Role of E2F1 and its involving pathway in esophageal squamous

O R I G I N A L A R T I C L E

Role of E2F-1 and its involving pathway in esophagealsquamous cell carcinomaWen Wang1, Luyan Shen1, Yu Sun2, Bin Dong2 & Keneng Chen1

1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Thoracic Surgery I, Peking University Cancer Hos-pital & Institute, Beijing, China2 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Pathology, Peking University Cancer Hospital &Institute, Beijing, China

KeywordsE2F-1; esophageal squamous cell carcinoma;

survival.

CorrespondenceKeneng Chen, Key Laboratory ofCarcinogenesis and Translational Research(Ministry of Education), Department of ThoracicSurgery I, Peking University School of Oncology,Beijing Cancer Hospital & Institute, Beijing100142, China.Tel: +86 10 88196536Fax: +86 10 88196526Email: [email protected]

Received: 5 May 2013;accepted 13 June 2013.

doi: 10.1111/1759-7714.12061

AbstractBackground: Esophageal cancer is a lethal disease and the optimal therapy remainsunclear. Neoadjuvant chemotherapy provides an increased chance of survival; there-fore, we attempted to identify potential molecular markers that might improveevaluations of individual responses to therapy.Methods: We recruited 109 patients with resectable esophageal squamous cell car-cinoma. The patients underwent radical esophagectomy and did not receive anyother perioperative treatment. Expression of E2F-1 and molecules involved in itstargeted pathways, pERK, Bim, pRb, epidermal growth factor receptor, EZH2 andpAKT, was investigated immunohistochemically.Results: Correlations were observed between E2F-1 and pRb expression; EZH2

expression was significantly correlated with the degree of carcinoma differentiation(P = 0.01). Stage III patients were found to have longer survival if they did notexpress pERK than if they did (23 months vs. 11 months, P = 0.01). Patients withE2F-1 not expressing pRb were found to have longer survival times than those withE2F-1 who expressed pRb (18.8 months vs. 8.6 months, P = 0.021). Similarly, stageIII patients with E2F-1 but not expressing pERK also survived longer than thoseexpressing pERK (23.5 months vs. 3.9 months, P < 0.001).Conclusions: A high expression of pERK was significantly associated with poorsurvival in patients with locally advanced esophageal cancer. Expression of a combi-nation of molecules, rather than of individual molecules, was more predictive ofdisease prognosis. E2F-1 and molecules of its targeted pathways may be candidateproteins as markers of chemosensitivity in esophageal cancer patients.

Introduction

Esophageal cancer is one of the most common malignanciesin China, positioned fifth in terms of incidence and fourthwith respect to overall mortality.1 Surgery is generally consid-ered the standard treatment for this malignancy; however,such treatment is not feasible for all patients. Even for patientsundergoing surgery, the five-year survival rate remains un-satisfactory, because of metastasis and recurrence. Recently,survival has improved with multimodal therapy, includingchemotherapy and radiotherapy.2 At present, platinum-basedchemotherapy is predominantly used in the treatment ofesophageal cancer. Platinum interacts with DNA to formintra-strand, cross-linked DNA adducts that trigger a series

of intracellular events that ultimately result in cell death.However, an efficient method for evaluating responses toinduction therapy is currently lacking. Results of studiesinvestigating neoadjuvant treatment with chemoradio-therapy and chemotherapy, based on two- or three-yearsurvival rates, have shown similar benefits (13% and 7%,respectively).2

Although several studies have investigated the prognosticfactors for esophageal cancer (mainly in adenocarcinomasof the esophagus or gastroesophageal junction), few hasfocused on patients with squamous cell carcinoma. Conven-tional modalities for evaluating response include computedtomography, magnetic resonance imaging, ultrasonography,endoscopy, radiography, and endoscopic ultrasonography,

Thoracic Cancer ISSN 1759-7706

Thoracic Cancer 5 (2014) 139–148 © 2013 Tianjin Lung Cancer Institute and Wiley Publishing Asia Pty Ltd 139

which are based on anatomic, structural, and metabolicchanges. However, changes at the molecular level occurearlier, suggesting that the identification of genes sensitiveto anticancer drugs could help more accurately evaluateresponses to induction chemotherapy.

E2Fs activate and repress the transcription of many essen-tial genes involved in cell proliferation, apoptosis, DNArepair, and differentiation. Normally, E2F-1 combines withthe Rb tumor suppressor protein to inhibit the transcrip-tional activity of E2F-1, by inducing the phosphorylation ofRb by Cdks. The Rb/E2F compound released after the loss ofE2F-1inhibition and the recovery of E2F-1 transcriptionalactivity activate the expression of cell cycle-associated genesand promote cell proliferation.3 The binding of E2F-1 withRb occurs via two patterns: (i) Rb combines with the C-end ofE2F-1, present in all E2F genes, or (ii) Rb combines withE2F-1 at other areas, specific to E2F-1.

