ORIGINAL PAPER

Elevated expression patterns and tight correlation of the PLCE1and NF-jB signaling in Kazakh patients with esophagealcarcinoma

Xiao-bin Cui • Xue-lian Pang • Su Li • Jing Jin • Jian-ming Hu •

Lan Yang • Chun-xia Liu • Li Li • Shu Jun Wen •

Wei-hua Liang • Yun-zhao Chen • Feng Li

Received: 14 September 2013 / Accepted: 25 November 2013 / Published online: 5 December 2013

� Springer Science+Business Media New York 2013

Abstract This study investigated the expression of the

phospholipase C epsilon 1 (PLCE1) and nuclear factor-

kappaB (NF-jB)-related proteins in Kazakh patients with

esophageal squamous cell carcinoma (ESCC). Tissue

microarrays of 90 ethnic Kazakh patients with ESCC and

exhibiting clinical characteristics were analyzed for protein

expression of PLCE1, IKKb, IKBa, p50, and p65 by

immunohistochemistry. Correlations between histoscores of

PLCE1 and NF-jB-related proteins were determined using

Spearman’s rank correlation tests. Expression of PLCE1 and

NF-jB-related proteins significantly increased in tumor

tissues compared with normal esophageal tissues (P =

9.48 9 10-7, 1.24 9 10-5, 0.004, 0.003, and 2.83 9 10-5,

respectively). Upregulation of PLCE1 was significantly

correlated with advanced tumor-node-metastasis stages

(P = 0.018) and lymph node metastasis (P = 0.003).

Overexpression of IKKb and IKBa was associated with

ESCC stages I/II (P = 3.36 9 10-4 and 0.022, respec-

tively). Increased expression of p50 was significantly higher

in patients with lymph node metastasis than without lymph

node metastasis (P = 0.048). Elevated expression of p65

protein was significantly correlated with poor and moder-

ately differentiated ESCC and depth of tumor invasion

(P = 0.026 and 0.010, respectively). Significant positive

correlations were observed between the expression of

PLCE1 and NF-jB-related proteins, especially IKKb (r =

0.246 and P = 0.025) and p50 (r = 0.244 and P = 0.024).

These results suggest, for the first time, that upregulation of

PLCE1 is correlated with increased expression of NF-jB-

related proteins in Kazakh patients with ESCC, suggesting

that interaction between PLCE1 with the NF-jB signal

pathway may be responsible for the carcinogenesis of ESCC,

such as ESCC-related inflammation.

Keywords Esophageal squamous cell carcinoma �Kazakh � PLCE1 � NF-jB

Introduction

Esophageal squamous cell carcinoma (ESCC), a serious

malignant tumor, is one of the primary causes of death

worldwide [1] and ranks as the fourth leading causes of

cancer-related deaths in China [2]. The Kazakh population

is characterized by high incidence and mortality of ESCC

[3]. Three-scale genome-wide association studies of

Xiao-bin Cui and Xue-lian Pang have contributed equally to this

work.

X. Cui � X. Pang � S. Li � J. Jin � J. Hu � L. Yang � C. Liu �W. Liang � Y. Chen (&) � F. Li (&)

Department of Pathology and Key Laboratory for Xinjiang

Endemic and Ethnic Diseases, Shihezi University School of

Medicine, North 4th Road, Shihezi 832002, Xinjiang,

People’s Republic of China

e-mail: cyz0515@sina.com

F. Li

e-mail: lifeng7855@126.com

X. Cui � Y. Chen � F. Li

Department of Oncology, Tongji Hospital, Tongji Medical

College, Huazhong University of Science and Technology,

Wuhan, People’s Republic of China

X. Cui � L. Li � Y. Chen � F. Li

Department of Pathology, The First Affiliated Hospital, Shihezi

University School of Medicine, Shihezi,

People’s Republic of China

S. J. Wen

Operating Room, The First Affiliated Hospital, Shihezi

University School of Medicine, Shihezi,

People’s Republic of China

123

Med Oncol (2014) 31:791

DOI 10.1007/s12032-013-0791-5

Chinese Han populations have recently identified a new

susceptibility locus in phospholipase C epsilon 1 (PLCE1)

