Ann Surg Oncol
https://doi.org/10.1245/s10434-019-07172-7

ORIGINAL ARTICLE – GASTROINTESTINAL ONCOLOGY

Comprehensive Genetic Search to Clarify the Molecular
Mechanism of Drug Resistance Identifies ASCL2-LEF1/TSPAN8
Axis in Colorectal Cancer
Toshimichi Tanaka, MD1, Keita Kojima, MD, PhD1, Kazuko Yokota, MD1, Yoko Tanaka, MD1, Yosuke Ooizumi,
MD1, Satoru Ishii, MD, PhD1, Nobuyuki Nishizawa, MD, PhD1, Keigo Yokoi, MD, PhD1, Hideki Ushiku, MD, PhD1,Mariko Kikuchi, MD, PhD1, Ken Kojo, MD, PhD1, Naoko Minatani, MD, PhD1, Hiroshi Katoh, MD, PhD1,
Takeo Sato, MD, PhD1, Takatoshi Nakamura, MD, PhD1, Masakazu Sawanobori, MD, PhD2, Masahiko Watanabe,
MD, PhD, FACS1, and Keishi Yamashita, MD, PhD, FACS2,3
Department of Surgery, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan; 2Epigenetic Treatment
Research Group, Chiyoda-ku, Tokyo, Japan; 3Division of Advanced Surgical Oncology, Department of Research andDevelopment Center for New Medical Frontiers, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan
1

ABSTRACT
Background. Treatment-resistance genes limiting anticancer therapy have not been well clarified in colorectal
cancer (CRC). We explored gene expression profiles to
identify biomarkers for predicting treatment resistance toan anticancer drug in CRC.
Methods. Six CRC cell lines were treated with phenylbutyrate (PB). The gene expression profiles were thencompared using microarrays (harboring 54,675 genes), andgenes associated with PB resistance were identified. Candidate genes were functionally examined in cell lines andclinically validated for treatment resistance in clinicalsamples.
Results. Both DLD1 and HCT15 cells were PB resistant,while HCT116 cells were identified as PB sensitive. On
microarray analysis, among the PB resistance-related
genes, the expression of the genes ASCL2, LEF1, andToshimichi Tanaka and Keita Kojima contributed equally to thiswork.

Electronic supplementary material The online version of thisarticle (https://doi.org/10.1245/s10434-019-07172-7) contains
supplementary material, which is available to authorized users.
Ó Society of Surgical Oncology 2019
First Received: 22 October 2018
K. Yamashita, MD, PhD, FACS
e-mail: keishi23@med.kitasato-u.ac.jp

TSPAN8 was clearly associated with PB resistance. PBsensitive cells transfected with one of these three genes
exhibited significant (P \ 0.001) augmentation of PB
resistance; ASCL2 induced expression of both LEF1 andTSPAN8, while neither LEF1 nor TSPAN8 induced ASCL2.
RNA interference via ASCL2 knockdown made PB-resistant cells sensitive to PB and inhibited both genes. ASCL2
knockdown also played a critical role in sensitivity totreatment by 5-fluorouracil and radiotherapy in addition to
PB. Finally, ASCL2 expression was significantly correlatedwith histological grade of rectal cancer with preoperative
chemoradiation therapy.
Conclusions. ASCL2 was identified as a causative gene
involved in therapeutic resistance against anticancer treatments in CRC.

Colorectal cancer (CRC) has been treated withchemo(radio)therapy1 and molecular target therapy.2,3However, some cases exhibit treatment failure and/or
severe adverse effects associated with anticancer therapy.
Hence, the mechanisms involved in drug resistance must be
clarified to develop new therapeutic strategies andbiomarkers for primary CRC.
We hypothesized that the resistance mechanism ofanticancer treatments may be independent of the modalities
or kinds of drugs applied. To identify genes uniquely
involved in anticancer treatments in CRC, one must use
individual anticancer drugs such as 5-FU, oxaliplatin, andradiation, respectively, because of their daily use in

