathways, play an important role both in
embryogenesis and organogenesis and in the maintenance of adult tis­
sues via regulation of the balance between self-renewal and differenti­
ation of stem cells (Fig. 3). In the regulation of organs, these signaling
pathways promote mammary gland organogenesis, development, and
maintenance by regulating the balance between the differentiation and
self-renewal of stem cells [37]. Studies have demonstrated that dysre­
gulation of these pathways may cause malignant transformation [42].
Thus, these pathways constitute potential targets for elimination of
BCSCs [43].
X. Zeng et al.
5.1. Notch signaling
The Notch signaling pathway helps to determine stem cell fate [44]
and helps both normal and malignant stem cells to maintain their sur­
vival and activity [45,46]. A Notch transmembrane receptor is activated
by a ligand [Delta-like (DLS) 1, 3, and 4; Jagged 1 and 2] presented by a
neighboring cell and undergoes serial cleavage events mediated by
gamma secretase. The Notch intracellular domain is separated from the
Notch protein due to this cleavage and translocates to the nucleus,
leading to activation of downstream transcription factors such as Hes
and Hay [44]. Aberrant activation of the Notch signaling pathway,
which promotes chemo-/radioresistance, has been reported in breast
cancer cells and BCSCs [46,47]. Moreover, the levels of Notch and the
p53 inhibitor NUMB were found to be downregulated in approximately
50 % of human breast tumor samples [48]. Gamma secretase inhibitors
(GSIs) efficiently block the Notch signaling pathway. Indeed, a phase I/II
clinical trial showed that the GSI MK-0752 in combination with doce­
taxel significantly reduced the numbers of CD44+/CD24− stem cells and
ALDH+ stem cells and decreased the mammosphere formation efficiency
in breast tumors [49]. Approximately three other GSIs have completed
clinical trials or are currently in phase II clinical trials in breast cancer,
although none of these has been approved for treatment [19].
5.2. Wnt signaling
Members of the Wnt family, a family of secreted signaling proteins,
stimulate target cells by binding receptors on the cell surface. Canonical
Wnt signaling is triggered after Wnt proteins bind to receptors of the
Frizzled family and the receptors low-density lipoprotein-related pro­
teins LRP5 and LRP6. This Wnt/Frizzled/LRP5/6 complex transduces
signals to the β-catenin and WNT/stabilization of proteins (STOP)
signaling cascades. β-Catenin translocates into the nucleus and activates
LEF1/TCF family transcription factors. Noncanonical Wnt signaling
activates Wnt/planar cell polarity (PCP), Wnt/receptor tyrosine kinase
(RTK) and WNT/Ca2+ signaling cascades via FZD receptors and/or
ROR1/ROR2/RYK coreceptors [50,51]. The Wnt pathway has been
shown to be very important for the biological functions and mainte­
nance of CSCs [50]. Additionally, activated Wnt/β-Catenin signaling
plays a key role in epithelial-mesenchymal transition (EMT), in which
cellular plasticity induces the generation of CSCs [52]. Specifically, in
BCSCs, aberrant activation of the Wnt signaling pathway has been re­
ported [53,54] and is related to dysregulation of noncoding RNAs
[55–57]. Treatment with inhibitors of Wnt signaling has demonstrated
inhibition of BCSC growth in vitro and in a xenograft model [58].
Encouragingly, inhibitors targeting BCSCs are entering phase I clinical
trials [50].
5.3. Hh signaling
The Hh signaling pathway contributes widely to the regulation of cell
proliferation, migration, and differentiation in space-, time- and
concentration-dependent manners [59–61]. Three lipid-modified
secreted ligands—Sonic Hedgehog (SHH), Indian Hedgehog (IHH),
and Desert Hedgehog (DHH)—transduce signals through Patched
(Ptch1), a 12-pass transmembrane spanning receptor, to the trans­
membrane protein Smoothened (SMO). Activated SMO activates a series
of downstream intracellular proteins and eventually results in upregu­
lation of Hh target genes. Studies have indicated that Hh signaling helps
to regulate CSCs in many cancers, including breast cancer. Hh signaling
can act on the same tumor through various signaling pathways that
mediate interactions between tumor cell populations and their micro­
environment [62]. The expression of Ptch1 and the downstream proteins
Gli1 and Gli2 is upregulated in normal breast stem cells and BCSCs but is
downregulated during cell differentiation [63]. Hh signaling proteins
are potential therapeutic targets in many types of CSCs, including
BCSCs. Inhibitors of these signaling proteins have been reported to
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Pharmacological Research 163 (2021) 105320
eliminate BCSCs [64]. In addition, the combination of the Hh inhibitor
GDC-0449 (vismodegib) with paclitaxel, epirubicin, and cyclophos­
phamide has been evaluated for the treatment of triple-negative breast
cancer in a phase II clinical trial.
