Expression and significance of ASPP2 in squamous carcinoma of esophagus

Kaohsiung Journal of Medical Sciences (2018) 34, 321e329

Available online at www.sciencedirect.com

ScienceDirect journal homepage: http://www.kjms-online.com

Original Article

Expression and significance of ASPP2 in squamous carcinoma of esophagus Bo Liu a,*, Lv Yang b, Xiu-Juan Li b, Rou Li b, Wei Sun b, Xiao-Yi Chen b, Jun-Chao Liu a a

Department of Pathology, The First Affiliated Hospital of Hebei North University, Zhangjiakou, China b Department of Histology and Embryology, Hebei North University, Zhangjiakou, China Received 28 July 2017; accepted 27 December 2017

Available online 19 January 2018

KEYWORDS Esophageal squamous cell carcinoma; ASPP2; Immuno histochemistry; Overall survival; EC109

Abstract To study the significance of apoptosis stimulating protein of P53 2 (ASPP2) expression in esophageal squamous cell carcinoma (ESCC), immunohistochemistry S-P method was used to examine the expression of ASPP2 in 136 cases of ESCC, 35 cases of high grade intraepithelial neoplasia (HGIN), 29 cases of low grade intraepithelial neoplasia (LGIN) and 37 cases of normal esophageal epithelium (NEE). The associations of ASPP2 expression with clinicopathological data and overall survival (OS) were also analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate ASPP2 expression in a total of 20 matched human ESCC tumor tissues and normal adjacent tissues (NAT). In addition, EC109 cells were treated with cisplatin (CDDP) in vitro for 24 h (the intervention group) and the control group was set up at the same time. Western blot was used to examine the expression of ASPP2 protein between the two groups. The expression of ASPP2 decreased progressively from NEE to LGIN, to HGIN, and to ESCC, and it was related to TNM stage, histological differentiation and lymph node metastasis in ESCC (P < 0.05). ASPP2 was a protective factor of patients with ESCC (P Z 0.008). The relative expression of ASPP2 mRNA was markedly downregulated in ESCC compared with the paired NAT (P < 0.01). Western blot results showed that cells in the intervention group could express ASPP2 while there was no expression of ASPP2 in the control group. Taken together, these results indicate that the abnormal expression of ASPP2 may play an important role for development and metastasis in ESCC. Copyright ª 2018, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. 14 Changqing Road, Zhangjiakou, Hebei, China. E-mail address: [email protected] (B. Liu). https://doi.org/10.1016/j.kjms.2017.12.011 1607-551X/Copyright ª 2018, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

322

Introduction Esophageal carcinoma is the eighth most prevalent cancer and the seventh leading cause of cancer-related death worldwide [1]. In China, the morbidity and mortality of esophageal carcinoma accounted for the fifth and fourth of malignancy, respectively [2]. As the most common histological type of esophageal carcinoma in China, the 5-year overall survival (OS) rate of esophageal squamous cell carcinoma (ESCC) patients after esophagectomy has significantly increased over the past several decades but remains approximately 20% [3e6]. However, the molecular mechanism of ESCC genesis and progression is not well understood, and biomarkers for predicting clinical outcome of ESCC patients are unavailable. Therefore, there is an urgent need to identify sensitive and specific biomarkers for ESCC. It is generally known that p53 is one of the mostly studied tumor suppressors, and it is closely related to the Apoptosis-Stimulating of p53 Protein (ASPP) family according to the recent studies. ASPP2 belongs to the evolutionarily conserved ASPP family of proteins, alongside ASPP1 and iASPP. It is commonly considered a tumor suppressor, and its expression is often reduced in malignant tumors [7e9]. Recent studies have also shown that ASPP2 is a key mediator of RAS-induced senescence, a property independent of p53 [10e12], suggesting that ASPP2 can suppress tumor growth through p53-dependent and -independent pathways. However, the role of it in ESCC is still not clear. In this study, we investigated the expression pattern of ASPP2 in ESCC, and evaluated its relationship with clinicopathological features and survival. In addition, EC109 cells were treated with cisplatin (CDDP) in vitro and the expression change of ASPP2 protein in EC109 cells was also detected.