related to ESCC [4–6], especially associated with ESCC in

the Kazakh population [5, 7]. Previous studies have dem-

onstrated that PLCE1 is associated with a variety of human

cancers, such as colorectal cancer [8–10], bladder cancer

[10, 11], [11, 12], head and neck cancers [13, 14], and skin

and intestine-related cancer induced by inflammation [15,

16]. It has been reported that PLCE1 promotes intestinal

tumorigenesis in Apc(Min/?) mice through augmentation of

inflammation and angiogenesis, dependent on the different

tumor stage [17]. PLCE1 also performs a significant

function in ultraviolet B-induced neutrophil-associated skin

inflammation by regulating CXCL1/KC expression [18].

Similarly, protein kinase C (PKC) and Ras GRP3, two

tetradecanoylphorbol-13-acetate (TPA) targets, participate

in TPA-induced inflammation by PLCE1 activation, lead-

ing to TPA-dependent tumor promotion [19].

Nuclear factor-kappa B (NF-jB), a key transcription

factor, is activated by phosphorylation and degradation of

IKB by the IKB kinase (IKK) complex [20, 21]. This process

leads to nuclear translocation of NF-jB and subsequent

transcription of NF-jB-dependent genes, such as TNF-a, IL-

1b, and IL-8. NF-jB performs important roles in the control

of cell growth, differentiation, apoptosis, and uncontrolled

inflammation of the malignant transformation regulation

network. NF-jB is also involved in the initiation and pro-

gression of several human inflammation-related malignan-

cies, including ESCC, by constitutive activation [22, 23].

Activation of NF-jB is stimulated by several mecha-

nisms, including signal transduction pathways involving

NF-jB inducing kinase, IKK, PI3 K, and, especially, the

PKC gene. Studies have shown that PLCE1 is an upstream

regulator of PKC [24]. PLCE1 protein can catalyze the

hydrolysis of phosphatidylinositol, 4,5-bisphosphate to

generate intracellular secondary messengers, such as ino-

sitol-1,4,5 trisphosphate (IP3) and diacylglycerol. Together

with calcium released through the activated IP3R, diacyl-

glycerol can activate conventional PKC [25, 26], which

often contributes to IKB phosphorylation and NF-jB

activation [27, 28]. PLCE1 reportedly has a crucial func-

tion in the development of bladder cancer cells by

increasing PKCa activity, thereby promoting cell prolif-

eration. However, ESCC is a phenotype resulting from

inflammation and alterations in multiple genes, with their

proteins involved in complex and interactive networks.

Studies focusing on the associations between the PLCE1

and NF-jB signaling pathway in ESCC are rarely reported.

The involvement of the co-activation of the PLCE1–NF-

jB signaling pathway in the carcinogenesis of Kazakh

ESCC remains undetermined.

To address this problem, we assessed the expression of

PLCE1- and NF-jB-related proteins (IKKa, IKBb, p50,

and p65) by immunostaining of tissue microarrays (TMAs)

in 90 Kazakh ESCC samples. In addition, we evaluated the

association of PLCE1 and NF-jB-related proteins with

various clinical characteristics, including sex, age, and

differentiation, depth of tumor invasion, lymph node

metastasis, and tumor-node-metastasis (TNM) stage. The

relationship between PLCE1 and NF-jB was also mea-

sured to determine whether the activation of the PLCE1–

NF-jB signaling pathway is involved in Kazakh ESCC

carcinogenesis.