T. Tanaka et al.

concurrent multimodality treatments for CRC such as
neoadjuvant chemoradiation therapy (radiation and 5-FU)4or FOLFOX (5-FU and oxaliplatin).5
We recently constructed a model using phenylbutyrate(PB) to identify genes involved in PB resistance in breast
cancer, and identified the ZEB1 gene,6 which has been
confirmed to be related to resistance to chemoradiation
therapy (multiplex chemotherapy drug and radiation) inbreast cancer.7,8 So, in the current study, we applied thesame model to identify genes involved in drug resistance in
CRC.
MATERIALS AND METHODS
Cell Lines
We used six CRC cell lines (DLD1, HCT15, COLO320,
LOVO, COLO205, and HCT116) as described previously.9
The cell lines excluding LOVO and HCT116 cells weremaintained in Roswell Park Memorial Institute (RPMI)
1640 medium (GIBCO, Carlsbad, CA). The LOVO cells
were maintained in 50:50 RPMI 1640:F-12 Ham’s medium(Sigma-Aldrich N6658, St Louis, MO, USA), and the
HCT116 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM, Sigma-Aldrich D6429). All
media contained 10% fetal bovine serum.
PB Treatments in CRC Cell Lines
One tablet of PB (1 g, triButyrateÒ; Fyrklövern Scandinavia AB, Sweden) was used as previously described.6
Serum PB concentrations reached 0.5–3 mM in humans
when administered at dosages of 27 or 36 g/day.10 Hence,
we added 1-, 2-, 4-, 10-, and 20-fold dosages of PB solution(onefold dosage = 0.5 mM, 0.5–10 mM) as previously
described.6
Expression Microarrays
Messenger RNA (mRNA) was extracted from the PBsensitive strain (HCT116) and the PB-resistant strains(DLD1 and HCT15) using an RNeasy Mini Kit (QIAGEN
Sciences, MD, USA). The gene profiles were analyzed
using Affymetrix 30 IVT Express Kit microarrays (harboring 54,675 genes) according to the manufacturer’s
instructions.
Semiquantitative RT-PCR
Total RNA from cell lines was reverse-transcribed usinga SuperScript III reverse transcriptase kit (Invitrogen) as
described previously.9 The thermal cycling conditions wereas follows: an initial hold at 95 °C for 3 min and 30 cyclesof 1 min at 95 °C, 1 min at 56 °C, 1 min at 72 °C, and10 min at 72 °C. PCR primer sequences are presented in
Supplementary Table S1.
5-Aza-dC and TSA Treatments in CRC Cell Lines
Cells from the PB-resistant strains (1 9 106 cells/T-75
flask) were treated with 1 or 5 lM of the demethylation
agent 5-aza-dC (Sigma-Aldrich) dissolved in 50% acetic
acid (Wako Pure Chemical Industries, Osaka, Japan) once
every 24 h for 4 days followed by 300 nM of the HDAC
inhibitor TSA (Sigma-Aldrich) for the final 24 h, as
described previously.6 On day 5, the cells were detachedusing Trypsin-ethylenediaminetetraacetic acid (EDTA),
and mRNA was extracted using the RNeasy Mini Kit(QIAGEN).
Plasmid and Transfection
The full-length cDNA sequences of the TSPAN8 and
LEF1 genes were isolated using PCR and subcloned into a
pcDNATM3.1D/V5-His-TOPO vector (Invitrogen). A vector with self-ligation was used as control. The ASCL2
plasmid was purchased from OriGene Technologies(Rockville, MD, USA).
The cells were transfected with 4 lg plasmid vector
using Lipofectamine 2000 reagent (Invitrogen) in OPTIMEM medium (GIBCO) according to the manufacturer’s
instructions. At 3 days after transfection, the viable cells
were counted and used for RT-PCR.
siRNA and Transfection
siRNA targeting human ASCL2 was prepared using themethod described by Jubb et al.11 A scramble siRNA wasprepared according to the manufacturer’s instructions as
control: sense, 50 -agggucagacggauagcaa-30 ; antisense, 50 uugcuauccgucugacccu-30 (Sigma-Aldrich).
The cells were seeded in six wells overnight to reach
30–40% confluence, then transfected with 100 pmol
siRNA using Lipofectamine 2000 reagent in OPTI-MEM
medium according to the manufacturer’s instructions. After
24 h, the cells were treated with a single administration of
tenfold PB solution, 1 lg/mL of 5-FU (Wako), 4 lg/mL of
L-OHP (Sigma-Aldrich), or 3 Gy/day of radiation for
2 days. At 3 days after transfection, the cells were counted
and used for RT-PCR.

