Cancer Letters 372 (2016) 147–156

Contents lists available at ScienceDirect

Cancer Letters
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Mini-review

Lung cancer stem cells: The root of resistance

Lauren MacDonagh a, Steven G. Gray a, Eamon Breen b, Sinead Cuffe a, Stephen P. Finn a,c,
Kenneth J. O’Byrne d, Martin P. Barr a,*
a Thoracic Oncology Research Group, School of Clinical Medicine, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James’s Hospital and

Trinity College Dublin, Ireland

b Flow Cytometry Core Facility, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James’s Hospital and Trinity College Dublin, Ireland
c Department of Histopathology, St. James’s Hospital and Trinity College Dublin, Ireland
d Cancer & Ageing Research Program, Queensland University of Technology, Brisbane, Australia

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

Article history: In the absence of specific treatable mutations, platinum-based chemotherapy remains the gold stan-
Received 10 December 2015 dard of treatment for lung cancer patients. However, 5-year survival rates remain poor due to the
Received in revised form 6 January 2016 development of resistance and eventual relapse. Resistance to conventional cytotoxic therapies pres-
Accepted 7 January 2016
ents a significant clinical challenge in the treatment of this disease. The cancer stem cell (CSC) hypothesis
suggests that tumors are arranged in a hierarchical structure, with the presence of a small subset of stem-
Keywords:
like cells that are responsible for tumor initiation and growth. This CSC population has a number of key
Lung cancer
properties such as the ability to asymmetrically divide, differentiate and self-renew, in addition to having
Cancer stem cells
Chemotherapy increased intrinsic resistance to therapy. While cytotoxic chemotherapy kills the bulk of tumor cells, CSCs
Resistance are spared and have the ability to recapitulate the heterogenic tumor mass. The identification of lung
Markers CSCs and their role in tumor biology and treatment resistance may lead to innovative targeted thera-
pies that may ultimately improve clinical outcomes in lung cancer patients. This review will focus on
lung CSC markers, their role in resistance and their relevance as targets for future therapies.
© 2016 Elsevier Ireland Ltd. All rights reserved.

Introduction
Lung cancer and resistance
Lung cancer is the leading cause of cancer-related death world-
wide and is classified into two main subtypes; non-small cell lung
cancer (NSCLC) which accounts for approximately 85% of all lung
cancers and small-cell lung cancer (SCLC) which is diagnosed in 15%
of cases [1,2]. Histologically, NSCLC is further divided into three sub-
types; adenocarcinoma, squamous-cell carcinoma and large cell
carcinoma [3]. Smoking is a major risk factor for lung cancer and
is associated with all histological subtypes, in particular, squa-
mous cell carcinomas. Adenocarcinoma on the other hand, is the
predominant lung cancer subtype commonly seen in never smokers.
Despite improved advances in the treatment of lung cancer, the
5-year survival rate remains low, largely due to the emergence of
resistance prior to and during the course of treatment with che-
motherapy and radiation therapy. This resistance to therapy
represents a significant clinical challenge in the treatment of lung
cancer and contributes largely to disease progression, recurrence
* Corresponding author. Tel.: +353 1 8963620; fax: +353 1 8963503.
E-mail address: mbarr@stjames.ie; barrma@tcd.ie (M.P. Barr).
and mortality [4,5]. Despite intense efforts to overcome such re-
sistance in lung cancer and other cancer types using novel agents,
alone and in combination with chemo- and radiotherapy, the un-
derlying mechanisms conferring this resistant phenotype in lung
cancer remain largely unknown [6]. It is now well established that
CSCs constitute a unique subset of cells which are distinct from the
bulk of tumor cells by their exclusive ability to perpetuate the growth
of a malignant population of cells, indefinitely. This may explain the
ineffectiveness of many conventional therapies and patient relapse
[7]. These important clinical observations have stimulated intense
interest in experimental approaches for further investigation of CSCs
and their role in the treatment of drug resistant lung cancer.
The cancer stem cell hypothesis
The CSC hypothesis is now a well-accepted and widely studied
field within oncology. Cellular heterogeneity is a histological hall-
mark of many solid tumors and the CSC hypothesis suggests that
this heterogeneity within a tumor is due to a hierarchical cell
dynamic and the presence of a small subpopulation of cells that
display the properties of normal somatic stem cells [8]. These CSCs
are multipotent, and have two key characteristics of stem cells; the
abilities to self-renew and to differentiate, properties necessary for
tumor initiation and progression (Fig. 1A). CSCs can asymmetri-