Materials and methods

Patients and tissue specimens

A total of 90 ESCC tissue samples excised between 1984

and 2010 were selected from the First University Hospital,

Shihezi University School of Medicine, Xinjiang Yili

Prefecture Friendship Hospital, and the People’s Hospital

of Xinjiang Uyghur Autonomous Region. No patients had

received prior surgery other than diagnostic biopsies,

chemotherapy, or radiation therapy. Data on clinicopatho-

logical variables (Table 1) were collected. All cases with

pathologic diagnoses for TNM stages were evaluated in

accordance with the Cancer Staging Manual (7th Edition,

Table 1 Clinicopathological demographics for the 90 patients with

ESCC

Patient characteristic Number of patients (%)

(n = 90)

Gender

Male 59 (65.6)

Female 31 (34.4)

Age (years)

\65 58 (64.4)

C65 32 (35.6)

Median 59

Range 34–76

Differentiation

G1 26 (28.9)

G2–G3 64 (71.1)

Invasion depth

T1–T2 46 (51.1)

T3–T4 44 (48.9)

Lymphatic invasion

N0 39 (43.3)

N1–N3 51 (56.7)

TNM stage

I/II 60 (66.7)

III/IV 30 (33.3)

791 Page 2 of 9 Med Oncol (2014) 31:791

123

2009) of the American Joint Committee on Cancer. Among

the 90 ESCC tissues samples, 48 matched normal esoph-

ageal tissue and were used as controls.

Expression of PLCE1 and IKKb, IKBa, p50, and p65

proteins as detected by immunohistochemical (IHC)

staining of TMAs

IHC staining was conducted using the streptavidin–perox-

idase (S–P) and Envision methods for TMAs. Sections of

each specimen (4-lm thick) were prepared on glass slides.

Sections were deparaffinized by three consecutive treat-

ments with xylene and rehydrated by sequential immersion

in graded alcohol. Endogenous peroxidase activity was

blocked by treatment with 3 % hydrogen peroxide in

methanol for 10 min. Sections were rinsed first with tap

water and then with distilled water, before they were

submitted for antigen retrieval. Heat-induced antigen

retrieval was performed for all antibodies (Table 2). Non-

specific staining for sections treated using the S–P method

was blocked by 10 % normal goat serum (Invitrogen,

Carlsbad, CA, USA) at 37 �C for 10 min. Table 2 shows

the primary antibodies used in this study, including their

respective dilutions immunostaining methods and sources.

The primary antibody in control experiments was replaced

with PBS. Tissue sections were then incubated overnight at

4 �C, washed in PBS, and incubated with Envision or S–P

secondary antibodies for 30 min at 37 �C. Sections that had

been washed in PBS were incubated with HRP–Streptavi-

din for 30 min at 37 �C for S–P method. Subsequently, 3,3-

diamino benzidine was used to visualize the binding of

various antibodies, and the tissue sections were counter-

stained with hematoxylin.

Semi-quantitative assessment and scoring

Expression levels of PLCE1, IKKb, IKBa, p50, and p65

were scored semi-quantitatively according to the percent-

age of positive cells and the cytoplasmic staining intensity.

Scores were given as follows: 0 (0–5 % positive cells); 1

(6–25 % positive cells); 2 (26–50 % positive cells); 3

(51–75 % positive cells), or 4 ([75 % positive cells).

The IHC staining intensities of tumor cells were scored

on a scale from 0 to 3 as follows: 0 (negative); 1 (buff); 2

(yellow); and 3 (brown). Percentages of positive epithelial

cells and the staining intensities were then multiplied to

generate a immunoreactivity score (IS) for each case. For

example, if the staining intensity was brown (3) and the

percentage of positive cells was greater than 75 % (4), the

IS would be determined as 3 9 4 = 12. Five random fields

were selected for scoring from each slide, and the mean

score for each slide was used for final analysis. For

example, the IS would range between 0 and 12. Optimal

cutoff values for this scoring system were identified as

follows: high expression of PLCE1, IKKb, and IKBa was

defined as an expression index score [4; and low expres-

sion of PLCE1, IKKb, and IKBa was defined as an

expression index score \4. In addition, high expression of

p50 and p65 was assigned an expression index score of 3,

and low expression of p50 and p65 was assigned an

expression index score of B4. IHC scoring was performed

independently by two observers. The inter-observer vari-

ability was \3 %.