ASCL2 Pathway Causes Treatment-Resistance in CRC

ASCL2 Genomic Gain Status in Primary CRC
and Liver Metastasis
The genomic gain status of ASCL2 gene was investigated in a total of 33 primary CRC patients with liver
metastasis who underwent surgical resection for their primary tumors and liver metastases at Kitasato University
Hospital in 2000 as previously described for PRL3 genomic
amplification12 (Supplementary Table S2). This study was
conducted in accordance with the Declaration of Helsinki
and was approved by the Research Ethics Committee ofKitasato University School of Medicine (number B17355). All patients agreed to use of their pathological
specimens for research purposes. Q-PCR with a TaqMan
probe was performed in triplicate samples using iQTM
Supermix (Bio-Rad Laboratories, Hercules, CA) and theiCycler iQTM Real-Time PCR Detection system (Bio-Rad).
To normalize each gene copy number per cell, b-actin wasused as endogenous reference, as described previously.13
The copy number of the tumor tissues relative to corresponding normal tissues was determined as 2-DDCt, where
DDCt = DCt(tumor) - DCt(corresponding normal). The
ASCL2 primer and probe were prepared using Primer 3
software (Supplementary Table S1). A DNA ratio in thetumor (primary CRC or liver metastasis) tissues (T) relative to the corresponding normal tissues (N) (T/N ratio) thatwas equal to or greater than twofold was defined as positive
genomic gain.
Immunohistochemistry
Immunohistochemical staining for ASCL2 was investigated initially for tumor tissues (primary CRC and liver
metastasis of CRC) and noncancerous mucosa tissues fromtwo CRC patients with positive ASCL2 genomic gain, thenfor before neoadjuvant chemoradiation therapy (NCRT)
biopsy samples from 57 primary rectal cancer patients who
underwent NCRT followed by surgical resection at Kitasato University Hospital between 2004 and 2014
(Supplementary Table S3). This study was conducted inaccordance with the Declaration of Helsinki and wasapproved by the Research Ethics Committee of Kitasato
University School of Medicine (number B17-355).
Tissues from the patients with CRC were immunohistochemically stained for ASCL2 using Anti-Achaete Scutehomolog 2, clone 8F1 (mouse monoclonal IgG1 k; Merck
Millipore Corp., Darmstadt, Germany) at antibody concentration of 10 lg/mL. This antibody has been
demonstrated to provide strong contrast between positive
and negative controls.14

Statistical Analysis
Continuous variables were evaluated using the Student
t test, and categorical variables were evaluated using the
Fisher exact test or the Chi square test, as appropriate.
P \ 0.05 was considered to indicate statistical significance.
All calculations were performed using JMPÒ 11 software(SAS Institute Inc., Cary, NC, USA).
RESULTSClassification of PB Resistance in CRC Cell Lines
Cell proliferation was assessed at various concentrations, ranging from 0.5 to 10 mM, of PB in the six CRC
cell lines. Viable cells were counted on day 7 and compared with the control cells (Fig. 1a). The numbers of
HCT116, COLO205, COLO320, and LOVO cells werelower after treatment with 0.5 mM PB than those of corresponding control cells (reduction rates of 45%, 35%,
26%, and 26%, respectively). The numbers of DLD1 and
HCT15 cells, however, were similar after treatment with
0.5 mM or 1 mM PB, while they were reduced in number
by more than 80% after treatment with 5 mM or
10 mM PB. In 0.5 mM PB treatments, both DLD1 and
HCT15 cells (PB-resistant CRC cells) did not show
reduced numbers, while HCT116 cells (PB-sensitive CRC
cells) showed significantly (P = 0.005) reduced numbers(Fig. 1b). The increase of DLD1 cells in the 0.5 mM
treatment was not statistically significant, but epigenetic
treatments increase both oncogenic and tumor suppressor
genes,15 thus specific conditions of PB may affect oncogenic genes more than tumor suppressor genes. Based on
the results of these experiments, it was considered thatdifferential PB sensitivity was seen in the six CRC celllines, whereas cell proliferation could always be suppressed by high concentration of PB in all six cell lines,
consistent with results in breast cancer.6 Hence, theHCT116 cells were designated as a PB-sensitive strain,
while both the DLD1 and HCT15 cells were designated as
PB-resistant strains throughout the remainder of the study.
Identification of Genes Whose Expression Is
Significantly Associated with PB Sensitivity and PB
Resistance Using Expression Microarrays
To clarify the differential gene expression profiles thatsignificantly differed between PB sensitivity and PB residence, Affymetrix expression microarrays harboring
54,675 genes were used. ‘‘High’’ expression in CRC celllines by microarrays was initially defined as a raw signal
value of 100 or beyond, because RT-PCR can steadily
amplify such expression levels, as previously experienced.6

T. Tanaka et al.

(a)
(%)

140

Percentage of the number of living cells

DLD1
DLD1
HCT15
HCT15

120

COLO320
COLO320
LOVO
LOVO

100

COLO205
COLO205
HCT116
HCT11680

IC50 (mM)
3.7
2.3
1.6
1.4
0.9
0.6

60

40

20

0
0

0.5

1

2

5

10

Concentration of PB (mM)

<DLD1>

150
100
50
0

mock

PB
(0.5 mM)(%)<HCT15>

150
100
50
0

mock

PB
(0.5 mM)

Percentage of the number of living cells(%)

Percentage of the number of living cells

Percentage of the number of living cells

(b)
(%)

