J Cancer Res Clin Oncol (2010) 136:587–594 DOI 10.1007/s00432-009-0693-2
ORIGINAL PAPER
Expression of Nucleostemin, epidermal growth factor and epidermal growth factor receptor in human esophageal squamous cell carcinoma tissues Gongyuan Zhang Æ Qiao Zhang Æ Qinxian Zhang Æ Lei Yin Æ Shenglei Li Æ Kuisheng Cheng Æ Yunhan Zhang Æ Honghui Xu Æ Weidong Wu
Received: 20 May 2008 / Accepted: 21 September 2009 / Published online: 13 October 2009 Ó Springer-Verlag 2009
Abstract Objective To determine the expression of nucleostemin (NS), epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) mRNA in human esophageal squamous cell carcinoma (ESCC) tissues and their association in a human ESCC cell line. Methods The expression of NS, EGF and EGFR mRNA was determined in paired normal esophageal and ESCC tissues of 62 patients using in situ hybridization. The association between NS and EGF or EGFR was examined using immunoblotting and real time polymerase chain reaction in a human ESCC cell line transfected with NS siRNA or treated with a selective EGFR inhibitor. Results In normal esophageal and ESCC tissues, the positive detection rates were 21.0% (13/62) and 69.4% (43/62) for NS mRNA staining, 40.3% (25/62) and 77.4% (48/62) for EGF mRNA staining, and 30.6% (19/62) and 75.8% (41/62) for EGFR mRNA staining, respectively. These results indicated that NS, EGF and EGFR mRNA expression was upregulated mostly in ESCC tissues.
G. Zhang and Q. Zhang contributed equally to this work. G. Zhang Q. Zhang (&) L. Yin Department of Histology and Embryology, College of Basic Medical Sciences, Zhengzhou University, 100 Kexue Boulevard, 450001 Zhengzhou, People’s Republic of China e-mail:
[email protected] Q. Zhang H. Xu W. Wu College of Public Health, Zhengzhou University, 450001 Zhengzhou, People’s Republic of China S. Li K. Cheng Y. Zhang Department of Pathology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, People’s Republic of China
Moreover, the expression of NS, EGF and EGFR mRNA was positively correlated with tumor grade, invasion and lymphatic metastasis of ESCC cells. NS mRNA was co-expressed with EGF and EGFR mRNA in ESCC tissues. The in vitro studies using a human ESCC cell line showed that knockdown of NS with NS siRNA significantly reduced EGF and EGFR expression. However, inhibition of the EGFR kinase activity with a specific EGFR kinase inhibitor had minimal effect on NS expression. Conclusion The upregulation of NS, EGF and EGFR mRNA frequently occurs in ESCC tissues and is associated with malignancy of human esophageal squamous tumors. NS is required for EGF and EGFR expression. Keywords Esophageal squamous cell carcinoma Epidermal growth factor Epidermal growth factor receptor Nucleostemin
Introduction Nucleostemin (NS) is a nucleolar protein located in the nucleoli of embryonic stem cells, adult central nervous system stem cells, primitive cells in the bone marrow and cancer cells including prostatic carcinoma cells, breast carcinoma cells, renal carcinoma and lung cancer cells, but not in the differentiated cells of most adult tissues (Tsai and Mckay 2002; Liu et al. 2004a, b). NS contains a basic domain, which is essential for its nucleolar localization and also for its interaction with tumor suppressor p53 under physiological conditions. Also, it contains a coiled-coil domain, G1 and G4 GTP-binding domains, a carboxy-terminal acidic domain, and nuclear localization signals (Tsai and McKay 2005). Deletion of the basic domain results in an increase in number of cells entering the cell cycle and fewer
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cells undergoing apoptosis, whereas deletion of G1 or G4 GTP-binding domain causes cell cycle arrest and apoptosis. Deletion of both G1 and basic domain rescues some cells from cell death via apoptosis (Tsai and Mckay 2002; Dai et al. 2008). Thus, NS plays an important role in controlling the cell-cycle progression in stem cells and cancer cells. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases in mammals is composed of four members: EGFR (ErbB1), ErbB2, ErbB3, and ErbB4 (Salomon et al. 1995). Of these, EGFR, an 1,186-amino acid residue transmembrane glycoprotein, is the prototypal member of this superfamily and is expressed in many cell types. Ten different ligands including EGF and transforming growth factor a, and among others, can selectively bind to each receptor. After a ligand binds to a single-chain EGFR, the receptor forms a dimer that signals within the cell by inducing receptor autophosphorylation through tyrosine kinase activity. Autophosphorylation triggers a series of intracellular pathways that may result in cancercell proliferation, apoptosis arrest, active invasion and metastasis, and stimulation of tumor-induced neovascularization (Edwin et al. 2006). Human esophageal carcinomas express multi-autocrine growth factors and hormones including EGF, TGFa and b, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) and sex hormones (Tahara 1990). Overexpression of EGF, TGFa and EGFR by tumor cells is closely correlated with the tumor invasion and patient prognosis (Yoshida et al. 1993). As depicted previously, abnormal expression of NS, EGF, and EGFR has been linked to carcinogenesis of a variety of cancers. However, the expression of NS in human esophageal squamous cell carcinoma (ESCC) tissues has not been examined. Thus, the purpose of this study was to determine the expression of NS mRNA in paired normal esophageal and ESCC tissues of 62 patients. Moreover, this study also investigated the co-expression of EGF and EGFR with NS in these tissues. In addition, the association between NS and EGF or EGFR expression was explored using a human ESCC cell line.