Statistical analysis

All statistical analyses were performed using SPSS 17.0

and GraphPad Prism 5.01. Categorical data were analyzed

using Mann–Whitney U tests. The Spearman’s rank cor-

relation test was used to determine the correlation of

PLCE1 with IKKb, IKKa, and p50. The results are

expressed as mean ± SD. P value of less than 0.05 was

considered statistically significant.

Results

Overexpression of PLCE1 and NF-jB-related proteins

in Kazakh ESCC tissue samples

A total of 90 Kazakh ESCC tissue samples and 48 NETs

were used in the study. The expression of PLCE1 and NF-

jB-related proteins (IKKb, IKBa, p50, and p65) was

detected (Fig. 1). Figures A1–A4 show diffused and strong

Table 2 Dilution, pre-treatment, immunostaining, positive controls, and source for the primary antibodies

Antibody Dilution and condition Pretreatment Immunostaining Positive control Source

PLCE1 1:50 4 �C overnight PCA-EDTA Envision Colon cancer Sigma, St. Louis, USA

IKKb 1:50 4 �C overnight PCA-CB Envision Cervical cancer Abcam, Hong Kong, China

IKBa 1:50 4 �C overnight PCA-CB Envision Prostatic cancer Abcam, Hong Kong, China

p50 1:50 4 �C overnight MWO-Tris–EDTA Envision Testicle Maixin, Fuzhou, China

p65 1:50 4 �C overnight MWO-Tris–EDTA SP Known ESCC Santa Cruze, Oregon, USA

PCA-EDTA, pressure cooker heating in ethylene diamine tetraacetic acid buffer (0.01 M, pH 8.5); PCA-CB, pressure cooker heating in citrate

buffer (0.01 M, pH 6.0); MWO-Tris–EDTA, microwave oven heating in Tris–ethylene diamine tetraacetic acid buffer (0.01 M, pH 8.5)

Med Oncol (2014) 31:791 Page 3 of 9 791

123

IS for PLCE1 (7.727 ± 3.461) in the cytoplasm of the

carcinoma cells. About 80.56 % (a) of the carcinoma cells

were highly stained for PLCE1, and 78.89 % (31 of 48) of

the NETs exhibited no or low PLCE1 expression, with an

average IS of 4.739 ± 3.289. Similarly, diffuse and strong

IS for IKKb (3.983 ± 1.403), IKBa (3.817 ± 1.403), p50

Table 3 The correlations between PLCE1, IKKa, IKBb, p50, p65 expression of squamous cell carcinoma and clinicopathological factors

Parameters PLCE1 expression IKKa expression IKBb expression

Low (%) High (%) P value Low (%) High (%) P value Low (%) High (%) P value

Age (years)a

\65 10 (52.6) 48 (67.6) 0.226 26 (61.9) 32 (66.7) 0.638 33 (61.1) 25 (69.4) 0.418

C65 9 (47.4) 23 (32.4) 16 (38.1) 16 (33.3) 21 (38.9) 11 (30.6)

Sex

Male 11 (57.9) 48 (67.6) 0.429 31 (73.8) 28 (58.3) 0.123 35 (64.8) 24 (66.7) 0.856

Female 8 (42.1) 23 (32.4) 11 (26.2) 20 (41.7) 19 (35.2) 12 (33.3)

Differentiationb

G1 7 (36.8) 19 (26.8) 0.389 16 (38.1) 10 (20.8) 0.071 14 (25.9) 12 (33.3) 0.448

G2–G3 12 (63.2) 52 (73.2) 26 (61.9) 38 (79.2) 40 (74.1) 24 (66.7)

Invasion depth (T)

T1–T2 9 (47.4) 37 (52.1) 0.713 25 (59.5) 21 (43.8) 0.181 31 (57.4) 15 (41.7) 0.143

T3–T4 10 (52.6) 34 (47.9) 17 (40.5) 27 (56.3) 23 (42.6) 21 (58.3)