5.4. Her2 signaling
Overexpression of the HER-2 gene, a member of the epidermal
growth factor receptor family, is an early event in the formation of
sporadic breast cancer [65]. Approximately 25 %–30 % of breast cancer
patients have tumors of the HER-2-amplified subtype, which is corre­
lated with a low survival rate and a high risk of relapse [66]. Emerging
studies have suggested that HER2 regulates BCSC activities, including
self-renewal and radioresistance [67–69]. HER2-targeted therapies are
very important and successful breast cancer therapeutics, especially in
the treatment of HER-2-overexpressing and HER-2+ breast cancers.
Clinical data has shown that patients treated with HER2-targeted anti­
bodies and agents such as lapatinib, Kadcyla and trastuzumab had
increased progression-free survival and overall survival rates among
patients with advanced -stage ER+ cancer that was resistant to hormone
therapy [70]. Moreover, trastuzumab contributes to reducing cancer
recurrence [71]. If BCSCs are the root cause of breast cancer recurrence,
the significant clinical efficacy of HER-2 inhibitors may result from
direct targeting of BCSCs by these drugs. Emerging studies have con­
nected the efficacy of HER-2-targeting agents to their ability to eliminate
BCSCs [72]. For example, trastuzumab has been reported to reduce the
CSC population in HER2-overexpressing breast cancer cell lines [73]. In
addition, lapatinib, an EGFR and HER-2/neu (ErbB-2) dual tyrosine ki­
nase inhibitor, reduced the percentage of the CD44+/CD24low popula­
tion and the ability for self-renewal, as assessed by mammosphere
formation, in ALDH1+/HER2+ BCSCs.
5.5. PTEN and AKT signaling
The PTEN tumor suppressor gene encodes a lipid and protein phos­
phatase (PTEN). Downstream of PTEN is protein kinase B (Akt), which is
a key regulator of Wnt and PI3K signaling pathways and plays a crucial
role in energy homeostasis [74]. PTEN regulates PI3K signaling by
dephosphorylating PIP3, a product of PI3K. Mutations in PTEN are
usually found in human breast cancers, and these mutations usually lead
to accumulation of PIP3, subsequently activating a series of signaling
cascades including AKT and other kinases [75]. Activated PTEN down­
regulates p27, leading to cell cycle progression and downregulation of
proapoptotic factors such as caspase-9 and BAD. Loss of PTEN was found
in 40 % of human breast cancers, and dysregulation of PI3K/AKT
signaling was found in up to 70 % of cancers [76,77]. PTEN has previ­
ously been reported to regulate the self-renewal of neuronal stem cells
and hematopoietic stem cells [78]. Later, evidence emerged indicating
that repressing PTEN contributes to BCSC self-renewal and survival via
the AKT signaling pathway [79]. These findings support the develop­
ment of inhibitors of PTEN/AKT signaling for targeting BCSCs and breast
cancer. Korkaya et al. reported that perifosine, an AKT inhibitor, inhibits
the formation of mammospheres in vitro and eradicates breast tumor
xenografts [80]. However, a phase II clinical trial evaluating targeting of
the AKT protein in advanced breast cancer patients with dysregulation
of PTEN/AKT/PI3K has been completed, but the effect of this mono­
therapy was not significant [81].
