Med Oncol (2013) 30:583 DOI 10.1007/s12032-013-0583-y

ORIGINAL PAPER

Expression and clinical significance of apolipoprotein E in pancreatic ductal adenocarcinoma Jiong Chen • Long-Jiang Chen • Ren-Bao Yang Yun-Lian Xia • Hang-Cheng Zhou • Wen Wu • Yin Lu • Li-Wei Hu • Yue Zhao

•

Received: 5 March 2013 / Accepted: 11 April 2013 Ó Springer Science+Business Media New York 2013

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a poor prognosis. Our previous proteomic analysis found apolipoprotein E (ApoE) protein to be up-regulated in the sera of patients with PDAC. In this study, we sought to confirm this finding and investigate the relationship between ApoE and PDAC. We measured ApoE expression in tissues from PDAC patients and normal controls (NC) by real-time PCR, western blot, and immunohistochemistry. Enzyme-linked immunosorbent assay (ELISA) was applied to measure the levels of ApoE and carbohydrate antigen 19-9 (CA19-9) in the sera from patients with PDAC and NC. Real-time PCR and western blots showed that the ApoE mRNA and protein levels were up-regulated in PDAC tissues. The immunohistochemical results revealed that overexpression of ApoE was detected in 43 of 55 (78.2 %) PDAC cases and 3 of 20 (15 %) NC. High levels of ApoE were more likely in PDAC patients with advanced T status and TNM stages (p = 0.023 and p = 0.018, respectively). The ELISA results also confirmed that ApoE levels were elevated in the sera of PDAC patients. The sensitivity and specificity for distinguishing PDAC from NC were 76.2 and 71.4 %, respectively, for ApoE, 66.7 and 85.7 %, respectively, for CA19-9, and 81.0 and 85.7 %, respectively, for their combination. These results suggest that ApoE may be a potential PDAC-related biomarker and alone or in combination with other markers

Jiong Chen and Long-Jiang Chen contributed equally to this work. J. Chen (&)  L.-J. Chen  R.-B. Yang  Y.-L. Xia  H.-C. Zhou  W. Wu  Y. Lu  L.-W. Hu  Y. Zhao Department of General Surgery, Anhui Provincial Hospital Affiliated with Anhui Medical University, 17 Lujiang Road, Hefei 230001, Anhui Province, People’s Republic of China e-mail: [email protected]

may provide additional information for the diagnosis and clinical management of PDAC. Keywords Apolipoprotein E  Pancreas  Adenocarcinoma  Biomarker  Proteomics

Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid malignancies and the fourth leading cause of cancer-related deaths, accounting for an estimated 43,140 new cases and 36,800 deaths in the United States [1]. Due to the aggressive nature of this disease, most patients present with locally advanced cancers and distant metastasis at the time of diagnosis [2]; therefore, PDAC tends to show poor prognosis. Fewer than 20 % of patients are indicated for radical surgical resection, and the overall 5-year survival rate is below 5 % [3, 4]. Although use of PDAC-related biomarkers such as carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen, cancer antigen (CA) 50, and CA125 has become routine in the clinic, their sensitivity and specificity are relatively low in fully reflecting the characteristics of PDAC patients, which makes treatment of PDAC quite difficult. For example, the median sensitivity of serum CA19-9 for diagnosis is 79 % (range, 70–90 %) and median specificity 82 % (range, 68–91 %) [5]. Moreover, CA19-9 is also elevated in some benign diseases and other types of gastrointestinal cancers, and 10–15 % of individuals do not secrete CA19-9 due to their Lewis antigen status [6]. Hence, it is necessary to establish new biomarkers for the diagnosis and clinical management of PDAC. In our previous study [7], we compared the protein expression profiles of serum from PDAC patients, chronic