Material and methods Patients and samples All patients were treated at the No.1 Affiliated Hospital of HeBei North University for ESCC from January 1, 2008 to December 31, 2009 and those with complete clinical data were enrolled. All procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the Helsinki Declaration and its later amendments or comparable ethical standards. A total of 136 ESCC patients were included in the present study and they were diagnosed based on pathological findings. These patients were 91 men and 45 women, ranging in age from 27 to 80 years (mean
SD: 61.2
8.9 years). The tumor-nodemetastasis (TNM) stage of the ESCC patients was defined according to the 7th edition of the UICC-AJCC TNM staging system [13]. For 35 cases of high grade intraepithelial neoplasia (HGIN) patients, they were 24 men and 11 women, ranging in age from 34 to 78 years (mean
SD: 57.8
8.1 years). For 29 cases of low grade intraepithelial neoplasia (LGIN) patients, they were 19 men and 10 women, ranging in age from 32 to 72 years (mean
SD: 55.9
7.8 years). For 37 cases of normal esophageal

B. Liu et al. epithelium (NEE), they were 20 men and 17 women, ranging in age from 43 to 71 years (mean
SD: 59.5
8.0 years). Statistics showed that the general data were no different among the 4 groups. Informed consent was obtained from all individual participants included in the study.

Follow up For 136 cases of ESCC patients, they were followed up from operation time to December 31, 2016 by telephone and return visits, with an interval of 3 months, and death was considered an event. Post-operative metastasis and recurrence were diagnosed on the basis of clinical examination, imaging assessment, operative and pathologic examination. Clinicopathologic data were obtained from pathologic reports, laboratory examination, medical records, and imaging, primarily including information of gender, age, tumor location, tumor size, histological differentiation, invasive depth, TNM stage, lymph node metastasis, postoperative radiotherapy and chemotherapy.

Immunohistochemistry (IHC) The appropriate paraffin-embedded specimen blocks of each case were obtained from the Department of Pathology. Tissue sections (4 mm thick) were dried at 60  C for 3e4 h, deparaffinized with three 10-min washes in xylene, and rehydrated in decreasing concentrations of ethanol in distilled water. Next, the sections were soaked in boiling sodium citrate buffer (ZSGB-BIO) for 20 min in microwave oven. When cooled to room temperature, the sections were washed 5 min in phosphate buffer solution (PBS, ZSGB-BIO, Beijing, China) for three times. Then, the tissue sections were treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. After washing 5 min in PBS for three times, the sections were incubated overnight at 4  C with rabbit monoclonal anti-ASPP2 antibody (ab181377; dilution, 1:100; Abcam) and followed by incubation with reagentⅠ(ZSGB-BIO) for 30 min at 37  C. The sections were washed 5 min in PBS for three times. After incubating with reagentⅡ(ZSGB-BIO) at 37  C for 30 min and washing with PBS for 5 min (three times), sections were counterstained with 5% hematoxylin for 5 min. Finally, the sections were blued in 1% hydrochloric-acid alcohol, dehydrated in increasing concentrations of ethanol, cleared with xylene, and mounted in neutral gum under a coverslip. The sections treated without primary antibody (use PBS as a substitute) were used as negative control.

Evaluation of immunohistochemistry Using a high-power (400) microscope (BX53, Olympus, Japan), ASPP2 expression as evaluated by two experienced pathologists independently, without knowledge of the clinical information. For each section, five random nonoverlapping fields containing at least 200 cells per field were observed and scored based on the percentage of positively stained cells (score 0 for negative, 1 for <10%, 2 for 10e50%, 3 for 51e80%, 4 for >80%) and the staining intensity (score 0 for negative staining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining).

Expression and significance of ASPP2

323

The intensity and proportion scores were multiplied to generate the IHC index. The expression level was considered as low (IHC index < 6), and as high (IHC index 6) [14].

