Y-box binding protein-1 (YB-1) contributes to both HER2...

YB-1-HER2 axis in gastric cancer cells

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Y-box binding protein-1 (YB-1) contributes to both HER2/ErbB2 expression and

lapatinib sensitivity in human gastric cancer cells

Tomohiro Shibata1*, Hitoshi Kan1*, Yuichi Murakami1, 2, Hiroki Ureshino1, Kosuke

Watari1, Akihiko Kawahara3, Masayoshi Kage3, Satoshi Hattori4, Mayumi Ono1 and

Michihiko Kuwano5✝

1) Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical

Sciences, Kyushu University, Fukuoka, Japan

2) St. Mary’s Hospital, Kurume, Japan

3) Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan

4) Biostatistics Center, Kurume University, Kurume, Japan

5) Laboratory of Molecular Cancer Biology, Graduate School of Pharmaceutical

Sciences, Kyushu University, Fukuoka, Japan

* These authors equally contributed.

Running title: YB-1-HER2 axis in gastric cancer cells

Keywords: Gastric cancer, HER2, lapatinib, trastuzumab, YB-1.

on June 14, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 27, 2013; DOI: 10.1158/1535-7163.MCT-12-1125

http://mct.aacrjournals.org/


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Financial Support: This work is supported by the 3rd Term Comprehensive Control

Research for Cancer from the Ministry of Health, Labour and Welfare, Japan and by

Grant-in-Aid for Challenging Exploratory Research from Japan Society for the

Promotion of Science (JSPS), KAKENHI Grant Number 24650646 (M. Ku.).

✝Corresponding author:

Michihiko Kuwano, M.D., Ph.D.

Laboratory of Molecular Cancer Biology

Graduate School of Pharmaceutical Sciences, Kyushu University

3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

Phone/Fax:+81-92-642-6139

E-mail: [email protected]

Disclosure of Conflict of Interest:

The authors declare there is no conflict of interest.

Word count: 4471 words





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Total number of figures: 5

Total number of table: 1

Total number of supplementary table: 1





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Abstract

Gene amplification of HER2/ErbB2 occurs in gastric cancer, and the

therapeutic efficacy of the HER2-targeted antibody, trastuzumab, has recently been

improved against HER2-positive advanced stomach cancer. Here we examined whether

Y-box-binding protein-1 (YB-1) could selectively control HER2 gene expression and

cellular sensitivity to epidermal growth factor receptor (EGFR) family protein-targeted

drugs in human gastric cancer cells. HER2 expression was specifically downregulated

by YB-1 silencing using its cognate siRNA, whereas there was less change in the

expression of EGFR and HER3. A chromatin immunoprecipitation assay revealed the

specific binding of YB-1 to its consensus sequence on the 5′-regulatory region of HER2.

YB-1 knockdown induced drug resistance to lapatinib, a dual EGFR and HER2 kinase

inhibitor, and also to erlotinib, an EGFR kinase inhibitor. Moreover, phosphorylation of

protein kinase B (Akt) was not markedly affected by lapatinib or erlotinib when YB-1

was silenced. Nuclear YB-1 expression was significantly (p=0.026) associated with

HER2 expression, but not with EGFR or HER3, in patients with gastric cancer (n=111).

The YB-1-HER2 axis might therefore be useful for the further development of

personalized therapeutics against gastric cancer by HER2-targeted drugs.





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Introduction

Overexpression of the HER2 is predictive for overall survival and disease-free

survival in node-positive breast cancer (1, 2). The humanized anti-HER2 antibody

trastuzumab improves the therapeutic efficacy of HER2-positive breast cancer (3-5).

HER2 overexpression is also predictive of poor prognosis in patients with gastric cancer

(6-10). A trastuzumab phase III study in HER2-positive advanced gastric cancer (the

ToGA trial) reported a survival benefit when trastuzumab was added to chemotherapy in

HER2-overexpressing gastric cancer patients (11), and trastuzumab was approved for

treatment of HER2-positive metastatic gastric and gastroesophageal junction cancer. On

the other hand, small-molecule tyrosine kinase inhibitors that simultaneously target

HER2 and other EGFR family proteins have been developed as an alternative

anti-HER2 therapeutic strategy. Lapatinib (Tykerb) is a potent ATP-competitive dual

kinase inhibitor that targets both inhibits EGFR and HER2 (12), and demonstrates

anti-proliferative activity against human HER2-amplified breast cancer cell lines in

culture (12). It also shows improved therapeutic efficacy in trastuzumab-refractory

breast cancer patients (13, 14). Consistent with the hypothesis that HER2-directed

therapy is clinically effective against HER2-amplified gastric cancer, lapatinib

selectively inhibits the proliferation of HER2-amplified human gastric cancer cells, and





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this effect is synergistic with trastuzumab both in vitro and in vivo (15).