<HCT116>
p = 0.005

150
100
50
0

mock

PB
(0.5 mM)

FIG. 1 Classification of PB sensitivity in CRC cell lines.
a Percentage of number of living cells in CRC cell lines according
to concentration of PB treatment. The number of HCT116 cells was
reduced by a onefold PB dose, while the numbers of DLD1 and

HCT15 cells were barely reduced by either a onefold or even twofold
PB dosage. b Reduction rate of viable cells in three cell lines. The
number of HCT116 cells was significantly reduced by onefold PB
treatment. Error bars indicate respective standard deviation

Genes with the top 40 highest expression ratios between
sensitive and resistant strain were categorized as PB sensitivity related, and those with the top 26 highest
expression ratios between resistant and sensitive strain as
PB resistance related. Heat maps of such genes according
to their expression ratio between PB-resistant strains(DLD1 and HCT15) and PB-sensitive strain (HCT116) are
shown in Fig. 2a. Semiquantitative RT-PCR confirmed thatthe microarray results were almost accurate (representative
genes are shown in Fig. 2b). The top 40 genes that werehighly expressed in the PB-sensitive strains included BCL2
interacting protein 3 (BNIP3), cysteine-rich intestinal
protein 1 (CRIP1), and retinol binding protein 1 (RBP1),
which have been previously reported as being associatedwith tumor suppressor activity.15,16 On the other hand, the26 genes that were highly expressed in the PB-resistantstrains included achaete-scute like 2 (ASCL2), lymphoidenhancer-binding factor 1 (LEF1), and Tetraspanin8(TSPAN8), which are known to be involved in Wnt pathway activation.11,17
Demethylation Treatment Results in Differential
Expression in Either PB Sensitivity- or ResistanceRelated Genes
Simultaneous treatment with 5-aza-20 -deoxycytidine (5aza-dC) and trichostatin A (TSA), a highly effective
demethylation method, was used to evaluate whether epigenetic factors affect the differential expression of thegenes identified above.15 Almost all the PB sensitivity-related genes were reactivated after demethylation treatment,
while the gene expressions of almost all the PB resistancerelated genes showed no change after the same treatment(Fig. 2c). These findings suggest that the expression of the
PB-resistant genes is regulated in a manner different fromthose of the PB-sensitive genes, and the molecular mechanism of PB resistance is likely to be mediated genetically,

ASCL2 Pathway Causes Treatment-Resistance in CRC
DLD1

5-Aza(x5)5-Aza(x5) + TSA

5-Aza(x1)5-Aza(x1) + TSA

mock

TSA alone

5-Aza(x5)5-Aza(x1)mock

TSA alone

DLD1

low

HCT15

high

HCT116

HCT116 HCT15 DLD1ANTXR1B3GNT7
BEX2
BNIP3
BST2
C10orf116
CD109
CHST15CRIP1CSAG2DNAJA4ELOVL5
FAM101B
FAM127B
FERMT2
FHL1
FNBP1
GJC1
GPAT2
GPR87
HBE1
HS6ST2
IFI27
IFI44L
KRT23
L1CAM
LHX2
MAGEA2MAGEA3MAGEA6MAGEB2
MIR100HG
NPW
RBP1
SLC10A4
SLCO3A1
SMARCA1STC2SYT1ZNF385B
ACOT1

HCT15

(c)

(b)

PB-resistantstrains

5-Aza(x5) + TSA

PB-sensitive
strain5-Aza(x1) + TSA(a)

ANTXR1
BNIP3
CRIP1

PB
sensitivityrelated genes(n = 40)

C10orf116
ELOVL5
FAM127B
HBE1
KRT23
RBP1
SMARCA1

AFAP1-AS1
ASCL2
AUTS2
C4BPB
C9orf64
CADM1
CBR1
DDX3Y
DLC1
EIF1AY
GALC
GLS2
GSPT2
HDGFRP3
LEF1
NNT
OLR1
PRTFDC1QPCT
SLC2A10
TMEM45B
TSPAN8TXLNG2P
VIL1
ZNF260

PB
resistancerelated genes(n = 26)

ASCL2
LEF1
TSPAN8B-actin

FIG. 2 Gene profiles of PB-resistant and PB-sensitive strains using
an expression microarray. a Heat maps of gene expression. A red barshows a high level of expression, while a green bar shows a low level.
b mRNA expression profiles of PB sensitivity- and resistance-related
genes using semiquantitative RT-PCR. The expression patterns were

almost the same as the microarray results. c Changes in expression
after demethylation treatment. Some PB sensitivity-related genes
were recognized after demethylation treatment, but none of the PB
resistance-related genes showed any change after the same treatment.
Representative genes are shown

which is different from the epigenetic regulation of ZEB1
in breast cancer.6 Actually, ZEB1, which is associated with
PB resistance in breast cancer, was silenced in all three
CRC cell lines, and no association between ZEB1 expression and PB resistance was observed in CRC. Considering
these unexpected results, we conclude that PB resistancerelated genes are regulated by genetic aberrations unique to
CRC. Importantly, CRC is well known to be generated
from adenomatous polyposis coli (APC)/b-catenin Wnt
genetic pathway activation differently from breast cancer,18 and ASCL2, LEF1, and TSPAN8 genes are wellknown to be involved in Wnt pathway activation, as
described above.