6. DNA damage response in BCSCs and therapeutic strategies
Cells must repair various genotoxic injuries, such as single and strand
breaks, intrastrand and interstrand crosslinks with bulky adducts, base
mismatches, insertions and deletions and base alkylation, due to
endogenous and exogenous causes via multiple DNA damage responses
(DDRs). Normal stem cells have a strong DDR to prevent the accumu­
lation of genetic damage and prevent mutations from passing to
X. Zeng et al.
daughter cells, which is particularly harmful to the whole organism
[82]. CSCs originate from normal stem cells (as mentioned above, the
CSCs are more likely to originate from normal stem cells than from
differentiated cancer cells) and have a strong DDR, which is stronger
than that in normal breast cancer cells [83,84]. Accumulating research
demonstrates that CSCs and cancer cells resist chemo-/radiotherapies
via the DDR [85]. As a result, theoretically, DDR inhibitors can enhance
efficiency of the therapies that aim to induce DNA damage in both of
cancer cells and CSCs. DDR inhibitors have been shown to inhibit DDRs
of breast cancer cells due to defects in DNA damage repair pathways
[82] and to inhibit DDRs in some CSCs such as glioblastoma [86–88] and
colon cancer [89,90]. A PARP1 inhibitor combined with the
cyclin-dependent kinase inhibitor dinaciclib limited the growth of
BCSCs [91]. BCSCs in TNB were resistant to a PARP1 inhibitor mediated
by RAD51 [92]. In addition to PARPs, BCSCs respond to DNA damage by
ATM kinase activation [93,94]. In summary, new DDR inhibitors for
BCSCs need to be screened.
7. The BCSC microenvironment and related therapies
The microenvironment surrounding stem cells has been termed the
stem cell niche. BCSCs are regulated by extrinsic factors within their
niche. The BCSC niche contains various cells, including immune cells,
adipocytes, mesenchymal stem cells, endothelial cells and fibroblasts
[95]. These other cells interact with CSCs through the cytokine network
via paracrine pathways [96]. Several cytokines secreted mainly from
immune cells, such as IL-6, IL-8, and the receptor CXCR1, have been
demonstrated to regulate BCSC activity in vitro and in xenograft models
[97,98]. CXCR1 expression is higher in ALDH+ cells than in ALDH– cells
of various breast cancer cell lines, and IL-8 increases the number of CSCs
[99]. Furthermore, reports indicate that chemotherapy-induced cyto­
toxicity may increase the local IL-8 level. The most likely outcome of this
increase may be an increase in the proportion of CSCs. The levels of IL-6
and IL-8 in the serum of patients have been correlated with the devel­
opment of metastasis and low therapeutic efficacy in advanced breast
cancers [97,100]. In summary, cytokines and their receptors play
important roles in the regulation of CSC biological characteristics by
changing the cell niche, and inhibitor treatments have been designed to
block inflammatory cytokines and/or their receptors in BCSCs. In
addition, the complex BCSC niche could be targeted for treatment.
7.1. Transforming growth factor-β (TGF-β)
The TGF-β signaling pathway plays an essential role in the regulation
of stem cell activity. On the one hand, TGF-β induces epithelial cells to
undergo EMT, leading to the expression of specific proteins in stem cells
and CSCs, which was found to lead in turn to increases in mammosphere
formation in vitro and tumorigenicity in a mouse model [34]. TGF-β
signaling is activated in CD44+/CD24–/low BCSCs, leading to upregula­
tion of vimentin, CTGF, SERPINE1, SPARC and TGFBR2 expression in
these cells [101]. Notably, inhibition of TGF-β can suppress the occur­
rence of breast cancer by affecting CSCs or early progenitor cells in
xenotransplantation models [102]. In addition, the crosstalk between
ER signaling activity and TGF-β signaling has been reported in the
regulation of breast stem cell maintenance as well as cell proliferation
and epithelial cell apoptosis. The anti-TGF-β/PD-1 bifunctional fusion
protein M7824 is in a phase II clinical trial for the treatment of small cell
lung cancer. However, to date, no phase II-IV clinical trials have been
conducted and no drugs targeting TGF-β in breast cancer have been
approved.