Immunofluorescence (IF) Immunofluorescence analysis was employed to investigate the expression and localization of ASPP2 protein in esophageal cancer tissues. Tissue sections (4 mm thick) were dried at 60  C for 3e4 h, deparaffinized with three 10-min washes in xylene, and rehydrated in decreasing concentrations of ethanol in distilled water. After endogenous peroxidase activity was quenched with 3% H2O2 for 30 min, tissue slide was washed with PBS, permeabilized with 0.2% Triton X-100 in PBS buffer for 2 min at room temperature, then washed with PBS again, blocked with normal goat nonimmune serum for 60 min. After that, the tissues were incubated with rabbit monoclonal anti-ASPP2 antibody (ab181377; dilution, 1:50; Abcam) at 4  C for 24 h. Next, they were stained with FITC-conjugated anti-Rabbit secondary antibody (Anti-rabbit IgG (h þ l) Ab, Dl488 072-0315-06, KPL) in the dark at room temperature for 60 min and washed with PBS three times. Then, we added 10 mL of 4, 6-diamidino-2-phenylindole (DAPI; D9542, Sigma) counterstain into the area of the specimen. Negative controls were also employed to offset the disturbance of the primary or secondary antibody. The results were observed and recorded by fluorescence microscopy (Leica TCS-ST2 Instrument, Japan).

Quantitative real-time PCR (qRT-PCR) A total of 20 matched human ESCC tumor tissues and normal adjacent tissues (NAT) were collected directly after surgical resection was performed at the No.1 Affiliated Hospital of Hebei North University (China). All of the tissue samples from patients with no prior neoadjuvant treatment were immediately frozen in liquid nitrogen and stored at 80  C until mRNA was extracted. Clinicopathological information for all of the samples was available. Our research protocol was approved by the Ethics Review Committee of the Institutional Review Board of the hospital. Standard written consent was obtained from each patient. Total RNA was extracted from tissue samples using TRIzol reagent (Invitrogen, China) and RNAfast200 Kit (Fastagen, China) according to the manufacturer’s protocol. RNA was then reverse transcribed into cDNA using M-MLV RTase cDNA Synthesis Kit (Invitrogen, China). Then the conditions were as follows: 95  C for 30 s, 40 cycles of 95  C for 5 s, 58  C for 35 s and last stage at 95  C for 15 s, 60  C for 1 min, 95  C for

Table 1

Cell culture The EC109 cell line was obtained from the Life Science Centre of Hebei North University (Zhangjiakou, China). This study has been approved by Ethical Committee at Hebei North University of Science and Technology (China). The cells were cultured in RPMI-1640 medium (corning, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/mL of penicillin and 100 mg/mL of streptomycin at 37  C in a humidified air atmosphere containing 5% CO2. When the density of the cell colonies reached approximately 90% confluence, the cells were trypsinized with 0.25% trypsin (Amresco, USA) and transferred to fresh flasks at a ratio of 1:2. The cells used in this study were required to stay in log phase.

Grouping and cell proliferation assay EC109 cells in log phase were added to 96-well plates at a density of 5  104 cells/mL, and then the cultivated cells were randomly assigned to two groups. The intervention group was treated with 3 mg/mL cisplatin (CDDP) in vitro for 24 h while the control group was treated with equivoluminal serum-free-medium, instead of CDDP. Then the effect of CDDP on cell viability was evaluated by MTT method. After culturing for 24 h, cells were stained with 20 mL of sterile MTT dye (5 mg/mL, Qilu Pharmaceutical Co., Ltd. China) for 4 h at 37  C. The culture medium was removed, and 150 mL of dimethyl sulfoxide (DMSO) was added. The 96-well plates were shaken until the formazan crystals dissolved completely. Then the absorbance value was measured on a microplate reader (SpectraMax M2, Molecular Devices, USA) at 490 nm.

Western blot Western blot analyses were performed on cell lysates prepared from EC109 of the intervention group and the control group as described previously. Transfected cells were lysed

ASPP2 protein expression during cancer progression by IHC analysis.

