Original Article

Yonago Acta medica 2016;59:93–99

Wnt/Beta-Catenin Signal Inhibitor HC-1 Sensitizes Oral Squamous Cell Carcinoma
Cells to 5-Fluorouracil through Reduction of CD44-Positive Population
Satoshi Yokogi,*† Toshiaki Tsubota,* Keita Kanki,‡ Junya Azumi,* Noriko Itaba,* Hiroyuki Oka,§ 		
Minoru Morimoto,§ Kazuo Ryoke† and Goshi Shiota**Division of Molecular and Genetic Medicine, Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of
Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan, †Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago 6838503, Japan, ‡Department of Biomedical Engineering, Faculty of Engineering, Okayama University of Science, Okayama 700-0005,
Japan and §Division of Instrumental Analysis, Research Center for Bioscience and Technology, Tottori University, Tottori 680-8550,
Japan.

ABSTRACT

Oral squamous cell carcinoma (OSCC) is a major prevalent malignancy throughout the world, and its mortality rate is approximately 50%.1–3 Despite advances inconventional treatments including surgery, radiotherapy
and chemotherapy, the survival rate for OSCC has not
improved significantly over the past few decades.4 Highmortality seems to be induced by the emergence of therapy-resistant local recurrences and the development of
distant metastases.5, 6 Thus, more effective therapeuticstrategy is urgently required for improving the clinicaloutcome of OSCC.
Recent data have demonstrated that multiple cancers, including OSCC, contain a small subpopulation ofcells that have stem cell-like properties, so-called cancer
stem cells (CSCs).7–9 CSCs have the unique features ofself-renewal and asymmetrical division, and show resistance to radio- and chemotherapy.10, 11 CSCs in OSCC
were first isolated using cell surface marker CD44.5 The
existence of CSCs may explain the high rate of recurrences after conventional therapies. Therefore, targetingof CSCs is considered to provide a new strategy to improve therapeutic effects on many cancers.
Wnt/beta-catenin signal is one of the crucial signaling pathways for self-renewal and differentiation of
CSCs.12, 13 Expression levels of stem cell related genes,
including CD44, are regulated by this pathway. We
have previously shown that Wnt/beta-catenin signal issuppressed during the hepatic differentiation process ofhuman mesenchymal stem cells (MSCs),14, 15 and recently developed small molecule compounds, derivativesof Wnt/beta-catenin signal inhibitors, that efficiently

Background

Oral squamous cell carcinoma is a prevalent and frequently lethal malignancy worldwide. Existence of treatment-resistant cancer stem cells is considered to be associated with tumor formation, recurrence
and metastasis. Wnt/beta-catenin signal is one of the
crucial signaling pathways for cancer stem cells. Wnt/
beta-catenin signal inhibitor may reduce the populationof cancer stem cells and improve therapeutic effects on
the cancers.
Methods The effects of three derivatives of Wnt/
beta-catenin signal inhibitors, HC-1, IC-2 and PN3-13,
which we recently developed, on oral squamous cell carcinoma cell line HSC2, were examined by luciferase reporter assay, WST assay, cell sorting assay and apoptosis
assay.
Results The reporter assay showed that these small
molecule compounds reduced Wnt/beta-catenin transcriptional activity in HSC2 cells. Of these compounds,
IC-2 and PN3-13 inhibited cell viability in a dose-dependent manner, whereas HC-1 did not at even higher
concentrations. Notably, however, the cell-sorting assay
revealed that HC-1 significantly reduces the CD44-positive population of oral squamous cell carcinoma cells,
compared to other compounds without affecting cell
viability. In addition, HC-1 increases the cytotoxicity of
HSC2 cells to 5-fluorouracil. The combination treatmentof HC-1 with 5-fluorouracil significantly increased theapoptotic cells whereas treatment by either compound
did not.
Conclusion These data suggest that HC-1 is an effective compound to target cancer stem cells, and the
combination treatment of HC-1 and 5-fluorouracil canstimulate the tumor suppressive effect on oral squamouscell carcinoma cells.

