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Cancer Cell & Microenvironment 2015; 2: e701. doi: 10.14800/ccm.701; © 2015 by Takahito Fukusumi, et al.
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REVIEW
Novel markers of cancer stem cells in head and neck
squamous cell carcinoma
Takahito Fukusumi1, 2, Hideshi Ishii2, Jun Koseki2, Atsushi Hanamoto1, Susumu Nakahara1, Yoshihumi Yamamoto1,
Hidenori Inohara1
1
Department of Otorhinolaryngology - Head and Neck Surgery, Osaka University, Graduate School of Medicine, Suita, 2-2
Yamadaoka, Osaka 565-0871, Japan
2
Department of Cancer Profiling Discovery, Osaka University, Graduate School of Medicine, Suita, 2-2 Yamadaoka, Osaka
565-0871, Japan
Correspondence: Hidenori Inohara
E-mail: [email protected]
Received: March 09, 2015
Published online: May 07, 2015
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, and its prognosis remains
poor. The poor prognosis results from recurrence, metastasis, and therapeutic resistance. Recently, cancer stem
cells (CSCs) were shown to result in treatment failure, but their function and identification are not well
understood in HNSCC. Previously, several markers of CSCs in HNSCC have been reported from 2007 to 2009.
However, whether these markers serve as true markers of CSCs remains controversial. Thus, there is a need to
explore novel CSC markers in HNSCC. Since 2013, several novel HNSCC CSCs markers have been reported,
and in this review we describe and discuss these markers.
Keywords: cancer stem cells; CD44v; CD98; CD271; CD200; CD10; head and neck squamous cell carcinoma
To cite this article: Takahito Fukusumi, et al. Novel markers of cancer stem cells in head and neck squamous cell carcinoma.
Can Cell Microenviron 2015; 2: e701. doi: 10.14800/ccm.701.
Introduction
Head and neck squamous cell carcinoma (HNSCC) is the
sixth most common cancer[1]. HNSCC management largely
consists of surgical resection, chemotherapy, and radiation
therapy[2]. Although recent progress of its management, the
prognosis remains poor[3], and only 50%−60% of the patients
are cured[4, 5]. Additionally, HNSCC treatment can be
traumatic and affect patient quality of life, specifically
speech and swallowing[6, 7]. The poor prognosis of HNSCC
results from high rates of recurrence and metastases.
Currently, there are no suitable prognosis markers; gene
expression profiling from biopsies did not show gene sets
with predictive significance[8].
Recently, the theory of cancer stem cells (CSCs) was
developed[9]. CSCs are defined as cells that have
tumorigenicity and self-renewal properties. In terms of
clinical characteristics, CSCs are capable of metastasizing[10]
and contribute to therapeutic resistance, such as
chemoresistance and radio-resistance[11]. Thus, the
identification of true CSC markers is the first step for the
establishment of prognostic biomarkers and target-specific
drugs. Previously, CD44[12], CD133[13], and aldehyde
dehydrogenase 1 (ALDH1)[14] were reported as CSC markers
in HNSCC from 2007 to 2009 (Table 1). Several reports also
revealed that expression of these markers in HNSCC cells
exhibit CSC properties. For example, CD44(+) cells from
primary tumors can give rise to tumors more rapidly in
xenograft models compared to CD44(−) cells, and these
xenograft tumors subsequently reproduce the original tumor
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Table 1. Previous CSCs markers in HNSCC and the conflicting data
Marker
CD44
Year
2007
CD133
2008
ALDH1
2009
Contrary opinions
CD44 expression in normal head and neck epithelium is as same
as in HNSCC.
CD44(+) and CD44(−) cells derived from squamous-spheres are
capable of regenerating these spheres from single cell
suspensions.
The CD44(−) cells have sphere-formation ability and
tumor-initiating capacity similar to the CD44(+) cells.
Reference
18
In clinical specimens of hypopharyngeal cancers, CD133
expression was negative, as determined by flow cytometry
analysis and immunohistochemistry.
Poor prognosis linked to decreased rather than increased
expression of ALDH1
21
heterogeneity observed in the primary tumor. Importantly,
CD44(+) cells have also been discovered to have a greater
capacity to handle oxidative stress and exhibit
radio-resistance[15]. This population has also been shown to
have a significantly greater ability to metastasize to regional
lymph nodes in animal models[16], and patients whose tumors
have greater percentages of CD44(+) cells have significantly
poorer clinical outcome[17].