Cancer progression NEE① LGIN② HGIN③ ESCC④

15 s by using an Applied Biosystems 7300 real-time PCR system (Applied Biosystems, USA). The housekeeping gene GAPDH was used as an internal control. Primers used for real-time PCR were listed below: ASPP2 forward 50 -GAAGACTCGGTGAGCATGCG-30 , reverse 50 -GCGATACGCTCTGAG CCAGT-30 and GAPDH forward 50 -CGACCACTTTGTCAAGC TCA-30 , reverse 50 -ACTGAGTGTGGCAGGGACTC-30 . Cycle threshold (Ct) was analyzed. The fold-change between ESCC and NAT for ASPP2 gene was calculated as mean of 2DDCt
standard deviation (SD).

ASPP2 expression [cases (%)] Low

High

6 (16.2) 8 (27.6) 14 (40.0) 69 (50.7)

31 21 21 67

(83.8) (72.4) (60.0) (49.3)

P value ①:② P Z 0.262 ①:④ P Z 0.000 ②:④ P Z 0.023

①:③ P Z 0.024 ②:③ P Z 0.298 ③:④ P Z 0.257

324

B. Liu et al.

Figure 1. Representative immunohistochemical staining of ASPP2,  200. (Positive expression(/) in normal esophageal epithelia (A), Positive expression(/) in low grade intraepithelial neoplasia (B), Positive expression(/) in high grade intraepithelial neoplasia (C), Positive expression(/) in ESCC (D), Negative expression in normal esophageal epithelia (E), Negative expression in low grade intraepithelial neoplasia (F), Negative expression in high grade intraepithelial neoplasia (G), Negative expression in ESCC (H).).

in RIPA lysis buffer (Beyotime, China). Cell protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membranes (Immobilon 0.45 mm, Millipore, USA), and immersed in a blocking solution containing 5% fat-free milk and 0.1%

Tween-20 for 1 h. After blocking, membranes were incubated with ASPP2 antibody (ab181377; dilution, 1:5000; Abcam) overnight at 4  C and then with diluted horseradish peroxidase-conjugated secondary antibody (sc-2004, dilution, 1:1000, Santa Cruz) for 2 h at room temperature. After

Expression and significance of ASPP2

325

washing, the resulting bands were visualized using the standard ECL procedure, quantified by densitometry, and normalized to the corresponding b-actin bands.

Statistical analysis Statistical analysis was performed using the SPSS v.17.0 software (USA). The relationships of ASPP2 expression between ESCCs and noncancerous tissue, ASPP2 expression with the clinicopathologic parameters were all assessed using Chi-square and Fisher’s exact tests. KaplaneMeier survival analysis and log-rank tests were used to evaluate the 5-year OS rates of ESCC patients. Cox univariate analysis was used to determine the prognostic significance of variables, and Cox multivariate analysis was applied to identify independent prognostic factors for ESCC. The oneway ANOVAs were used to analyze the results of MTT and the differences in ASPP2 mRNA expression between ESCC and NAT. For all results, differences were considered to be statistically significant at P < 0.05 with a 95% confidence interval.

Results Association between ASPP2 expression and clinicopathological parameters of ESCC by IHC analysis ASPP2 was measured in NEE, LGIN, HGIN and ESCC. IHC analysis showed that the frequency of ASPP2 protein expression was greatest in NEE (83.8%) and decreased gradually during the evolution of esophageal carcinogenesis, with only 49.3% of ESCC showing high expression of ASPP2 protein (Table 1, Fig. 1). Furthermore, a c2 test showed that there was a significant difference when comparing the prevalence of ASPP2 suppression in various levels of cancer progression (c2 Z 16.904, P Z 0.001) (Fig. 2). Moreover, the abnormal expression of ASPP2 was related to TNM stage, differentiation degree and lymph node metastasis (P < 0.05) (Table 2).