Materials and methods Reagents Fetal bovine serum was purchased from Hyclone Company (USA). Heochst 33342 and PI dyes, and trypsin–EDTA were obtained from Sigma Company (USA). RPMI1640 was purchased from Gibco Company (USA). Anti-human nucleostemin antibody was purchased from R & D Systems (Minneapolis, MN, USA). Trizol reagent and LipofectamineTM 2000 transfection kit were obtained from Invitrogen Company (USA). pGEM-T Easy vector, Taq
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DNA polymerase, T4 DNA polymerase, and avian myeloblastosis virus (AMV) reverse transcriptase were purchased from Promega Company (USA). Tissue samples Tissue samples were obtained from 62 patients undergoing esophagectomy at the Municipal Cancer Hospital of Anyang, Henan Province from 26 February to 16 March 2006. Among them, 33 patients were older than 60 years and 36 were male. The patients with chemotherapy, radiotherapy or immunotherapy were excluded from this study. The ESCC tissue specimen without necrosis and the normal esophageal tissue specimen were collected from the same patient within 30 min after esophagectomy and then fixed in 40 g/l paraformaldehyde solution, followed by dehydration and paraffin embedment prior to in situ hybridization. The normal and ESCC tissues (total pairs 62) were classified by two authoritative pathologists according to American Joint Committee on Cancer (AJCC) classification. In detail, 7 cases (11.3%) in stage I, 19 cases (30.7%) in stage IIA, 9 cases (14.5%) in stage IIB, 26 cases (41.9%) in stage III, and 1 case (1.6%) in stage IVA. Of these 62 ESCC patients, 20 cases were with lymphatic metastasis. Based on the depth of cancer-cell invasion, 7 cases were categorized as the superficial muscularis invasion group, and 55 cases as the deep muscularis or fibrous membrane invasion group. In situ hybridization Paraffin-embedded sections were dewaxed and immersed in 0.5% hydrogen peroxide for 30 min. After digestion in pepsin solution at 37°C for 10 min, the sections were rinsed 4 times in deionized water. The slides were dried in air for 15 min. Then one drop (approximately 20 lL) of prehybridization solution was applied to the sections and incubated at 42°C for 4 h. Slides were transferred to a prewarmed humid chamber containing hybridization solution with probes at 42°C for 12–16 h. Following hybridization, the slides were washed by 0.19 SCC for 30 min and further incubated with SA-Bio-AP at 37°C for 10 min. BCIP/ NBT substrate solution was applied to cover each tissue section. The slides were further incubated at room temperature for 0.5–2 h. The hybridization solution without probes was used as a negative control. Biotinylated NS, EGF, EGFR mRNA probes were purchased from AOKE Biotechonology Company. The probe sequences used in this study were as follows: NS, 50 -CAGCAAAGCCAAGAAGCACCTGGAG-30 ; EGF, 50 -GAGGTCTTGCCCACACACAGTGCTGC-30 ; EGFR, 50 -CGATCCCCAGGGCCACCACCAGCAGC-30 .