When a cell is exposed to external stimuli, such as ionizingradiation, ultraviolet radiation, or chemotherapy, DNAdamage may be induced. Under these circumstances, thecharacteristic pattern of E2F-1 is abated, and the E2F-1/Rbcompound inhibits the cell cycle genes involved in the tran-scription of cyclin A2 and promotes the transcription of theapoptosis genes, thereby activating p73 and caspase-7 toinduce apoptosis.3 Therefore, E2F-1 participates in the regu-lation of both cell proliferation and apoptosis.

In various types of tumors, E2F-1 shows an increased levelof abnormal expression and gene amplification. High E2F-1expression is associated with the degree of malignancyand metastasis.4 Literature reports suggest that E2F-1 canimplement this process by inducing increased epidermalgrowth factor receptor (EGFR) expression and activatingthe mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase[PI3K]/pAKT signaling pathways that promote cancerprogression.4,5

Chemotherapy, a double-edged sword, can induce bothapoptosis and resistance to cell death, especially in advancedtumors where chemotherapy-induced DNA damage mayinduce chemotherapy drug resistance. In tumor cells resistantto chemotherapy, E2F-1 is observed to be upregulated, allow-ing tumor cells to escape the lethal effects of the chemothera-peutic drug.6 E2F-1 can also induce the expression of EZH2,which inhibits downstream expression of Bim genes and pro-motes tumor cell metastasis.7,8

This study examined potential molecular markers thatcould be used to evaluate chemotherapeutic effects. Thepotential markers had to satisfy two conditions: they had tobe associated with chemotherapy resistance or sensitivity andthe molecules could not have any significant influence on thesurvival of patients with esophageal cancer, so that it wouldnot be a confounding factor in the evaluation of chemo-therapy effectiveness. Therefore, we evaluated E2F-1 expres-

sion and the regulation of cell proliferation and apoptosis atthe molecular level in esophageal cancer tissues and analyzedits relationship with clinicopathological factors and its influ-ence on patient survival.

Methods

Patients

We recruited 109 patients with resectable esophageal squa-mous cell carcinoma (ESCC) who had been referred to theDepartment of Thoracic Surgery, Peking University Schoolof Oncology, between January 2001 and December 2009.The study population comprised 142 men and 52 women,ranging in age from 34 to 76 years (median, 59 years). Thepatients underwent radical esophagectomy and did notreceive any other treatment before or after surgery (Table 1).

Table 1 Patients’ clinicopathological details (n = 109)

Item No.

GenderMale 79Female 30

Age≤63 55>63 54

Tumor locationL0 4L1 11L2 56L3 38

Tumor cell differentiationG1 18G2 55G3 36

Tumor invasion (T)Tis 3T1 19T2 26T3 49T4 12

Lymph nodes metastasis (N)N0 72N1 23N2 10N3 4

TNM stage0 3Ia 1Ib 21IIa 21IIb 27IIIa 20IIIb 8IIIc 8

TNM, tumor node metastasis.

Role of E2F-1 in ESCC W. Wang et al.

140 Thoracic Cancer 5 (2014) 139–148 © 2013 Tianjin Lung Cancer Institute and Wiley Publishing Asia Pty Ltd

The clinicopathological characteristics and follow-up datawere obtained from our prospective database and clinicalrecords. These patients, like all postoperative patients, werescheduled for regular visits to our outpatient clinic everythree months during the first two years after surgery, thenevery six months for the third to fifth years, and annually,thereafter. Both the Ethics and the Academic committees ofthe Peking University School of Oncology approved thisstudy; informed consent was obtained from all patients par-ticipating in the study.

Immunohistochemistry

The tissue slides were deparaffinized with xylene and rehy-drated through a series of decreasing alcohol concentrations.Endogenous peroxidase activity was blocked by incubation ofthe sections with 3% hydrogen peroxide/methanol buffer,and antigen retrieval was carried out by microwave treatmentof the slides in sodium citrate buffer (pH 6.0) for 10 minutes.The slides were rinsed in phosphate-buffered saline (PBS)and incubated with blocking serum to block nonspecificstaining. Immunostaining for E2F-1, pRb, EGFR, pERK,pAKT, EZH2, and Bim was performed by incubating with theprimary antibody overnight at 4°C. PBS was used as a nega-tive control. After washing, the sections were incubated withthe secondary antibody (Vector Labs, Burlingame, CA, USA)for 15 minutes. A streptavidin/peroxidase amplification kit(Zymed, South San Francisco, CA, USA) was used to detectthe antigen-antibody reactions; peroxidase activity wasdeveloped with diaminobenzidine. All sections were counter-stained with hematoxylin.

Two observers, blinded to both the clinical and pathologicdata, independently examined the slides. Protein expressionwas quantified using a visual grading system based on theextent of staining (the percentage of positive tumor cells wasgraded on a scale of 0 to 4: 0, none; 1, 1–10%; 2, 11–50%; 3,50–75%; 4, >75%. Four high-power fields (200×) per slidewere evaluated, and the results were averaged.