Relationships between ASPP2 expression and survival of the patients The association between ASPP2 protein expression and OS of ESCC was estimated using log-rank test and multivariable Cox proportional hazard regression analysis (Table 3). The 5-year OS rate for all 136 ESCC patients was 13.2% (Fig. 3A). KaplaneMeier survival analysis showed that ESCC patients with low or loss expression of ASPP2 protein had a significantly worse prognosis than ESCC with higher expression. The 5-year OS rate in ESCC patients with low (n Z 69) and high ASPP2 protein (n Z 67) were 26.1% and 10.4%, respectively (log-rank test, c2 Z 52.306, P Z 0.000, Fig. 3B). Furthermore, the 5-year OS rate was significantly correlated with histological grade, TNM, invasion depth, lymph node metastasis and ASPP2 status. Multivariate analysis demonstrated that histological grade, TNM, invasion depth and ASPP2 status were all

Figure 2. The expression of ASPP2 in ESCC and noncancerous tissues. (HGIN vs ESCC, P Z 0.257(A); LGIN vs ESCC, P Z 0.023(B); NEE vs ESCC, P Z 0.000(C)) (Chi-square and Fisher’s exact tests).

significant prognostic factors for 5-year OS rate of patients with ESCC. Furthermore, there were no LGIN became ESCC during follow-up period. But in 35 cases of HGIN patients, 16 cases of them became ESCC. KaplaneMeier survival analysis showed that HGIN patients with low or loss expression of ASPP2 protein had a significantly worse prognosis than HGIN patients with higher expression of ASPP2 protein (log-rank test, c2 Z 12.540, P Z 0.000).

Distribution of ASPP2 protein in ESCC tissues by IF In order to show the distribution patterns of ASPP2 protein in ESCC tissues, immunofluorescence stainings were performed. Under the confocal-microscope observation (Fig. 4), ASPP2 protein was stained with green fluorescence, which was expressed in the perinuclear cytoplasm and/or the nucleus in ESCC cells.

326

B. Liu et al.

Table 2 Associations between the expression of ASPP2 and clinicopathologic characteristics of the 136 cases of ESCC patients. Characteristic

Total Gender Male Female Age (years) n  30 30  n  60 n>60 Histological differentiation Well Moderate Poor Invasive depth T1e2 T3 T4 pTNM stage I Ⅱ Ⅲ Ⅳ Lymph node metastasis Absence Presence

ASPP2 expression [cases (%)]

P value

Low

High

69

67

46 (66.7) 23 (33.3)

45 (67.2) 22 (32.8)

0 (0) 35 (50.7) 34 (49.3)

1 (1.5) 31 (46.3) 35 (52.2)

Effects of CDDP on morphological alteration of EC109 cells 0.951

0.541

0.001 29 (42.0) 35 (50.7) 5 (7.3)

10 (14.9) 43 (64.2) 14 (20.9)

6 (8.7) 26 (37.7) 37 (53.6)

6 (9.0) 17 (25.4) 44 (54.6)

5 (7.2) 27 (39.1) 34 (49.3) 3 (4.4)

6 (9.0) 15 (22.4) 33 (49.2) 13 (19.4)

0.292

0.021

0.000 57 (82.6) 12 (17.4)

the relative expression of ASPP2 mRNA was markedly downregulated in ESCC samples compared with the paired tumor adjacent tissues (0.482
0.04 vs 1.000
0.0), (P Z 0.000; Fig. 5).

29 (43.3) 38 (56.7)

Compared with the control group, cells treated with CDDP for 24 h significantly exhibited apoptosis-related morphological changes, such as cell shrinkage and rounding up, cell membrane blebbing, nuclear fragmentation, and condensation (Fig. 6).

Effects of CDDP on EC109 cell proliferation and viability The effects of CDDP on EC109 cell proliferation and viability were measured using MTT assay whose measurement is represented as absorbance value. The proliferation of EC109 cells was inhibited by CDDP. Twenty-four hours after treatment with 3 mg/mL CDDP, the inhibiting rate of cell proliferation in the intervention group [(25.2
2.6)%] was increased compared with the control group. Statistical analysis showed that cell viability was significantly decreased (t Z 14.985, P Z 0.001) after treatment with CDDP. Then the cells in each group were used for Western blot.

Expression of ASPP2 on EC109 cell of each group by western blot The results of Western blot showed that EC109 cells in the intervention group could express ASPP2 while there was no expression of ASPP2 in the control group (Fig. 7).

The ASPP2 mRNA expression between ESCC and NAT by qRT-PCR

Discussion

Relative ASPP2 expression was detected by qRT-PCR between paired tumor tissues and normal adjacent tissues from 20 patients with ESCC. Our results demonstrated that

In the present study, we demonstrated that ASPP2 expression was significantly decreased in ESCC and was associated with TNM stage, histological differentiation and lymph node

Table 3

Associations between the expression of ASPP2 and 5-year overall survival rate of the 136 cases of ESCC patients.