The Y-box binding protein-1 (YB-1) containing a conserved cold-shock domain

is a candidate regulatory molecule for HER2 expression in human cancer cells (16, 17).

YB-1 also plays a key role in embryogenesis and differentiation (18-20), and cell

proliferation and malignant transformation (18, 19, 21, 22) .YB-1 expression is also

significantly correlated with the acquirement of global drug resistance to various

anticancer agents in human cancers (23). YB-1 knockdown inhibits the cell proliferation

of human breast cancer, and suppresses the expression of various cell cycle/DNA

replication-related genes including cell-division control protein 6 homolog (CDC6)

(24-26). Astanehe et al. demonstrated that YB-1 activation mediated drug resistance to

trastuzumab through the mitogen-activated protein kinase-interacting kinase in breast

cancer cells, suggesting a critical role for YB-1 in the acquirement of drug resistance to

trastuzumab (27). YB-1 was initially reported to interact with the Y-box element in the

5′ regulatory element of the EGFR and HER2 genes (28), and it had a critical role in the

expression of both genes as shown by the effects of its knockdown in breast (29-31) and

lung (32, 33) cancer cells in culture. HER2 expression is more specifically susceptible

to YB-1-dependent regulation than EGFR in cultured breast and lung cancer cells (31,

32, 34). Consistent with these in vitro studies, nuclear YB-1 expression was more





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closely correlated with HER2 than EGFR expression in patients with breast (31) and

lung (32) cancer.

HER2 could play a pivotal role in the tumor growth of both breast and gastric

cancer, and its expression is related to the therapeutic efficacy of molecular-targeted

drugs such as trastuzumab and lapatinib in gastric cancer cells (11, 15). However,

trastuzumab alone has only marginal cytotoxicity against various cancer cell lines in

vitro. Here we asked whether YB-1 plays a specific regulatory role in the expression of

HER2 in gastric cancer, and whether the YB-1-HER2 axis modulates cellular sensitivity

to lapatinib and erlotinib. The association of YB-1 and HER2 expression in patients

with gastric cancer was also considered.





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Materials and Methods

Cell lines and culture, and reagents

The human gastric cancer cell lines MKN7, MKN28, MKN45, MKN74,

KATO3, and NUGC3 were purchased from Health Science Research Resources Bank

(Osaka, Japan). The human duodenal cancer cell line, AZ521, was also purchased from

Health Science Research Resources Bank. NCI-N87 was purchased from the American

Type Culture Collection, and SNU216 was from the Korean Cell Line Bank (Seoul,

Korea). 44As3 was kindly provided by Dr. K. Yanagihara (National Cancer Center

Research Institute, Japan) (35). All cell lines were purchased more than 6 months ago

and were not further tested or authenticated by the authors. And these cell lines were

cultured in RPMI-1640 supplemented with 10% FBS in a humidified atmosphere

containing 5% CO2 at 37 °C.

Erlotinib was kindly provided by F. Hoffmann-La Roche Ltd. (Basel,

Switzerland), cisplatin was by Bristol-Myers Squibb K.K (New York, NY), and CPT-11

was by Yakult Honsha Co., Ltd. (Tokyo, Japan). Lapatinib was purchased from Toronto

Research Chemicals (Toronto, Canada), and SU11274 and anti-phospho-EGFR

(pEGFR) antibodies were from Calbiochem (San Diego, CA). Anti-HER2 and

anti-phospho-HER2 (pHER2) antibodies were purchased from Upstate Biotechnology





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(Lake Placid, NY). Anti-EGFR, anti-phospho-Met (pMet), anti-phospho-HER3

(pHER3), anti-extracellular-signal-regulated kinase 1/2 (Erk1/2), anti-phospho-Erk1/2

(pErk1/2), anti-protein kinase B (Akt), anti-phospho-Akt (pAkt), and anti-CDC6

antibodies were purchased from Cell Signaling Technology (Beverly, MA), anti-Met

and anti-HER3 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA),

anti-α-tubulin antibody was purchased from Sigma-Aldrich (St Louis, MO),

anti-GAPDH antibody was from Trevigen (Gaithersburg, MD). Anti-YB-1 antibody

(st1968) was generated by immunization of New Zealand white rabbits with synthetic

YB-1 peptide (carboxy-terminal amino acids 299-313) as described previously (36).