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589
Histological and histochemical scoring
95°C incubation for 10 s, followed by 40 cycles of 60°C for 60 s. Quantitative PCR was performed using an ABI Prism 5700 sequence detector (Applied Biosystems, Foster City, CA). NS mRNA levels were normalized using b-actin mRNA levels. Relative amounts of NS and b-actin mRNA were based on standard curves prepared by five serial dilutions of cDNA from pGEM-T-NS and pGEM-Tb-actin. The following oligonucleotide primers and probes were employed:
The stained NS, EGF and EGFR mRNA was in blue and located in the cytoplasm and nucleus of tumor cells. Under high-power magnification, five fields of vision (FOVs) were randomly selected (for each FOV, there were more than 200 cells). The staining was scored based on the percentage of positively stained cells and density of cell staining. The total score was calculated as a product of A and B, wherein A was the density of cell staining: 0, no cell staining; 1, light blue; 2, purple blue; 3, purple brown. B was the percentage of positively stained cells in the same view field: 1, \30%; 2, 30–70%; 3, [70%. The total score between 0 and 1 was designated as the negative score (-), and C2 as the positive score (?).
NS: 50 -AAAGCCATTCGGGTTGGAGT-30 (sense), 50 -ACCACAGCAGTTTGGCAGCAC-30 (antisense), 50 -CAGCAAAGCCAAGAAGCACCTGGAG-30 (probe), b-actin: 50 -GATCATTGCTCCTCCTGAGC-30 (sense), 50 -ACTCCTGCTTGCTGATCCAC-30 (antisense).
Cell culture Immunoblotting The ESCC cell line EC9706 was cultured in RPMI 1640 (pH 7.0–7.2) supplemented with 10% fetal bovine serum (FBS), 100 lg/ml streptomycin and 100 U/ml penicillin (Gibco, USA) at 37°C with 5% CO2 and saturated moisture. Transfection of EC9706 cells with NS siRNA NS siRNA has been constructed previously in our laboratory and recombinated into pRNAT-U6.1 plasmids (pRNAT-U6.1-siNS) (Yue et al. 2007). EC9706 cells grown to 60–70% confluence were transfected with pRNAT-U6.1-siNS or non-specific siRNA (pRNATU6.1-siC) with the transfection reagent for 6 h, respectively. mRNA and proteins were extracted from untransfected and transfected EC9706 cells using RIPA buffer (19 PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors: 20 g/ml leupeptin, 20 g/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 200 lM sodium orthovanadate, and 20 mM sodium fluoride) or TRIZOL reagent (Invitrogen Corporation, Carlsbad, CA). mRNA was reversely transcribed into cDNA. Real time PCR and immunoblotting were used to examine the expression levels of NS mRNA and protein in EC9706 cells, respectively. Real-time polymerase chain reaction (RT-PCR)
EC9706 cells treated with DMSO or the EGFR kinase inhibitor PD153035 (1 lM) for different times, respectively. Cells were washed twice with PBS, and then lysed in RIPA buffer. Cell lysates were subjected to SDS-PAGE. Proteins were transferred onto nitrocellulose membrane. Membrane was blocked with 5% BSA, washed briefly, incubated with primary antibody at 4°C overnight, followed by incubation with corresponding HRP-conjugated secondary antibody for 1 h at room temperature. Immunoblotting images were detected using chemiluminescence’s reagents and the Gene Gynome Imaging System (Syngene, Frederick, MD, USA). Statistical analysis SPSS 10.0 was used for data analysis. Two-related samples rank sum test was used to compare the positive detection rate of NS mRNA staining in normal and ESCC tissues (Table 1). v2 or Fisher’s exact probability was used to analyze the association of NS, EGF, and EGFR mRNA expression with pathological features of ESCC (Table 2).
Table 1 The detection rates of positive NS mRNA staining in normal and ESCC tissues ESCC tissues
The real time PCR kit was purchased from Takara Company. RT-PCR was conducted according to the manufacture’s instructions. The reaction mixture (50 ll) included: 25 ll 29 SYBR premix Ex TaqTM, 1 ll NS forward primer, 1 ll NS reverse primer, 1 ll 509 ROX reference dye, 4 ll cDNA, 18 ll dH2O. The PCR scheme consists of
Normal tissues
Total
?
-
?