http://dx.doi.org/10.1016/j.canlet.2016.01.012
0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.
148 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156
Fig. 1. Division strategies of cancer stem cells. (A) Cancer stem cells (purple) must
accomplish the dual task of self-renewal and generation of differentiated progeny
(blue). (B–D) Strategies that maintain a balance of stem cells and differentiated
progeny. (B) Symmetric cell division. Each cancer stem cell can give rise to two cancer
stem cells or to two differentiated progeny. (C) Asymmetric cell division, each cancer
stem cell gives rise to one cancer stem cell and one differentiated cell. (D) Cancer
stem cell maintenance may be a combination of symmetric and asymmetric divi-
sion strategies. A population strategy provides dynamic control over the balance
between stem cells and differentiated progeny, a capacity necessary for the main-
tenance or expansion of cancer stem cells.
cally divide (Fig. 1C), which enables them to simultaneously self-
perpetuate and to generate differentiated progeny and give rise to
heterogenic tumor, with a consistently maintained CSC subpopu-
lation [9,10]. Although asymmetric division allows for both self-
renewal and differentiated progeny, it does not allow the CSC
subpopulation to expand. However, the stem cell niche has been
shown to expand during development. Similarly, the CSC popula-
tion expands during periods of stress, suggesting that they can also
symmetrically divide (Fig. 1B) [11–13]. CSCs can rely on either asym-
metric or symmetric division or a combination of the two (Fig. 1D).
Previous studies have shown that a pure lung CSC population is
capable of giving rise to a heterogenic progeny of CSCs and non-
CSC cells through asymmetric cell division, whereas non-CSC lung
cancer cells undergo only symmetric division resulting in a pure non-
CSC cell population [14].
Cellular stress, such as chemotherapy treatment, can induce sym-
metric division of rare CSC populations and apoptosis of non-CSC
cells, therefore enriching the population [15]. Symmetric division
of the CSC population during initial cycles of chemotherapy may
trigger relapse in the form of a chemoresistant tumor [16]. Despite
an initial response to anti-cancer treatments, chemotherapeutic treat-
ment can lead to the symmetric propagation of a small subpopulation
of drug-tolerant cells with stem cell features [17–20]. Cisplatin treat-
ment causes the lung CSC population to expand in patient ex-
plants and in H460-xenografted nude mice. Similar studies have also
shown side-population CSC expansion following treatment with cy-
totoxic drugs such as 5’fluorouracil, an anti-metabolite commonly
used in the treatment of several tumor types [21,22].
Lung cancer stem cells
Conventional anti-cancer therapies kill the bulk of the tumor,
however, CSCs exhibit robust intrinsic resistance and survive therapy
due to increased telomere length, activation of anti-apoptotic path-
ways, increased membrane transporter activity and their ability to
migrate and metastasize [23].
Telomeres are comprised of a repetitive G-rich sequence and an
abundance of associated proteins that form an effective cap that pro-
tects chromosome ends. Telomerase, also known as telomere
terminal transferase, is a ribonucleoprotein complex that adds the
telomere DNA sequence repeats to lengthen the telomere. It confers
limitless proliferative capacity to cells through its ability to elon-
gate telomeres [24,25]. Telomerase activity is readily detected in most
cancers but not in somatic tissues and is essential for stem cell in-
tegrity and longevity [26]. Increased telomere length has previously
been shown to confer resistance to a number of anti-cancer thera-
pies; inversely, shortened telomere length is associated with drug
sensitivity [27]. Telomerase inhibition enhanced the pro-apoptotic
response to anti-cancer therapies resulting in delayed relapse fol-
lowing chemotherapeutic treatment [27–29]. Lung cancer stem cells
display longer telomeres than their non-CSC counterparts. Treat-
ment with the specific telomerase inhibitor, MST312, has a strong
and preferential anti-proliferative effect on the lung CSC popula-
tion in vitro and in vivo [14]. Inhibition of telomerase using Imetelstat,
either alone or in combination with Trastuzumab, decreases breast
CSCs and inhibits their capacity to self-renew in HER2 positive breast
cancer cells [30].
Several mechanisms have been postulated to play a role in re-
sistance, including impaired apoptotic machinery, increased DNA-
repair mechanisms, up-regulation of multidrug resistance proteins
and membrane efflux transporters [31]. Such mechanisms may be
responsible, at least in part, for driving CSCs into a phenotype of
reduced apoptotic cell death, which in turn forms the basis of tumor
progression. ATP-binding cassette (ABC) transporters, such as
p-glycoprotein and multidrug resistant associated protein (MRP1),
are membrane transporters that can pump structurally unrelated
small molecules, such as cytotoxic chemotherapeutic drugs out of
the cell. Normal stem cells and lung CSCs express high levels of ABC-
transporters resulting in low intracellular drug concentrations [32,33].
For example, the ABCG2 gene is highly expressed in hematopoi-
etic and lung cancer stem cells but is switched off in most terminally
differentiated progeny [34].
Cancer stem cells are chemoresistant and metastatic, two fea-
tures that correlate with poor prognosis and tumor recurrence [35].
Lung adenocarcinoma CSCs have increased expression of galectin-
1, a multifunctional protein which promotes invasiveness and
metastasis and is associated with poor overall survival and lymph
node metastasis [36]. Epithelial to mesenchymal transition (EMT)
is an evolutionary conserved developmental process. Studies indi-
cate that metastatic cancer cells, cells which have undergone EMT,
exhibit a CSC phenotype [37,38]. Numerous pathways, including the
Wnt/β-catenin [39–42], Notch [43,44], Hedgehog [45–47] and NFκB
[48] regulate CSC maintenance as well as EMT and may prove to
be key targets in eradicating the CSC population and therefore the
root of resistance and metastasis [37]. Induction of EMT confers re-
sistance of NSCLC cells to EGFR-tyrosine kinase inhibitors, however,
enforced inhibition of the Hedgehog pathway re-sensitizes the NSCLC
cells to EGFR-tyrosine kinase inhibitors through mediation of the
EMT process [47].
Previously identified lung CSC subsets have been shown to confer
resistance to conventional chemotherapeutics, biological mol-
ecules, targeted therapies and radiotherapy used in the current
management of lung cancer. The elimination of lung CSCs is of
utmost importance at the time of therapeutic intervention in order
to prevent CSC expansion and subsequent tumor recurrence, relapse
 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156
Fig. 2. Anti-cancer therapies induce CSC expansion. Treatment with conventional chemotherapeutic agents or radiotherapy kills the bulk of tumor cells (blue). However
the intrinsically resistant CSCs (purple) survive. Cellular stress caused by chemotherapeutic treatment can induce expansion of these CSCs. The survival and expansion of
CSCs promotes tumor relapse and metastasis.
and metastasis (Fig. 2). Identifying a lung CSC population and elu-
cidating its mechanisms of intrinsic resistance may highlight a key
molecule or pathway targeting strategy that may prevent tumor re-
sistance and recurrence at its source, leading to more effective lung
cancer treatments. While little is currently known regarding lung
cancer stem cell biology, a number of CSC markers have be iden-
tified and studied. These include ALDH1, CD133, side population
(Hoechst-negative), CD44, CD87 and CD117, all of which have been
linked to chemoresistance in a number of first line anti-cancer
therapies.
Aldehyde dehydrogenase and resistance
The aldehyde dehydrogenase (ALDH) family members are cy-
tosolic isoenzymes responsible for oxidizing intracellular aldehydes,
including the oxidation of retinol (vitamin A) to retinoic acid, an
important modulator of gene expression and cell differentiation.
ALDH is highly expressed in hematopoietic stem cells [49,50]. In
recent years ALDH1 has emerged as a prominent marker for CSCs
in a number of solid tumors such as breast, colon, head and neck,
prostate [51–55], and lung cancer [56]. ALDH1 is expressed in a
number of NSCLC cell lines, where ALDH1-positive cell subpopu-
lations show distinct stem-like characteristics and gene expression
profiles. Low numbers of ALDH1-positive cells, when injected into
nude mice have the ability to initiate tumor growth and generate
heterogenic tumors. On the other hand, their ALDH1-negative coun-
terparts do not demonstrate this ability, indicating that the ALDH1-
positive fraction is capable of self-renewal and can give rise to a
differentiated cell population. ALDH1-positive cells are highly re-
sistant to chemotherapeutic agents commonly used as first-line
therapy in the clinical setting, such as cisplatin, gemcitabine, doxo-
rubicin, vinorelbine and docetaxel, whereas ALDH1-negative cells
are sensitive to the cytotoxic activity of these drugs [56]. ALDH1
overexpression is associated with poor prognosis in NSCLC pa-
tients, where high ALDH1 expression is significantly associated with
a more aggressive and advanced pathological grade and stage [56,57].
Furthermore, increased ALDH1 expression has been associated with
increased metastasis in multiple cancers [58,59].
149
The Notch pathway is a highly conserved embryogenesis sig-
naling pathway involved in the determination of cell fate [60].
This pathway has been identified as a key pathway associated
with the CSC maintenance of ALDH1-positive lung CSCs. Suppres-
sion of Notch signaling with a γ-secretase inhibitor results in a
much reduced ALDH1 expressing population and decreased pro-
liferation and clonogenic capacity of tumor cells [59]. In a study
by Barr et al., an isogenic panel of cisplatin resistant NSCLC cell
lines was generated by chronic exposure to cisplatin. Increased
ALDH1 activity correlated with significantly increased expression
of CSC and EMT markers, Nanog, Oct-4, Sox-2, c-Met and β-catenin
in the cisplatin resistant cell lines relative to their sensitive
counterparts [61].
Treatment with paclitaxel has also been shown to induce a greater
ALDH1 CSC population in H460 and H1299 lung cancer cell lines,
whereas treatment with the selective CSC inhibitor, salinomycin, de-
creased the presence of the ALDH1 population and reduced the
tumorsphere-forming potential of these cells, with no effect on the
ALDH1-negative population [62]. While paclitaxel treatment in an
in vivo model decreased tumor volume, it increased the number of
metastatic nodules, most likely due to the expansion of an ALDH1-
positive tumor initiating CSC population. Salinomycin alone did not
reduce the size of the primary tumor but decreased metastasis [62].
In addition to chemotherapy, radiotherapy has also been shown to
induce and expand the ALDH1-positive CSC population in lung ad-
enocarcinoma A549 cells. Repeated 4 Gy radiation treatment was
used to generate an A549 radioresistant cell line, which showed
greater proliferative and clonogenic capacity when challenged with
radiation, compared to their parental counterparts. Expansion of the
ALDH1-positive subpopulation correlated with the development of
radioresistance and increased doses of radiation [63].
Resistance to epidermal growth factor receptor (EGFR) tyro-
sine kinase inhibitors is a major problem in the treatment of lung
cancer. A model of acquired resistance to gefitinib was estab-
lished in a number of EGFR mutated cell lines, EGFR-mutant HCC827
(exon19del E746–A750), PC-9 (exon19del E746–A750), HCC4006
(exon19del L747–E749), and HCC4011 (L858R) cells [64]. Cell lines
were treated using a stepwise dose escalation or high-dose gefitinib
150 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156