This antibody could detect both cytoplasmic and nuclear YB-1. Anti-YB-1 antibody

(EPITOMICS) was purchased from EPITOMICS (Burlingame, CA). The siRNA

corresponding to YB-1 (5’-GGUUCCCACCUUACUACAU-3’) was purchased from

QIAGEN Inc. (Valencia, CA), corresponding to HER2

(5’-AAACGUGUCUGUGUUGUAGGUGACC-3’), HER3

(5’-GGCAGUGUAUAAUCUAUCUCCACUA-3’) were purchased from Invitrogen,

and corresponding to EGFR (5’-GAGGAAAUAUGUACUACGA-3’) was purchased

from Sigma-Aldrich. Cells were transfected with siRNA duplexes using Lipofectamine

RNAiMAX and Opti-MEM (Invitrogen) according to the manufacturer's





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recommendations.

Western blot analysis

Cells were rinsed in ice-cold PBS and lysed in Triton X-100 buffer (50 mmol/L

HEPES, 150 mmol/L NaCl, 50 mmol/L NaF, 1% Triton X-100, and 10% glycerol

containing 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL

aprotinin, 10 µg/mL leupeptin, and 1 mmol/L sodium orthovanadate). Cell lysates were

separated by SDS-PAGE and transferred to Immobilon membranes (Millipore Corp.,

Bedford, MA). After transfer, membranes were incubated in blocking solution, probed

with various antibodies, washed, and visualized by using horseradish

peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ) and

enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Forty-eight hours after siRNA transfection, total RNA was isolated from cell

cultures by using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan), according to the

manufacturer’s instructions. The RNA concentration was assessed by spectrophotometry

at 260 nm. RNA was reverse-transcribed from random hexamers using AMV reverse





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transcriptase (Promega, Madison, WI), and qRT-PCR was performed by using the

Real-Time PCR system 7300 (Applied Biosystems, Foster City, CA).

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was performed using the EZ ChIP kit (Millipore Corp.),

according to the manufacturer’s recommendations. Briefly, soluble chromatin from 1 ×

106 cells was incubated with 1 µg anti-YB-1 antibody. Purified DNA was dissolved in

50 µl H2O, and 4 µl DNA was used for PCR analysis (40 cycles) with the following

primer pairs: HER2#1, 5′-GCTCCAAATTGCCTTTCCAA-3′ (forward) and

5′-CAGTTCCGGCTTTGAAGACG-3′ (reverse); HER2#2,

5′-AAAAGGCTCATGTCCCCGTG-3′ (forward) and

5′-GGTGTACCGCGACTGCGTAC-3′ (reverse); and HER2#3,

5′-AGGGGCTCCAATAGAA-3′ (forward) and 5′-AATTTGGGAGGAGACAG-3′

(reverse). PCR products were analyzed on 1% agarose gels and stained with ethidium

bromide.

Cell proliferation assay

Cells (5 × 103) were seeded in 24-well plates, and the cell numbers in each well





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were counted by a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter.,

Fullerton, CA) at 5 days after siRNA transfection. Results were expressed as the mean ±

SD of triplicate wells.

Immunohistochemistry (IHC)

Tissue sections were taken from 111 gastric cancer patients who underwent

radical surgery at the Department of Surgery, Kurume University Hospital, Japan,

between 2001 and 2004. Patient characteristics are summarized in Table 1. The 4-μm

tissue sections were deparaffinized, and the slides were heated in a cell conditioning

solution buffer for 60 min at 100 °C. The sections were immunohistochemically stained

with anti-YB-1 antibody (st1968), anti-CDC6, anti-HER2, anti-HER3, and

anti-EGFR antibodies by using the BenchMark XT (IHC Automated System, Tucson,

AZ) and ChemMate ENVISION method (Dako Corporation, Carpinteria, CA). Samples

were viewed by using a BX51 fluorescence microscope (Olympus, Tokyo, Japan), and

the extent of YB-1 staining was classified according to the percentage of cells with

strongly stained nuclei (≥10% indicated that a gland was positive). All IHC studies were

evaluated by two experienced observers who were blind to the conditions of the

patients.





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Statistical analysis

The associations between YB-1 and clinicopathological findings were tested by

Fisher’s exact test or the Chi-squared test depending on the type of data. p<0.05 was

regarded as significant unless otherwise indicated.





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Results

Effects of lapatinib and erlotinib on the phosphorylation of EGFR family proteins

and their downstream signaling molecules in human gastric cancer cell lines

We initially examined protein expression levels of HER2 and other

growth-factor receptors in 10 human gastric cancer cell lines using western blot analysis.

As shown in Fig. 1A, HER2 expression was the highest in NCI-N87, whereas MKN7

and SNU216 showed relatively high HER2 expression, which is consistent with the

previous studies (15, 37). HER2 was expressed at lower levels in MKN45, AZ521, and

KATO3 cells, whereas Met levels were the highest in MKN45 cells (Fig. 1A).