Statistical analysis

Statistical analysis of the data was performed using IBMSPSS version 19.0 (SPSS, Chicago, IL, USA). Univariateanalyses of the associations were determined using Pear-son’s χ2 test. Survival rates were calculated according to thenumber of patients alive at the end of the follow-up period,and Kaplan–Meier curves were plotted; survival data werecensored if the patient was still alive. The log-rank test wasused to determine any statistically significant differencesbetween survival rates. A P-value of <0.05 was consideredstatistically significant.

Results

Examination of protein expression

Expression of the following seven proteins was observed inthe tissue samples from the patients: E2F-1 (28.4%, 31/109)in the nucleus, pERK (35.8%, 39/109) in the nucleus andcytoplasm, Bim (43.1%, 47/109) in the cell membrane, pRb(21.1%, 23/109) in the nucleus, EGFR (45.0% 49/109) in thecell membrane, EZH2 (35.8%, 39/109) in the nucleus, andpAKT (72.5%, 79/109) in the nucleus and cytoplasm (Table 2and Fig 1).

Association of protein expression withclinicopathological characteristics

The associations between protein expression in ESCC and theseven clinicopathological characteristics of the patients(gender, age, tumor location, differentiation, tumor invasion,lymph nodes metastasis, and tumor node metastasis [TNM]stage) are shown in Table 2. Only EZH2 expression wassignificantly correlated with the degree of cancer differen-tiation (P = 0.01). The expression of other proteins wasnot significantly associated with patient clinicopathologicalcharacteristics.

Association of protein expression withoverall survival

E2F-1, pERK, Bim, pRb, EGFR, EZH2, and pAKT expressionlevels were not associated with overall survival (P > 0.05).After stratifying the patients according to disease stage,patient survival was found to be significantly longer in thosenot expressing pERK, than in those expressing pERK (23months vs. 11 months, P = 0.01) among patients with stage IIIdisease (Table 3 and Fig 2).

When combining E2F-1 with other molecules, amongpatients with E2F-1 expression, survival was longer in thosewho did not express pRb, than in those with pRb expression(18.8 months vs. 8.6 months, P = 0.021) for patients withstage III disease. Similarly, among patients with E2F-1 expres-sion, survival was longer in those who did not express pERK,than in those who expressed pERK (23.5 months vs. 3.9months, P < 0.001) among patients with stage III disease(Table 4 and Figs 3, 4).

Correlation analysis between E2F-1 andother molecules

The correlations between E2F-1 and other molecules are sum-marized in Table 5. Positive correlations were only observedbetween E2F-1 and pRb (r = 0.272, P = 0.004).

W. Wang et al. Role of E2F-1 in ESCC


Tab

le2

Ass

ocia

tion

betw

een

the

seve

nm

arke

rsex

pres

sion

and

clin

ical

char

acte

ristic

sof

patie

nts

with

esop

hage

alsq

uam

ous

cell

carc

inom

a(E

SCC

)(n

=10

9)

Clin

icop

atho

logi

cch

arac

teris

tics

E2F-

1ex

pres

sion

P-va

lue

pERK

expr

essi

on

P-va

lue

pRB

expr

essi

on

P-va

lue

Bim

expr

essi

on

P-va

lue

Posi

tive

(%)

Neg

ativ

e(%

)Po

sitiv

e(%

)N

egat

ive

(%)

Posi

tive

(%)

Neg

ativ

e(%

)Po

sitiv

e(%

)N

egat

ive

(%)

Age ≤6

313

(24.

1)41

(75.

9)0.

317

17(3

1.5)

37(6

8.5)

0.35

410

(18.

5)44

(81.

5)0.

512

24(4

4.4)

30(5

5.6)

0.78

2>6

318

(32.

7)37

(67.

3)22

(40.

0)33

(60.

0)13

(23.

6)42

(76.

4)23

(41.

8)32

(58.

2)G

ende

rM

ale

24(3

0.4)

55(6

9.6)

0.46

628

(35.

4)51

(64.

6)0.

912

16(2

0.3)

63(7

9.7)

0.73

634

(43.

0)45

(57.

0)0.

978

Fem

ale

7(2

3.3)

23(7

6.7)

11(3

6.7)

19(6

3.3)

7(2

3.3)

23(7

6.7)

13(4

3.3)

17(5

6.7)

Tum

orlo

catio

nL0

2(5

0.0)

2(5

0.0)

0.08

32

(50.

0)2

(50.

0)1

(25.

0)3

(75.

0)2

(50.

0)2

(50.

0)L1

0(0

.00)

11(1

00.0

)6

(54.

5)5

(45.

5)0.

100

2(1

8.2)

9(8

1.8)

0.99

03

(27.

3)8

(72.

7)0.

242

L215

(26.

8)41

(73.