Corresponding author: Toshiaki Tsubota, PhD
ttsubota@med.tottori-u.ac.jp
Received 2016 January 13
Accepted 2016 February 4
Abbreviations: 5-FU, 5-fluorouracil; ATRA, all-trans retinoic acid;
CSC, cancer stem cell; DMSO, dimethylsulfoxide; HMBA, hexamethylamine bisacetamide; IC50, inhibitory concentration 50; MSC,
mesenchymal stem cell; OSCC, Oral squamous cell carcinoma;
PMA, phorbol myristate acetate

Key words

5-fluorouracil; cancer stem cells; oral
squamous cell carcinoma; small molecule compound;Wnt/beta-catenin signal inhibitor
93

S. Yokogi et al.

suppress Wnt/beta-catenin transcriptional activity in human MSCs.16, 17 In the present study, we found that thesecompounds also suppress the Wnt/beta-catenin signal
in human OSCC cell line HSC2. Of these compounds,
HC-1 effectively reduced the rate of CD44-positive cells
without affecting cell viability. In addition, HC-1 sensitized HSC2 cells to 5-fluorouracil (5-FU), a DNA/RNA
synthesis inhibitor,18 by virtue of enhancement of apoptotic cell death.

x(50-C)/(D-C) + LOG(B)] - A, a higher concentration of
two values that sandwich IC50; B, a lower concentrationof two values that sandwich IC50; C, cell viability (%) at
B; D, cell viability (%) at A.
Flow cytometry

HSC2 cells were seeded onto 10 cm plate at a densityof 5 × 105 cells/well. After overnight incubation, thecells were treated with indicated concentrations of compounds for 48 h. Then cells were sorted by a cell sorting
system (FACS-Aria, Becton Dickinson and Company,
Franklin Lakes, NJ) using CD44 mouse antibody (Cell
Signaling Technology, Danvers, MA) and goat-antimouse IgG Alexa 488 (Life Technologies, Carlsbad,
CA).

MATERIALS AND METHODS
Compounds

We recently developed new small molecular compounds,
the derivatives of the compounds which were originallyreported to be Wnt/beta-catenin signal inhibitors of colorectal cancer cells,19–21 for human MSCs.17 These compounds include HC-1, a derivative of hexachlorophene;
IC-2, a derivative of ICG-001; and PN-3-13, a derivativeof PNU-74654. The compounds were dissolved in dimethylsulfoxide (DMSO) whose final concentration was
1%, and used in the experiments.

Apoptosis assay

HSC2 cells were seeded onto a 6-well plate at a density of 1 × 105 cells/well. After overnight incubation,
the cells were treated with 0.1 µM 5-FU and/or 0.1 µM
HC-1 for 48 h. Then, the cells were stained by annexin-V and PI using an annexin-V-FLOUS Staining Kit(Roche Diagnostics, Indianapolis, IN), photographedwith a microscope (IX-71) (Olympus, Tokyo, Japan) andanalyzed using an image analysis system (inForm 2.0)
(PerkinElmer, Norwalk, CT).

Cells and culture

Human OSCC cell line, HSC2 were kindly provided by
Dr. H. Kugoh (Tottori Univ., Yonago, Japan). The cells
were incubated in DMEM (Life Technologies, Carlsbad,CA) supplemented with 10% FBS, 1% penicillin streptomycin, 2 mM L-glutamine, 0.2% NaHCO3 and 3.5 g/L
D-glucose with or without chemical compounds.

Western blot analysis

Cell lysis proteins (12.5 µg) were subjected to Western
blot assay. The rabbit polyclonal antibody against beta-catenin (Cell Signaling Technology, Danvers, MA)
and goat polyclonal antibody against Actin (Santa Cruz
Biotechnology, Santa Cruz, CA) were used. Actin wasserved as an internal control.

Luciferase reporter assay

HSC2 cells were seeded onto a 24-well plate at a density of 5 × 104 cells/well. After overnight incubation, thecells were transfected with pTCF4-CMVpro-Fluc andpRL-CMV-Rluc plasmids14 using Lipofectamine 2000
(Invitrogen, Carlsbad, CA) for 4 h, and then treated withvarious concentrations of compounds for 48 h. The luciferase activity was measured by Dual-LuciferaseR Reporter assay system (Promega, Madison, WI) and MiniLumat LB 9506 (Berthold Technologies, Bad Wildbad,
Germany).