Cellular sensitivity to anticancer drugs targeting growth-factor receptors and

cytotoxic drugs, such as cisplatin and CPT-11, was compared among 10 cell lines, and

IC50 values for each drug were presented based on dose-response curves for various

drugs (Supplementary Table S1). Of the 10 cell lines, NCI-N87 and SNU216 were

highly susceptible to lapatinib, whereas MKN45 was the most sensitive to SU11274. By

contrast, all 10 cell lines showed similar drug sensitivities to cisplatin and CPT-11

(Supplementary Table S1). Trastuzumab had only a slight cytotoxic effect on NCI-N87

(data not shown), but not on the other cell lines.

We further examined the effect of erlotinib and lapatinib on activation of EGFR





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family proteins and downstream signaling molecules, Akt and Erk, in SNU216, MKN45,

and NCI-N87 cells (Fig. 1B, C). Western blot analysis showed that 10μM erlotinib

blocked the phosphorylation of EGFR, HER2, and Akt in SNU216 and NCI-N87 (Fig.

1B). By contrast, 10μM erlotinib only slightly, if any, inhibited phosphorylation of

HER2, HER3, and Akt in MKN45. On the other hand, phosphorylation of EGFR, HER2,

HER3, Akt and Erk was inhibited by lapatinib at 1 and 10μM in SNU216 and NCI-N87,

but not in MKN45 (Fig. 1C). Akt and Erk phosphorylation was more sensitive to the

inhibitory effects of lapatinib and erlotinib in SNU216 and NCI-N87 cells than MKN45,

suggesting that EGFR and its family proteins are more closely linked to cell growth and

survival in the former two cell lines.

YB-1 knockdown suppresses HER2 expression in gastric cancer cells

We next examined the effect of YB-1 knockdown on the expression of EGFR

family proteins in MKN45, SNU216, and NCI-N87 cell lines (Fig. 2A, B). YB-1 siRNA

suppressed HER2 expression in all three cell lines, whereas there was almost no or

much less suppression of EGFR. HER3 expression was not at all affected by YB-1

knockdown. To determine whether HER2 mRNA levels were affected by YB-1

knockdown, we used qRT-PCR in YB-1 siRNA-treated cells. HER2, but not EGFR nor





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HER3, mRNA was decreased following treatment with YB-1 siRNA (Fig. 2C), which

was consistent with the western blot analysis.

Y-box-like elements are located in the 5′-regulatory promoter region of HER2

(28, 29). As HER2 has two transcription variants (NM_001005862 and NM_004448),

we analyzed the binding of YB-1 to the two putative promoters (1 and 2) of HER2. Of

the three putative YB-1-responsive elements in promoters 1 and 2, YB-1 bound directly

to the #2 and #3 elements, but not to the #1 element (Fig. 2D).

YB-1 knockdown alters sensitivity to lapatinib and erlotinib

We next examined whether YB-1 knockdown altered sensitivity to lapatinib

and erlotinib in MKN45, SNU216, and NCI-N87 cells (Fig. 3). We previously

demonstrated that YB-1 knockdown confers drug resistance to cisplatin, which is a

DNA-damaging anticancer agent, in both cancer cells and normal cells (19, 36, 38). We

also examined the effect of YB-1 knockdown on sensitivity to cisplatin in gastric cancer

cells. There was no marked change in sensitivity to erlotinib and lapatinib when YB-1

was silenced in MKN45 (Fig. 3A). By contrast, treatment with YB-1 siRNA resulted in

varying degrees of decreased sensitivity to both erlotinib and lapatinib in the two gastric

cancer cell lines, SNU216 and NCI-N87 (Fig. 3 B, C), suggesting that the suppression





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of HER2 expression by YB-1 knockdown is closely associated with sensitivity to the

above mentioned drugs. Of the three cell lines, YB-1 knockdown enhanced sensitivity

to cisplatin only in MKN45 cells.

We further examined the effects of erlotinib and lapatinib on the

phosphorylation of Akt, Erk1/2, EGFR, HER2, and HER3 in a gastric cancer cells

treated with or without YB-1 siRNA. Treatment of NCI-N87 cells with YB-1 siRNA

suppressed HER2 expression without affecting EGFR and HER3 expression (Fig. 4A,

C). Erlotinib inhibited the phosphorylation of EGFR, HER3, Akt, and Erk both with and

without YB-1 siRNA (Fig. 4A). HER2 phosphorylation was only slightly inhibited by

erlotinib, whereas Erk and Akt phosphorylation was highly susceptible to its inhibition.