2)14

(25.

0)42

(75.

0)12

(21.

4)44

(78.

6)21

(37.

5)35

(62.

5)L3

14(3

6.8)

24(6

3.2)

17(4

4.7)

21(5

5.3)

8(2

1.1)

30(7

8.9)

21(5

5.3)

17(4

4.7)

Tum

orce

lldi

ffer

entia

tion

G1

7(3

8.9)

11(6

1.1)

0.14

13

(16.

7)15

(83.

3)0.

114

6(3

3.3)

12(6

6.7)

0.30

39

(50.

0)9

(50.

0)0.

551

G2

18(3

2.7)

37(6

7.3)

24(4

3.6)

31(5

6.4)

9(1

6.4)

46(8

3.6)

25(4

5.5)

30(5

4.5)

G3

6(1

6.7)

30(8

3.3)

12(3

3.3)

24(6

6.7)

8(2

2.2)

28(7

7.8)

13(3

6.1)

23(6

3.9)

Tum

orin

vasi

on(T

)Ti

s0

(0.0

)3

(100

.0)

2(6

6.7)

1(3

3.3)

0(0

.0)

3(1

00.0

)1

(33.

3)2

(66.

7)T1

4(2

1.1)

15(7

8.9)

0.68

17

(0.0

)12

(100

.0)

0.45

14

(0.0

)15

(100

.0)

0.83

26

(0.0

)13

(100

.0)

0.25

4T2

7(2

6.9)

19(7

3.1)

11(4

2.3)

15(5

7.7)

7(2

6.9)

19(7

3.1)

15(5

7.7)

11(4

2.3)

T316

(32.

7)33

(67.

3)17

(34.

7)32

(65.

3)10

(20.

4)39

(79.

6)18

(36.

7)31

(63.

3)T4

4(3

3.3)

8(6

6.7)

2(1

6.7)

10(8

3.3)

2(1

6.7)

10(8

3.3)

7(5

8.3)

5(4

1.7)

Lym

phno

des

met

asta

sis

(N)

N0

20(2

7.8)

52(7

2.2)

0.83

925

(13.

5)47

(86.

5)0.

847

15(1

3.5)

57(8

6.5)

0.87

233

(13.

5)39

(86.

5)0.

768

N1

8(3

4.8)

15(6

5.2)

9(3

9.1)

14(6

0.7)

4(1

7.4)

19(8

2.6)

9(3

9.1)

14(6

0.7)

N2

2(2

0.0)

8(8

0.0)

3(3

0.0)

7(7

0.0)

3(3

0.0)

7(7

0.0)

3(3

0.0)

7(7

0.0)

N3

1(2

5.0)

3(7

5.0)

2(5

0.0)

2(5

0.0)

1(2

5.0)

3(7

5.0)

2(5

0.0)

2(5

0.0)

TNM

stag

e0

0(0

.0)

3(1

00.0

)2

(66.

7)1

(33.

3)0

(0.0

)3

(100

.0)

1(3

3.3)

2(6

6.7)

Ia0

(0.0

)1

(100

.0)

0.87

40

(0.0

)1

(100

.0)

0.81

50

(0.0

)1

(100

.0)

0.90

01

(100

.0)

0(0

.0)

0.87

8Ib

8(3

8.1)

13(6

1.9)

8(3

8.1)

13(6

1.9)

4(1

9.0)

17(8

1.0)

9(4

2.9)

12(5

7.1)

IIa6

(28.

6)15

(71.

4)9

(42.

7)12

(57.

1)4

(19.

0)17

(81.

0)8

(38.

1)13

(61.

9)IIb

7(2

5.9)

20(7

4.1)

10(3

7.0)

17(6

3.0)

8(3

0.0)

19(7

0.0)

11(4

0.7)

16(5

9.3)

IIIa

5(2

5.0)

15(7

5.0)

5(2

5.0)

15(7

5.0)

4(2

0.0)

16(8

0.0)

9(4

5.0)

11(5

5.0)

IIIb

2(2

5.0)

6(7

5.0)

3(3

7.5)

5(6

2.5)

2(2

5.0)

6(7

5.0)

3(3

7.5)

5(6

2.5)

IIIc

3(3

7.5)

5(6

2.5)

2(2

5.0)

6(7

5.0)

1(1

2.5)

7(8

7.5)

5(6

2/5)

3(3

7.5)

TNM

,tum

orno

dem

etas

tasi

s.



Tab

le2

Con

tinue

d

Clin

icop

atho

logi

cch

arac

teris

tics

EZH

2ex

pres

sion

P-va

lue

EGFR

expr

essi

on

P-va

lue

pAK

Tex

pres

sion

P-va

lue

Posi

tive

(%)

Neg

ativ

e(%

)Po

sitiv

e(%

)N

egat

ive

(%)

Posi

tive

(%)

Neg

ativ

e(%

)

Age ≤6

320

(37.

0)34

(63.