Statistical analysis

All the values were expressed as mean ± SD. Multiple
comparison was performed by a one-way ANOVA followed by a Tukey HSD test or Games-Howell test using
predictive analytics software (SPSS, Chicago, IL). The
differences between the two groups were analyzed by an
unpaired two-tailed Student’s t-test. A P-value less than
0.05 was considered to be significant.

WST assay

HSC2 cells were seeded onto a 96-well plate at a densityof 2.5 × 103 cells/well. After overnight incubation, thecells were treated with various concentrations of compounds for 48 h. Cell viability was analyzed by WST
assay using Cell Counting kit-8 (Dojindo, Kumamoto,Japan) and Micro plate reader (Tecan, Männedorf, Switzerland). IC50 values are 50% cell growth inhibitory
concentrations of each compound, which are obtained
from the following equation: IC50 = 10^[LOG(A/B)

RESULTS
Small molecule compounds inhibit TCF4/beta-catenin transcriptional activity in OSCC HSC2 cells

To examine the effect of derivatives of Wnt/beta-cateninsignal inhibitors on TCF4/beta-catenin transcriptional
activity in OSCC cells, a luciferase reporter assay was
performed using HSC2 cells. The cells were treatedwith various concentrations of HC-1, IC-2, PN3-13 or
94

1.4

Relative reporter activity(Fluc/Rluc)

Relative reporter activity(Fluc/Rluc)

Sensitization of cancer cells to 5-FU by HC-1

1.2
1.0
0.8

* * * *

0.6
0.4
0.2
0.0

1.2
1.0

* *

0.8
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0.0

0

0.1 0.5510

50 1000

15-FU (µM)
1.2

*

1.0
0.8

*

0.6

*

0.4
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5

10

25

50

* *

1.2
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* *

0.8
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0.4

*

0.2
0.0

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0

1

5

10

25

100

HC-1 (µM)

Relative reporter activity(Fluc/Rluc)

Relative reporter activity(Fluc/Rluc)* *0

50 100

1

5

10

15

25

*
50

PN3-13 (µM)

IC-2 (µM)

Fig. 1. Small molecule compounds inhibit TCF4/beta-catenin transcriptional activity in OSCC HSC2 cells. TCF4/beta-catenin transcriptional activity was examined by luciferase reporter assay with indicated concentrations of 5-FU (0–100 µM), HC-1 (0–100 µM), IC-2
(0–100 µM) and PN3-13 (0–50 µM). The cells were transfected with pTCF4-CMVpro-Fluc plasmid and internal control pRL-CMVRluc plasmid, and luciferase activity of pTCF4-CMVpro-Fluc (Fluc) was normalized by the activity of pRL-CMV-Rluc (Rluc).*P < 0.05,
compared to 0 µM of each compound. The statistics were examined by unpaired Student’s t-test. Data are shown as mean ± SD of threeexperiments. 5-FU, 5-fluorouracil; OSCC, Oral squamous cell carcinoma.

5-FU for 48 h (Fig. 1). All the derivatives significantly
suppressed the luciferase activity in a dose-dependent
manner, indicating that these compounds possess an inhibitory effect on Wnt/beta-catenin signal in HSC2 cells(Fig. 1)

HC-1 reduces CD44-positive population in HSC2
cells

Growing evidence suggests that CSCs are highly resistant to conventional therapies and are responsible forrecurrence and metastasis.10, 11 Thus, the suppression of
CSCs seems to be important for cancer therapy. To examine the effect of Wnt/beta-catenin inhibitors on CSC
population, we next performed FACS analysis using antibody specific to CD44, a CSC marker for OSCC (Fig.
3A).5 HSC2 cells were treated with these compounds for
48 h at two different concentrations (lower and higher)
whose values are close to the IC50 of the cell viability
assay (Fig. 2). Both concentrations displayed similareffects on the CD44-positive population. IC-2 showed amodest decrease of CD44-positive cells, whereas PN313 and 5-FU did not significantly change the rate, com-Cell viability assay of HSC2 cells treated withchemical compounds