Currently, therapies specifically targeting the CSC niche are being
developed. As a receptor for hyaluronic acid and other extracellular
matrix proteins, as well as a cofactor for growth factors and cytokines,
CD44 regulates cell migration, proliferation, differentiation and survival
by transducing signals from the extracellular matrix to the intracellular
environment [103]. In BCSCs, CD44 is a marker and regulates various
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Pharmacological Research 163 (2021) 105320
CSC biological activities, such as self-renewal. An anti-CD44 antibody
was found to significantly reduce the growth of human breast tumors in
a xenograft model [104]. Integrins, a family of membrane receptors,
mediate cell-extracellular matrix and cell-cell interactions in BCSCs
[105]. In a mouse model, the number of BCSCs was reduced by ablation
[105] or inhibition [106] of focal adhesion kinase (FAK), a key mediator
of integrin signaling, suggesting that FAK is an oncogenic protein [105].
Although several FAK inhibitors have been developed, none have been
used to treat breast cancer.
In addition, reports indicate that antibodies targeting the IL-8 receptor
CXCR1 and the small molecule CXCR1/CXCR2 inhibitor repertaxin can
target BCSCs in xenograft models, inhibiting tumor growth and metastasis
[80]. The effect of these inflammatory cytokines on BCSCs might contribute
to these clinical effects. Recently, overexpression of Snail or Twist, two
inducible EMT transcription factors, was found to increase the
CD44+/CD24− subpopulation in immortalized human breast epithelial cells.
The cells that underwent EMT exhibited increased mammosphere-forming
capacity and tumor initiation potential in NOD/SCID mice. These results
suggested a relationship between EMT and the CSC phenotype. Subse­
quently, specific screened small molecules targeting EMT cells, such as sali­
nomycin, etoposide, abamectin and nigericin, were confirmed to have the
greatest effect in vitro in follow-up studies. Collectively, these findings suggest
that the cytokine network plays an important role in regulating CSCs in their
niche and that developing strategies to interfere with these loops may provide
a novel approach to target CSC populations.
8. The UPS in BCSCs and UPS-targeted therapies
The UPS plays an important role in cell proliferation, transcription,
the immune response, DNA damage repair, apoptosis, neoplastic growth
and metastasis. It is also crucial for the regulation of cell cycle protein
degradation [107]. Ubiquitination modifies target proteins through a
series of enzymes, including ubiquitin-activating (E1) enzymes,
ubiquitin-conjugating (E2) enzymes and ubiquitin ligase (E3) enzymes
[108,109]. E3s, which function as key enzymes in this process by
recruiting specific target proteins, are classified as RING finger, HECT,
Ubox, PHD finger and RBR E3s based on their structures [110] (Fig. 4).
A zinc finger transcription factor, kruppel-like factor 4 (KLF4, GKLF),
regulates cellular responses to various cellular stresses and signals,
including DNA damage, oxidative stress, an inflammatory environment
and stem cell renewal-related signals [111]. KLF4 is a fast-turnover
protein, and its turnover half-life is governed by the UPS [112,113].
In breast cancer, the oncogenic effect of KLF4 has been extensively re­
ported [113–117]. Notably, other reports have shown the tumor sup­
pressor function of KLF4 [118,119]. As KLF4 is essential for induced
pluripotent stem (iPS) cell generation, it is believed to play a critical role
in mammary stem cell and BCSC maintenance and differentiation. For
example, Ai et al. reported that KLF4 is essential for the maintenance,
migration and invasion of BCSCs [120]. Estrogen signals can lead to
KLF4 protein accumulation by inhibiting its CUL2/VHL-mediated pro­
tein degradation to further trigger estrogen-induced signaling [121].
Furthermore, KLF4 is a key node for crosstalk between TGF-β signaling
and ER signaling. These results further reveal KLF4 as a critical factor
that controls normal and CSC maintenance and differentiation and plays
an essential role in breast cancer tumor occurrence and progression,
suggesting that KLF4 may serve as a biomarker or therapeutic target for
BCSCs. F-box and WD repeat domain-containing 7 (FBW7) is an
important substrate adaptor for ubiquitin E3 ligases, which act mainly
on many important oncogenic proteins, including Notch, c-Myc, KLF5,
Mcl1, mTOR, presenilin, cyclin E and c-Jun, for ubiquitin-mediated
proteolysis. In addition, FBW7 was confirmed to act as a tumor sup­
pressor in a gene knockout mouse model. In various stem cells, such as
hematopoietic stem cells, hepatic stem cells and neural stem cells, FBW7
also regulates self-renewal, differentiation and maintenance. Therefore,
FBW7 could be targeted in human diseases and for BCSC-targeted
therapy [122].