However, whether these markers are true CSC markers
remains controversial (Table 1). For example, CD44
expression in normal head and neck squamous epithelium is
as same as in HNSCC[18]. Lim et al. found that both CD44(+)
and CD44(−) cells derived from squamous-spheres are
capable of regenerating these spheres from single cell
suspensions[19]. Oh et al. showed that CD44(−) cells have
stem-cell like traits. In their report, CD44(−) cells showed
sphere-formation ability and tumor-initiating capacity similar
to CD44(+) cells[20]. In clinical specimens from
hypopharyngeal cancers, CD133 expression was negative, as
determined by flow cytometry and immunohistochemistry[21].
Furthermore, poor prognosis linked to decreased rather than
increased expression of ALDH1[22]. Because of these
discrepancies, the novel CSCs marker in HNSCC is needed
to explore. Since 2013, several novel HNSCC CSCs markers
have been reported. The methods used to find these markers,
and the phenotypes of these proposed CSCs differ. In this
review, we explain and explore these markers in
chronological order.
CD44v
Yoshikawa et al. reported that CD44v(+) cells have CSC
properties in HNSCC[23]. CD44 is a major marker for CSCs
in many epithelial tumors[24-26] and is implicated in tumor
growth, invasion, metastasis[27, 28]. CD44 have variant
isoforms generated by alternative mRNA splicing[29].
Previously, their group showed that CD44v8-10(+)
gastrointestinal cancer cells have CSC phenotypes, and
CD44v interacts with the cystine transporter subunit xCT.
xCT reduces glutathione (GSH) and promotes reactive
19
20
22
oxygen species (ROS) resistance in cancer cells[30].
Sulfasalazine is a specific inhibitor of cysteine transport that
is mediated by xCT, and suppresses the proliferation,
metastasis, and invasion of several cancers[31, 32]. They also
showed that sulfasalazine treatment suppressed tumor growth
and chemoresistance of CD44v(+) gastrointestinal cancer
cells[30]. Similar to gastrointestinal cancer, Yoshikawa et al.
explored whether the CD44v(+) cells in HNSCC have
various CSC properties and investigated their relationship
with xCT. To explore the clinical characteristics of CD44v(+)
cells in HNSCC patients, CD44v9 expression in tumor
tissues of patients treated with or without neoadjuvant
chemotherapy were evaluated. CD44v(+) areas are increased
in HNSCC tumors of patients who received neoadjuvant
chemotherapy, suggesting that the CD44v(+) cells have
chemoresistance compared to the CD44v(−) cells. Moreover,
expression levels of the differentiation marker involucrin
tended to be reduced in tumor tissue resected after
neoadjuvant chemotherapy compared with patients not
subjected to chemotherapy. The GSH-dependent antioxidant
system promotes resistance of CD44v(+) gastrointestinal
cancer cells[30]. Thus, to investigate the chemoresistant
mechanism in HNSCC cells, they examined the effect of
cisplatin, which induces cytotoxity mediated by ROS[33]. The
results show that CD44v expression status of HNSCC cell
lines is related to cisplatin resistance. Additionally, the
GSH-dependent system plays a key role in cisplatin
resistance as a result of its ability to scavenge ROS[34];
therefore, they also examined GSH levels in HNSCC cell
lines. As expected, intracellular GSH levels correlated with
cisplatin resistance. Given that xCT promotes GSH synthesis,
and that CD44v stabilizes xCT and ROS defense in cancer
cells[30], they next examined the effect of sulfasalazine on
HNSCC cells. The results showed that CD44v(+) cells are
sensitive to sulfasalazine in vivo. Moreover, the tumors
formed by the CD44v(+) cell line comprised a heterogeneous
population of CD44v(+) and CD44v(−) cells. Furthermore,
CD44v(−) cells in established tumors expressed involucrin.
Given that the expression level of the epidermal growth
factor receptor (EGFR) affects the squamous cell carcinoma
differentiation[35], Yoshikawa et al. investigated cellular
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EGFR expression and revealed that it was lower in the
CD44v(+) cells than in CD44v(−) cells. These results suggest
that EGFR signaling is suppressed in CD44v(+) cells.