To investigate the effect of Wnt/beta-catenin signal
inhibitors on proliferation of HSC2 cells, cell viability
was analyzed by WST assay. The cells were treated withvarious concentrations of compounds for 48 h. IC-2,
PN3-13 and 5-FU significantly reduced cell viability ina dose-dependent manner with IC50 at 31 µM, 9.6 µM
and 4.1 µM, respectively (Fig. 2). HC-1 had little effecton cell viability up to 50 µM, whereas it reduced that toabout 50% at 100 µM.
95

120

140

100

120

*

80

* *

60
40
20

* *

Viability (%)	Viability (%)	S. Yokogi et al.

*

100

*

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5-FU (µM)	

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Viability (%) 	
 

*	
 *	

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Viability (%)

*

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*	

* *

0

100

0

IC-2 (µM)	

1

5

7.5

10

25

50

PN3-13 (µM) 	
 Fig. 2. Cell viability assay of HSC2 cells treated with small molecule compounds. Cells were treated with increasing concentrations of
5-FU, HC-1, IC-2 and PN3-13 for 48 h. Cell viability was analyzed by WST assay. *P < 0.05, compared to 0 µM of each compound. The
statistics were examined by unpaired Student’s t-test. Data are shown as mean ± SD of three experiments. 5-FU, 5-fluorouracil.

pared to DMSO treatment (Fig. 3B). Interestingly, HC-1
clearly and more effectively reduced the CD44-positive
population, compared to other compounds (Fig. 3B),
suggesting that HC-1 efficiently targets CSCs of OSCC
cells.

or without HC-1 (Fig. 4B). Annexin -V and PI staining
revealed that apoptosis increased when the cells weretreated with 5-FU in combination with HC-1, compared
to DMSO treatment (Figs. 4B and C). No induction of
apoptosis was observed in the cells treated with HC-1
or 5-FU alone, which is consistent to the cell viability
assay (Figs. 2 and 4A). These results suggest that HC-1
increases the cytotoxic effect of 5-FU through enhancement of apoptosis in HSC2 cells.

Combined treatment using 5-FU and HC-1 increases the rate of apoptotic cell death

The decrease of the CD44-positive population by HC-1
treatment raised the possibility that HC-1 increases thecytotoxicity of conventional anticancer drugs, such as
5-FU. To test the effect of the combination treatment,
HSC2 cells were treated with 0 to 100 µM 5-FU alone
or in combination with HC-1 (Fig. 4A). The concentration of HC-1 used was 50 µM, which showed no inhibitory effect on HSC2 proliferation (Fig. 2). Notably, HC-1
significantly enhanced the cytotoxicity of 5-FU even at
0.1 µM (Fig. 4A). To investigate how HC-1 enhanced thecytotoxicity of 5-FU, we examined whether apoptosisof the cells is induced by the treatment of 5-FU withDISCUSSION
CSCs have self-renewal ability, higher tumor forming
capacity, and show resistance to chemotherapy.10, 11
Thus, CSCs are thought to be a key factor for tumorigenesis, progression, metastasis and recurrence after
treatments. To improve therapeutic effect on cancers,
targeting the CSCs is considered to provide an effective
strategy. CSCs use many of the same signaling pathways
that have been found in normal stem cells, such as Wnt/
beta-catenin, Notch and Hedgehog.22–24 Wnt/beta-cat96

Yokogi et al., Fig.3, up↑

Sensitization of cancer cells to 5-FU by HC-1

A

CD44 (+) cells (%)

B
100
80

**
*	
*	
*
**	

60

*

*	
*

40
20
0

CD44 (+) cells (%)

Lower concentration

Lower concentration

Higher concentration

100
80
60

*	
*
*	
*	
*	
*	

*
*	
*
*	
*	

40
20
0

Higher concentration

Fig. 3. HC-1 reduces CD44-positive population in HSC2 cells. Cells were treated by 5-FU, HC-1, IC-2 and PN3-13 for 48 h with indicatedconcentrations and sorted by cell sorting system using CD44 antibody.
A: Representative sorting images of the cells treated with lower or higher concentration, of which value is close to IC50 of the compounds(Fig. 2), with the ratio of CD44-positive cells. B: Quantification of CD44-positive cells treated with lower or higher concentration, of
which value is close to IC50. *P < 0.05, **P < 0.01, compared between two groups. The statistics were examined by one-way ANOVA,
followed by Tukey HSD test. Data are shown as mean ± SD of three experiments. 5-FU, 5-fluorouracil; DMSO, dimethylsulfoxide; IC50,
inhibitory concentration 50.