Transfection of ASCL2, LEF1, and TSPAN8 Genes
into PB-Sensitive CRC Cells
We next made expression plasmids of ASCL2, LEF1,
and TSPAN8 genes from CRC cell lines. Transient transfection of a plasmid vector with the full-length ASCL2 gene
into HCT116 cells (PB-sensitive cells) induced mRNA
expression of both LEF1 and TSPAN8 genes (Fig. 3a). Thistransient transfection of an ASCL2-expressing vector significantly (t test, P \ 0.001) augmented PB resistance tothe same level as observed in PB-resistant cells (DLD1mock cells) after 5 mM PB treatment.
On the other hand, transient transfection of a plasmid
vector with the full-length LEF1 or TSPAN8 gene into
HCT116 cells (PB-sensitive cells) did not induce expression of the two other genes (Fig. 3b, c). The transienttransfection of either an LEF1- or TSPAN8-expressing
vector significantly (t test, P \ 0.001 for both vectors)

DLD1

transfection

mock

HCT116(a)B-actin
ASCL2
transfection

ASLC2
LEF1

Percentage of the number ofliving cells in PB treatment

T. Tanaka et al.

(%)

80
60
40
20
0
HCT116TSPAN8LEF1

Percentage of the number ofliving cells in PB treatment

DLD1

mock

transfection

ASLC2

100
80
60
40
20
0
HCT116mock

LEF1

DLD1+mock

DLD1

transfection

mock

HCT116ASLC2
LEF1
TSPAN8Percentage of the number ofliving cells in PB treatment****; p <0.001

B-actin
TSPAN8transfection

DLD1+mock****

TSPAN8

(c)

ASCL2

(%)

B-actin
LEF1
transfection

mock****; p <0.001

HCT116

(b)

****

100

100

****

(%)

80
60
40
20
0
HCT116mock

TSPAN8 DLD1+mock****; p <0.001

FIG. 3 EMT-related gene transfection experiments in PB-sensitive
CRC cells. a ASCL2 transfected into HCT116. Transfection of ASCL2
induced expression of LEF1 and TSPAN8 and significantly
augmented PB resistance. b LEF1 transfected into HCT116.

Transfection of LEF1 significantly augmented PB resistance.
c TSPAN8 transfected into HCT116. Transfection of TSPAN8
significantly augmented PB resistanceaugmented PB resistance to a level similar to that observedin DLD1 mock cells after 5 mM PB treatment.
These findings suggest that ASCL2 is an upstream activator of either LEF1 or TSPAN8, and we proceeded toperform detailed experiments focusing on ASCL2 among
the three PB-resistance-related genes.

also reduced the expression of TSPAN8 gene, whereas
LEF1 gene was little changed (Fig. 4b). Under PB treatment, the transient transfection of ASCL2 siRNA
remarkably (t test, P = 0.006) increased PB sensitivity in
DLD1 cells.
As PB is not a drug used in daily clinical practice to treat
CRC, we further investigated the associations between thegene expression of ASCL2 and anticancer treatments otherthan PB treatment, viz. 5-fluorouracil (5-FU), oxaliplatin(L-OHP), and radiation. Under 5-FU treatment, transienttransfection of ASCL2 siRNA into HCT15 and DLD1
significantly (t test, P \ 0.0001 and P = 0.008, respectively) increased 5-FU sensitivity. Under L-OHP treatment,on the other hand, transient transfection of ASCL2 siRNA
into HCT15 and DLD1 did not cause any changes in
L-OHP sensitivity. Under radiation treatment, transienttransfection of ASCL2 siRNA into HCT15 and DLD1
significantly (t test, P \ 0.0001 and P = 0.0002,

ASCL2 Gene Knockdown by Transfection of Small
Interfering RNA (siRNA) into PB-Resistant CRC Cells
We then performed knockdown of ASCL2 gene in the
PB-resistant cell lines (DLD1 and HCT15 cells). Transienttransfection of ASCL2 siRNA into HCT15 cells reduced theexpression of LEF1 and TSPAN8 genes as expected(Fig. 4a). Under PB treatment, the transient transfection of
ASCL2 siRNA significantly (t test, P = 0.002) increased
PB sensitivity in HCT15 cells. On the other hand, thetransient transfection of ASCL2 siRNA into DLD1 cells