Moreover, the effect of the EGFR inhibitor increased
significantly followed CD44 ablation. They also investigated
the effect of combined treatment consisting of the EGFR
inhibitor cetuximab and sulfasalazine on mouse xenograft
tumor; sulfasalazine significantly increased the anti-cancer
effect of cetuximab. Therefore, they concluded that
CD44v(+) HNSCC cells are undifferentiated, are
chemoresistant, stabilize xCT, and maintain low ROS levels.
However, CD44v(+) cells are sensitive to the xCT inhibitor
sulfasalazine. Thus, they suggest targeting the CD44v(+)
CSC population using sulfasalazine.
CD98
Squamous epithelium stem cells are hypothesized to exist
in the basal cell layer[36]. Moreover, mutations of normal
epithelium stem cells may be the first step of oncogenesis[37].
Kemp et al. generated a monoclonal antibody for squamous
cell carcinoma surface antigens[38]. This monoclonal antibody
K984 targeted undifferentiated cells in the basal layer. To
determine what was recognized by K984, they performed
immunoprecipitation. Analysis of this immunoprecipitation
product obtained from this study indicated that this antibody
combines CD98. CD98 expression has been shown to relate
to the survival rate of several cancers[39-43]; these studies
indicate that higher CD98 expression in tumors is related to
poor prognoses. In head and neck carcinoma, CD98
expression is an independent prognostic marker[44]; however,
the underlying mechanisms of this relationship is not clear.
CD98 consists of heavy chains and light chains. The CD98
heavy chain was shown to mediate integrin signals[45],
leading to AKT phosphorylation and stimulate cell survival
and metastasis. On the other hand, its light chain can import
essential amino acids[46], which activate the mTOR pathway.
This pathway stimulates cell proliferation and protein
synthesis. Kemp et al. reported that CD98 is specifically
expressed on the basal layer of normal mucosa and HNSCC,
and assumed that CD98 is a novel marker of HNSCC
CSCs[47]. To confirm this hypothesis, they first examined
CD98 and CD44 expression by immunohistochemistry and
found that the expression of CD98 overlaps with CD44v6 in
HNSCC tumors. Next, they evaluated the tumorigenicity of
the CD98(+) cells by means of transplantation into
immunodeficient mice. Mice injected with unselected cells
showed one tumor following six injections of 25,000 cells.
CD98(−) cells did not result in any tumor growth, whereas
CD98(+) cells grew into tumors in four out of six injections
of 25,000 cells. These tumors arising from the CD98(+) cells,
were passaged by injection of 25,000 sorted cells from the
CD98(+) and CD98(−) population. The CD98(−) cells form
no tumors, whereas CD98(+) cells developed tumors. From
these observations, they concluded that CD98(+) cells have
more tumorigenicity than the CD98(−) cells. To further
investigate the difference between CD98(+) and CD98(−)
cells at the molecular level, they isolated these cell
populations from mice tumors made by a HNSCC cell line,
and analyzed gene expression using a microarray. 292 genes
were expressed significantly higher in CD98(+) cells than
CD98(−) cells. They used a database and found that cell
cycle and DNA integrity genes were significantly enriched in
this dataset. In this result, CD98(+) cells were indicated to
control cell cycle that is tightly regulated by CSCs.
Conversely, 387 genes that were expressed significantly
higher in CD98(−) cells than CD98(+) cells. Using a
database, they found that the dataset was significantly
enriched for genes related to cellular differentiation,
including keratinocyte differentiation. This result implies that
CD98(−) cells are differentiated and therefore not able to
produce progeny. However, stem cell related genes, such as
OCT3/4, SOX2, KLF4, and Nanog, did not show significant
differences between CD98(+) and CD98(−) cell populations.