enin signal is an evolutionary conserved developmental
pathway, which is crucial for self-renewal and differentiation of CSCs.12, 13 Since we have recently developed
small molecule compounds, the derivatives of Wnt/beta-catenin signal inhibitors that efficiently suppress Wnt/
beta-catenin transcriptional activity of human MSCs,16,
17
we tested the effects of these inhibitors on HSC2OSCC cells. Here, we showed that HC-1, IC-2 and PN313 reduced Wnt/beta-catenin signal in HSC2 cells. Of
these compounds, HC-1 showed efficient reduction of
CD44-positive population without affecting cell viability. In addition, HC-1 sensitized HSC2 cells to 5-FU,
which inhibits the synthesis of DNA/RNA in non-CSCs.

These results suggest that HC-1 is an effective compound to enhance the cytotoxicity of the cells by shifting
CSCs to non-CSCs (Fig. 4E).
It has been reported that one of the first successesof differentiation therapy is to use all-trans retinoic acid
(ATRA), which was given to patients suffering fromacute promyelocytic leukemia.25 ATRA treatment stimulates the shift of abnormal leukemic promyelocytes
into mature granulocytes, and markedly improves patient survival.26 The success of this therapy has induced
the concept that changes of the cell state are effectively
used to treat other forms of cancers. The other agentsthat have similar effects to change the leukemic cell97

S. Yokogi et al.

A

B
160

*

Viability (%)

140
120
100

5-FU

*

DMSO

5-FU + HC-1(50 µM)

* *

80

HC-1 50 µM

5-FU 0.1 µM
HC-1 50 µM

Phasecontrast*

P = 0.066 	

*Annexin-V

60
40
20

PI

0
0

0.1

0.5

1

5

10

50

100

5-FU concentration (µM)

*
18
16
14
12
10
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6
4
2
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D
*

**	
	
*
PI (+) cells (%)

C
Annexin-V (+) cells (%)

5-FU 0.1 µM

16
14
12
10
8
6
4
2
0

HC-1
DMSO

50 µM

100 µM

Beta-catenin

*

Actin

E
5-FU

5-FU
Wnt/beta-cateninsignal inhibitor

resistant

CD44(+)(CSCs)

HC-1

sensitive(more apoptosis)

CD44(-)(non-CSCs)

Oral squamous cell carcinoma cells

Fig. 4. Combined treatment using 5-FU and HC-1 increases the rate of apoptotic cell death.
A: HSC2 cells were treated with increasing concentration of 5-FU alone or in combination with 50 µM HC-1 for 48 h. Cell viability was
analyzed by WST assay. *P < 0.05, compared between with and without HC-1. The statistics were examined by unpaired Student’s t-test.
Data are shown as mean ± SD of three experiments. At 10 µM 5-FU, P value was 0.066. B: Cells were treated with DMSO, 0.1 µM 5-FU,
50 µM HC-1 or combination of 5-FU and HC-1 for 48 h. The representative cell images are shown [phase contrast (upper), annexin-V
staining (middle) and PI staining (lower)]. Bar = 500 µm. C: Quantification of annexin-V (+) and PI (+) cells. Ten fields were randomly
chosen, photographed from each group, and statistically analyzed. *P < 0.05, **P < 0.01, compared between two groups. The statistics
were examined by one-way ANOVA, followed by Games-Howell test. Data are shown as mean ± SD of 10 fields. D: Western blot analysis of beta-catenin using HSC2 cells treated with DMSO, 50 µM or 100 µM HC-1 for 48 h. Actin was used as an internal control. E:
Model of the chemotherapeutic effect of combined treatment with HC-1 and 5-FU to CD44-positive OSCC cells. Wnt/beta-catenin inhibitor HC-1 stimulates shift of CD44-positive cells (CSCs) to CD44-negative cells (non-CSCs), and sensitizes HSC2 cells to 5-FU throughenhancement of apoptotic cell death. 5-FU, 5-fluorouracil; CSC, cancer stem cell; DMSO, dimethylsulfoxide; OSCC, Oral squamous cell
carcinoma.