Lymphatic invasion (N)

N0 14 (73.7) 25 (35.2) 0.003 22 (52.4) 17 (35.4) 0.105 22 (40.7) 17 (47.2) 0.543

N1–N3 5 (26.3) 46 (64.8) 20 (47.6) 31 (64.6) 32 (59.3) 19 (52.8)

UICC staging (TNM)c

I/II 17 (89.5) 43 (60.6) 0.018 20 (47.6) 40 (83.3) 3.36 9 10-4 41 (76.0) 19 (52.8) 0.022

III/IV 2 (10.5) 28 (39.4) 22 (52.4) 8 (16.7) 13 (24.0) 17 (47.2)

Parameters p50 expression p65 expression

Low (%) High (%) P value Low (%) High (%) P value

Age (years)a

\65 11 (64.7) 47 (64.4) 0.980 28 (71.79) 30 (58.8) 0.203

C65 6 (35.29) 26 (35.6) 11 (28.21) 21 (41.2)

Sex

Male 9 (53.0) 50 (68.5) 0.224 27 (69.23) 32 (62.8) 0.153

Female 8 (47.0) 23 (31.5) 12 (30.77) 19 (37.3)

Differentiationb

G1 6 (35.29) 20 (27.4) 0.518 16 (41.03) 10 (19.6) 0.026

G2-G3 11 (64.71) 53 (72.6) 23 (58.97) 41 (80.4)

Invasion depth (T)

T1–T2 10 (58.82 36 (49.3) 0.480 26 (66.67) 20 (39.2) 0.010

T3–T4 7 (41.18) 37 (50.7) 13 (33.33) 31 (60.8)

Lymphatic invasion (N)

N0 11 (64.71) 28 (38.4) 0.048 19 (48.72) 20 (39.2) 0.367

N1–N3 6 (35.29) 45 (61.6) 20 (51.28) 31 (60.8)

UICC staging (TNM)c

I/II 12 (70.59) 48 (65.8) 0.703 28 (71.79) 32 (62.8) 0.340

III/IV 5 (29.41) 25 (34.2) 11 (28.21) 19 (37.3)

Bold values indicate significant correlation between protein expression and clinicopathological featurea Mean ageb Histologic grade was with reference to WHO classification published in 2009c TNM stage was based on the UICC criteria published in 2009

791 Page 4 of 9 Med Oncol (2014) 31:791

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(5.652 ± 2.840), and p65 (3.906 ± 2.383) was observed in

carcinoma cells. About 53.33 % (48 of 90), 60.00 % (54 of

90), 81.11 % (73 of 90), and 57.78 % (52 of 90) of these

carcinoma cells were highly stained for IKKb, IKBa, p50,

and p65, respectively. However, 81.25 % (39 of 48),

83.3 % (40 of 48), 52.08 % (25 of 48), and 62.50 % (30 of

Fig. 1 Representative photographs of the immunohistochemical

features of proteins in ESCC and adjacent normal tissue. ESCC

tissues show strong positive staining. A1/2 PLCE1, C1/2 IKBa, and

E1/2 p65 demonstrating diffuse cytoplasmic staining of tumor cells,

and B1/2 IKKb and D1/2 p50 demonstrating diffuse cytoplasmic and

nuclear staining of tumor cells. A3, B3, C3, D3, and E3) Negative

expression of PLCE1, IKKb, IKBa, p50, and p65 in normal epithelial

cells, respectively (940 magnification). A4, B4, C4, D4, E4 Boxplot

showing significantly higher expression of PLCE1, IKKb, IKBa, p50,

and p65 in ESCC tissue than that in normal esophageal tissue

Med Oncol (2014) 31:791 Page 5 of 9 791

123

48) of the NETs showed no or low expression, having low

average IS for IKKb (2.445 ± 1.332), IKBa (2.018 ±

1.506), p50 (4.621 ± 1.883), and p65 (2.826 ± 2.070),

respectively. IKBa and p65 were observed in the cyto-

plasm, whereas IKKb and p50 were observed in the

cytoplasm and/or the nucleus of the carcinoma cell

(Fig. B1–E4). Comparison of PLCE1, IKKb, IKBa, p50,

and p65 protein expression between ESCC and NETs

samples indicated that these proteins exhibited higher IS in

tumors group than the control group (P = 9.48 9 10-7,

1.24 9 10-5, 0.004, 0.003, and 2.83 9 10-5, respectively).