Variables Univariate analysis Age Gender Histological grade Invasion depth pTNM Lymph node metastasis ASPP2 Multivariate analysis Histological grade Invasion depth pTNM Lymph node metastasis ASPP2

Subset

HR

95% CI

P

60 vs. >60 Male vs. Female G1 vs. G2eG3 T1þT2 vs. T3þT4 No vs. Yes IeII vs IIIeIV Low vs. High

1.070 1.221 2.381 2.307 1.733 1.732 4.004

(0.713e1.604) (0.808e1.844) (1.402e4.403) (1.371e3.879) (1.178e2.551) (1.053e2.820) (2.652e6.044)

0.745 0.343 0.001 0.002 0.005 0.030 0.000

G1 vs. G2eG3 T1þT2 vs. T3þT4 IeII vs IIIeIV No vs. Yes Low vs. High

0.506 0.391 2.407 0.770 2.177

(0.301e0.853) (0.185e0.827) (1.264e4.582) (0.459e1.290) (1.229e3.859)

0.011 0.014 0.007 0.320 0.008

Expression and significance of ASPP2

327

Figure 3. KaplaneMeier survival curves of ESCC patients with low and high expression of ASPP2. (The 5-year OS rates of a total of 136 ESCC patient (A); Patients with high ASPP2 expression have higher 5-year OS rates than those with low ASPP2 expression (B)).

metastasis. ASPP2 was an independent predictor of ESCC patients. As a tumor suppressor and an activator of p53 family, the expression of ASPP2 is often reduced in malignant tumors and it is involved in apoptosis in cancer cells [15,16]. For example, Song B et al. analyzed the expression level of ASPP2 in pancreatic cancer tissue samples with qRT-PCR, Western Blot and immunohistochemistry staining. Their results showed that expression of ASPP2 was downregulated in cancerous tissues in comparison with para-cancerous tissues [17]. However, the inconsistent results or even opposite results were found in data analysis of different study, which may be related to the experimental condition and experimental methods. For instance, Xie XF et al.

examined the protein expression of ASPP1, ASPP2, and P53 in 175 specimens of ESCC by immunohistochemical staining. Their results showed that the protein expression level of ASPP2 was significantly higher in cancerous tissues than in paired noncancerous tissues [9]. In our study, the frequency of ASPP2 protein expression was greatest in NEE and decreased gradually during the evolution of esophageal carcinogenesis, with only 49.3% of ESCC showing high expression of ASPP2 protein by IHC. Moreover, a c2 test showed that there was a significant difference when comparing the ASPP2 expression in various levels of cancer progression. Furthermore, our results showed that ASPP2 abnormal expression was associated with TNM stage, histological differentiation and lymph node metastasis of ESCC, suggesting that ASPP2 may repress the progression and play a role for development and metastasis in ESCC. As a member of ASPP family, the function of ASPP2 is potentially controlled by its binding partners and localisation. When ASPP2 locates at the cytosol/nucleus, ASPP2 enhances p53-induced apoptosis in cancer cells [15,18,19], whereas in the cell junctions, it binds and maintains the

Figure 4. The distribution of ASPP2 protein in ESCC tissues by immunofluorescence stainings,  400. (ASPP2 protein was stained with green fluorescence(/), which was expressed in the perinuclear cytoplasm and/or the nucleus in ESCC cells; Nuclei were counterstained with DAPI).

Figure 5. Relative expression of ASPP2 mRNA by qRT-PCR between paired ESCC tissues and normal adjacent tissues. (The relative expression of ASPP2 was markedly downregulated in ESCC samples compared with the paired tumor adjacent tissues, P Z 0.000) (one-way ANOVA).