ASCL2 Pathway Causes Treatment-Resistance in CRC

(a)

ASCL2
suppressionin HCT15

ASLC2
LEF1
TSPAN8

(%)

100
80
60
40
20
0

60

Percentage of the numberof living cells in HCT15

siRNA

mock

HCT15

B-actin

(%)

80
***

40
20
0
HCT15

mock

(%)

100
80
60
40
20
0

100
80
60
40
20
0
mock siASCL2䕔PB 䕕5FU

(b)

ASCL2
suppressionin DLD1

ASLC2
LEF1
TSPAN8

(%)

100
80
60
40
20
0

20
10
0
DLD1

mock

(%)

100
80
60
40
20
0

100
80
60
40
20
0
mock siASCL2䕔PB 䕕5FU

mock siASCL2

*

DLD1

siASCL2

(%)

DLD1

****

HCT15

(%)

***

mock siASCL2

L-OHP Radiation***; p <0.005, ****; p <0.001

40
30Percentage of the numberof living cells in DLD1

siRNA

mock

DLD1

B-actin

HCT15

siASCL2

(%)

HCT15

****

mock siASCL2

****

DLD1

mock siASCL2

L-OHP Radiation*; p <0.05, ***; p <0.005, ****; p <0.001

FIG. 4 Knockdown experiments in PB-resistant CRC cells.
Transfection of ASCL2 siRNA into HCT15 (a) and DLD1 (b).
Suppression of ASCL2 gene expression reduced expression of LEF1

and TSPAN8 and significantly increased the sensitivities of both
HCT15 and DLD1 cells to PB, 5-FU, and radiation treatment

respectively) increased radiation sensitivity. 5-FU andradiation treatments are main components of NCRT in
advanced rectal cancer treatment at present,4 raisinginterest in the clinical association between ASCL2 expression and NCRT response in rectal cancer.

(CRC tumor or liver metastasis tissue)/N (noncancerous
mucosa tissue) ratios of the ASCL2 DNA are shown in
Fig. 5a. All 33 cases had both primary CRC and liver
metastasis. An ASCL2 genomic gain (defined as T/N ratioof 2 or above) was seen in three cases, composed of one
liver metastasis (case 1) and two primary CRC (cases 2 and5), which is much less than in a previous report.19 In case 1,
ASCL2 genomic gain was only found in liver metastasis but
not in primary CRC tumor. In cases 2 and 5, ASCL2
genomic gain was seen only in primary CRC tumors. A
representative genomic quantification (case 5) is shown in
Fig. 5b. A positive correlation of ASCL2 genomic gain wasseen between the primary CRCs and corresponding liver
metastases (Fig. 5c, P = 0.02). The expression of ASCL2
protein was finally investigated in two cases with genomic
gain (cases 1 and 2) in immunohistochemistry (Fig. 5d).
ASCL2-positive cells were observed in the intestinal crypts
of normal colon mucosa as previously described,11 while

Genomic Amplification of ASCL2 and Immunostainingof ASCL2 in CRC Clinical Materials
The above results suggest that genomic changes in
ASCL2 may be involved in the sensitivity of cells to avariety of anticancer treatments for CRC. Gene copy
number gain of ASCL2 gene has been reported in liver
metastasis of CRC.19 We thus investigated the genomic
gain status of ASCL2 gene in primary CRC tumor tissues
and corresponding liver metastases with respect to corresponding noncancerous mucosa tissues.
Quantitative PCR (Q-PCR) for ASCL2 DNA was initially performed to assess the gene copy number. The T

T. Tanaka et al.

(a)

(b)
2.5

Liver metastasis

5000

2

4000
3000

DNA T/N ratio

RFU

Primary

2000
1000
0
0

10

20

30

40

CyclesPrimary ASCL2

Metastasis ASCL2

Normal ASCL2

Primary beta-actin

Metastasis beta-actin

Normal beta-actin

1.5

1

0.5

(c)

case 1case 2case 3case 4case 5case 6case 7case 8case 9case 10case 11case 12case 13case 14case 15case 16case 17case 18
case 19case 20case 21case 22case 23case 24case 25case 26
case 27case 28case 29
case 30case 31case 32case 330(d)(case 1 normal x400)(case 1 liver x400)(case 2 normal x400)(case 2 primary x400)

2.5case 2

primary2
1.5
1case 1

0.5
R2 = 0.154
0
0

0.5

1
1.5
liver metastasis

2

2.5

FIG. 5 Genomic amplification and immunostaining of ASCL2 inprimary CRC and corresponding liver metastasis. a Representative
genomic quantification (case 5). b T/N ratio of ASCL2 gene between
primary CRC and corresponding liver metastasis. c T/N ratio of

ASCL2 gene. d Immunostaining of ASCL2 in case 1 and case 2.
ASCL2-positive cells are clearly localized in the crypts of normalcolon tissue and are increased in primary CRC and liver metastasis

higher numbers of ASCL2-positive cells were seen in thecorresponding primary CRC and liver metastasis.