13
30
-
0
19
19
13
49
62
Total
43
Two-related samples rank sum test: v2 = 28.033, P \ 0.001
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Table 2 Correlation of NS, EGF, and EGFR mRNA expression with pathological features of ESCC Pathological parameters
n
Positive NS mRNA staining (%)
v2
P
0.006
0.937
Positive EGF mRNA staining (%)
v2
P
27 (81.8)
0.781
0.377
Positive EGFR mRNA staining (%)
v2
P
25 (75.8)
\0.001
0.992
0.601
0.438
6.867
0.032
12.722
0.001
9.422
0.001
Age (years) \60
33
23 (69.7)
C60 Gender
29
19 (65.5)
Male
36
23 (63.9)
Female
26
19 (73.1)
I
15
8 (53.3)
II
25
15 (60.0)
20 (80.0)
III
22
19 (86.4)
21 (95.5)
21 (72.4) 0.239
0.625
28 (77.8)
22 (75.9) 0.006
0.937
20 (76.9)
26 (72.2) 21 (80.8)
Histological grade 5.602
0.061
7 (46.7)
8 (53.3) 12.303
0.002
19 (76.0) 20 (90.9)
Invasion depth Superficial layer
7
1 (14.3)
55
41 (74.5)
No
42
25 (59.5)
Yes
20
17 (85.0)
Deep layer
7.745
0.005
3 (42.9)
3.394
0.005
45 (81.8)
1 (14.3) 46 (83.6)
Lymphatic metastasis 4.024
0.045
29 (69.0) 19 (95.0)
3.841
0.05
27 (64.3) 20 (100.0)
Pearson contingency coefficients (rP) were calculated to reveal the association strength of NS mRNA, EGF mRNA and EGFR mRNA (Tables 3, 4). The data in Tables 5 and 6 was presented as X S, and least significant difference
was used to compare the difference between any two groups. All the significant level was 0.05 (two-tail). The data presented in Fig. 4 was analyzed using two-tailed Student’s t test.
Table 3 Correlation of NS mRNA expression with EGF and EGFR mRNA expression in ESCC tissues
Results
NS mRNA
EGF mRNA ?
EGFR mRNA
-
rP
P
?
0.394
\0.05
40
3
7
12
?
38
5
-
10
9
Table 4 Correlation of EGF and EGFR expression in ESCC tissues
EGF mRNA
rP
P
0.604
\0.05
EGFR mRNA
Total
?
-
?
42
6
48
-
5
9
14
47
15
62
Total
rP = 0.506, P \ 0.05
-
Table 5 Effect of NS siRNA on EGFR mRNA expression X S in EC9706 cells Treatment
Vectors
EGFR mRNA
NS siRNA
pRNAT-U6.1-siNS2
0.07 ± 0.02#
Non-specific siRNA
pRNAT-U6.1-siC
0.09 ± 0.03
Control
No
0.10 ± 0.03
#
Compared to control siRNA, P \ 0.05
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Expression of NS, EGF, and EGFR mRNA in Human Esophageal Tissues As shown in Fig. 1, stained NS mRNA was mainly located in the cytoplasm and nucleus of tumor cells in purple blue or purple brown. As shown in Table 1, the detection rate of positively stained NS mRNA in ESCC tissues is 69.4% (43/62), which is significantly higher than in normal esophageal tissues [21% (13/62), P \ 0.05]. As shown in Fig. 2, EGF mRNA staining was mainly observed in the cytoplasm of ESCC cells in purple blue or purple brown. The positive detection rate of EGF mRNA in ESCC tissues is 77.4% (48/62), which is significantly higher than in normal esophageal tissues [40.3% (25/62), P \ 0.05]. EGFR mRNA positive staining was mainly observed in the cytoplasm of ESCC cells and also in purple blue or purple brown, as shown in Fig. 3. The positive detection rate of EGFR mRNA in ESCC tissues is 75.8% (47/62), which is significantly higher than in normal esophageal tissues [30.6% (19/62), P \ 0.05]. In summary, these data indicated that expression of NS, EGF, and EGFR mRNA occurs frequently in ESCC tissues.