X. Zeng et al.
Fig. 4. Pathway of UPS-mediated degradation in CSCs. Proteins are targeted for degradation by the UPS through enzymatic cascades.
Accumulating findings have shown that inhibition of the proteasome
is another potential therapeutic approach for cancer. The proteasome
inhibitor PS-341 is an example of a novel class of potent and effective
antitumor agents [107]. Chemotherapeutic agents, such as ifosfamide
and cisplatin, downregulate ubiquitin–proteasome-mediated proteolysis
by inhibiting the expression of proteasomal subunits. Given its ability to
control stem cell differentiation, regulation of the UPS by novel ap­
proaches is likely to significantly impact the design of new
disease-specific therapies for cancer, including therapies targeting
BCSCs.
9. Noncoding RNAs and BCSCs
MiRNAs are small noncoding regulatory RNAs that control mRNA
translation and posttranslational modification of the encoded proteins
via inhibition of ribosomal function, dealkylation of the poly (A) tail,
uncapping of the 5′ -cap structure and degradation of the target mRNA.
Many miRNAs play essential roles in a wide range of tissues and tumors,
while other miRNAs may have functions only in specific tissues, specific
tumors or specific tumor progression processes [123]. The miRNA 200
family (miR-200a, 200b, 200c, 141, 182, 183, and 96), the let-7 family
(let-7a, b, c, d, e, f, g, and i) and miR-30e suppress the self-renewal of
BCSCs and suppress their chemoresistance and differentiation
[124–126]. Furthermore, the miR-200 family suppresses the expression
of ZEB1 and ZEB2, resulting in inhibition of EMT [127]. However,
miR-181a, miRNA181 and miR-155 were found to enhance cell
self-renewal, sphere formation, or tumor progression in breast cancer. In
addition to the tumor suppressor and oncogenic miRNAs discusses
above, at least 19 other miRNAs or miRNA families, such as miRNA 600
and 760, have been identified to be tumorigenesis-related factors in
BCSCs [128]. Recently, a study reported that miRNA-140 inhibits the
proliferation of BCSCs by downregulating the expression of Wnt (Fig. 5)
[129].
Long noncoding RNAs (lncRNAs) are a population of noncoding
RNAs with a length of more than 200 nucleotides compared with the
20–24 nucleotides in miRNAs. LncRNAs regulate diverse cellular pro­
cesses, as well as tumorigenesis, usually as mediators in signaling
pathways, and have been demonstrated to play important roles in CSC
plasticity and EMT programs. Upregulation of the lncRNA HOTAIR in
BCSCs contributes to downregulation of miRNA-7, which is a suppressor
7
Pharmacological Research 163 (2021) 105320
of EMT [130]. Additionally, lncRNA-Hh enhances EMT via the Hh
signaling pathway [131]. In addition, 16 other lncRNAs have been found
to be involved in the formation of BCSCs and/or maintenance of these
cells [132].
Accumulating evidence indicates that circular RNAs (circRNAs),
characterized by their covalently closed circular structure containing
approximately 1000 nucleotides, play oncogenic roles in breast cancer
development. CircRNAs have been reported to be associated with EMT
in breast cancer [133–137]. Moreover, novel EMT-related circRNAs, for
example, SCYL2 [138] and ANKS1B [139], have been identified in
breast cancer tissue. Recently, a profile of a set of circRNAs in BCSCs was
defined; a total of 19 differentially expressed circRNAs were identified,
among which VRK1 was identified as a suppressor of BCSCs [140].