Table 1 conditions select undifferentiated CD133-positive cells and promote

Potential strategies targeting CSC markers. their enrichment and expansion and induce sphere formation [93,94].
CSC Marker Drug Reference Overexpression of the embryonic stem cell transcription factors
ALDH1 Salinomycin [62] Oct-4 and Nanog confirmed the undifferentiated immature phe-
Silibinin [67] notype of CD133-positive cells [93]. In the presence of serum, tumor-
DEAB [68,69] initiating spheres adhered to the plastic and acquired the
Disulfiram [70–73] morphological features of differentiated cells. CD133 expression
CD133 Pantoprazole [21]
Neuropeptide antagonists [21]
was lost during differentiation. Subcutaneous injection of lung
Side population Verapamil [74] sphere-forming cells or pure CD133-positive populations into im-
EGFR Inhibitors [75,76] munodeficient mice resulted in the growth of tumors with
Obatoclax [77] morphological features similar to those seen in the human tumor
GDC-0449 [78]
from which they were derived, confirming the tumorigenic poten-
Mithramycin A [79]
Nicardipine [80] tial of these CD133 expressing sphere-forming cells [93]. Injection
Pravastatin [80] of differentiated cells or CD133-negative subpopulations using the
Secalonic Acid D [81] same animal model resulted in decreased tumorigenic potential
Low Weight Molecular [82] and loss of key stemness characteristics. Lung tumor CSC-spheres
Heparin
Axitinib [83]
and CD133-positive cells showed resistance to cisplatin, gemcitabine,
CD117 Imatinib [84] paclitaxel, doxorubicin and etoposide when treated with peak plasma
Axitinib [83] concentrations. Resistance was directly correlated with increased
A number of cancer stem cell (CSC) markers identified to date may offer potential expression of the ABC-transporter, ABCG2 [93,95]. Knock-down of
as specific targets for therapy, particularly in chemoresistant lung cancer. Combi- the embryonic cell marker Oct-4 specifically blocked the ability of
nation treatments using novel agents or drugs targeting stem-like cells with CD133-positive cells to form spheres and further promoted their
conventional anti-cancer treatments may further enhance the efficacy of chemo- differentiation into CD133-negative cells, thereby reducing the
therapeutic drugs. As such, co-treatment strategies may re-sensitize drug resistant
lung cancer cells to therapy, while at the same time inhibiting expansion of the CSC
CD133-positive population. Oct-4 inhibition decreased the inva-
population within the tumor. sive and tumorigenic potential of CD133-positive cells in vivo and
increased apoptosis of the CD133-positive cells in response to ra-
diotherapy in vitro [95].
to generate resistant sublines. Treatment with high-dose gefitinib Platinum-based chemotherapy, such as cisplatin and carboplatin,
induced ALDH1 overexpression, increased EMT-associated markers is an integral first-line therapy for patients with NSCLC. However,
and self-renewal capabilities. These results suggest that acquired tumor recurrence and resistance is commonly seen during the course
resistance is based, at least in part, on the expansion of a stem cell- of treatment with these platinum agents. Cisplatin treatment, either
like population [64,65]. Gefitinib resistant cells exhibit a higher acute or chronic, resulted in an 8-fold enrichment of CD133-
tumor-initiating capability than their parental controls when in- positive fractions. Analysis of CD133 in patient tumor resections
jected subcutaneously into NOD/SCID mice. Gefitinib resistant identified a CD133-positive subtype as a biomarker for predicting
sublines displayed resistance to the commonly used chemothera- shorter progression-free survival [96]. This enrichment was thought
peutic agents, docetaxel and paclitaxel, an effect that may be due to be due to drug resistance and recurrence. Low-dose cisplatin treat-
to the expansion of the ALDH1 CSC population [64]. Isolated ALDH1 ment, sufficient to cause DNA-damage but not cell death, expanded
cells from gefitinib resistant PC-9 cells showed increased resis- the CD133-positive population and up-regulated ABCG2 and ABCB1
tance to cisplatin, etoposide and fluorouracil compared to ALDH1- drug efflux-associated proteins in H460 and H661 cell lines [21].
negative cells [66]. A similar induction of an ALDH1-positive CD133 expansion and stem-cell associated gene expression was de-
subpopulation was observed during the development of NSCLC re- pendent on cisplatin concentration and exposure time. Cisplatin-
sistance to the EGFR tyrosine kinase inhibitor, erlotinib. ALDH1- primed cells show increased tumor-sphere formation in soft agar
positive cells showed a refractory, inherent resistance to this EGFR colony-forming assays. Cells pre-treated with cisplatin showed in-
TKI. Treatment with a natural polyphenol, silibinin, specifically targets creased cross-resistance to doxorubicin and paclitaxel. Treatment
erlotinib-refractory ALDH1-positive cells, reducing population size of side population cells with a specific ABCG2 inhibitor
and circumventing erlotinib resistance in vivo, minimizing the ability (pantoprazole) or a pan-inhibitor of ABC transporters (Verapamil)
of the CSCs to evade death and therefore reducing the possibility completely abrogated cisplatin-induced resistance [21].
of recurrence [67]. Targeting ALDH1, in addition to one or more CSC Tumor-hypoxia has long been considered a therapeutic problem
surface markers, may offer potential as novel strategies for over- in many solid tumors. Recent studies have shown that hypoxic con-
coming the development of treatment resistance in lung cancer ditions significantly increase CD133-positive and Oct-4 expressing
(Table 1). cells, which correlate with increased gefitinib resistance. Hypoxic
expansion of gefitinib resistant cells was mediated through the up-
CD133 and resistance regulation of HIF-1α and insulin-like growth factor-1 expression and
the subsequent activation of insulin-like growth factor-1 receptor
The CD133 antigen, known as prominin 1, is a 120 kDa pentaspan [97]. Inhibition of HIF-1α or insulin-like growth factor-1 receptor
transmembrane protein, encoded by the prom1 gene. It is highly significantly decreases CD133 and Oct-4 positive populations and
expressed on undifferentiated cells, such as hematopoietic stem overcomes gefitinib resistance [97]. Elevated CD133 expression is
cells, endothelial progenitor cells and neural stem cells [85–87]. associated with intrinsic chemotherapy and radiotherapy resis-
CD133 is a well-documented CSC marker that represents a tumor- tance due to reduced apoptotic response, diminished cell cycle arrest
initiating cell subset in breast, colon, prostate, liver and ovarian and enhanced DNA repair capacity [98]. SCLC chemoresistant CD133-
solid tumors, as well as SCLC and NSCLC [88–93]. Immunohisto- positive cells show increased expression of the mitogenic
chemical analysis of patient-derived tumors showed variation in neuropeptide receptor for gastrin-releasing peptide and arginine va-
CD133 positivity among patients, but was not detectable in normal sopressin. These CD133-positive cells show increased sensitivity to
lung tissues [93]. SCLC and NSCLC tumors were dissociated and the growth inhibitory and pro-apoptotic effects of broad spec-
cultured at a low density in serum-free media supplemented with trum neuropeptide antagonists, highlighting a potential role for
epidermal growth factor and basic fibroblast growth factor. Such neuropeptide antagonists as CSC targeting agents in SCLC [99].
 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156
A meta-analysis of 11 studies, consisting of 1004 NSCLC patient
tissues, further confirmed that high CD133 expression correlates with
significantly worse 5-year overall survival compared to those with
low CD133 expression. CD133 expression was associated with a
number of clinical parameters such as tumor stage, grade, differ-
entiation status and lymph node metastasis [100,101].
Side population and resistance
The flow cytometry based side population strategy was initially
conceived to enrich or isolate hematopoietic stem cells. It is based
on the ability of stem cells to efficiently efflux Hoechst 33342, and
therefore present as a Hoechst-negative population on a dual wave-
length flow cytometry plot. This technique has been used to isolate
putative cancer stem cells from a number of solid tumor types, in-
cluding prostate, breast, malignant pleural mesothelioma and lung
[74,102–105]. Tumor side population cells pump out fluorescent
Hoechst 33342 dye (Hoechst-negative), whereas the main bulk pop-
ulation, retain the dye (Hoechst-positive). The efflux capacity of the
side population phenotype is determined by ATP-binding cassette
membrane transporter G2 (ABCG2), also known as breast cancer re-
sistance protein 1 (BCRP1) and is associated with tumor growth,
progression and metastasis [106]. ABCG2 actively effluxes a wide
variety of chemically and structurally unrelated compounds from
cells; ABCG2 overexpression in tumor cells confers multidrug re-
sistance to a multitude of anti-cancer drugs [107]. Hoechst staining
of NSCLC cell lines confirmed the presence of a Hoechst-negative
side population. However, treatment of cells with verapamil, a
calcium-channel blocker and inhibitor of ABCG2, completely elimi-
nated the presence of the side population, therefore confirming the
transporter ABCG2 as an integral efflux pump in CSCs. NSCLC side
population cells show increased proliferative capacity, tumorige-
nicity and overexpression of ABCG2 compared to non-side population
cells [33,74].
NSCLC side population cells are enriched in the cell cycle G0/G1
phase, have higher ALDH1 activity, higher clonogenic survivability
and increased self-renewal and differentiation capacities compared
to the main cell population. Treatment of NSCLC cell lines (A549, H1650
and H1975) with EGFR tyrosine kinase inhibitors resulted in de-
creased side population frequency and ABCG2 expression. Downstream
regulation of the EGFR-Src-Akt signaling axis resulted in significant
inhibition of Sox-2. This inhibition resulted in the abrogation of the
self-renewal and metastatic abilities of the side population cells [75,76].
β-arrestin is involved in the progression of NSCLC, where its inhibi-
tion has been shown to significantly decrease the presence of the side
population and inhibit the growth and expansion of A549 cells. The
Bcl-2 family member, MCL-1, is overexpressed in the side popula-
tion and is associated with the self-renewal of CSCs. MCL-1 specific
inhibition, with Obatoclax, prevented self-renewal of EGFR–resistant
NSCLC cells [77]. The Hedgehog pathway has long been implicated
in the maintenance of CSCs. Treatment of adenocarcinoma cells
(HCC) and SCLC cells (H1339) with the Hedgehog inhibitor GDC-
0449, inhibited cell growth and significantly decreased side population
frequency. Hedgehog inhibition in combination with cisplatin in-
creased the cytotoxic effects of cisplatin when compared to cisplatin-
only treatment [78]. Small molecule inhibition or gene silencing of
CK2α resulted in the down-regulation of the Hedgehog pathway and
subsequently induced a reduction of the side population percent-
age [108].
Cigarette smoking at diagnosis or during lung cancer treat-
ment is associated with poor prognosis and cigarette smoke has now
been shown to promote drug resistance via the expansion of the
CSC side population [109]. Treatment of A549 adenocarcinoma cells
with cigarette smoke condensate resulted in a significant increase
in side population frequency, ABCG2 expression and doxorubicin
efflux. This effect was mediated by PI3K/Akt signaling. PI3K inhi-
bition and subsequent ABCG2 down-regulation and decreased side
151
population occurrence has been shown to re-sensitize adenocar-
cinoma (A549) and malignant pleural mesothelioma (ZL55 and
SDM103T2) cell lines to doxorubicin and mitoxantrane, respective-
ly [109,110]. In a similar study, treatment of A549 and Calu-6 cell
lines with mithramycin A (a selective inhibitor of the binding of tran-
scription factor Sp1 to the ABCG2 promoter region) repressed smoke-
induced expression of ABCG2 and side population frequency [79,111].
Occurrence of the side population has been associated with re-
sistance to many chemotherapeutic agents and targeted therapies
used today due to its characteristic excessive efflux capacity. Tar-
geting side population cells may prove to be an important mechanism
to overcome resistance to a number of therapies by reducing drug
efflux from cells, thereby increasing drug retention within the cells.
In a recent study by Wu et al., NSCLC patients exhibiting chemo-
resistance to platinum-based chemotherapy had elevated serum
levels of cholesterol and increased expression of ABCG2 [80]. In an
in vitro model of NSCLC using A549 cells, cholesterol increased che-
moresistance while inhibition of ABCG2 with Nicardipine reversed
cholesterol-induced platinum-resistance. Similarly, treatment with
the statin Pravastatin, re-sensitized A549 cells to cisplatin, carboplatin
and oxaliplatin. This study suggests that lung cancer patients with
high cholesterol may respond to platinum-based therapy when com-
bined with a statin [80]. Secalonic acid D, a marine fungal-derived
metabolite, has a potent anti-cancer effect through its inhibition of
ABCB1, ABCC1 and most significantly ABCG2, in multidrug resis-
tant cells. Treatment with Secalonic acid D significantly increased
ABCG2 degradation via the activation of caplain 1, decreased the
percentage of side population cells and inhibited the sphere-
forming abilities of the side population CSC cells [81]. Low weight
molecular heparin has been suggested to affect the biological prop-
erties of side population cells. Treatment of side population cells
with low weight molecular heparin decreased the sphere-forming
abilities of the CSC population through the degradation of ABCG2.
While treatment of the cisplatin resistant side population cells with
low weight molecular heparin alone did not affect their prolifera-
tive capacity, the side population cells were re-sensitized to cisplatin-
induced apoptosis when used in combination with cisplatin [82].
CD44 and resistance
CD44 is an integral membrane glycoprotein that functions as a
receptor for the extracellular matrix glycan, hyaluronan. CD44 is ex-
pressed on the surface of specific glandular, fibroblastoid and
hematopoietic cells [112]. CD44 has been identified as an impor-
tant marker of CSCs in breast, prostate, pancreatic, squamous head
and neck, and in more recent years, lung cancer [113–116]. NSCLC
cells expressing CD44 are enriched for stem-like properties. CD44-
positive cells showed increased spheroid body formation, enhanced
self-renewal abilities and increased tumor initiating capacity in vivo
compared to their negative counterparts, and the heterogenic CD44
population from which they were derived [117]. CD44-positive cells
showed increased expression of pluripotency markers (Nanog, Oct-
4, Sox-2) and EMT markers (SNAI1, CDH2 and VIM) and increased
resistance to cisplatin [117]. NSCLC cells surviving 5 Gy radiation
therapy were enriched for CD44 expressing cells, and showed in-
creased tumor initiating potential, indicating an inherent
radioresistance within the CD44-positive subpopulation [118]. Both
lung cancer and malignant pleural mesothelioma cells co-expressing
CD44 with ALDH1 showed increased drug resistance, sphere-
forming capacity and tumorigenicity [15,68].
Immunohistochemical staining of 159 resected NSCLC tumors
showed that CD44 expression is more frequently detected in squa-
mous cell carcinoma compared to adenocarcinoma histology. High
CD44 expression was also significantly correlated with advanced
regional lymph node metastasis and is a significant predictor of a
more advanced tumor status in squamous cell carcinomas [119].
152 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156