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591
Table 6 Effect of NS siRNA on EGF mRNA expression X Sin EC9706 cells Treatment
Vectors
NS siRNA
pRNAT-U6.1-siNS2
EGFR mRNA 46.80 ± 20.11#
Non-specific siRNA
pRNAT-U6.1-siC
114.60 ± 18.60
Control
No
114.00 ± 20.40
#
Co-expression of NS mRNA with EGF and EGFR mRNA in ESCC tissues
Compared to control siRNA, P \ 0.05
The correlation of NS mRNA expression with EGF or EGFR mRNA expression was evaluated. As shown in Table 3, NS mRNA expression in ESCC tissues was positively correlated with EGF or EGFR mRNA expression. In addition, the expression of EGF mRNA was also correlated with EGFR mRNA expression in ESCC tissues (Table 4). These data indicated that NS, EGF, and EGFR mRNA were frequently co-expressed in ESCC tissues. NS was required for EGF and EGFR mRNA expression in ESCC cell line
Fig. 1 Staining of NS mRNA in normal (left) and ESCC tissue (right)
Fig. 2 Staining of EGF mRNA in normal (left) and ESCC tissue (right)
As demonstrated previously, NS, EGF, and EGFR mRNA were frequently co-expressed in ESCC tissues. To further characterize the intrinsic association between NS and EGF or EGFR mRNA expression, specific NS siRNA constructed in our laboratory was transfected into EC9706 cells. RNA was extracted from cells and RT-PCR used to examine the levels of EGF and EGFR mRNA. As expected, expression of NS siRNA abrogated NS mRNA in EC9706 cells (data not shown). Interestingly, expression of NS siRNA significantly suppressed EGF as well as EGFR mRNA expression in EC9706 cells (Tables 5, 6), implying that NS was required for both EGF and EGFR mRNA expression. EGFR kinase activity was not required for NS expression in EC9706 cells
Fig. 3 Staining of EGFR mRNA in normal (left) and ESCC tissue (right)
EC9706 cells were treated with the selective EGFR kinase inhibitor (1 lM), PD153035, for 4, 8 and 24 h. Cell lysates were subjected to immunoblotting using an anti-NS antibody. mRNA was extracted and reverse transcribed into cDNA for RT-PCR to determine NS mRNA expression. As shown in Fig. 4a, b, PD153035 had minimal effect on both NS protein and mRNA expression, implying that the EGFR kinase activity was dispensable for the regulation of NS expression.
Correlation of NS, EGF and EGFR mRNA expression with pathological features of ESCC tissues
Discussion
As shown in Table 2, there was no significant difference in the positive detection rate of NS, EGF, or EGFR mRNA in ESCC tissues between different age groups (P [ 0.05), and between male and female patients (P [ 0.05). However, the positive staining rates of NS, EGF, and EGFR mRNA in ESCC tissues were positively correlated with the malignancy of ESCC including histological cancer grade, invasion depth, and lymph node metastasis of cancer cells.
Human esophageal carcinoma, especially ESCC, is one of the most common causes of cancer death worldwide. It occurs at a very high frequency in China with a generally poor prognosis (Parkin et al. 2005; Enzinger and Mayer 2003). Among the newly diagnosed esophageal carcinoma patients worldwide, more than 1/2 cases were reported in China. Esophageal carcinoma is a disease involving multi-factors and multi-stages including pure hyperplasia,
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J Cancer Res Clin Oncol (2010) 136:587–594 1.00
A
DMSO
0.75
density
NS mRNA band
PD153035
0.50
0.25
0.00 4
8
24
time (h) 0.75
B
DMSO
0.50
density
NS protein band
PD153035
0.25
0.00 4
8
24
time (h) Fig. 4 Effect of the EGFR kinase inhibitor, PD153035, on expression of NS mRNA (a) and proteins (b) in EC9706 Cells. Data shown are representative of three separate experiments
atypical hyperplasia, carcinoma in situ and infiltrating carcinoma. The mechanisms for the pathogenesis of esophageal carcinoma are still unclear. Molecular studies of human esophageal tumors have revealed frequent genetic abnormalities. Regardless of patient origin and suspected etiological factors, genetic changes that are consistently observed in ESCC are: (1) alterations in tumor suppressor genes, specifically p53, leading to altered DNA replication and repair, cell proliferation and apoptosis; (2) disruption of the G1/S cell cycle checkpoint and loss of cell cycle control; (3) alterations in oncogene function leading to deregulation of cell signaling cascades (Lam 2000; Mandard et al. 2000). In this present study, we demonstrated that NS, a p53binding protein, was highly expressed in ESCC tissues. In addition, NC expression was positively correlated with cancer grade, cancer-cell invasion and lymphatic metastasis of ESCC, implying that NS expression was closely associated with ESCC pathogenesis. How NS regulates carcinogenesis of NSCC remains elusive. Previous studies have demonstrated that NS might play an important role in the proliferative regulation of cancer cells (Liu et al. 2004a, b). NS accumulates predominantly in the nucleolus and localizes into nucleoplasm after binding with GTP