328

B. Liu et al.

Figure 6. Effects of CDDP on morphological alteration of EC109 cells (Cells in the intervention group exhibited apoptosis-related morphological changes, such as cell shrinkage and rounding up(/), cell membrane blebbing, nuclear fragmentation, and condensation (A); Cells in the control group were mostly fusiform or polygonal. It often had an oval nucleus which located in the center(/).

integrity of cell polarity and adherence junction via its N-terminus [20,21]. In this study, ASPP2 protein was stained with green fluorescence, which was expressed in the perinuclear cytoplasm and/or the nucleus in ESCC cells by immunofluorescence. This result indicated that ASPP2 mainly plays the role of apoptosis in ESCC. Studies show that cell autophagy increased and apoptosis decreased when ASPP2 was silenced. This phenomenon indicated that reduced ASPP2 in cells created conditions for tumour formation [15]. It reduced damaged cell death and accumulated injuries in cells, which was beneficial for tumour survival. According to our results, low ASPP2 expression independently predicted poor prognosis of ESCC patients, which is consistent with previous reports of other cancers [22,23]. The results of qRT-PCR in this study demonstrated that the relative expression of ASPP2 mRNA was markedly downregulated in ESCC samples compared with the paired tumor adjacent tissues. Furthermore, after treatment with CDDP for 24 h, EC109 cells could express ASPP2 while there was no expression of ASPP2 in the control group. In this context, more aggressive therapy modality

such as radiotherapy and chemotherapy, may be necessary for patients with low ASPP2 expression. A growing number of researchers believe that ASPP2 is considered a key mediator in Ras oncogene induced senescence and apoptosis response [10]. Ras isoforms are mutated in the 30% of human cancers and are major drivers of tumourigenesis [24]. Cells have evolved safety mechanism to shut down overactive Ras. And ASPP2 downregulation in cancer has been associated with increased tumourigenesis [10,11]. However, in this study, we detected ASPP2 expression mainly in ESCC tissue by immunohistochemistry, qRT-PCR and Western blot, so the results need further experiments to verify and explore.

Conclusions Taken together, the data provide that the abnormal expression of ASPP2 may play a role for development and metastasis in ESCC. It was a potential prognostic biomarker and examination of ASPP2 may be useful for diagnosis and guiding the clinical therapy in ESCC.

Acknowledgments We thank the grant from the Science and Technology Supporting Plan in Hebei Province (NO. 15277707D) and the science and technology guidance project of Health and Family Planning Commission of Hebei Province(NO.ZD20140220) for support of the study.

References

Figure 7. Western blot results of ASPP2 protein of EC109 cells in the intervention group and the control group.

[1] Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin 2015; 65:87e108. [2] Chen W, Zheng RS, Zhang SW, Zeng HM, Zuo TT, Jia MM, et al. Report of cancer incidence and mortality in China, 2012. China Coal 2016;25:1e8 [In Chinese, English abstract]. [3] Worni M, Martin J, Gloor B, Pietrobon R, D’Amico TA, Akushevich I, et al. Does surgery improve outcomes for esophageal squamous cell carcinoma? An analysis using the

Expression and significance of ASPP2

[4]

[5]

[6]

[7]

[8] [9]

[10]

[11]

[12]

[13]

[14]