CD87 and resistance
Numerous solid tumors, including lung cancer, show increased
expression of urokinase plasminogen activator (uPA) and its recep-
tor, urokinase plasminogen activator receptor (uPAR), otherwise
known as CD87. CD87 is highly involved in migration and in the reg-
ulation of cell adhesion. High expression of CD87 is strongly
correlated with an unfavorable clinical outcome and a signifi-
cantly shorter overall survival in SCLC [120,121]. In an in vivo model
of NSCLC, simultaneous inhibition of CD87 and MMP-9 inhibited
tumor growth, invasion, metastasis and angiogenesis [122]. Reduc-
tion of CD87 signaling increased cisplatin-induced apoptosis and
anoikis, a method of programmed cell death induced by anchorage-
dependent cells detaching from the extracellular matrix [123]. Gutova
et al. identified a rare population of CD87-positive cells in a panel
of human SCLC cell lines (H1688, H1417, H69AR, H211, H1882 and
H250). Isolation of these CD87-positive cells confirmed the expres-
sion of stemness and CSC markers, such as CD44 and MDR1,
respectively. The CD87-positive population demonstrated high
sphere-forming capabilities, increased tumor initiating potential, as
well as significant resistance to numerous traditional chemothera-
peutic agents, including cisplatin, etoposide and paclitaxel
[120,124,125].
CD117 and resistance
CD117 (KIT, c-KIT) is a type III receptor tyrosine kinase that is
phosphorylated upon binding of its ligand, stem cell factor (SCF),
leading to a signaling cascade that culminates in the alteration of
a number of biological processes, such as apoptosis, cell differen-
tiation, cell adhesion and proliferation [126]. SCF and CD117 are
overexpressed in a number of human malignancies, including gas-
trointestinal stromal tumors (GIST) [127], breast cancer [128], acute
myeloid leukaemia (AML) [129] and lung cancer [130]. High SCF ex-
pression in lung adenocarcinoma is strongly associated with smoking
status and poor prognosis [131]. Overexpression of CD117 within
lung tumors is also associated with poor prognosis, lower survival
rates and chemoresistance [84]. NSCLC cell lines, H460 and A549,
when grown in CSC selective media, gave rise to tumorspheres that
exhibit increased expression of CD133, CD117, Oct-4, TRA-1-81, in-
creased nuclear expression of β-catenin and resistance to
chemotherapy [84]. CSC-spheres also showed high expression of SCF,
whereas SCF was not detectable in the bulk cell population. Exog-
enous SCF expression increased CSC-sphere size and stimulated their
proliferation. Treatment of the CD117-expressing CSC-spheres with
Imatinib (Gleevec), an FDA-approved tyrosine kinase CD117 inhib-
itor commonly used in the treatment of AML and GIST, dramatically
reduced CSC-sphere size and number. Imatinib given in combina-
tion with cisplatin or doxorubicin led to a potent elimination of both
CSC-sphere forming cells and the bulk cell population [84].
Targeting CSCs to overcome therapeutic resistance in lung cancer
Specific targeting of cancer stem cells in combination with first-
line chemotherapeutic agents holds great promise as a strategy to
overcome chemoresistance, tumor relapse and metastasis (Fig. 3).
However, such specific targeting of CSCs has not been extensively
explored as a therapeutic option in the treatment of lung cancer.
Inhibition of CSC markers, such as ABCG2, has been shown to
enhance the efficacy of chemotherapeutic agents in CSC cells [83].
Axitinib is an oral, potent, small molecule ATP competitive multi-
target tyrosine kinase inhibitor. It inhibits CSC markers CD117 and
ABCG2. Axitinib has also been shown to reverse multidrug resis-
tance via ABCG2 inhibition both in vitro and in vivo. Axitinib–
doxorubicin combination treatment promoted intracellular
accumulation of doxorubicin within side population CSC cells, and
significantly enhanced the cytotoxic effects of doxorubicin [83].
An increasing body of evidence is beginning to emerge in rela-
tion to the inhibition of ALDH1 in stem-like tumor cells that are
resistant to therapy [132]. Conventional chemotherapy has had little
effect in the treatment of malignant pleural mesothelioma. However,
combination treatment of ALDH-high, CD44-positive subpopula-
tions isolated from three mesothelioma cell lines (H28, H2052 and
Meso4) with the ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB),
increased cisplatin sensitivity and significantly reduced cell viabil-
ity [68]. Knockdown (siRNA) or chemical inhibition (using DEAB or

Fig. 3. Future implications for CSC-targeted therapies. (A) Current treatment strategies focus on debulking the tumor (blue) but not the intrinsically resistant CSC subset
(purple). Chemotherapy kills the majority of cells within the main tumor cell population. However the CSC subset survives and gives rise to new, therapeutically resistant
heterogenic tumors and metastases. (B) Combining current cytotoxic therapies with novel CSC-targeted therapies may eradicate this CSC subset and therefore inhibit the
root of chemoresistance, tumor recurrence and metastasis, thereby improving clinical outcomes.
 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156 153