Quantitative analysis showed that Akt phosphorylation was relatively less sensitive to

erlotinib under YB-1 knockdown condition (Fig. 4B). Phosphorylation of EGFR, HER3,

Akt, and Erk was suppressed by lapatinib both with and without YB-1 knockdown (Fig.

4C). Quantitative analysis showed that Akt phosphorylation was relatively less sensitive

to lapatinib under YB-1 knockdown compared with under control conditions (Fig. 4D).

Correlation of YB-1 with HER2 expression in gastric cancer

Use of a specific YB-1 antibody recognizing YB-1 in both the nucleus and





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cytoplasm allowed us to perform IHC of surgically resected gastric tumors. Fig. 5A

shows typical nuclear YB-1 immunostaining in gastric cancer. IHC profiles of YB-1

nuclear expression were summarized by dividing all samples into five groups (0-4%,

5-9%, 10-29%, 30-39%, and >40%) (Table 1) .Of all 111 samples, approximately 60%

showed relatively low expression (0-4% and 5-9%). We used a cut-off value of 10% to

divide the samples into decreased (<10%) and increased (≧10%) expression, which

were evaluated as negative and positive, respectively. Fig. 5B shows representative

images for EGFR, HER2, HER3, and CDC6 expression. Fig. 5C shows the typical IHC

images of both nuclear YB-1 expression and HER2 expression in cancer cells of the

serial sections. Two clinical samples (case 1 and 2) showed higher expression of HER2

with nuclear YB-1 expression, but the other two samples (case 3 and 4) did not show

apparent expression of HER2 without nuclear YB-1 expression.

We classified gastric cancer samples into high (2+, 3+) and low (0, 1+)

expression of EGFR, HER2, HER3, and CDC6. Consistent with the association of YB-1

with CDC6 in breast cancer patients (26), nuclear YB-1 expression was found to be

significantly associated with CDC6 expression (p<0.0027) in gastric cancer in the

present study (Table 1). Of the three EGFR family proteins, HER2 expression was

significantly (p=0.026) correlated with YB-1. By contrast, EGFR expression was closely,





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but not significantly (p=0.097), associated with YB-1. There was no association of

nuclear YB-1 expression with HER3 expression.





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Discussion

The present study demonstrated that YB-1 knockdown specifically suppressed

the expression of HER2 and induced drug resistance to lapatinib in gastric cancer cells.

Lapatinib has previously been shown to be effective against HER2-positive human

breast and gastric cancer cells in vitro and in vivo (12, 15, 39). Higher expression of

YB-1 in the nucleus of cancer cells was significantly correlated with HER2, but not

EGFR or HER3, expression in gastric cancer (Table 1). And nuclear YB-1 expression

was found to be significantly associated with poor prognosis (p<0.01) and with

metastasis to the lymph node and peritoneum (p<0.005) in gastric cancer patients

(unpublished data). Recent study by Wu et al. has also demonstrated the close

association of high YB-1 expression with liver metastasis and poor survival in advanced

gastric cancer (40).

Of the three cell lines used in this study, SNU216 and NCI-N87 cells showed

higher HER2 expression than MKN45 cells. In particular, NCI-N87 cells expressed the

highest level of HER2 expression and showed constitutive activation of both EGFR and

HER2. It was highly susceptible to the cytotoxic effect of lapatinib, and SNU216 cells

showed similar cellular sensitivity to lapatinib as NCI-N87 cells (Supplementary Table

S1). The phosphorylation of EGFR, HER2 and HER3 was similarly suppressed by

lapatinib in both SNU216 as NCI-N87 cells (Fig. 1C). Treatment of SNU216 and





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NCI-N87 cells with lapatinib thus disrupts the heterodimer formation of EGFR/HER2

and HER2/HER3, and inactivates their downstream signaling pathways involving Akt

and Erk.

Akt and Erk activation was markedly blocked by erlotinib or lapatinib in

SNU216 and NCI-N87 cells (Fig. 1B, C), suggesting a close association of EGFR

family proteins, including HER2, with cell growth and survival. Following YB-1

knockdown, Akt phosphorylation was less susceptible to the inhibitory effects of

lapatinib or erlotinib in NCI-N87 cells when compared with under control conditions

(Fig. 4). Together with these findings, our present study suggests that YB-1

knockdown-induced drug resistance to lapatinib is mainly caused by decreased HER2

expression without affecting EGFR and/or HER3 expression. HER2 might play

essential roles in the determination of sensitivity to EGFR-targeted drugs in gastric

cancer cells as seen in lung cancer cells (41-44). The close link between YB-1 and

HER2 might therefore limit sensitivity to EGFR-targeted drugs not only in lung cancer

cells but also in gastric cancer cells.