0)0.

790

25(4

6.3)

29(5

3.7)

0.78

639

(72.

2)15

(27.

8)0.

95>6

319

(34.

5)36

(65.

5)24

(43.

6)31

(56.

4)40

(72.

7)15

(27.

3)G

ende

rM

ale

27(3

4.2)

52(6

5.8)

0.57

340

(50.

6)39

(49.

4)0.

052

61(7

7.2)

18(2

2.8)

0.07

0Fe

mal

e12

(40.

0)18

(60.

0)9

(30.

0)21

(70.

0)18

(60.

0)12

(40.

0)Tu

mor

loca

tion

L01

(25.

0)3

(75.

0)2

(50.

0)2

(50.

0)3

(75.

0)1

(25.

0)L1

7(6

3.6)

4(3

6.4)

0.09

06

(54.

5)5

(45.

5)0.

644

10(9

0.9)

1(9

.1)

0.47

3L2

15(2

6.8)

41(7

3.2)

27(5

1.0)

29(4

9.0)

38(7

0.4)

18(2

9.6)

L316

(42.

1)22

(57.

9)14

(38.

9)24

(61.

1)28

(73.

7)10

(26.

3)Tu

mor

cell

diff

eren

tiatio

nG

17

(38.

9)11

(61.

1)0.

010

8(4

4.4)

10(5

5.6)

0.38

213

(72.

2)5

(27.

8)1.

000

G2

13(2

3.6)

42(7

6.4)

28(5

0.9)

27(4

9.1)

40(7

2.7)

15(2

7.3)

G3

15(4

1.7)

21(5

8.3)

13(3

6.1)

23(6

3.9)

26(7

2.2)

10(2

7.8)

Tum

orin

vasi

on(T

)Ti

s2

(66.

7)1

(33.

3)0

(0.0

)3

(100

.0)

2(6

6.7)

1(3

3.3)

T13

(15.

8)16

(84.

2)0.

213

10(5

2.6)

9(4

7.4)

0.47

511

(57.

9)8

(42.

1)0.

550

T29

(34.

6)17

(65.

4)10

(38.

5)16

(61.

5)20

(76.

9)6

(23.

1)T3

19(3

8.8)

30(6

1.2)

23(4

6.9)

26(5

3.1)

36(7

3.5)

13(2

6.5)

T46

(50.

0)6

(50.

0)6

(50.

0)6

(50.

0)10

(83.

3)2

(16.

7)Ly

mph

node

sm

etas

tasi

s(N

)N

025

(34.

7)47

(65.

3)0.

893

34(4

7.2)

38(5

2.8)

0.62

151

(70.

8)21

(29.

2)0.

564

N1

9(3

9.1)

14(6

0.9)

11(4

7.8)

12(5

2.2)

19(8

2.6)

4(1

7.4)

N2

3(3

0.0)

7(7

0.0)

3(3

0.0)

7(7

0.0)

6(6

0.0)

4(4

0.0)

N3

2(5

0.0)

2(5

0.0)

1(2

5.0)

3(7

5.0)

3(7

5.0)

1(2

5.0)

TNM

stag

e0

2(6

6.7)

1(3

3.3)

0(0

.0)

3(1

00.0

)2

(66.

7)1

(33.

3)Ia

1(1

00.0

)0

(0.0

)0.

244

0(0

)1

(100

.0)

0.14

20

(0)

1(1

00.0

)0.

195

Ib4

(19.

0)17

(81.

0)13

(61.

9)8

(38.

1)12

(57.

1)9

(42.

9)IIa

9(4

2.9)

12(5

7.1)

7(3

3.3)

14(6

6.7)

17(6

6.7)

4(3

3.3)

IIb10

(37.

0)17

(63.

0)14

(51.

9)13

(48.

1)22

(81.

5)5

(18.

5)III

a6

(30.

0)14

(70.

0)10

(50.

0)10

(50.

0)15

(75.

0)5

(25.

0)III

b2

(25.

0)6

(75.

0)1

(12.

5)7

(87.

5)4

(50.

0)4

(50.

0)III

c5

(62.

5)3

(37.

5)4

(50.

0)4

(50.

0)7

(87.

5)1

(12.

5)

EGFR

,epi

derm

algr

owth

fact

orre

cept

or;T

NM

,tum

orno

dem

etas

tasi

s.



a b

c d

e f

g h

Figure 1 Positive expression of the seven proteins (a) E2F-1, (b) pRb, (c) EGFR, (d) pERK, (e) pAKT, (f) EZH2, (g) Bim, (h) negative control ×200.