disulfiram) of ALDH1A1 in A549 and H522 lung cancer cells de- many have been shown to be well tolerated and show enhanced
creased the cells proliferative and migratory capabilities [69]. Of efficacy when used in combination with conventional cytotoxic
interest is the recent re-purposing of the FDA-approved pan- chemotherapies.
ALDH1 inhibitor, disulfiram (Antabuse), originally used in the
treatment of chronic alcoholism [133]. In more recent years, a Acknowledgments
number of studies have demonstrated the potential use of disulfi-
ram in targeting ALDH1 expressing CSCs in breast, cervical, prostate, This work was funded by Molecular Medicine Ireland (MMI), as
melanoma and lung tumors [134–138]. As such, disulfiram was used part of the Clinical & Translational Research Scholars Programme
to examine its effects in re-sensitizing cells to current therapies or (CTRSP).
enhancing the cytotoxic effects of platinum-based chemotherapy
and radiation therapy [70,71]. When disulfiram is administered in
combination with copper chloride in vitro, it acts as an effective Conflict of interest
proteasome inhibitor and as a copper ionophore. Disulfiram–
copper complexes are formed and induce copper-dependent The authors have no conflict of interest to declare.
oxidative stress, exerting its potent apoptotic and anti-cancer effects
[72]. In a small phase IIb trial, disulfiram in combination with References
cisplatin and vinorelbine was well-tolerated and significantly pro-
longed overall patient survival in metastatic NSCLC patients [73]. [1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer
Interestingly, of the forty patients included in the trial, there were statistics, CA Cancer J. Clin. 61 (2011) 69–90.
[2] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, et al.,
two long-term survivors, both of whom were in the disulfiram com- Cancer incidence and mortality worldwide: sources, methods and major
bination treatment group. patterns in GLOBOCAN 2012, Int. J. Cancer 136 (2015) E359–E386.
The presence of hyper-malignant or CSCs in lung cancer and other [3] R.S. Herbst, J.V. Heymach, S.M. Lippman, Lung cancer, N. Engl. J. Med. 359
(2008) 1367–1380.
tumor types are largely responsible for the failure of current anti- [4] F.H. Sarkar, Y. Li, Z. Wang, D. Kong, S. Ali, Implication of microRNAs in drug
cancer treatments with metastasis and relapse accounting for a high resistance for designing novel cancer therapy, Drug Resist. Updat. 13 (2010)
percentage of patients. In a recent study by Li et al., the small mol- 57–66.
[5] K.E. Allen, G.J. Weiss, Resistance may not be futile: microRNA biomarkers for
ecule inhibitor BBI608 inhibited gene transcription driven by the chemoresistance and potential therapeutics, Mol. Cancer Ther. 9 (2010)
signaling transducer and activator of transcription 3 (STAT3) [139]. 3126–3136.
BBI608 abrogated cancer stem cell properties including tumorsphere [6] G. Szakacs, J.K. Paterson, J.A. Ludwig, C. Booth-Genthe, M.M. Gottesman,
Targeting multidrug resistance in cancer, Nat. Rev. Drug Discov. 5 (2006)
formation and self-renewal capabilities through the depletion of
219–234.
cancer stem cells. BBI608 treatment effectively blocks cancer relapse [7] L.V. Nguyen, R. Vanner, P. Dirks, C.J. Eaves, Cancer stem cells: an evolving
and metastasis in mice and holds great promise as a future anti- concept, Nat. Rev. Cancer 12 (2012) 133–143.
stemness therapy [139]. [8] T. Reya, S.J. Morrison, M.F. Clarke, I.L. Weissman, Stem cells, cancer, and cancer
stem cells, Nature 414 (2001) 105–111.
[9] S.J. Morrison, J. Kimble, Asymmetric and symmetric stem-cell divisions in
Conclusion development and cancer, Nature 441 (2006) 1068–1074.
[10] C. Cocola, S. Sanzone, S. Astigiano, P. Pelucchi, E. Piscitelli, L. Vilardo, et al., A
rat mammary gland cancer cell with stem cell properties of self-renewal and
Despite the development of targeted therapies, there has been multi-lineage differentiation, Cytotechnology 58 (2008) 25–32.
little improvement in the prognosis of lung cancer patients. The [11] M. Diehn, R.W. Cho, N.A. Lobo, T. Kalisky, M.J. Dorie, A.N. Kulp, et al., Association
of reactive oxygen species levels and radioresistance in cancer stem cells,
cancer stem cell hypothesis has provided a new avenue of focus in
Nature 458 (2009) 780–783.
overcoming resistance to therapy, preventing tumor relapse and im- [12] A.B. Hjelmeland, Q. Wu, J.M. Heddleston, G.S. Choudhary, J. MacSwords, J.D.
proving progression-free and overall survival rates in lung cancer Lathia, et al., Acidic stress promotes a glioma stem cell phenotype, Cell Death
Differ. 18 (2011) 829–840.
patients. Signaling pathways associated with stem cell properties,
[13] J. Tower, Stress and stem cells, Wiley Interdiscip. Rev. Dev. Biol. 1 (2012)
such as differentiation and self-renewal capacities in addition to the 789–802.
identification of CSC markers, offer potential targets for novel anti- [14] D. Serrano, A.M. Bleau, I. Fernandez-Garcia, T. Fernandez-Marcelo, P. Iniesta,
cancer strategies. While a number of pre-clinical and clinical studies C. Ortiz-de-Solorzano, et al., Inhibition of telomerase activity preferentially
targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer,
(Table 2) examining CSC-targeted agents are still in their infancy, Mol. Cancer 10 (2011) 96.
[15] J. Liu, Z. Xiao, S.K. Wong, V.P. Tin, K.Y. Ho, J. Wang, et al., Lung cancer
tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi)
tumor initiating cells, Oncotarget 4 (2013) 1698–1711.
Table 2 [16] G. Hamilton, U. Olszewski, Chemotherapy-induced enrichment of cancer stem
Clinical trials targeting CSCs. cells in lung cancer, J. Bioanal. Biomed. S9 (2013) 003, doi:10.4172/1948-
593X.S9-003.
Target Agent Phase Clinical Trials
[17] S.V. Sharma, D.Y. Lee, B. Li, M.P. Quinlan, F. Takahashi, S. Maheswaran, et al.,
KRAS Defactinib II NCT01951690 A chromatin-mediated reversible drug-tolerant state in cancer cell
CD117 Imetelstat II NCT01197968 subpopulations, Cell 141 (2010) 69–80.
DLL4 Demcizumab I/II NCT01189968 [18] M. Wintzell, L. Lofstedt, J. Johansson, A.B. Pedersen, J. Fuxe, M. Shoshan,
Repeated cisplatin treatment can lead to a multiresistant tumor cell population
NCT02259582
with stem cell features and sensitivity to 3-bromopyruvate, Cancer Biol. Ther.
STAT3 Methformin II NCT01717482
13 (2012) 1454–1462.
STAT3 BBI608 I/II NCT02347917
[19] V. Levina, A.M. Marrangoni, R. DeMarco, E. Gorelik, A.E. Lokshin, Drug-selected
NCT02432326 human lung cancer stem cells: cytokine network, tumorigenic and metastatic
NCT02467361 properties, PLoS ONE 3 (2008) e3077.
NCT01775423 [20] A.M. Calcagno, C.D. Salcido, J.P. Gillet, C.P. Wu, J.M. Fostel, M.D. Mumau, et al.,
Notch RO49290971 I/II NCT01193868 Prolonged drug selection of breast cancer cells and enrichment of cancer stem
NCT01193881 cell characteristics, J. Natl Cancer Inst. 102 (2010) 1637–1652.
CSC Immunotherapy Vaccine I/II NCT02115958 [21] Y.P. Liu, C.J. Yang, M.S. Huang, C.T. Yeh, A.T. Wu, Y.C. Lee, et al., Cisplatin selects
NCT02084823 for multidrug-resistant CD133+ cells in lung adenocarcinoma by activating
Sp1 Mithramycin A II NCT01624090 Notch signaling, Cancer Res. 73 (2013) 406–416.
ALDH1 Disulfiram II/III NCT00312819 [22] M.M. Shi, Y.L. Xiong, X.S. Jia, X. Li, L. Zhang, X.L. Li, et al., Fluorouracil selectively
enriches stem-like cells in the lung adenocarcinoma cell line SPC, Tumour Biol.
Therapeutics specifically targeting CSC-associated markers are currently being as- 34 (2013) 1503–1510.
sessed as individual therapies and in combination with conventional chemotherapies [23] J. Sleeman, P.S. Steeg, Cancer metastasis as a therapeutic target, Eur. J. Cancer
in lung cancer. 46 (2010) 1177–1180.
154 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156
[24] S.M. Bailey, J.P. Murnane, Telomeres, chromosome instability and cancer,
Nucleic Acids Res. 34 (2006) 2408–2417.
[25] A. Tomas-Loba, I. Flores, P.J. Fernandez-Marcos, M.L. Cayuela, A. Maraver, A.