The Met gene is amplified in MKN45 cells (Fig. 1A), which are highly

susceptible to the cytotoxic effects of SU11274, a Met-targeted drug (Supplementary

Table S1). Therefore, Met driven signaling pathway in MKN45 cells limit cytotoxicity





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to EGFR or HER2 targeted drugs (42).YB-1 knockdown decreased HER2 expression

without affecting Met expression (data not shown). Consistent with these findings,

YB-1 knockdown did not affect sensitivity to lapatinib or erlotinib in MKN45 cells (Fig.

3).

The ChIP assay in the present study revealed an interaction of YB-1 with

Y-boxes in the promoter region of HER2. Previously, Wu et al. used microarray analysis

to establish a close correlation between YB-1 and EGFR or HER2 expression in tumor

samples from breast cancer patients (29), and this was also observed by IHC analysis.

We previously demonstrated the selective association of YB-1 and HER2 expression in

human breast cancer cell lines (31) as well as in human lung cancer cells both in vitro

and in vivo (32). These studies suggest that YB-1 expression selectively controls the

expression of HER2, rather than EGFR and HER3, in human gastric cancer cells and

breast and lung cancer cells.

Finally, one could ask whether the association of nuclear YB-1 expression and

HER2 expression is differentially affected by tumor types, including breast cancer (31),

lung cancer (32) and gastric cancer (cf. this study). YB-1 knockdown reduced

expression of HER2 in 2 of 4 breast cancer cell lines (31), 2 of 5 lung cancer cell lines

(32), and 3 of 3 gastric cancer cell lines (Fig. 2), respectively in vitro. In patients with





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negative nuclear YB-1 expression, frequencies of relatively higher HER2 expression

(+2 and +3) were less than 10% for breast cancer (4/43 = 9.3%) and lung cancer

(adenocarcinoma) (2/47 = 4.3%), and relatively high for gastric cancer (12/64 = 18.8%).

Although there is such a difference in baseline frequencies, frequencies of high HER2

expression in patients with positive nuclear YB-1 expression were found to be about

20% higher than those in patients with negative nuclear YB-1 expression consistently in

these three different tumor types (10/30 = 33.3% versus 9.3% for breast cancer; 4/19 =

21.1% versus 4.3% for lung cancer; and 18/47 = 38.3% versus 18.8% for gastric cancer).

Taken together, nuclear YB-1 expression may have similar effects on HER2 expression

in breast, lung and gastric cancer.

In conclusion, HER2 and HER3 appear to play an important role in the

activation of human gastric cancer cell-signaling pathways for survival and proliferation.

YB-1 specifically controls the expression of HER2, rather than EGFR and HER3,

affecting drug sensitivity to HER2- and/or EGFR-targeted anticancer drugs in gastric

cancer cells. As YB-1 nuclear expression is significantly correlated with HER2

expression in human gastric cancer, the YB-1-HER2 axis could contribute to the further

development of personalized therapeutics by EGFR-targeted drugs against human

gastric cancer.





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Acknowledgements

We thank Drs. I. Okamoto and K. Nakagawa at Kinki University for fruitful

discussion. We also thank E. Kajita at Kyushu University for preparing this manuscript.

Grant Support

This work is supported by the 3rd Term Comprehensive Control Research for

Cancer from the Ministry of Health, Labour and Welfare, Japan and by Grant-in-Aid for

Challenging Exploratory Research from Japan Society for the Promotion of Science

(JSPS), KAKENHI Grant Number 24650646 (M. Ku).





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Table 1. Correlation between nuclear YB-1 expression and expression of three

EGFR family proteins and CDC6 in gastric cancer patients (n=111) a

a Determined by Fisher’s exact test

Nuclear YB-1

Variable n negative positive p-value

CDC6 0.0027

Low 39 30 (46.90%) 9 (19.10%)

High 72 34 (53.10%) 38 (80.90%)

EGFR 0.097

0 24 11 (17.20%) 13 (27.70%)

+1 41 27 (42.20%) 14 (29.80%)

+2 38 24 (37.50%) 14 (29.80%)

+3 8 2 (3.100%) 6 (12.80%)

HER2 0.026

0 42 29 (45.30%) 13 (27.70%)

+1 39 23 (35.90%) 16 (34.00%)

+2 16 4 (6.300%) 12 (25.50%)

+3 14 8 (12.50%) 6 (12.80%)

HER3 0.593

0 42 24 (37.50%) 18 (38.30%)

+1 28 19 (29.70%) 9 (19.10%)

+2 20 10 (15.60%) 10 (21.30%)

+3 21 11 (17.20%) 10 (21.30%)





35

Figure legends

Figure 1. Expression of EGFR family proteins and effects of erlotinib and lapatinib in

gastric cancer cell lines. (A) Western blots showing the expression of EGFR, pEGFR,

HER2, pHER2, HER3, pHER3, Met, pMet, Erk, pErk, Akt, and pAkt proteins and

α-tubulin as a loading control in 10 human gastric cancer cell lines. The inhibitory effect

of erlotinib (B), and lapatinib (C), on the expression of pEGFR, pHER2, pAkt, and pErk

in MKN45, SNU216, and NCI-N87 cells. Cells were treated with erlotinib or lapatinib

for 6 hours, then analyzed by western blotting.