Correlation between PLCE1 and NF-jB-related protein

expression with clinicopathological characteristics

We further analyzed the expression of PLCE1, IKKb, IKBa,

and p50 in 90 ESCC tissue samples against their clinical

characteristics. Overexpression of PLCE1 was significantly

correlated with lymph node metastasis (P = 0.003) and

advanced TNM stage (P = 0.018) (Table 3). Expression of

both IKKb and IKBa proteins was correlated with the TNM

stage (P = 3.36 9 10-4, 0.022, respectively). Overexpres-

sion of p50 was correlated with lymph node metastasis

(P = 0.048). Overexpression of p65 was associated with

moderate/poor-differentiation and depth of tumor invasion

(P = 0.026, 0.010, respectively) (Table 3).

Association of PLCE1 expression with NF-jB

expression in ESCC

We further determined whether a significant relationship

existed between protein expression of PLCE1 and the NF-

jB signaling pathway. Our data suggested a significant

correlation between PLCE1 and IKKb expression (r =

0.246, P = 0.025) (Table 4; Fig. 2a). The histoscores of

PLCE1 expression were higher in ESCC tissues with high

levels of IKKb expression than in those ESCC tissue

samples with low levels of IKKb expression (8.489 ±

2.999 vs 6.758 ± 3.579, P \ 0.05) (Fig. 2c). The expres-

sion of PLCE1 in ESCC samples was also associated with

p50 (r = 0.244, P = 0.024) (Table 4; Fig. 2b). Similarly,

the PLCE1 histoscore was higher in the ESCC tissue

samples with high levels of p50 expression

(8.188 ± 3.196) than in those ESCC tissue samples with

low levels of p50 expression (5.706 ± 3.584; P \ 0.05)

(Fig. 2c).

Discussion

ESCC is a malignant tumor with a variety of abnormal

gene expression characteristics and signal pathway inter-

actions, such as PLCE1, NF-jB, and p53. The precise

mechanism(s) underlying the development of ESCC are

largely undetermined, although recent epidemiologic and

etiologic studies have revealed that the carcinogenesis of

ESCC involves multiple factors, stages, and alterations in

gene expression [29, 30]. In the present study, we observed

the overexpression of PLCE1, IKKb, IKBa, p50, and p65

proteins in Kazakh ESCC. In addition, PLCE1 was posi-

tively associated with the NF-jB signaling pathway in

Kazakh patients with ESCC.

Our findings indicated that PLCE1 protein expression

increased in Kazakh patients with ESCC, supporting our

previous studies that suggest upregulated expression of the

PLCE1 gene in ESCC cells lines, such as TE1, EC109,

EC9706, and KYSE105 [31]. Our observations are con-

sistent with a study by Wang et al. [5] that focused on

Chinese Han patients with ESCC, but contradict a study by

Hu et al. Hu et al. [32] found no difference in PLCE1

expression between ESCC tissue and the matching adjacent

NETs. The distinct differences among those studies could

be attributed to the diversity of tumor types, the geo-

graphical area under study, and population heterogeneity.

Variations need to be clarified in future studies using uni-

form ethnic groups with a larger sample size.