state include phorbol myristate acetate (PMA), hexamethylamine bisacetamide (HMBA), DMSO and vitamin
D3.27–30 Since HC-1 efficiently shifted CSCs to nonCSCs in OSCC cells, HC-1 may also be beneficial forcancer therapy. HC-1 is a derivative of hexachlorophene.
Hexachlorophene inhibits Wnt/beta-catenin signal bypromoting E3 ubiquitin-protein ligase SIAH1-dependent degradation of beta-catenin.21 Consistent with this,Western blot analysis showed that HC-1 also decreased
protein levels of beta-catenin (Fig. 4D), suggesting that
HC-1 inhibits Wnt/beta-catenin signal through beta-catenin reduction. Therefore, beta-catenin degradation
may be associated with the reduction of CSC population. Further studies are required to reveal the crucial
function of HC-1 in shifting CSCs to non-CSCs, which
would also facilitate understanding of CSC property and98Sensitization of cancer cells to 5-FU by HC-1

development of cancer chemotherapy.

Murai R, et al. Hepatic differentiation of human bone marrow-derived UE7T-13 cells: Effects of cytokines and CCN
family gene expression. Hepatol Res. 2007;37:1068-79. PMID:
17627621.
16 Itaba N, Matsumi Y, Okinaka K, Ashla AA, Kono Y, Osaki M,
et al. Human mesenchymal stem cell-engineered hepatic
cell sheets accelerate liver regeneration in mice. Sci Rep.
2015;5:16169. PMID: 26553591.
17 Itaba N, Sakabe T, Kanki K, Azumi J, Shimizu H, Kono Y,
et al. Identification of the small molecule compound which
induces hepatic differentiation of human mesenchymal stem
cells. Regenerative Therapy. 2015;2:32-41. DOI: 10.1016/
j.reth.2015.10.001.
18 Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer.
2003;5:330-8. PMID: 12724731.
19 Lepourcelet M, Chen YN, France DS, Wang H, Crews P,
Petersen F, et al. Small-molecule antagonists of the oncogenic
Tcf/beta-catenin protein complex. Cancer cell. 2004;5:91-102.
PMID: 14749129.
20 Voronkov A, Krauss S. Wnt/beta-catenin signaling and small
molecule inhibitors. Curr Pharm Des. 2013;19:634-64. PMID:
23016862.
21 Park S, Gwak J, Cho M, Song T, Won J, Kim DE, et al. Hexachlorophene inhibits Wnt/beta-catenin pathway by promoting
Siah-mediated beta-catenin degradation. Mol Pharmacol.
2006;70:960-6. PMID: 16735606.
22 Takebe N, Miele L, Harris PJ, Jeong W, Bando H, Kahn M,
et al. Targeting Notch, Hedgehog, and Wnt pathways in cancer
stem cells: clinical update. Nat Rev Clin Oncol. 2015;12:44564. PMID: 25850553.
23 Espinoza I, Miele L. Notch inhibitors for cancer treatment.
Pharmacol Ther. 2013;139:95-110. PMID: 23458608.
24 Beachy PA, Hymowitz SG, Lazarus RA, Leahy DJ, Siebold C.
Interactions between Hedgehog proteins and their binding
partners come into view. Genes Dev. 2010;24:2001-12. PMID:
20844013.
25 Warrell RP Jr., de Thé H, Wang ZY, Degos L. Acute promyelocytic leukemia. N Engl J Med. 1993;329:177-89. PMID:
8515790.
26 Mi JQ, Li JM, Shen ZX, Chen SJ, Chen Z. How to manage
acute promyelocytic leukemia. Leukemia. 2012;26:1743-51.
PMID: 22422168.
27 Carey JO, Posekany KJ, deVente JE, Pettit GR, Ways DK.
Phorbol ester-stimulated phosphorylation of PU.1: association
with leukemic cell growth inhibition. Blood. 1996;87:4316-24.
PMID: 8639791.
28 Wu H, Scher BM, Chu CL, Leonard M, Olmedo R, Scher GS,
et al. Reduction in lactate accumulation correlates with differentiation-induced terminal cell division of leukemia cells.
Differentiation. 1991;48:51-8. PMID: 1683843.
29 Arcangeli A, Carlà M, Del Bene MR, Becchetti A, Wanke E,
Olivotto M. Polar/apolar compounds induce leukemia celldifferentiation by modulating cell-surface potential. Proc Natl
Acad Sci USA. 1993;90:5858-62. PMID: 8516337.
30 Olsson I, Gullberg U, Ivhed I, Nilsson K. Induction of differentiation of the human histiocytic lymphoma cell line
U-937 by 1 alpha, 25-dihydroxycholecalciferol. Cancer Res.
1983;43:5862-7. PMID: 6315218.