Figure 2. Effect of YB-1 knockdown on EGFR, HER2, HER3, Akt, pAkt, Erk and pErk

expression. (A) MKN45, SNU216, and NCI-N87 cells were treated with YB-1 siRNAs

for 48 hours, and western blotting was used to analyze the expression of EGFR, HER2,

HER3, Akt, pAkt, Erk, and pErk. (B) Quantitative analysis of the effect of YB-1

knockdown on expression of EGFR, HER2 and HER3 by YB-1 siRNA (as indicated in

Fig. 2A) when normalized in the absence of the siRNA. (C) Effect of YB-1 siRNA on

EGFR, HER2, and HER3 mRNA levels as determined by qRT-PCR in MKN45 cells.

Each bar is an average ± SD of triplicate experiments, *p<0.01. (D) Schematic

representation of potential YB-1 binding sites and primer locations used for ChIP assay.





36

Figure 3. Effect of YB-1 knockdown on sensitivity to erlotinib, lapatinib, and cisplatin

in MKN45, SNU216, and NCI-N87 cells. MKN45 (A), SNU216 (B), and NCI-N87 (C)

cells were treated with YB-1 siRNA for 48 hours, exposed to drugs, and a

cell-proliferation assay was carried out. Each value is an average ± SD of triplicate

dishes. Values expressed as % normalized to the value with no drugs (100%).

Figure 4. Effect of erlotinib and lapatinib on EGFR, pEGFR, HER2, pHER2, HER3,

pHER3, Akt, pAkt, Erk, and pErk expression in the presence or absence of YB-1 siRNA

in NCI-N87 cells. (A) Effect of erlotinib on expression of growth-factor receptors and

their downstream-regulatory molecules with or without YB-1 siRNA treatment for 48

hours, followed by erlotinib exposure and western blot analysis. (B) Quantitative

analysis of the effect of erlotinib on Akt and Erk phosphorylation in the presence of

YB-1 siRNA. Akt and Erk phosphorylation in the presence of YB-1 siRNA is

normalized by pAkt and pErk protein levels, respectively, in the absence of erlotinib.

(C) Effect of lapatinib on expression of growth-factor receptors and their

downstream-regulatory molecules with or without YB-1 siRNA, followed by lapatinib

exposure and western blot analysis. (D) Quantitative analysis of the effect of lapatinib





37

on Akt and Erk phosphorylation. Akt and Erk phosphorylation in the presence of YB-1

siRNA is normalized by pAkt and pErk protein levels in the absence of the drug.

Figure 5. IHC images for expression of nuclear YB-1, EGFR, HER2, HER3 and CDC6

in gastric tumor specimens. (A) Representative images of nuclear YB-1 expression.

Positive (2 cases on the left) and negative (2 cases on the right) cases are presented. (B)

Representative images for low (0, +1) and high (2+, 3+) expression of EGFR, HER2,

HER3, and CDC6 in various tumor samples. (C) Representative images of HER2 and

nuclear YB-1 expression in the serial section. Of 4 clinical samples of the serial section,

2 cases (case 1 and 2) show higher expression of both HER2 (3+) and nuclear YB-1,

and the other 2 cases (case 3 and case 4) show no apparent expression of both HER2 (0,

+1) and nuclear YB-1 in cancer cells.




Figure 1

A

EGFRpEGFRHER2

pHER2HER3

pHER3Met

pMetErk

B

ErkpErkAkt

pAktα-tubulin

B

Erlotinib (μM)