Discussion

In the past, the only treatment for advanced esophagealcancer was surgical resection, but the five-year survival ratewas less than 30%. During the last 20 years, with the develop-ment of neoadjuvant chemoradiotherapy, great improve-ments in patient postoperative survival rates have beenrealized. The common consensus is that patients undergoingcomplete resection after these treatments achieve five-yearsurvival rates of up to 70%.9 However, only patients who

Table 3 Association of E2F-1, pRb, EGFR, pERK, pAKT, EZH2, Bim expres-sion with survival time in stage III (n = 36)

No. (%)

Mediansurvivaltime(Months)

95%confidenceinterval P-value

E2F-1 + 10 (27.8) 8.6 (4–13) 0.071− 26 (72.2) 23 (16–31)

pRb + 7 (19.4) 19 (0–49) 0.537− 29 (80.6) 23 (13–24)

EGFR + 15 (41.7) 20 (6–34) 0.494− 21 (58.3) 19 (7–31)

pERK + 10 (27.8) 11 (7–14) 0.010− 26 (72.2) 23 (18–29)

pAKT + 11 (30.5) 12 (5–19) 0.298− 25 (69.5) 23 (12–35)

EZH2 + 13 (36.1) 23 (7–38) 0.240− 23 (63.9) 40 (25–56)

Bim + 17 (47.2) 23 (17–29) 0.235− 19 (52.8) 15 (7–24)

EGFR, epidermal growth factor receptor.

Figure 2 Overall survival of pERK expression in stage III patients (n = 36)., 0; , 1; , 0-censored; , 1-censored.

Table 4 Association of E2F-1 and other molecular combination expres-sions with survival time in stage III (n = 36)

No. (%)

Mediansurvivaltime(Months)

95%confidenceinterval P-value

E2F-1/pRb 0.0210 22 (61.1) 18.8 (9.3–28.3)1 11 (30.6) 23.3 (14.1–32.5)2 3 (8.3) 8.6 (7.1–10.1)

E2F-1/pERK <0.0010 20 (55.6) 23.5 (9.2–37.7)1 12 (33.3) 12.1 (3.3–20.9)2 4 (11.1) 3.9 (0–8.6)

E2F-1/Bim 0.9860 15 (41.7) 23.5 (8.9–38.0)1 15 (41.7) 18.8 (6.8–30.8)2 6 (16.7) 17.1 (0.2–34.0)

E2F-1/pAKT 0.2270 21 (58.3) 30.3 (20.3–40.3)1 9 (25.0) 15.2 (6.2–24.2)2 6 (16.7) 7.3 (1.6–12.9)

E2F-1/EGFR 0.0850 15 (41.7) 23.3 (16.4–30.3)1 17 (47.2) 20.1 (0.0–44.3)2 4 (11.1) 3.9 (0.0–17.8)

E2F-1/EZH2 0.2150 16 (44.4) 23.3 (17.5–29.1)1 17 (47.2) 10.6 (6.2–15.1)2 3 (8.3) 22.7 (13.7–31.7)

0 = −/−,1 = −/+ or +/−,2 = +/+.

Figure 3 Overall survival of E2F-1+pRb expression in stage III patients(n = 36). , .00; , 1.00; , 2.00; , .00-censored; , 1.00-censored; , 2.00-censored.



experience tolerable toxicity and positively respond to preop-erative therapy benefit from this approach. Moreover, the roleof adjuvant chemotherapy in patients who have receivedneoadjuvant chemotherapy is uncertain, particularly in thepatients (50–60%) who do not respond to preoperativechemotherapy.

Reliable evaluation of tumor response in surgical resectionspecimens would be helpful in planning postoperativechemotherapy. In addition, predictive markers of responsewould be valuable for individualizing patient treatment, asit would enable discrimination of those patients likely torespond to combination therapy. Changes at the molecularand cellular levels would naturally be assumed to occurearlier than those that occur at the morphological level.Therefore, effective molecular markers may allow a moreaccurate judgment of the curative effects of a specific preop-erative induction therapy. This would help avoid both exces-sive and insufficient treatments.

Preoperative chemotherapy mainly involves platinum-based therapies that are based on the drugs’ ability to induceDNA damage. Therefore, we chose relevant E2F-1 and related

pathways for the current research; E2F-1 has a dual mecha-nism and is involved in regulating cell proliferation and apop-tosis. Thus, we hypothesized that chemotherapy-inducedchanges would result in E2F-1 regulation of cell developmentin two different ways. First, when DNA damage was irrepa-rable, the E2F-1 induction of downstream apoptosis geneswould make the cells sensitive to the chemotherapeuticdrug(s). Second, if the DNA damage was repaired, E2F-1would regulate cell proliferation, allowing the cell cycle tocontinue and drug resistance to develop.