Two therapeutic methods—viral vector or nanoparticle (liposome,
polyethylenimine, or dendrimer) delivery systems and antagonists to
eliminate oncogenic miRNAs or miRNA mimics to restore tumor sup­
pressor miRNAs—have been demonstrated to prevent tumor occurrence
and progression in vitro and in mouse tumor models [141]. In BCSCs,
miRNA-34a mimics were reported to be able to eliminate BCSCs directly
by inhibiting the accumulation of C22ORF28 [142]. In addition,
miRNA-200c mimics improved the targeting effect of paclitaxel to
BCSCs in vitro [143]. Based on these findings, the development of
BCSC-targeted noncoding RNA therapy combined with other therapies is
a future direction for breast cancer eradication.
10. Differentiation therapy in BCSCs
Cell cycle- or DNA damage-dependent chemotherapeutic agents are
toxic to rapidly dividing cells, but long-lived and relatively quiescent
CSCs are more resistant to these cytotoxicities. Therefore, via a method
inducing cell differentiation, CSCs can transform to an active state to
divide, differentiate and lose their self-renewal potential, an effect that
can increase their sensitivity to chemo-/radiotherapy. Differentiation
therapy has been successfully used to treat specific hematological can­
cers. However, the application of differentiation therapy specifically
targeting BCSCs needs further definition. One differentiation agent, all-
trans retinoic acid (RA), can promote the differentiation of BCSCs or
suppress the stemness of breast cancer cells, leading to a reduction in
their invasiveness and metastasis and an increase in their sensitivity to
chemotherapy. A recent report indicated that biological fluids, culture
X. Zeng et al. Pharmacological Research 163 (2021) 105320

Fig. 5. A summary of miRNA regulators and biomarkers in the processes of normal mammary gland maintenance and breast cancer initiation, metastasis, and
therapeutic resistance. In these four processes, mammary stem cells (MaSCs), breast cancer stem cells (BCSCs), metastatic CSCs (MCSCs), and therapy-resistant CSCs
(TRCSCs) are pivotal originators and/or critical cellular players. The miRNA regulators are listed as suppressor miRs (in green), oncomiRs (in red), and their targeted
genes (in blue). The miRNA biomarkers are listed as upregulated miRNAs (in red), downregulated miRNAs (in green), and related target genes (in blue).

supernatant from replicative senescent fibroblasts, human ovarian
follicular fluid and supernatant from fibroblasts subjected to serum
starvation for 16 h, regulate Oct4, Sox2, Nanog, and C-myc expression,
resulting in increased differentiation of BCSCs [144].
11. Therapies targeting drug transporters
Overexpression of ABC transporters is not only a property of stem
cells but also a feature of CSCs, including BCSCs [145]. ABC transporters
display strong drug efflux capacity; thus, these transporters provide a
protective mechanism against xenobiotic toxins and are partially
responsible for the resistance of CSCs to traditional therapies [145].
Inhibition of drug transporters may constitute a novel therapeutic means
of resensitizing CSCs. Pheophorbide, a chlorophyll catabolite, is a spe­
cific probe for ABCG2 that inhibits the efflux properties of ABCG2 [146].
Combined treatment with ABC transporter inhibitors and chemotherapy
could be used to increase the efficiency of chemotherapeutic drugs to kill
BCSCs [147]. Therefore, effective targeting of these molecules could be
vital since they play an important role in the resistance of BCSCs to
treatment.
12. Immunotherapy targeting BCSCs
The purpose of immunotherapy is to stimulate the immune response
or help the immune system to recognize and eliminate various patho­
genic factors, including cancer cells. Current potential immunothera­
peutic strategies targeting solid tumors, including breast cancer, are
common cancer vaccines and monoclonal antibodies. Adoptive immu­
notherapy has been successfully applied as chimeric antigen receptor T
(CAR-T) or TCR-T cell therapy in leukemia; thus, both treatments should
be considered in breast cancer.