surveillance epidemiology and end results registry from 1998 to 2008. J Am Coll Surg 2012;215:643e51. Dubecz A, Gall I, Solymosi N, Schweigert M, Peters JH, Feith M, et al. Temporal trends in long-term survival and cure rates in esophageal cancer: a SEER database analysis. J Thorac Oncol 2012;7:443e7. Luo LL, Zhao L, Xi M, He LR, Shen JX, Li QQ, et al. Association of insulin-like growth factor-binding protein-3 with radiotherapy response and prognosis of esophageal squamous cell carcinoma. Chin J Cancer 2015;34:514e21. Qi YJ, Wang M, Liu RM, Wei H, Chao WX, Zhang T, et al. Downregulation of 14-3-3s correlates with multistage carcinogenesis and poor prognosis of esophageal squamous cell carcinoma. PLoS One 2014;9:e95386. Turnquist C, Wang YH, Severson DT, Zhong S, Sun B, Ma JY, et al. STAT1-induced ASPP2 transcription identifies a link between neuroinflammation, cell polarity, and tumor suppression. Proc Natl Acad Sci USA 2014;111:9834e9. Sullivan A, Lu XASPP. A new family of oncogenes and tumour suppressor genes. Br J Canc 2007;96:196e200. Xie XF, Yang Q, Chi J, Yang XZ, Wang HY, Xu GL. Prognostic values of apoptosis-stimulating P53-binding protein 1 and 2 and their relationships with clinical characteristics of esophageal squamous cell carcinoma patients: a retrospective study. Chin J Cancer 2017;36:15. Wang Y, Godin-Heymann N, Wang XD, Bergamaschi D, Llanos S, Lu X. ASPP1 and ASPP2 bind active RAS, potentiate RAS signalling and enhance p53 activity in cancer cells. Cell Death Differ 2013;20:525e34. Wang YH, Wang XD, Lapi E, Sullivan A, Jia W, He YW, et al. Autophagic activity dictates the cellular response to oncogenic RAS. Proc Natl Acad Sci USA 2012;109:13325e30. Posada IM, Serulla M, Zhou Y, Oetken-Lindholm C, Abankwa D, Lectez B. ASPP2 is a novel Pan-Ras nanocluster scaffold. PLoS One 2016;11:e0159677. Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, TrottiA, editors. AJCC cancer staging manual. 7th ed. New York: Springer-Verlag; 2010. Wang CN, Li ZT, Shao F, Yang XY, Feng XL, Shi SS, et al. High expression of Collagen Triple Helix Repeat Containing 1

329

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

(CTHRC1) facilitates progression of oesophageal squamous cell carcinoma through MAPK/MEK/ERK/FRA-1 activation. J Exp Clin Canc Res 2017;36:84. Shi Y, Han Y, Xie F, Wang A, Feng XK, Li N, et al. ASPP2 enhances Oxaliplatin (L-OHP)-induced colorectal cancer cellapoptosis in a p53-independent manner by inhibiting cell autophagy. J Cell Mol Med 2015;19:535e43. Chen R, Wang H, Liang BB, Liu GK, Tang M, Jia RJ, et al. Downregulation of ASPP2 improves hepatocellular carcinoma cells survival via promoting BECN1-dependent autophagy initiation. Cell Death Dis 2016;7:e2512. Song B, Bian Q, Zhang YJ, Shao CH, Li G, Liu AA, et al. Downregulation of ASPP2 in pancreatic cancer cells contributes to increased resistance to gemcitabine through autophagy activation. Mol Canc 2015;14:177. Liu K, Shi Y, Guo X, Wang S, Ouyang Y, Hao M, et al. CHOP mediates ASPP2-induced autophagic apoptosis in hepatoma cells by releasing Beclin-1 from Bcl-2 and inducing nuclear translocation of Bcl-2. Cell Death Dis 2014;5:e1323. Liu ZY, Qiao LX, Zhang YL, Zang YJ, Shi Y, Liu K, et al. ASPP2 plays a dual role in gp120-induced autophagy and apoptosis of neuroblastoma cells. Front Neurosci 2017;11:150. Royer C, Koch S, Qin X, Zak J, Buti L, Dudziec E, et al. ASPP2 links the apical lateral polarity complex to the regulation of YAP activity in epithelial cells. PLoS One 2014;9:e111384. Wang XW, Yu M, Zhao KM, He MM, Ge WJ, Sun YH, et al. Upregulation of MiR-205 under hypoxia promotes epithelialemesenchymal transition by targeting ASPP2. Cell Death Dis 2016;7:e2517. Schittenhelm MM, Illing B, Ahmut F, Rasp KH, Blumenstock G, Do ¨hner K, et al. Attenuated expression of apoptosis stimulating protein of p53-2 (ASPP2) in human acute leukemia is associated with therapy Failure. PLoS One 2013;8:e80193. Wu ZY, Wang SG, Xue P, Wang SL, Wang GC, Zhang W. Inhibitory member of the apoptosis-stimulating protein of p53 is overexpressed in bladder cancer and correlated to its progression. Medicine (Baltimore) 2017;96:e6640. Alberto FM, Eugenio S. Ras in cancer and developmental diseases. Genes Cancer 2011;2:344e58.