Acknowledgments: We thank Dr. T. Sakabe for helpful discussion
on this manuscript. This work was supported by a Management
Expenses Grant from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
The authors declare no conflict of interest.

REFERENCES
1 Ferlay J, Shin HR, Bray F, Forman D, Mathers C,
Parkin DM. Estimates of worldwide burden of cancer in 2008:
GLOBOCAN 2008. Int J Cancer. 2010;127:2893-917. PMID:
21351269.
2 Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K,
Sivachenko A, et al. The mutational landscape of head andneck squamous cell carcinoma. Science. 2011;333:1157-60.
PMID: 21798893.
3 Hunter KD, Parkinson EK, Harrison PR. Profiling early headand neck cancer. Nat Rev Cancer. 2005;5:127-35. PMID:
15685196.
4 Gupta S, Kong W, Peng Y, Miao Q, Mackillop WJ. Temporal
trends in the incidence and survival of cancers of the upper
aerodigestive tract in Ontario and the United States. Int J Cancer. 2009;125:2159-65. PMID: 19569190.
5 Prince ME, Sivanandan R, Kaczorowski A, Wolf GT,
Kaplan MJ, Dalerba P, et al. Identification of a subpopulation of cells with cancer stem cell properties in head andneck squamous cell carcinoma. Proc Natl Acad Sci USA.
2007;104:973-8. PMID: 17210912.
6 Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecularbiology of head and neck cancer. Nat Rev Cancer. 2011;11:922. PMID: 21160525.
7 Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells,
cancer, and cancer stem cells. Nature. 2001;414:105-11. PMID:
11689955.
8 Shackleton M, Quintana E, Fearon ER, Morrison SJ. Heterogeneity in cancer: cancer stem cells versus clonal evolution.
Cell. 2009;138:822-9. PMID: 19737509.
9 Meacham CE, Morrison SJ. Tumour heterogeneity and cancer
cell plasticity. Nature. 2013;501:328-37. PMID: 24048065.
10 Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl
J Med. 2006;355:1253-61. PMID: 16990388.
11 Cabrera MC, Hollingsworth RE, Hurt EM. Cancer stem cell
plasticity and tumor hierarchy. World J Stem Cells. 2015;7:2736. PMID:16990388.
12 Takahashi-Yanaga F, Kahn M. Targeting Wnt signaling:
can we safely eradicate cancer stem cells?. Clin Cancer Res.
2010;16:3153-62. PMID: 20530697.
13 Takebe N, Harris PJ, Warren RQ, Ivy SP. Targeting cancer
stem cells by inhibiting Wnt, Notch, and Hedgehog pathways.
Nat Rev Clin Oncol. 2011;8:97-106. PMID: 21151206.
14 Yoshida Y, Shimomura T, Sakabe T, Ishii K, Gonda K,
Matsuoka S, et al. A role of Wnt/beta-catenin signals in hepatic fate specification of human umbilical cord blood-derived
mesenchymal stem cells. Am J Physiol Gastrointest Liver
Physiol. 2007;293:G1089-98. PMID: 17884977.
15 Shimomura T, Yoshida Y, Sakabe T, Ishii K, Gonda K,

99