We also observed that high PLCE1 expression in Ka-

zakh patients with ESCC was associated with lymph node

metastasis and the TNM stage, indicating that PLCE1

affects the metastasis and the aggressiveness of Kazakh

ESCC. Studies have reported that silencing PLCE1 in

bladder cancer decreases the invasive power of bladder

cancer T24 cells by downregulating the level of MMP2,

MMP9, and Bcl-2 expression, thereby inhibiting tumor

development [12]. However, the precise mechanism by

which the PLCE1 gene influences the invasion of ESCC

remains unknown. Aberrant activation of PLCE1 induces

the PLCE1–Ca2? signaling pathway, causing the upregu-

lation of Ca2?/calmodulin-dependent kinase II and the

subsequent phosphorylation of the cytoskeletal protein fil-

amin. This process promotes tumor cell migration and

progression by binding Ras family small GTPases [13].

Similarly, PLCE1 has a crucial function in bladder cancer

cells by increasing PKCa activity, which can regulate the

expression of cell cycle regulatory protein and promote cell

Table 4 Spearman’s correlations between PLCE1 and NF-jB-rela-

ted proteins

PLCE1 IKKb IKBa p50 p65

PLCE1 1

IKKb 0.246* 1

IKBa 0.180 0.409*** 1

p50 0.244* 0.206 0.088 1

p65 0.161 0.223* 0.151 0.406*** 1

* P \ 0.05, *** P \ 0.0001

791 Page 6 of 9 Med Oncol (2014) 31:791

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proliferation [11]. On the basis of these findings, we infer

that PLCE1 overexpression can alter the motility of ESCC

cells through the same signaling pathway. This goes some

way to our findings that overexpressed PLCE1 may be

involved in metastasis and aggressiveness. Detailed in vitro

studies should be performed to confirm this hypothesis.

The NF-jB-related proteins—IKKa, IKBb, p50, and

p65—are overexpressed in Kazakh ESCC and are mainly

located in the cytoplasm. This observation agrees with a

study by Su that showed high expression of NF-jB in

ESCC tumors [33]. In addition, overexpression of p50, p65,

IKBa, and IKKb, which are mainly located in the cyto-

plasm, was confirmed in two human ESCC cell lines,

Eca109, and EC9706, and in ESCC tissues [23, 34]. Our

observations of NF-jB-related proteins showed that IKBaand p65 increased in the cytoplasm, and IKKb and p65

increased in the cytoplasm and/or the nucleus in Kazakh

ESCC tissue samples, as was observed in other studies.

Notably, p50 expression was correlated with lymph node

metastasis of ESCC, with high expression in ESCC with

lymph node metastasis but low expression in ESCC with-

out lymph node metastasis.

P65 expression was correlated with the differentiation

and depth of tumor invasion of ESCC and high expression

in poor/moderate differentiated groups of ESCC. Indirect

evidence for this is provided by a report that suggests that

downregulation of p65 and p-IKBa proteins and elevated

IKBa protein expression of the NF-jB pathway, accom-

panied with decreases in Bcl-2 and MMP-9 protein

expressions and increases in Bax protein levels and cas-

pase-3 activities, result in the inhibition of cell proliferation

and invasion and induce apoptosis of ESCC [35]. Previous

studies have reported that p65 was overexpressed in human

ESCC tissues, especially in ESCC tissues with deep inva-

sion and lymph node metastasis, whereas suppression of

p65 led a lower cell viability and higher apoptosis of ESCC

cell lines with the downregulation of MMP-9 and E-Cad-

herin, a hallmark of invasive carcinomas that have acquired

epithelial-mesenchymal transition phenotypes [36, 37].

Specifically, HtrA1 overexpression inhibited the phos-

phorylation of IKBa and p65 subunit of the NF-jB sig-

naling pathway. In contrast, Htr A1 overexpression

increased total IKBa expression, coupled with decrease in

Ki-67, Bcl-2, Bcl-xL, cyclinD1, and MMP-9 proteins and

increase in caspase-3 activity. Such overexpression reduces

the invasion in ESCC [38] (Fig. 3). An increasing number

of studies have also suggested that overexpression of NF-

jB and the activation of the NF-jB signaling pathway in

Kazakh ESCC tumors can influence metastasis and

aggressiveness.

Inflammation is another vital constituent of the local

environment that sustains tumor development [39].