Evaluation of the effects of induction chemotherapy onpathological responses and postoperative survival timesrequires that the molecular marker not influence the patho-logical stage or survival. Thus, the correlation of pathologicstaging and patient survival with the responses of weakmolecular markers may achieve these goals. In this study, weselected individual surgery patients and evaluated the asso-ciation of the expression of E2F-1 and related molecules withclinicopathological factors and patient survival. The resultsshowed that E2F-1 does not have an obvious correlation withclinicopathological factors or survival. The choice of E2F-1 asthe research molecule was feasible because Rb deficiencies arepresent in most tumor cells, leading to abnormal expressionof E2F-1.10 Whether E2F-1 plays a role in transcriptionalactivation or inhibition depends on the cell’s environment.Further, we analyzed the effects of pRb and E2F-1 on down-stream molecules that promote cell proliferation andapoptosis, EGFR, EZH2, pAKT, pERK, and Bim, and theirrelationship with clinicopathological factors and survival.We found that none of these markers correlated with clinico-pathological factors or survival, except that EZH2 expressionwas significantly correlated with the degree of tumor differ-entiation (P = 0.01).

The relationship of pERK and Bim with ESCC patientsurvival has not been previously reported. However, westratified the patients according to disease stage andobserved that the survival of patients with stage III diseasenot expressing pERK was significantly longer than that ofpatients expressing pERK (23 months vs. 11 months, P =0.01). In patients with stage I or II disease, the survival ofpatients expressing Bim was longer than that of patients notexpressing Bim (114 months vs. 34 months), but the differ-ence was not significant (P = 0.088).

In previous studies, pRb,11–14 EGFR,15 E2F-1, EZH2, pERK,and pAKT have been reported to be related to patient sur-vival; this is inconsistent with our results, which showedthat pRb and EGFR are not independent prognostic factorsfor patients with esophageal cancer. Some studies havereported that E2F-1 expression is significantly related topathological staging and that high expression levels pre-dicted the prognosis of patients with ESCC.16–19 In ourstudy, we examined the patients with E2F-1 separately, andfound that the survival of patients with E2F-1 expression

Figure 4 Overall survival of E2F-1+pERK expression in stage III patients(n = 36). , .00; , 1.00; , 2.00; , .00-censored; , 1.00-censored; , 2.00-censored.

Table 5 E2F-1 expression association with pRB in esophageal squamouscell carcinoma (ESCC)

E2F-1expression

pRb expression

r P-valuePositive Negative

Positive 12 19 0.272 0.004Negative 11 67



who did not express pRb was longer than that of patientsexpressing pRb (18.8 months vs. 8.6 months, P = 0.021)among patients with stage III disease. Similarly, the survivalof E2F-1-positive patients with stage III disease who didnot express pERK was longer than that of patients whoexpressed pERK (23.5 months vs. 3.9 months, P < 0.001).Thus, prognosis appears to be better predicted by a combi-nation of molecules, than by a single molecule.

Ha et al.20 suggested that the expression of EZH2 was not anindependent prognostic factor for these patients, consistentwith our results; co-expression with Bmi-1 was, however, anindependent prognostic factor. Yamada et al.21 showed thathigh EZH2 expression was correlated with tumor infiltrationdepth and predicted a poor prognosis. In another study,Yamada et al.22 also found an obvious relationship betweenthe elevated expression of EZH2 and survival in patients whohad undergone preoperative chemoradiotherapy. Therefore,the expression of EZH2 may be a biomarker for predicting thecurative effects of preoperative chemoradiotherapy. pAKT, asa component of the PI3K/pAKT/mammalian target of therapamycin cell proliferation pathway, plays an importantrole in chemotherapeutic drug resistance. Some studies havereported that pAKT expression is higher in patients whoreceive preoperative chemotherapy than in those who do notreceive such treatment. The effect of high pAKT expressionon patient survival was not significantly associated withsurgery, but pAKT could be an independent prognostic factorfor patients receiving chemoradiotherapy. Accordingly,pAKT may act as an index to evaluate responses to preopera-tive chemotherapy.23

Conclusion

In a follow-up study, we will continue to explore the expres-sion of E2F-1, pERK, Bim, pRb, EGFR, EZH2, and pAKT inpatients with ESCC who have received preoperative chemo-therapy. We intend to analyze the chemotherapeutic effectsand evaluate the feasibility of molecular markers in deter-mining the effect of preoperative chemotherapy.

Acknowledgments

We thank Drs Zhen Liang, Hongchao Xiong, Bin Qin, WanpuYan, Yongbo Yang, Xiaozheng Kang, Liang Dai, and Heli Yangfor helping with patient care, and PhD candidates Hui Li,Yabing Du, Mengying Fan, and MD candidates Hao Fu, andChuan Huang, for data collection. This work was supportedby the Beijing Academic Leaders Program (Grant 2009-2-17),Beijing Natural Science Foundation (Grant 7102029), CapitalMedical Development Research Fund (Grant 2007-1023),New Scholar Star Program of Ministry of Education, andNational Basic Research Program of China (973 programs)(Grant 2011CB504300).

Disclosure

No authors report any conflict of interest.

References1 Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med 2003;

349: 2241–52.2 Gebski V, Burmeister B, Smithers BM et al. Survival benefits

from neoadjuvant chemoradiotherapy or chemotherapy inoesophageal carcinoma: a meta-analysis. Lancet Oncol 2007; 8:226–34.