Tejera, et al., Telomerase reverse transcriptase delays aging in cancer-resistant
mice, Cell 135 (2008) 609–622.
[26] M. Armanios, C.W. Greider, Telomerase and cancer stem cells, Cold Spring Harb.
Symp. Quant. Biol. 70 (2005) 205–208.
[27] R.J. Ward, C. Autexier, Pharmacological telomerase inhibition can sensitize
drug-resistant and drug-sensitive cells to chemotherapeutic treatment, Mol.
Pharmacol. 68 (2005) 779–786.
[28] M.A. Cerone, J.A. Londono-Vallejo, C. Autexier, Telomerase inhibition enhances
the response to anticancer drug treatment in human breast cancer cells, Mol.
Cancer Ther. 5 (2006) 1669–1675.
[29] C. Bruedigam, F.O. Bagger, F.H. Heidel, C. Paine Kuhn, S. Guignes, A. Song, et al.,
Telomerase inhibition effectively targets mouse and human AML stem cells
and delays relapse following chemotherapy, Cell Stem. Cell 15 (2014) 775–790.
[30] J.E. Koziel, B.S. Herbert, The telomerase inhibitor imetelstat alone, and in
combination with trastuzumab, decreases the cancer stem cell population and
self-renewal of HER2(+) breast cancer cells, Breast Cancer Res. Treat. 149 (2015)
607–618.
[31] M. Signore, L. Ricci-Vitiani, R. De Maria, Targeting apoptosis pathways in cancer
stem cells, Cancer Lett. 332 (2013) 374–382.
[32] K. Chen, Y.H. Huang, J.L. Chen, Understanding and targeting cancer stem cells:
therapeutic implications and challenges, Acta Pharmacol. Sin. 34 (2013)
732–740.
[33] M.M. Ho, A.V. Ng, S. Lam, J.Y. Hung, Side population in human lung cancer
cell lines and tumors is enriched with stem-like cancer cells, Cancer Res. 67
(2007) 4827–4833.
[34] M. Dean, T. Fojo, S. Bates, Tumour stem cells and drug resistance, Nat. Rev.
Cancer 5 (2005) 275–284.
[35] R. Perona, B.D. Lopez-Ayllon, J. de Castro Carpeno, C. Belda-Iniesta, A role for
cancer stem cells in drug resistance and metastasis in non-small-cell lung
cancer, Clin. Transl. Oncol. 13 (2011) 289–293.
[36] X. Zhou, D. Li, X. Wang, B. Zhang, H. Zhu, J. Zhao, Galectin-1 is overexpressed
in CD133+ human lung adenocarcinoma cells and promotes their growth and
invasiveness, Oncotarget 6 (2015) 3111–3122.
[37] A. Singh, J. Settleman, EMT, cancer stem cells and drug resistance: an emerging
axis of evil in the war on cancer, Oncogene 29 (2010) 4741–4751.
[38] J.J. Wamsley, M. Kumar, D.F. Allison, S.H. Clift, C.M. Holzknecht, S.J. Szymura,
et al., Activin upregulation by NF-kappaB is required to maintain mesenchymal
features of cancer stem-like cells in non-small cell lung cancer, Cancer Res.
75 (2015) 426–435.
[39] I. Malanchi, H. Peinado, D. Kassen, T. Hussenet, D. Metzger, P. Chambon, et al.,
Cutaneous cancer stem cell maintenance is dependent on beta-catenin
signalling, Nature 452 (2008) 650–653.
[40] I.H. Oh, Microenvironmental targeting of Wnt/beta-catenin signals for
hematopoietic stem cell regulation, Expert Opin. Biol. Ther. 10 (2010)
1315–1329.
[41] H. Wang, G. Zhang, H. Zhang, F. Zhang, B. Zhou, F. Ning, et al., Acquisition of
epithelial-mesenchymal transition phenotype and cancer stem cell-like
properties in cisplatin-resistant lung cancer cells through AKT/beta-catenin/
Snail signaling pathway, Eur. J. Pharmacol. 723 (2014) 156–166.
[42] Y. Teng, X. Wang, Y. Wang, D. Ma, Wnt/beta-catenin signaling regulates cancer
stem cells in lung cancer A549 cells, Biochem. Biophys. Res. Commun. 392
(2010) 373–379.
[43] A.W. Duncan, F.M. Rattis, L.N. DiMascio, K.L. Congdon, G. Pazianos, C. Zhao,
et al., Integration of Notch and Wnt signaling in hematopoietic stem cell
maintenance, Nat. Immunol. 6 (2005) 314–322.
[44] K.A. Hassan, L. Wang, H. Korkaya, G. Chen, I. Maillard, D.G. Beer, et al., Notch
pathway activity identifies cells with cancer stem cell-like properties and
correlates with worse survival in lung adenocarcinoma, Clin. Cancer Res. 19
(2013) 1972–1980.
[45] V. Justilien, A.P. Fields, Molecular pathways: novel approaches for improved
therapeutic targeting of Hedgehog signaling in cancer stem cells, Clin. Cancer
Res. 21 (2015) 505–513.
[46] M. Zuo, A. Rashid, C. Churi, J.N. Vauthey, P. Chang, Y. Li, et al., Novel therapeutic
strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract
cancer, Br. J. Cancer 112 (2015) 1042–1051.
[47] A. Ahmad, M.Y. Maitah, K.R. Ginnebaugh, Y. Li, B. Bao, S.M. Gadgeel, et al.,
Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies
through modulation of EMT-regulating miRNAs, J. Hematol. Oncol. 6 (2013)
77.
[48] M. Kumar, D.F. Allison, N.N. Baranova, J.J. Wamsley, A.J. Katz, S. Bekiranov, et al.,
NF-kappaB regulates mesenchymal transition for the induction of non-small
cell lung cancer initiating cells, PLoS ONE 8 (2013) e68597.
[49] M.B. Kastan, E. Schlaffer, J.E. Russo, O.M. Colvin, C.I. Civin, J. Hilton, Direct
demonstration of elevated aldehyde dehydrogenase in human hematopoietic
progenitor cells, Blood 75 (1990) 1947–1950.
[50] L.C. Hsu, W.C. Chang, I. Hoffmann, G. Duester, Molecular analysis of two closely
related mouse aldehyde dehydrogenase genes: identification of a role for Aldh1,
but not Aldh-pb, in the biosynthesis of retinoic acid, Biochem. J. 339 (Pt 2)
(1999) 387–395.
[51] C. Ginestier, M.H. Hur, E. Charafe-Jauffret, F. Monville, J. Dutcher, M. Brown,
et al., ALDH1 is a marker of normal and malignant human mammary stem
cells and a predictor of poor clinical outcome, Cell Stem. Cell 1 (2007) 555–
567.
[52] A. Shenoy, E. Butterworth, E.H. Huang, ALDH as a marker for enriching
tumorigenic human colonic stem cells, Methods Mol. Biol. 916 (2012) 373–
385.
[53] M.R. Clay, M. Tabor, J.H. Owen, T.E. Carey, C.R. Bradford, G.T. Wolf, et al.,
Single-marker identification of head and neck squamous cell carcinoma cancer
stem cells with aldehyde dehydrogenase, Head Neck 32 (2010) 1195–1201.
[54] C. van den Hoogen, G. van der Horst, H. Cheung, J.T. Buijs, J.M. Lippitt, N.
Guzman-Ramirez, et al., High aldehyde dehydrogenase activity identifies
tumor-initiating and metastasis-initiating cells in human prostate cancer,
Cancer Res. 70 (2010) 5163–5173.
[55] T. Li, Y. Su, Y. Mei, Q. Leng, B. Leng, Z. Liu, et al., ALDH1A1 is a marker for
malignant prostate stem cells and predictor of prostate cancer patients’
outcome, Lab. Invest. 90 (2010) 234–244.
[56] F. Jiang, Q. Qiu, A. Khanna, N.W. Todd, J. Deepak, L. Xing, et al., Aldehyde
dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer, Mol.
Cancer Res. 7 (2009) 330–338.
[57] K. Okudela, T. Woo, H. Mitsui, T. Suzuki, M. Tajiri, Y. Sakuma, et al.,
Downregulation of ALDH1A1 expression in non-small cell lung carcinomas–its
clinicopathologic and biological significance, Int. J. Clin. Exp. Pathol. 6 (2013)
1–12.
[58] E. Charafe-Jauffret, C. Ginestier, F. Iovino, C. Tarpin, M. Diebel, B. Esterni, et al.,
Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and
poor clinical outcome in inflammatory breast cancer, Clin. Cancer Res. 16
(2010) 45–55.
[59] J.P. Sullivan, M. Spinola, M. Dodge, M.G. Raso, C. Behrens, B. Gao, et al., Aldehyde
dehydrogenase activity selects for lung adenocarcinoma stem cells dependent
on notch signaling, Cancer Res. 70 (2010) 9937–9948.
[60] J. Wang, Z.H. Li, J. White, L.B. Zhang, Lung cancer stem cells and implications
for future therapeutics, Cell Biochem. Biophys. 69 (2014) 389–398.
[61] M.P. Barr, S.G. Gray, A.C. Hoffmann, R.A. Hilger, J. Thomale, J.D. O’Flaherty, et al.,
Generation and characterisation of Cisplatin-resistant non-small cell lung
cancer cell lines displaying a stem-like signature, PLoS ONE 8 (2013) e54193.
[62] L. Larzabal, N. El-Nikhely, M. Redrado, W. Seeger, R. Savai, A. Calvo, Differential
effects of drugs targeting cancer stem cell (CSC) and non-CSC populations on
lung primary tumors and metastasis, PLoS ONE 8 (2013) e79798.
[63] J. Mihatsch, M. Toulany, P.M. Bareiss, S. Grimm, C. Lengerke, R. Kehlbach, et al.,
Selection of radioresistant tumor cells and presence of ALDH1 activity in vitro,
Radiother. Oncol. 99 (2011) 300–306.
[64] K. Shien, S. Toyooka, H. Yamamoto, J. Soh, M. Jida, K.L. Thu, et al., Acquired
resistance to EGFR inhibitors is associated with a manifestation of stem cell-like