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(Bernardi and Pandolfi 2003). In the nucleoplasm, NS and p53 exist in a protein complex, and subsequently the growth-suppressive function of p53 was inhibited. During cell differentiation, when NS expression is downregulated, p53 is released from the protein complex, which allows the stabilization or activation of p53 and induction of target genes critical for cell cycle exit and differentiation, such as p21CIP. In addition, NS expression is required for HeLa cells to complete DNA synthesis and progress through S-phase since knockdown of NS expression leads to increased numbers of HeLa cells in G0/G1 phase and the cell proliferation rate and in vivo tumorigenic capacity reduced markedly (Liu et al. 2004a, b). Similar to NS expression, the expression of EGFR (proto-oncogen c-erbB-1) and its cognate ligand EGF was detected in more than 75% of ESCC tissues in this study. The association of EGFR with carcinogenesis has been well studied. Overexpression of the EGFR family members has been identified in a variety of human cancers such as gastrointestinal tract (Ghaderi et al. 2002; Kimura et al. 2004), colorectal (Hayashi et al. 1994; Porebska et al. 2000; Ooi et al. 2004), breast (Suo et al. 2002; Witton et al. 2003; Abd El-Rehim et al. 2004; DiGiovanna et al. 2005), lung (Hirsch et al. 2003), prostate (Di Lorenzo et al. 2002), and bladder cancers (Chow et al. 2001; Wester et al. 2002), and is correlated in a wide variety of tumors with the progression (Yarden and Sliwkowski 2001). Mostly in the squamous cell cancer, EGFR expression is associated with poor prognosis (Laimer et al. 2007). Recently, the overexpression of EGFR, partially accounted for by gene amplification, has been found in 50–70% of ESCC (Itakura et al. 1994; Hanawa et al. 2006), and is indicative of a poor prognosis (Ozawa et al. 1989; Yano et al. 1991). Activation of the EGFR family triggers a network of signaling pathways related to tumor cell proliferation, survival, invasion, and angiogenesis, including mitogen-activated protein kinase, phospholipase Cc, phosphoinositide 3-kinase, and signal transducers and activators of transcription (Yarden and Sliwkowski 2001; Grandis and Sok 2004). Overproduction of ligand (such as EGF) in conjunction with increased expression of EGFR in cancer cells facilitates the development of an autocrine and/or paracrine growth pathway (Grandis and Sok 2004). Given that NS, EGF and EGFR mRNA are co-expressed frequently in ESCC tissues, it is intriguing to uncover the possible association between NS and EGF or EGFR. It was found that inhibition of NS expression by overexpression of NS siRNA could block EGF and EGFR expression in ESCC cell line. However, the EGFR kinase inhibitor had minimal effect on NS expression in an ESCC cell line. These data strongly suggested that in ESCC tissues NS was required for expression of both EGF and EGFR during the pathogenesis of ESCC. Thus, in addition to modulating p53
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function, this study reveals that NS may also promote carcinogenesis through regulation of EGF and EGFR expression. The mechanisms underlying NS-regulated EGF and EGFR expression remains to be elucidated. In summary, the results from this study suggest that NS is frequently over-expressed in ESCC tissues. Importantly, this study reveals that NS over-expression is closely associated with the features of ESCC malignancy, such as tumor grade, invasion and lymphatic metastasis of esophageal squamous cell carcinoma. In addition, NS overexpression usually co-exists with EGF and EGFR expression in ESCC tissues. Therefore, the results from this study have important clinical implications. By measuring NS as well as EGF and EGFR expression in esophageal squamous cells collected using esophageal exfoliative cytology technique, we can evaluate ESCC prognosis and the efficacy of therapeutic scheme. Acknowledgment This work is partly supported by the National Natural Science Foundation of China no. 30872148. Conflict of interest statement All authors disclose no financial and personal relationships with other people or organisations that could inappropriately influence our work.
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