CD271
CD271 is known as the low affinity nerve growth factor
receptor (NGFR) or p75 neurotrophin receptor. In the
nervous system, CD271 has critical functions in cell
survival[48], differentiation[49], and migration[50] of neuronal
cells. Previously, CD271 was reported as a tissue stem cell
marker of laryngeal[51], oral[52], and esophageal squamous
epithelia[53]. CD271 is also reported to be a CSC marker in
malignant melanoma[54, 55], and esophageal squamous cell
carcinoma[56]. Given this background, Imai et al. speculated
that CD271 is a marker of CSCs in HNSCC[21]. To prove this
hypothesis, they used tumor specimens resected from
hypopharyngeal cancer (HPC) patients, and injected these
specimens into immunodeficient mice. From this, they
established three xenograft tumor lines. In these transplanted
tumor lines, the CD271 expression was analyzed. The result
showed that 2%−20% of cells were positive for CD271. Next,
they analyzed the location of CD271(+) cells in transplanted
tumors. The most of CD271(+) cells existed in the basal
layer of the tumor, and in the peripheral zone of the tumor.
CD271(+) cells were almost observed next to the stroma.
CD271(+) cells were also moderately positive for CK8,
which is a marker of undifferentiated squamous carcinoma
cells. They also examined CD271 expression within clinical
HPC specimens and normal hypopharyngeal mucosa. Similar
to transplanted tumors, CD271(+) cells exist in the basal
layer and in the invasive cancer front. These results suggest
that CD271(+) HPC cells exist in the basal layer where CSC
may reside, and they contain undifferentiated cells. They
further examined whether sphere formation culture would
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affect CD271 expression and showed that sphere formation
caused enrichment of the CD271(+) population.
Tumorigenicity analysis of the CD271(+)/(−) cells was also
performed; CD271(+) cells kept tumorigenic with as few as
30 cells, and possessed higher tumorigenicity than CD271(−)
cells. They next asked whether the CD271(+) cells had
characteristics associated with stemness, invasion, and
metastasis. They examined the expression of stem cell
related genes, Nanog, Oct3/4, and Sox2. Nanog expression
was significantly higher in the three CD271(+) HPC lines
compared to CD271(−) cells. However, Oct3/4 and Sox2
expression showed no significant difference between
CD271(+) cells and CD271(−) cells. Next, they examined the
expression of MMP1, MMP2, and MMP10 that were known
as invasion related genes. The CD271(+) cells of the HPC
lines showed a significantly higher expression of these genes
than
CD271(−)
cells.
They
also
examined
immunohistochemistry staining of these MMPs, and showed
the CD271(+) cells were positive for all of these genes.
Considering the localization of the CD271(+) cells at
invasive fronts, these results suggest that CD271(+) cells are
affected by MMPs that enable tissue invasion. To determine
whether CD271(+) cells have chemoresistant properties, the
effect of cisplatin on these cells was analyzed. They treated
mice bearing tumors with cisplatin. The tumors were
resected and analyzed by flow cytometry analysis on day 7.
The CD271(+) fraction increased from 16.3% to 35.2%. This
result indicated that the CD271(+) cells had cisplatin
resistance. The chemoresistance of CSCs is linked to higher
expression of the ATP binding transporter[57]. Therefore, they
compared the expression of ABCC2, ABCB5, and ABCG2
between CD271(+) and CD271(−) cells; the expression of
these ABC-transporter in CD271(+) cells was significantly
higher than that in CD271(−) cells. Next, they investigated
whether CD271 expression related to the prognosis of HPC
patients. They classified 83 patients as strong or
moderate-weak group according to the degree of CD271
expression. The 3-year survival rate of the CD271 strong
group was significantly lower than that of the CD271
moderate-weak group. These date indicate that CD271
expression is associated with poor clinical outcome.
Murillo-Sauca et al. also indicated that CD271 is a novel
HNSCC CSC marker[58]. They mainly used surgical
specimens and cell lines in oral squamous cell carcinoma.