Many vaccines for solid tumors have achieved different success rates
in preclinical or clinical trials, but only the HPV vaccine for cervical
carcinoma has been approved [148,149]. Tumors impair the immune
system, which contributes to cancer surveillance during breast tumor
progression and can suppress or eradicate tumors. Dendritic cell (DC)
vaccines are common vaccines in breast cancer treatment, and specific
antigens for BCSCs have been identified and targeted to elicit long-term
immune responses targeting CSCs. Ideal antigens should be exclusively
expressed on BCSCs and breast cancer cells but not on normal stem cells or
normal cells, although no ideal antigen has been found. The second-best
antigens are the BCSC markers that have been identified, such as CD44,
ALDH, Her-2 and CD49f. A HER-2/neu DC vaccine successfully induced
the production of anti-HER-2/neu antibodies, stimulated interferon-γ
expression and reduced the size of a spontaneous breast tumor in a mouse
model [150,151]. More potential onco-antigens in BSCs and breast cancer
cells as targets, including some cancer/testis antigens [152], TENM 4
[153], TLR2 and xCT, have been identified [154]. In addition to the DC
vaccine, adoptive HER-2-specific T cells have been reported to eliminate
specific breast tumor cells [155]. Determining the number of BCSCs in the
tumor is important; the extent of tumor regression depends on the escape
of BCSCs, which itself depends on the number of BCSCs in the tumor and
the ability to eradicate these cells after treatment.
In fact, each type of immunotherapy requires additional treatment,
such as targeting cytotoxic T lymphocyte antigen (CTLA)-4, PD-1 or PD-
L1, to block T cell regulation [156]. In recent years, immune checkpoint
inhibitors (ICIs) have shown positive effects in breast cancer treatment
alone or combination of other therapies. Many clinical trials on immu­
notherapy of the ICIs have been performed based on the data from
clinicaltrials.gov and PubMed. Combined therapy with atezolizumab, a

8
X. Zeng et al.
PD-L1 inhibitor, and albumin-bound paclitaxel is approved by the FDA
for the treatment of metastatic triple negative breast cancer (TNBC)
[153]. It has been reported that PD-L1 upregulates in BCSCs in vitro, but
further studies are still needed [157].
13. Perspective
Current cancer treatment methods have shown effectiveness in
removing a large number of differentiated cancer cells but fail to elimi­
nate CSCs that cause tumor recurrence. CSCs are resistant to the current
drugs, a property constituting an important obstacle for cancer treatment.
Targeting BCSCs is an extremely attractive approach for reducing breast
cancer metastasis and recurrence to markedly improve the remission rate
and quality of life of patients. Although many drugs and experimental
therapies have been developed for targeting CSCs, a main challenge is
distinguishing CSCs from normal stem cells. Breast cancer is a very het­
erogeneous disease, and no universal molecular, cellular or genetic sets of
markers for BCSCs have been identified. Therefore, the target on BCSCs
needs further clarification to effectively target the BCSC compartment,
thus avoiding the normal stem cell niche but destroying the CSC niche.
We must improve our understanding of both normal stem cell biology and
CSC biology in order to identify such targets. In particular, a deeper un­
derstanding of the control of stem cell self-renewal may lead to the
development of new therapies in the near future. In addition, approaches
for targeting BCSCs that would be fully optimal for combination therapies
with existing treatments need further development. Moreover, mini­
mizing or avoiding long-term and/or side effects should remain the ulti­
mate goal of targeting BCSCs.
Authors’ contributions
Xiaobin Zeng and Chenxiao Liu had performed and updated the
literature search, and drafted for the article. Nianhong Chen had the
idea, performed the literature search, data analysis, and drafted for the
article. Nianhong Chen and Yingpeng Li contributed to interpretation of
results and made critical revisions. Jie Yao, Haoqiang Wan and Guoqing
Wan gave some important suggestions, also made critical revisions. All
authors have reviewed the final version of the manuscript and approved
it for publication.
Funding
This work was supported by grants from the Natural Science Foun­
dation of China (81503221, 81703939), the Science and Technology
Planning Project of Shenzhen Municipality (JCYJ20170307095556333,
JCYJ20170413093108233, JCYJ20170307100726777), and the Natu­
ral Science Foundation of Guangdong Province (2017A030313659,
2014A030310365).
Declaration of Competing Interest
The authors reported no declarations of interest.
Acknowledgements
We thank Mr. Joe X. Qiao for his efforts in figure draft. We apologize
to colleagues in the field whose work was not included due to space
limitation.
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