properties in cancer cells, Cancer Res. 73 (2013) 3051–3061.
[65] R.R. Arasada, J.M. Amann, M.A. Rahman, S.S. Huppert, D.P. Carbone, EGFR
blockade enriches for lung cancer stem-like cells through Notch3-dependent
signaling, Cancer Res. 74 (2014) 5572–5584.
[66] C.P. Huang, M.F. Tsai, T.H. Chang, W.C. Tang, S.Y. Chen, H.H. Lai, et al., ALDH-
positive lung cancer stem cells confer resistance to epidermal growth factor
receptor tyrosine kinase inhibitors, Cancer Lett. 328 (2013) 144–151.
[67] B. Corominas-Faja, C. Oliveras-Ferraros, E. Cuyas, A. Segura-Carretero, J. Joven,
B. Martin-Castillo, et al., Stem cell-like ALDH(bright) cellular states in
EGFR-mutant non-small cell lung cancer: a novel mechanism of acquired
resistance to erlotinib targetable with the natural polyphenol silibinin, Cell
Cycle 12 (2013) 3390–3404.
[68] L. Cortes-Dericks, L. Froment, R. Boesch, R.A. Schmid, G. Karoubi, Cisplatin-
resistant cells in malignant pleural mesothelioma cell lines show
ALDH(high)CD44(+) phenotype and sphere-forming capacity, BMC Cancer 14
(2014) 304.
[69] J.S. Moreb, H.V. Baker, L.J. Chang, M. Amaya, M.C. Lopez, B. Ostmark, et al., ALDH
isozymes downregulation affects cell growth, cell motility and gene expression
in lung cancer cells, Mol. Cancer 7 (2008) 87.
[70] A. O’Brien, J.E. Barber, S. Reid, N. Niknejad, J. Dimitroulakos, Enhancement of
cisplatin cytotoxicity by disulfiram involves activating transcription factor 3,
Anticancer Res. 32 (2012) 2679–2688.
[71] Y. Wang, W. Li, S.S. Patel, J. Cong, N. Zhang, F. Sabbatino, et al., Blocking the
formation of radiation-induced breast cancer stem cells, Oncotarget 5 (2014)
3743–3755.
[72] J.L. Allensworth, M.K. Evans, F. Bertucci, A.J. Aldrich, R.A. Festa, P. Finetti, et al.,
Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent
oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer,
Mol. Oncol. 9 (2015) 1155–1168.
[73] H. Nechushtan, Y. Hamamreh, S. Nidal, M. Gotfried, A. Baron, Y.I. Shalev, et al.,
A phase IIb trial assessing the addition of disulfiram to chemotherapy for the
treatment of metastatic non-small cell lung cancer, Oncologist 20 (2015)
366–367.
[74] Y. Shi, X. Fu, Y. Hua, Y. Han, Y. Lu, J. Wang, The side population in human lung
cancer cell line NCI-H460 is enriched in stem-like cancer cells, PLoS ONE 7
(2012) e33358.
[75] S. Singh, J. Trevino, N. Bora-Singhal, D. Coppola, E. Haura, S. Altiok, et al.,
EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of
stem-like side-population cells in non-small cell lung cancer, Mol. Cancer 11
(2012) 73.
[76] R. Xiang, D. Liao, T. Cheng, H. Zhou, Q. Shi, T.S. Chuang, et al., Downregulation
of transcription factor SOX2 in cancer stem cells suppresses growth and
metastasis of lung cancer, Br. J. Cancer 104 (2011) 1410–1417.
[77] S. Singh, N. Bora-Singhal, J. Kroeger, H. Laklai, S.P. Chellappan, betaArrestin-1
and Mcl-1 modulate self-renewal growth of cancer stem-like side-population
cells in non-small cell lung cancer, PLoS ONE 8 (2013) e55982.
 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156
[78] F. Tian, J. Mysliwietz, J. Ellwart, F. Gamarra, R.M. Huber, A. Bergner, Effects of
the Hedgehog pathway inhibitor GDC-0449 on lung cancer cell lines are
mediated by side populations, Clin. Exp. Med. 12 (2012) 25–30.
[79] M. Zhang, A. Mathur, Y. Zhang, S. Xi, S. Atay, J.A. Hong, et al., Mithramycin
represses basal and cigarette smoke-induced expression of ABCG2 and inhibits
stem cell signaling in lung and esophageal cancer cells, Cancer Res. 72 (2012)
4178–4192.
[80] Y. Wu, R. Si, H. Tang, Z. He, H. Zhu, L. Wang, et al., Cholesterol reduces the
sensitivity to platinum-based chemotherapy via upregulating ABCG2 in lung
adenocarcinoma, Biochem. Biophys. Res. Commun. 457 (2015) 614–620.
[81] Y.P. Hu, L.Y. Tao, F. Wang, J.Y. Zhang, Y.J. Liang, L.W. Fu, Secalonic acid D reduced
the percentage of side populations by down-regulating the expression of
ABCG2, Biochem. Pharmacol. 85 (2013) 1619–1625.
[82] Q. Niu, W. Wang, Y. Li, D.M. Ruden, F. Wang, J. Song, et al., Low molecular
weight heparin ablates lung cancer cisplatin-resistance by inducing
proteasome-mediated ABCG2 protein degradation, PLoS ONE 7 (2012) e41035.
[83] F. Wang, Y.J. Mi, X.G. Chen, X.P. Wu, Z. Liu, S.P. Chen, et al., Axitinib targeted
cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via
inhibiting the drug transport function of ABCG2, Mol. Med. 18 (2012) 887–
898.
[84] V. Levina, A. Marrangoni, T. Wang, S. Parikh, Y. Su, R. Herberman, et al.,
Elimination of human lung cancer stem cells through targeting of the stem
cell factor-c-kit autocrine signaling loop, Cancer Res. 70 (2010) 338–346.
[85] P.A. Horn, H. Tesch, P. Staib, D. Kube, V. Diehl, D. Voliotis, Expression of AC133,
a novel hematopoietic precursor antigen, on acute myeloid leukemia cells,
Blood 93 (1999) 1435–1437.
[86] N. Sanai, A. Alvarez-Buylla, M.S. Berger, Neural stem cells and the origin of
gliomas, N. Engl. J. Med. 353 (2005) 811–822.
[87] A. Pircher, C.M. Kahler, S. Skvortsov, M. Dlaska, G. Kawaguchi, T. Schmid, et al.,
Increased numbers of endothelial progenitor cells in peripheral blood and
tumor specimens in non-small cell lung cancer: a methodological challenge
and an ongoing debate on the clinical relevance, Oncol. Rep. 19 (2008)
345–352.
[88] M.H. Wright, A.M. Calcagno, C.D. Salcido, M.D. Carlson, S.V. Ambudkar, L.
Varticovski, Brca1 breast tumors contain distinct CD44+/CD24- and CD133+
cells with cancer stem cell characteristics, Breast Cancer Res. 10 (2008) R10.
[89] C.A. O’Brien, A. Pollett, S. Gallinger, J.E. Dick, A human colon cancer cell capable
of initiating tumour growth in immunodeficient mice, Nature 445 (2007)
106–110.
[90] J. Miki, B. Furusato, H. Li, Y. Gu, H. Takahashi, S. Egawa, et al., Identification
of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized
primary nonmalignant and malignant tumor-derived human prostate epithelial
cell lines and in prostate cancer specimens, Cancer Res. 67 (2007) 3153–3161.
[91] S. Yin, J. Li, C. Hu, X. Chen, M. Yao, M. Yan, et al., CD133 positive hepatocellular
carcinoma cells possess high capacity for tumorigenicity, Int. J. Cancer 120
(2007) 1444–1450.
[92] M.D. Curley, V.A. Therrien, C.L. Cummings, P.A. Sergent, C.R. Koulouris, A.M.
Friel, et al., CD133 expression defines a tumor initiating cell population in
primary human ovarian cancer, Stem Cells 27 (2009) 2875–2883.
[93] A. Eramo, F. Lotti, G. Sette, E. Pilozzi, M. Biffoni, A. Di Virgilio, et al., Identification
and expansion of the tumorigenic lung cancer stem cell population, Cell Death
Differ. 15 (2008) 504–514.
[94] V. Tirino, R. Camerlingo, R. Franco, D. Malanga, A. La Rocca, G. Viglietto, et al.,
The role of CD133 in the identification and characterisation of tumour-
initiating cells in non-small-cell lung cancer, Eur. J. Cardiothorac Surg. 36 (2009)
446–453.
[95] Y.C. Chen, H.S. Hsu, Y.W. Chen, T.H. Tsai, C.K. How, C.Y. Wang, et al., Oct-4
expression maintained cancer stem-like properties in lung cancer-derived
CD133-positive cells, PLoS ONE 3 (2008) e2637.
[96] G. Bertolini, L. Roz, P. Perego, M. Tortoreto, E. Fontanella, L. Gatti, et al., Highly
tumorigenic lung cancer CD133+ cells display stem-like features and are spared
by cisplatin treatment, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 16281–16286.
[97] A. Murakami, F. Takahashi, F. Nurwidya, I. Kobayashi, K. Minakata, M.
Hashimoto, et al., Hypoxia increases gefitinib-resistant lung cancer stem cells
through the activation of insulin-like growth factor 1 receptor, PLoS ONE 9
(2014) e86459.
[98] L. Lundholm, P. Haag, D. Zong, T. Juntti, B. Mork, R. Lewensohn, et al., Resistance
to DNA-damaging treatment in non-small cell lung cancer tumor-initiating
cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest,
Cell Death Dis. 4 (2013) e478.
[99] S. Sarvi, A.C. Mackinnon, N. Avlonitis, M. Bradley, R.C. Rintoul, D.M. Rassl, et al.,
CD133+ cancer stem-like cells in small cell lung cancer are highly tumorigenic
and chemoresistant but sensitive to a novel neuropeptide antagonist, Cancer