EGFRpEGFR

HER2pHER2

HER3

0 0.1 1 10

SNU216MKN45 NCI-N87

0 0.1 1 10 0 0.1 1 10

AktpAktErk

pErkα-tubulin

HER3pHER3

C

pEGFRHER2

Lapatinib (μM) 0 0.1 1 10

SNU216MKN45 NCI-N87

0 0.1 1 10 0 0.1 1 10EGFR

pHER2

AktpAktErk

pErk

HER3pHER3

α-tubulin




MKN45siYB-1(nM) 0 2 10

YB-1

EGFR

HER2

SNU216

0 2 10

NCI-N87

0 10 50

Figure 2A

AktpAkt

ErkpErk

HER3

α-tubulin

MKN45

1

1.5

pres

sion

SNU216

YB-1 EGFR HER2 YB-1 EGFR HER2 HER3HER3

B

0

0.5

0 2 10

Rel

ativ

e ex

p

0 2 10 0 2 10 0 2 10 0 2 10 0 2 10

NCI-N87

siYB-1(nM) 0 2 10 0 2 10

0

0.5

1

1.5

0 10 50

Rel

ativ

e ex

pres

sion

siYB-1(nM) 0 10 50 0 10 50

YB-1 EGFR HER2

0 10 50

HER3

C

1

1.5

e YB

-1 m

RN

A pr

essi

on

0.02

0.04

EGFR

mR

NA

pres

sion

0.02

0.03

0.04

HER

2 m

RN

A pr

essi

on

0 005

0.01

0.015

e H

ER3

mR

NA

pres

sion

YB-1 EGFR HER2 HER3

*

0 10 50siYB-1(nM) 0 10 50 0 10 50 0 10 50

0

0.5

Rel

ativ

eex

p

0

Rel

ativ

e ex

p

0

0.01

Rel

ativ

e ex

p

0

0.005

Rel

ativ

eex

siCont siYB-1 siCont siYB-1 siCont siYB-1 siCont siYB-1

*

D#1start point of

start point of #1

#2

#3#2 #3

start point of transcription 2

transcription 1promoter 1

promoter 2

#1

: ATTG

: ATTGG

: CAAT




Figure 3

A MKN45si Control

si YB-1

406080100120140160

% o

f con

trol

406080100120140

% o

f con

trol

40

60

80

100

120

% o

f con

trol

02040

0 0.03 0.1 0.3 1 3 10

%

Lapatinib(μM)

02040

0 0.1 0.3 1 3 10

%

Cisplatin(μM)

B SNU216

0

20

0 0.1 0.3 1 3 10

%

Erlotinib (μM)

6080100120140

cont

rol

6080100120140

cont

rol

6080100120140

f con

trol

B SNU216

0204060

0 0.03 0.1 0.3 1 3 10

% o

f

Lapatinib(μM)

0204060

0 0.1 0.3 1 3 10

% o

f

Erlotinib(μM)

02040

0 0.1 0.3 1 3 10

% o

f

Cisplatin(μM)

60

80

100

120

ontr

ol

C NCI-N87

60

80

100

120

ontr

ol

80

100

120

140

ontr

ol

0

20

40

60

0 0.03 0.1 0.3 1 3 10

% o

f co

Lapatinib(μM)

0

20

40

60

0 0.1 0.3 1 3 10

% o

f co

Erlotinib (μM)

0

20

40

60

0 0.1 0.3 1 3 10

% o

f co

Cisplatin(μM)(μ ) p (μ )




pAkt

Cont siRNA YB-1 siRNA

B

Figure 4

A

100

120

Erlotinib (μM) 0 0.1 1 10

EGFR

pEGFR

HER2

YB-10 0.1 1 10

0

20

40

60

80

0 0 1 1 10

% o

f con

t

siCont

siYB-1

pErk

HER2pHER2

Akt

pAkt

HER3pHER3

60

80

100

cont

0 0.1 1 10Erlotinib(μM)

pAkt

Erk

pErk

α-tubulin 0

20

40

0 0.1 1 10%

of

Erlotinib(μM)

siContsiYB-1

pAktC D

Lapatinib (μM)

Cont siRNA YB-1 siRNA

0 0.1 1 10 0 0.1 1 10 80

100

120

cont

EGFRpEGFR

HER2

pHER2

YB-1

0

20

40

60

0 0.1 1 10

% o

f c

Lapatinib(μM)

siContsiYB-1

pErkpHER2

Akt

pAkt

HER3

pHER3

60

80

100

120

of c

ont

siContErk

pErk

GAPDH0

20

40

0 0.1 1 10

%

Lapatinib(μM)

siYB-1




Nuclear YB-1

Positive Negative

High(2+, 3+)

Low(0, 1+)

Figure 5

A B

Positive Negative

EGFR

HER2

HER3

CDC6

HER3

C

Case 1 Case 2 Case 3 Case 4

Nuclear YB-1

－－+ +

HER2

－－+ +




Published OnlineFirst February 27, 2013.Mol Cancer Ther Tomohiro Shibata, Hitoshi Kan, Yuichi Murakami, et al. cellsexpression and lapatinib sensitivity in human gastric cancer Y-box binding protein-1 (YB-1) contributes to both HER2/ErbB2

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