Esophageal cancer generally develops first by inflamma-

tion, followed by inflammatory hyperplasia, and atypical

hyperplasia, and finally evolving into a tumor [40–42]. In

the present study, we found that PLCE1 expression posi-

tively correlated with NF-jB-related proteins, such as p50

and p65. NF-jB is identified as a key orchestrator of innate

immunity/inflammation, and aberrant NF-jB regulation

has been observed in many cancers [43]. It has been

reported that NF-jB enhanced the expression of several T

cell chemokines, including Ccl2, Ccl5, and COX-2, which

are important inflammatory chemokines, in a lung cancer

model [44, 45]. Cancer-related inflammation is an essential

process in malignant disease. Until recently, the field has

been driven by the hypothesis that extrinsic inflammatory

pathways promoters, in some cases, initiate cancer—i.e.,

that inflammation causes or promotes cancer [39, 46].

PLCE1, through the inflammatory signaling pathways

and angiogenesis, promotes intestinal cancer formation,

Fig. 2 Correlation of IKKb and p50 expression with PLCE1 in

ESCC. a Protein expression of IKKa in ESCC tissue is positively

correlated with PLCE1 expression in tumors. b Expression of p50 in

ESCC tissue is positively associated with PLCE1 expression in

tumors. c Mean histoscore of IKKa expression is higher in ESCC

tissue with high PLCE1 expression (8.489 ± 2.999) than in tissue

with low PLCE1 expression (6.758 ± 3.597) (P \ 0.05). Similarly,

mean histoscore of p50 expression is higher in ESCC tissue with high

levels of PLCE1 expression (8.188 ± 3.196) than in tissue with low

PLCE1 expression (5.706 ± 3.584) (P \ 0.05). Dotted line denotes

mean histoscore of proteins

Med Oncol (2014) 31:791 Page 7 of 9 791

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with increased expression of Cxcl-1, Cxcl-2, and COX-2

[17]. PLCE1 exhibits oncogenic function in the carcino-

genesis of UBV neutrophil-associated skin inflammation

by upregulating the expression CLCL1/KC [18]. Our

findings identify NF-jB as the major effector molecule in

PLCE1 activation, which is also linked to inflammation.

Recent reports have suggested the significance of PLCE1–

NF-jB cascade. PLCE1 reportedly cooperates with the NF-

jB pathway to increase the expression of the tumor

necrosis factor (TNF) a-stimulated chemokine (C–C motif)

ligand 2/Monocyte chemoattractant protein-1 (CCL2/

MCP1) expression. PLCE1 also upregulates the proin-

flammatory cytokine production in human keratinocyte

cells [47]. As an upstream regulator of PKCe in the heart

[24], PLCE1 indirectly activates the conventional PKC

isoforms (a, bI, bII, c) [25, 26]. Subsequently, PKC con-

tributes to IKB phosphorylation and then to the NF-jB

activation [27, 28]. On the basis of these observations,

activation of the PLCE1–NF-jB cascade may occur during

the development of Kazakh ESCC. This requires validation

with future studies.

In conclusion, this study is the first to show that

PLCE1 and NF-jB proteins are overexpressed in Kazakh

patients with ESCC and that such overexpression is cor-

related with the depth of invasion or lymph node metas-

tasis of ESCC. Our findings demonstrate that high

expression of PLCE1 correlated with upregulated

expression of NF-jB-related proteins in Kazakh ESCC

patients. These findings indicate a positive relationship

between PLCE1 and NF-jB. Therefore, PLCE1 and NF-

jB signaling pathways exhibit co-activation roles in

cancer-related inflammation and cancer-induced metasta-

sis and aggressiveness.

Acknowledgments This study was supported by National Natural

Science Foundation of China (81160301, 81360358, 81260301),

Ministry of Science and Technology of China (Grant No.

2010DFB34100 and 2012AA02A503) and Maxin Pathology Fund

of China (m1108).

Conflict of interest The authors declare that they have no conflict

of interest.

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