ASCL2 IHC2?, compared with only 15% for those withhistological grade 3 (complete remission).

Prediction of Histological Gradeby Immunohistochemistry of ASCL2 in Rectal Cancer
Biopsy Samples

DISCUSSION

Finally, preoperative biopsy samples from 57 rectal
cancer patients who underwent NCRT were immunostained for ASCL2. The 57 rectal cancers showedhistological grade 1 (n = 23), 2 (n = 21), and 3 (n = 13)
after NCRT, and the complete remission (CR) rate was
13/57 (22.8%), consistent with our previous report.20
Immunohistochemistry of ASCL2 was classified into IHC0
(n = 7), IHC1? (n = 19), and IHC2? n = 31) (Fig. 6a).
Interestingly, ASCL2 immunostaining was significantly(P = 0.006) correlated with pathological histology grade(Fig. 6b). Among rectal cancer patients with NCRT
showing histological grade 1, 78% were immunostained for

We recently identified ZEB1-RAB25/ESRP1 as PB-resistance genes in breast cancer,6 and ZEB1 has received
wide attention with regard to chemoresistance in breast
cancer.7,8 Utilizing the same search strategy, we again used
the same model for molecules involved in PB chemoresistance in this work for CRC. Interestingly, ZEB1
expression was robustly suppressed by epigenetic(demethylating) treatments,6 and miRNA, e.g., the Mir200
family, which is epigenetically regulated in human cancer,
might play a causative role in ZEB1 overexpression inhuman cancers.21 In our CRC cell lines, on the other hand,
ZEB1 was not expressed in either PB-sensitive or PB-

ASCL2 Pathway Causes Treatment-Resistance in CRC
FIG. 6 Immunohistochemistry
for ASCL2 in CRC tissue withNCRT.
a Immunohistochemistry of
ASCL2 was classified as IHC0
for no staining, IHC1? formoderate staining, and IHC2?
for diffuse staining.
b Distribution of ASCL2
staining score in eachhistological grade(a)

IHC 0 (n=7)

(b)

×400

×400

×400

IHC 2+ (n=31)

IHC 1+ (n=19)(%)

p=0.006

100

15%

ASCL2 IHC2+

62%

ASCL2 IHC1+

23%

ASCL2 IHC 0

80
78%

52%

60

40
33%
20
0

17%
4%

14%

Grade 1

Grade 2

Grade 3(n=23)(n=21)(n=13)

Pathological histology grade

resistant cells (data not shown). This finding suggests that
ZEB1 is not associated with PB resistance in CRC, unlike
the situation in breast cancer.
In the current search, on the other hand, we discoveredthat ASCL2 plays a critical role in PB resistance in CRC.
ASCL2 was significantly associated with resistance against
various anticancer treatments, including PB, 5-FU, andradiation, but not L-OHP. ASCL2 was not altered by epigenetic treatments, unlike the situation of ZEB1 gene inbreast cancer,6 but we focused on ASCL2 gene because it iscritically involved in the Wnt pathway, together with LEF1
and TSPAN8, which have well-known genetic aberrationsin CRC. In a previous report, LEF1 gene was shown to act
upstream of ASCL2,11 while the results for TSPAN8 were
not informative with regard to its relation to ASCL2.
However, the results of this study reveal, for the first time,that both LEF1 and TSPAN8 are regulated by ASCL2.
ASCL2 and LEF1 are involved in the Wnt pathway, suggesting that genomic alterations play causative roles in the
Wnt activation phenotype linked to chemoresistance in
CRC, unlike the situation in breast cancer.
Dominant negative LEF1 was reported to inhibit ASCL2
expression, suggesting that LEF1 acts upstream of
ASCL2.11 However, in this study, this finding could not be
confirmed. ASCL2 induced expression of both LEF1 andTSPAN8 genes, whereas ASCL2 gene inhibition suppressed