Similar to Imai et al., CD271 expression was located in the
basal layer of malignant epithelium, and CD271(+) cells
exhibited greater tumorigenicity than CD271(−) cells in oral
squamous cell carcinoma. Additionally, they examined the
interdependence between CD271 and CD44; the CD271(+)
cells comprised a subpopulation among CD44(+) cells in
primary tumors, but there was no appreciable
CD44(−)CD271(+) subpopulation by flow cytometry. Thus,
they sorted the CD44(+)CD271(+) cells, CD44(+)CD271(−)
cells, and CD44(−)CD271(−) cells from primary human oral
SCC specimens and implanted them into immunodeficient
mice. Overall, the CD44(+) cells had a greater capacity to
form xenograft tumors compared to CD44(−) cells, but when
low numbers of cells were injected, it was clear that the
CD44(+)CD271(+) cells had the greatest capacity to form
tumors among these three populations. The tumors that
formed in mice implanted with CD44(+)CD271(+) cells
recapitulated the heterogeneity seen in the parental tumor. To
assess the function of CD271, they generated CD271
knockdown cells, and investigated subsequent tumor growth
and cell cycle. Their results showed that CD271 significantly
affected tumor growth and a partial cell cycle block during
G2-M transition. Based on these results, they suggested that
this molecule might be a therapeutic target in HNSCC. To
address this hypothesis, they focused on the fact that CD271
is known as low affinity NGFR. They targeted the receptor
with a monoclonal antibody specific for NGFR. Incubation
of oral squamous cancer cells with this antibody resulted in a
significant reduction in cell proliferation in vitro.
Furthermore, cells treated with the anti-NGFR antibody
resulted in a significant reduction in tumor growth in vivo.
CD271/NGFR activation has been previously shown to
work through downstream activation of both the MAPK and
PI3K pathways[59]. To determine the functional role of
CD271 in HNSCC, they treated cells with recombinant
human NGF in vitro and assessed activation of the MAPK
pathway. NGF treatment resulted in an increase in detectable
Erk phosphorylation. Furthermore, pre-incubation with the
monoclonal antibody against CD271 abrogated this increase
in Erk phosphorylation. However, there was no apparent
effect on the PI3K pathway, since p-Akt was not affected.
Their data indicate that CD271/NGFR is functionally active
in HNSCC and that targeting CD271 with a monoclonal
antibody is a potential therapeutic strategy in this cancer.
One of the significant adverse features seen with some
HNSCC tumors is perineural invasion (PNI), which has been
associated with local recurrence and metastases[60]. Because
Schwann cells around neurons and epithelium rich in neurons
express NGF[61, 62], they assumed that expression of CD271
might predispose tumor cells to PNI. Indeed, in malignant
melanoma, CD271 expression has been associated with
PNI[63], and in oral cancer[64, 65], NGF expression has also
been associated with PNI.
CD200
CD200 is a type-I transmembrane glycoprotein[66]. CD200
is expressed in kidney glomeruli, endothelial cells,
lymphocytes, neurons, hair follicle epithelial cells, and
thymocytes[67]. Several cancer cells and tissues express
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CD200, including melanoma, malignant mesothelioma,
neuroblastoma, chronic lymphocytic leukemia, ovarian,
testicular, renal cell carcinoma, prostate, breast, and colon
cancers[68]. CD200 is also interdependent on CSC markers of
prostate, colon cancer, glioblastoma, and breast[69], but there
are no studies focused on the role of CD200.
Jung et al. proposed CD200 as a HNSCC CSC marker[70].
First, to assess the expression of CD200, several HNSCC cell
lines of varying human papillomavirus (HPV) statuses were
analyzed; CD200 was expressed in all HNSCC cell lines
irrespective of HPV status. These results indicate that HPV
status does not related to CD200 expression. Next, they
analyzed the relationship between CD200 and the CSC
related markers, such as sonic hedgehog (Shh) and B
lymphoma Mo-MLV insertion region 1 homolog (Bmi-1).
Their result showed that Shh signal intensity was related to
CD200 signal intensity, and that overexpression of CD200
increased Shh and Bmi-1 expression. Next, they used tonsil
epithelial cells of wild-type mouse. They transfected HPV E6,
E7 protein and Ras, and immortalized these cells (MEER).
MEER were transfected to overexpress CD200 to investigate
whether CD200 expression results in phenotypic changes
associated with oncogenesis. CD200 overexpressing cells
demonstrated significantly slower proliferation than control
MEER cells. However, colony formation assays after
exposure to cisplatin and radiation revealed that CD200 did
not induce resistance in vitro. To evaluate if the expression
of CD200 affects the resistance of cisplatin and radiation in
vivo, they injected CD200 overexpressing and control MEER
cells (1 × 106 cells) into C57BL/6 mice, and treated mice
with cisplatin and radiation. CD200 overexpressed cells grew
significantly faster than control cells. Moreover, the survival
rate of CD200 overexpressed cells was significantly worse
than control cells. These data indicated that CD200
expression related to the resistance of chemo-radiation in
vivo.