3 Engelmann D, Pützer BM. The dark side of E2F1: in transitbeyond apoptosis. Cancer Res 2012; 72: 571–5.

4 Lee JS, Leem SH, Lee SY et al. Expression signature of E2F1and its associated genes predict superficial to invasiveprogression of bladder tumors. J Clin Oncol 2010; 28: 2660–7.

5 Chaussepied M, Ginsberg D. Transcriptional regulation ofAKT activation by E2F. Mol Cell 2004; 16: 831–7.

6 Andersen JB, Factor VM, Marquardt JU et al. An integratedgenomic and epigenomic approach predicts therapeuticresponse to zebularine in human liver cancer. Sci Transl Med2010; 2: 54ra77.

7 Min J, Zaslavsky A, Fedele G et al. An oncogene-tumorsuppressor cascade drives metastatic prostate cancer bycoordinately activating Ras and nuclear factor-kappaB. NatMed 2010; 16: 286–94.

8 Bug M, Dobbelstein M. Anthracyclines induce theaccumulation of mutant p53 through E2F1-dependent and-independent mechanisms. Oncogene 2011; 30: 3612–24.

9 Vallböhmer D, Hölscher AH, DeMeester S et al. A multicenterstudy of survival after neoajuvant radiotherapy/chemotherapy and esophagectomy for yp T0N0M0R0esophageal cancer. Ann Surg 2010; 252: 744–9.

10 Soengas MS, Capodieci P, Polsky D et al. Inactivation of theapoptosis effector Apaf-1 in malignant melanoma. Nature2001; 409: 207–11.

11 zur Hausen A, Sarbia M, Heep H, Willers R, Gabbert HE.Retinoblastoma-protein (pRb) expression and prognosis insquamous-cell carcinomas of the esophagus. Int J Cancer1999; 84: 618–22.

12 Ikeguchi M, Sakatani T, Ueta T, Kaibara N. Cyclin D1expression and retinoblastoma gene protein (pRb) expressionin esophageal squamous cell carcinoma. J Cancer Res ClinOncol 2001; 127: 531–6.

13 Ikeguchi M, Oka S, Gomyo Y, Tsujitani S, Maeta M, Kaibara.Combined analysis of p53 and retinoblastoma proteinexpressions in esophageal cancer. Ann Thorac Surg 2000; 70:913–7.

14 Ikeguchi M, Oka S, Gomyo Y, Tsujitani S, Maeta M, Kaibara.Clinical significance of retinoblastoma protein (pRb)expression in esophageal squamous cell carcinoma. J SurgOncol 2000; 73: 104–8.

15 Yu WW, Guo YM, Zhu M et al. Clinicopathological andprognostic significance of EGFR over-expression in



esophageal squamous cell carcinoma: a meta-analysis.Hepatogastroenterology 2011; 58: 426–31.

16 Yamazaki K, Hasegawa M, Ohoka I et al. Increased E2F-1expression via tumor cell proliferation and decreasedapoptosis are correlated with adverse prognosis in patientswith squamous cell carcinoma of the oesophagus. J ClinPathol 2005; 58: 904–10.

17 Mega S, Miyamoto M, Ebihara Y et al. Cyclin D1, E2F1expression levels are associated with characteristics andprognosis of esophageal squamous cell carcinoma. DisEsophagus 2005; 18: 109–13.

18 Ebihara Y, Miyamoto M, Shichinohe T et al. Over-expressionof E2F-1 in esophageal squamous cell carcinoma correlateswith tumor progression. Dis Esophagus 2004; 17: 150–4.

19 Yamazaki K, Hasegawa M, Ohoka I et al. Increased E2F-1expression via tumour cell proliferation and decreasedapoptosis are correlated with adverse prognosis in patients

with squamous cell carcinoma of the oesophagus. J ClinPathol 2005; 58: 904–10.

20 Ha SY, Kim SH. Co-expression of Bmi1 and EZH2 as anindependent poor prognostic factor in esophageal squamouscell carcinoma. Pathol Res Pract 2012; 208: 462–9.

21 Yamada A, Fujii S, Daiko H, Nishimura M, Chiba T, Ochiai A.Aberrant expression of EZH2 is associated with a pooroutcome and P53 alteration in squamous cell carcinoma ofthe esophagus. Int J Oncol 2011; 38: 345–53.

22 He LR, Liu MZ, Li BK et al. High expression of EZH2 isassociated with tumor aggressiveness and poor prognosis inpatients with esophageal squamous cell carcinoma treatedwith definitive chemoradiotherapy. Int J Cancer 2010; 127:138–47.

23 Yoshioka A, Miyata H, Doki Y et al. The activation of pAKTduring chemotherapy for esophageal cancer correlations withpoor prognosis. Oncol Rep 2008; 19: 1099–107.



Documents

Role of E2F1 and its involving pathway in esophageal squamous