Res. 74 (2014) 1554–1565.
[100] H. Qu, R. Li, Z. Liu, J. Zhang, R. Luo, Prognostic value of cancer stem cell marker
CD133 expression in non-small cell lung cancer: a systematic review, Int. J.
Clin. Exp. Pathol. 6 (2013) 2644–2650.
[101] W. Wang, Y. Chen, J. Deng, J. Zhou, Y. Zhou, S. Wang, The prognostic value of
CD133 expression in non-small cell lung cancer: a meta-analysis, Tumour Biol.
35 (2014) 9769–9775.
[102] L.E. Pascal, A.J. Oudes, T.W. Petersen, Y.A. Goo, L.S. Walashek, L.D. True, et al.,
Molecular and cellular characterization of ABCG2 in the prostate, BMC Urol.
7 (2007) 6.
[103] K.M. Britton, J.A. Kirby, T.W. Lennard, A.P. Meeson, Cancer stem cells and side
population cells in breast cancer and metastasis, Cancers (Basel) 3 (2011)
2106–2130.
155
[104] M.A. Goodell, K. Brose, G. Paradis, A.S. Conner, R.C. Mulligan, Isolation and
functional properties of murine hematopoietic stem cells that are replicating
in vivo, J. Exp. Med. 183 (1996) 1797–1806.
[105] K. Kai, S. D’Costa, B.I. Yoon, A.R. Brody, R.C. Sills, Y. Kim, Characterization of
side population cells in human malignant mesothelioma cell lines, Lung Cancer
70 (2010) 146–151.
[106] M. Christgen, M. Ballmaier, U. Lehmann, H. Kreipe, Detection of putative cancer
stem cells of the side population phenotype in human tumor cell cultures,
Methods Mol. Biol. 878 (2012) 201–215.
[107] B. Sarkadi, C. Ozvegy-Laczka, K. Nemet, A. Varadi, ABCG2—a transporter for
all seasons, FEBS Lett. 567 (2004) 116–120.
[108] S. Zhang, Y. Wang, J.H. Mao, D. Hsieh, I.J. Kim, L.M. Hu, et al., Inhibition of
CK2alpha down-regulates Hedgehog/Gli signaling leading to a reduction of
a stem-like side population in human lung cancer cells, PLoS ONE 7 (2012)
e38996.
[109] Y. An, A. Kiang, J.P. Lopez, S.Z. Kuo, M.A. Yu, E.L. Abhold, et al., Cigarette smoke
promotes drug resistance and expansion of cancer stem cell-like side
population, PLoS ONE 7 (2012) e47919.
[110] B. Fischer, C. Frei, U. Moura, R. Stahel, E. Felley-Bosco, Inhibition of
phosphoinositide-3 kinase pathway down regulates ABCG2 function and
sensitizes malignant pleural mesothelioma to chemotherapy, Lung Cancer 78
(2012) 23–29.
[111] W.J. Yang, M.J. Song, E.Y. Park, J.J. Lee, J.H. Park, K. Park, et al., Transcription
factors Sp1 and Sp3 regulate expression of human ABCG2 gene and
chemoresistance phenotype, Mol. Cells 36 (2013) 368–375.
[112] M.B. Penno, J.T. August, S.B. Baylin, M. Mabry, R.I. Linnoila, V.S. Lee, et al.,
Expression of CD44 in human lung tumors, Cancer Res. 54 (1994) 1381–
1387.
[113] T.M. Phillips, W.H. McBride, F. Pajonk, The response of CD24(-/low)/CD44+
breast cancer-initiating cells to radiation, J. Natl Cancer Inst. 98 (2006)
1777–1785.
[114] E.M. Hurt, B.T. Kawasaki, G.J. Klarmann, S.B. Thomas, W.L. Farrar, CD44+ CD24(-)
prostate cells are early cancer progenitor/stem cells that provide a model for
patients with poor prognosis, Br. J. Cancer 98 (2008) 756–765.
[115] C. Li, D.G. Heidt, P. Dalerba, C.F. Burant, L. Zhang, V. Adsay, et al., Identification
of pancreatic cancer stem cells, Cancer Res. 67 (2007) 1030–1037.
[116] V. Orian-Rousseau, CD44, a therapeutic target for metastasising tumours, Eur.
J. Cancer 46 (2010) 1271–1277.
[117] E.L. Leung, R.R. Fiscus, J.W. Tung, V.P. Tin, L.C. Cheng, A.D. Sihoe, et al., Non-small
cell lung cancer cells expressing CD44 are enriched for stem cell-like properties,
PLoS ONE 5 (2010) e14062.
[118] R. Gomez-Casal, C. Bhattacharya, N. Ganesh, L. Bailey, P. Basse, M. Gibson, et al.,
Non-small cell lung cancer cells survived ionizing radiation treatment display
cancer stem cell and epithelial-mesenchymal transition phenotypes, Mol.
Cancer 12 (2013) 94.
[119] Y.H. Ko, H.S. Won, E.K. Jeon, S.H. Hong, S.Y. Roh, Y.S. Hong, et al., Prognostic
significance of CD44s expression in resected non-small cell lung cancer, BMC
Cancer 11 (2011) 340.
[120] M. Gutova, J. Najbauer, A. Gevorgyan, M.Z. Metz, Y. Weng, C.C. Shih, et al.,
Identification of uPAR-positive chemoresistant cells in small cell lung cancer,
PLoS ONE 2 (2007) e243.
[121] C.E. Almasi, L. Drivsholm, H. Pappot, G. Hoyer-Hansen, I.J. Christensen, The
liberated domain I of urokinase plasminogen activator receptor—a new tumour
marker in small cell lung cancer, APMIS 121 (2013) 189–196.
[122] J.S. Rao, C. Gondi, C. Chetty, S. Chittivelu, P.A. Joseph, S.S. Lakka, Inhibition of
invasion, angiogenesis, tumor growth, and metastasis by adenovirus-mediated
transfer of antisense uPAR and MMP-9 in non-small cell lung cancer cells, Mol.
Cancer Ther. 4 (2005) 1399–1408.
[123] D. Alfano, I. Iaccarino, M.P. Stoppelli, Urokinase signaling through its receptor
protects against anoikis by increasing BCL-xL expression levels, J. Biol. Chem.
281 (2006) 17758–17767.
[124] T. Kubo, N. Takigawa, M. Osawa, D. Harada, T. Ninomiya, N. Ochi, et al.,
Subpopulation of small-cell lung cancer cells expressing CD133 and CD87 show
resistance to chemotherapy, Cancer Sci. 104 (2013) 78–84.
[125] X. Qiu, Z. Wang, Y. Li, Y. Miao, Y. Ren, Y. Luan, Characterization of sphere-
forming cells with stem-like properties from the small cell lung cancer cell
line H446, Cancer Lett. 323 (2012) 161–170.
[126] M. Miettinen, J. Lasota, KIT (CD117): a review on expression in normal and
neoplastic tissues, and mutations and their clinicopathologic correlation, Appl.
Immunohistochem. Mol. Morphol. 13 (2005) 205–220.
[127] H. Joensuu, Treatment of inoperable gastrointestinal stromal tumor (GIST) with
Imatinib (Glivec, Gleevec), Med. Klin. (Munich) 97 (Suppl. 1) (2002) 28–30.
[128] J. Liu, X. Liu, X. Feng, S. Lv, W. Zhang, Y. Niu, C-kit overexpression correlates
with KIT gene copy numbers increases in phyllodes tumors of the breast, Breast
Cancer Res. Treat. 149 (2015) 395–401.
[129] S. Ferrari, A. Grande, P. Zucchini, R. Manfredini, E. Tagliafico, E. Rossi, et al.,
Overexpression of c-kit in a leukemic cell population carrying a trisomy 4 and
its relationship with the proliferative capacity, Leuk. Lymphoma 9 (1993)
495–501.
[130] A.D. Donnenberg, L. Zimmerlin, R.J. Landreneau, J.D. Luketich, V.S. Donnenberg,
KIT (CD117) expression in a subset of non-small cell lung carcinoma (NSCLC)
patients, PLoS ONE 7 (2012) e52885.
[131] D. Perumal, S. Pillai, J. Nguyen, C. Schaal, D. Coppola, S.P. Chellappan, Nicotinic
acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote
stemness in an ARRB1/ beta-arrestin-1 dependent manner in NSCLC,
Oncotarget 5 (2014) 10486–10502.
156 L. MacDonagh et al./Cancer Letters 372 (2016) 147–156
[132] R. Januchowski, K. Wojtowicz, M. Zabel, The role of aldehyde dehydrogenase
(ALDH) in cancer drug resistance, Biomed. Pharmacother. 67 (2013) 669–680.
[133] R.G. Bell, H.W. Smith, Preliminary report on clinical trials of antabuse, Can.
Med. Assoc. J. 60 (1949) 286–288.
[134] D. Chen, Q.C. Cui, H. Yang, Q.P. Dou, Disulfiram, a clinically used anti-alcoholism
drug and copper-binding agent, induces apoptotic cell death in breast cancer
cultures and xenografts via inhibition of the proteasome activity, Cancer Res.
66 (2006) 10425–10433.
[135] P. Boyd, I. Major, W. Wang, C. McConville, Development of disulfiram-loaded
vaginal rings for the localised treatment of cervical cancer, Eur. J. Pharm.
Biopharm. 88 (2014) 945–953.
[136] C. Thoma, Prostate cancer: copper unlocks therapeutic potential of disulfiram,
Nat. Rev. Urol. 11 (2014) 664.
[137] B.W. Morrison, N.A. Doudican, K.R. Patel, S.J. Orlow, Disulfiram induces
copper-dependent stimulation of reactive oxygen species and activation of
the extrinsic apoptotic pathway in melanoma, Melanoma Res. 20 (2010) 11–20.
[138] L. Duan, H. Shen, G. Zhao, R. Yang, X. Cai, L. Zhang, et al., Inhibitory effect of
Disulfiram/copper complex on non-small cell lung cancer cells, Biochem.
Biophys. Res. Commun. 446 (2014) 1010–1016.
[139] Y. Li, H.A. Rogoff, S. Keates, Y. Gao, S. Murikipudi, K. Mikule, et al., Suppression
of cancer relapse and metastasis by inhibiting cancer stemness, Proc. Natl.
Acad. Sci. U.S.A. 112 (2015) 1839–1844.