However,
tumorigenicity without
treatment
demonstrated no difference between CD200 overexpressed
cells and control cells when injected into wild type or
immunodeficient mice.
CD10
CD10 was detected by an antigen array Lyoplate to
identify antigens refractory to cisplatin and radiation. The
BD Lyoplate Human Cell Surface Marker Screening Panel
consists of 242 purified monoclonal antibodies against cell
surface markers (CD1−CD340). This is the first research that
attempted to identify the marker of therapeutic resistance and
CSC properties using this array. The antigen expression
levels of three HNSCC cell lines that survived cisplatin or
radiation treatment were compared with untreated
counterparts using this array. The result showed that four cell
surface antigens, CD10, CD15s, CD146, and CD282, were
upregulated in all cell lines after cisplatin or radiation
exposure. To validate whether these antigens related to
therapeutic resistance, they used a cisplatin-resistant cell line,
and compared these four antigens expression. They found
that only CD10 expression was higher in cisplatin-resistant
cells than parental cells. Indeed, the CD10(+) subpopulation
accounted for 22.5% of cisplatin-resistant cells compared
with 1.4% of parental cells, respectively. They concluded
that CD10 is the marker of therapeutic resistance in HNSCC
cells[71].
CD10 is a zinc-dependent metalloendoprotease that
cleaves signaling peptides[72, 73]. CD10 has been shown to
expressed in various normal cells, and to be the tissue stem
cell marker of the bone marrow[74], adipose tissue[75], lungs
[76], and breasts[77]. CD10 is also expressed in several types of
cancers, such as the kidney, liver, skin, cervix, prostate, lung,
breast, pancreas, stomach, and bladder[78]. The association
between CD10 and metastasis have been shown by several
reports[78]. In HNSCC, the role of CD10 in tumor
differentiation and growth has been reported[79]; however, the
underlying mechanism is not clear. Fukusumi et al. focused
on the cell cycle, because slow-cell cycling cells are resistant
to chemotherapeutic agents[80], and most resistant to radiation
during G0/1 and S phases[81]. Using Hoechst33342 staining,
they revealed that CD10(+) cells were slow-cell cycling
compared to CD10(−) cells. Given that CSCs are responsible
for therapeutic resistance[11], and are slow-cell cycling and
mainly in G0/G1 phase[82-84], they hypothesized that CD10
serve as a CSC marker of HNSCC. First, they compared the
CD10 expression in spheroid cells and control adherent cells.
In two HNSCC cell lines, CD10 expression in spheroid cells
was significantly greater than in adherent cells. Next, they
compared sphere formation ability between CD10(+) and
CD10(−) cells; CD10(+) cells formed significantly more
spheroid colonies than CD10(−) cells. Moreover, CD10(+)
colonies were significantly larger than CD10(−) colonies.
They also examined whether CD10(+) cells had more
tumorigenicity than CD10(−) cells in vivo. They sorted
CD10(+)/(−) cells and individually injected into
immunodeficient mice. When 1,000 cells were injected, the
CD10(+) cells formed tumors in all mice, while the CD10(−)
cells formed tumors in one third of mice. Furthermore,
CD10(+) cells kept tumorigenic with only 100 cells. Based
on these results, they concluded that the CD10(+) cells had
sphere formation ability in vitro and tumorigenicity in vivo
compared to the CD10(−) cells. They also examined the
interdependence between CD10 and other HNSCC CSCs
markers, such as CD44 and ALDH1. They found no
significant interdependence between CD44 and CD10. On
the other hand, they revealed CD10(+) cells contained
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Table 2. Characteristics of current CSCs markers in HNSCC
Marker
Year
CD44v
2013
CD98
2013
CD271
2013
CD271
2014
CD200
2014
CD10
2014
Materials
cell line
cell line, clinical
specimen
clinical
specimen
cell line, clinical
specimen
cell line
cell line
Chemoresistance
CDDP
-
CDDP
-
CDDP*
CDDP, 5-FU
Radioresistance
-
-
-
-
Yes*
Yes
Sphere assay
Yes
-
Yes
-
-
Yes
Minimum cells
of tumorigenicity
Related genes
-
25,000
30
100 **
-
100
xCT
genes related to
cell cycle and
DNA integrity
Nanog, MMPs,
ABCC2,
ABCB5,
ABCG2
-
Shh, Bmi1
Oct3/4
Function
ROS resistance
-
-
cell cycle block
-
slow cell cycle
Reference
23
47
21
58
70
71
CDDP: cisplatin; 5-FU: fluorouracil; ROS: reactive oxygen species. *mouse tonsil epithelial cell line; **CD44(+)CD271(+) cells
significantly more ALDH1(+) fraction compared to CD10(−)
cells. To determine the mechanisms of tumorigenicity and
the self-renewing properties of CD10(+) cells, the expression
of OCT3/4, a tissue stem cell gene[85], and CSCs[86] was
compared between CD10(+) and CD10(−) cells. Chen et
al.[18, 87] showed that HNSCC CSCs upregulated OCT3/4. As
expected, the expression of OCT3/4 in the CD10(+) cells was
significantly increased compared to the CD10(−) cells. Of
note, CD10 knockdown using siRNA resulted in decreased
OCT3/4 expression.