either or both genes. These findings suggest that ASCL2 is
an upstream regulator of the critical Wnt pathway genes
LEF1 and TSPAN8. Experiments based on transfection ofboth LEF1 and TSPAN8 genes indicated that both genes
augmented PB resistance, while neither LEF1 nor TSPAN8
could induce vice versa. Thus, we speculated that LEF1
and TSPAN8 genes were also involved in chemoresistance,
and those actions are dependent on ASCL2 overexpression
in CRC.
LEF1 is a well-established molecule that converges with
the Wnt pathway, with downstream genes of LEF1
reportedly including cyclin D1, c-myc, and Bcl-xl.22 These
genes could be individually involved in chemoresistance.23,24 On the other hand, to the best of the authors’knowledge, TSPAN8 involvement in chemoresistance has
not been reported, while CD44 involved in the cancerinitiating process induces TSPAN8, which plays a criticalrole in cancer invasion.25 Although TSPAN8 was induced
by ASCL2 transfection in this study, the molecular mechanism has not yet been elucidated. Drug resistance capacityof cancer cells is often accompanied by augmented oncogenic capacity, because the molecular mechanisms that
explain these two different phenotypes are redundant.26
Although there are no reports describing the molecularmechanism of TSPAN8 in anticancer drug resistance inhuman cancer, it has been reported that TSPAN8 is actually

T. Tanaka et al.

correlated with drug resistance in various human cancers,17,27 and TSPAN8 may be involved in cancer
stemness28–30 which has been considered to be involved in
drug resistance in cancer cells. Based on these reports, theprecise molecular mechanism is likely to be at least partially explained by cancer-derived exosomes including
TSPAN8 to yield cancer-initiating cells.
The ASCL2 gene, which is for a basic helix–loop–helix
transcription factor, has been reported to be a major transcription factor active in intestinal stem cells that can
trigger formation of liver metastases if overexpressed.14,31
In a previous study, ASCL2, which is located on chromosome 11p15.5, was reported to be amplified by 25% in liver
metastases of CRC.19 In this study, however, ASCL2
genomic amplification was less than expected (Fig. 5a).
The Cancer Genome Network has also focused specificallyon ASCL2 genomic amplification, which was also less than
expected relative to the previous report,19 and that ASCL2
overexpression was unlikely to be associated with thefrequent genomic gain of the 11p15 locus in CRC.32
Among the genes at this locus, gene overexpression
accompanied by genomic gain was recognized for IGF2,
and IGF2 overexpression was exclusive for expression ofrelated genes, such as IRS2. These findings suggest that
ASCL2 is not dominantly responsible for the genomic gainof 11p15.
ASCL2 has been proposed to be a marker of dynamic
cancer-initiating cells, and it was ubiquitously expressed in
Lgr5-positive colon stem cells, but not in ?4 stem cells,
which are alternative stem cells which are positive forhomeodomain-only protein homeobox (HOPX). 14,33
Immunohistochemistry studies clearly showed the localization of ASCL2 in the crypts of normal colon tissue aspreviously shown (Fig. 5d). Simultaneously, ASCL2 isoverexpressed in primary CRC tumors and liver metastasis,suggesting that such cancer cells were derived from progenitor dynamic stem cells overexpressing ASCL2.
Inhibition of ASCL2 results in arrest at the G2/M
checkpoint of the cell cycle.11,14 Several recent reports
describe that sensitivity to several anticancer drugs such as
CPT11 or Cdk inhibitors is accompanied by G2/M cell
cycle arrest.34,35 In this study, ASCL2 was for the first time
confirmed to be involved in 5-FU or radiotherapy resistance, in addition to PB resistance, while L-OHP sensitivity
was not affected by the expression level of ASCL2. A direct
effect of L-OBP is cross-linking of double-stranded DNA,and DNA replication is inhibited, resulting in arrest at theG1/S rather than G2/M phase of the cell cycle.36 Such a
differential acting mechanism may be reflected in the typesof anticancer drug resistance observed in this study.
The results of this study also demonstrate that ASCL2
could be useful as a chemoresistance biomarker for actual
anticancer treatments targeting the cell cycle or damaging

DNA, such as PB, 5-FU, and radiation, even when using
biopsy samples prior to NCRT for locally advanced rectal
cancer. Recently, it was reported that ASCL2 expression
was mainly regulated by long noncoding RNA of
WiNTRLINC1 in CRC, which is a direct target of Wnt
pathways.37 These findings, together with our data (showing that ASCL2 genomic amplification is an infrequent
event), suggest that the WiNTRLINC1/ASCL2 axis could
contain key targets to control the Wnt pathway and CRC
disease.
CONCLUSIONS
A comprehensive search for PB-resistance genes identified ASCL2, which was genetically regulated possibly
through upstream Wnt pathway activation in CRC. The
ASCL2 gene was also involved in resistance to PB treatment as well as NCRT. The regulation of ASCL2 mayprovide a clue for the molecular understanding of CRC
treatment failure.
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