experiments using these markers. There are also reports that
performed experiments to compare expression of potential
CSC markers with previous HNSCC markers. For example,
the CD10(+) cells expressed significantly more ALDH1 than
CD10(−) cells[71]. Again, however, there are no studies
investigating CSC properties using a combination of these
recent markers. These facts indicate that further studies
combining various markers are needed. In fact, CSC markers
of other organs, such as AML[88] and breast cancer[24], are
defined by a combination of several CD markers.
Discussion
Table 2 shows the genes related to the investigated
markers. Each CD marker is related to different genes, and
almost all of these genes are related to stemness. The
relationship between CSCs and squamous epithelium stem
cells also remains unknown. As for squamous epithelium
stem cells in the head and neck region, these cells reside in
the basal cell layer[36]. Bmi-1[90] and Lgr5[36] were reported as
markers of squamous epithelium stem cells. Bmi-1 has been
already been examined with respect to a HNSCC CSC
context [87]: Bmi-1 overexpression ALDH1(+) HNSCC cells
resulted in increased colony formation, migration, and
invasion. Moreover, Bmi-1 over expression increased tumor
formation, radio-resistance, and distant metastasis[91].
CD98[44] and CD271[21, 58] also exist in the basal layer of
malignant epithelium; however, these reports did not show
the relationship or changes between normal epithelium and
cancerous tissue.
CSCs were first identified in acute myeloid leukemia;
acute myeloid leukemia cells with CD34(+)CD38(−) are
tumorigenic in immunodeficient mice[88]. Since this report,
several others have described CSC markers in several organs.
For instance, brain CD133(+) tumor cells[89] and breast
CD44(+)CD24(−) cancer cells [24] were identified as CSCs.
From 2007 to 2009, CSC markers in HNSCC have been
reported. However, whether these markers are true CSCs
markers remains controversial (Table 1). Thus, novel CSC
markers in HNSCC require further exploration.
Since 2013, several CSC markers, such as CD44v[23],
CD98[47], CD271[21, 58], CD200[70], and CD10[71] were
reported HNSCC (Table 2). The strategies for finding these
markers vary, and include investigating a CSC marker of
other organs, staining the basal cell layer, and using a cell
surface antigen array. These markers have also been studied
with respect to known CSC properties, such as
chemoresistance, radio-resistance, sphere formation assay,
tumorigenicity, and the expression of stem cell related genes.
However, to date, there is no report that performs all of these
Conclusions
Recently, several HNSCC CSC markers have been
reported. Identification of accurate HNSCC CSC markers is
imperative to reveal this disease characteristic. But, further
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Cancer Cell & Microenvironment 2015; 2: e701. doi: 10.14800/ccm.701; © 2015 by Takahito Fukusumi, et al.
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study about the mechanisms and functions of these markers
is needed. These studies may contribute to the diagnosis,
prognosis, and treatment of HNSCC patients.
Conflicting interests
Institutional endowments were received partially from
Taiho Pharmaceutical Co., Evidence Based Medical (EBM)
Research Center, Chugai Co., Ltd, Yakult Honsha Co., Ltd.,
and Merck Co., Ltd.
Author contributions
All authors conceived of the study, and participated in its
design and coordination and helped to draft the manuscript.
All authors read and approved the final manuscript.
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