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pancreatitis patients, and normal controls (NC) using twodimensional difference gel electrophoresis followed matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The proteomic results indicated that apolipoprotein E (ApoE) was overexpressed in the sera of PDAC patients. Based on these findings, together with published reports, we became interested in the expression and function of this protein in patients with PDAC. Apolipoprotein E is a secretory glycoprotein, mainly produced in the liver. Besides its well-known role in cholesterol transport and metabolism [8], ApoE appears to be involved in several pathophysiological processes. By binding to its receptor, ApoE may activate cellular changes in signal transduction resulting in anti-oxidant effects, platelet aggregation, immune-regulation, and cell proliferation [9–12]. Moreover, ApoE has been associated with tumor development in some malignant tumors such as thyroid carcinoma, gastric cancer, and head and neck cancer [13–15]. In the current study, we investigated the expression of ApoE in PDAC patients by employing real-time polymerase chain reaction (PCR), western blot, immunohistochemical, and enzyme-linked immunosorbent assay (ELISA) methods. We also analyzed the biological correlation between this protein and clinicopathological parameters of PDAC patients. Finally, we evaluated the sensitivity and specificity of ApoE in distinguishing patients with PDAC from normal individuals.

Materials and methods

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blocks (45 PDAC tissues and 10 NC tissues) were obtained from the Pathological Department for immunohistochemical analysis. In addition, serum samples were obtained from 40 patients with histologically confirmed PDAC and 40 age- and sex-matched NC. In each case, blood was drawn prior to selective surgery using red top tubes without anticoagulation and then centrifuged at 4,0009g for 10 min at 4 °C. Real-time PCR analysis Real-time PCR was performed to investigate the ApoE mRNA in pancreatic tissues. Total RNA was isolated from pancreatic tissue samples using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, USA), and cDNA was synthesized using Reverse Transcription System (Promega, USA). Then, PCR amplification was performed in 50 ll reaction mixture containing 500 ng cDNA template, 5 mM each dNTP, 0.1 lmol/l each forward and reverse primer, each probe 2.5U, Taq polymerase, and PCR buffer. The PCR conditions were as follows: initial denaturation (95 °C, 10 min), followed by 40 cycles of denaturation (95 °C, 30 s), annealing (57 °C, 30 s) and extension (72 °C, 30 s) and a final extension step of 72 °C for 10 min. The amount of starting cDNA was adjusted using b-actin intensity. The expression of the ApoE mRNA was calculated using the 2-DDCT method [16]. The genespecific primers were designed as follows: ApoE (sense 50 -CTGCGTTGCTGGTCA-30 , antisense 50 -GCTCCTCG GTGCTCT-30 ) and b-actin (sense 50 -CAACTTCATCCA CGTTCACC-30 , antisense 50 -GAAGAGCCAAGGACA GGTAC-30 ).

Patients and samples Western blot analysis This study was approved by the Ethics Committee of the Affiliated Provincial Hospital with Anhui Medical University for clinical investigation, and informed consent was obtained from all subjects. Fresh tissue samples were collected from the Affiliated Provincial Hospital with Anhui Medical University. Pathologically proven PDAC tissue samples were obtained from 10 patients with PDAC. We also collected 10 normal pancreatic specimens. The samples were collected within 30 min of surgical resection, snap-frozen in liquid nitrogen, and then kept in a freezer at -80 °C until further processing. It is difficult to obtain specimens from completely normal patients because they do not undergo surgery; thus, the 10 normal specimens were obtained from patients who had pancreatic trauma or benign pancreatic disease such as pseudocyst, cystadenoma, or microcystic adenoma. None of the patients had received any prior anticancer treatment before surgery. Another 55 paraffin-embedded tissue

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For western blot, total proteins from PDAC tissues and NC tissues were extracted, quantified, and subjected to 12 % (w/v) SDS–PAGE. The gel was then transferred onto a PVDF membrane (Millipore, Bedford, MA, USA) in a trans-blot electrophoresis transfer cell. Membranes were blocked overnight at 4 °C with blocking buffer (50 mM Tris; 150 mM NaCl; 5 % non-fat dried milk). Then, membranes were incubated in primary antibody diluted 1:2,000 in 0.59 Superblock overnight at 4 °C. The antiApoE antibody and b-actin monoclonal antibody were purchased from Santa Cruz Biotechnology. Then, membranes were washed in TBS-T (3 9 10 min) and probed with horseradish peroxidase-conjugated secondary antibody (diluted 1:10,000, Santa Cruz Biotechnology) for 1 h at room temperature. The signal on the blots was detected using an ECL Plus kit (Pierce). The expression of b-actin was used as loading control.

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Immunohistochemical analysis

Results

A total of 75 tissue samples (55 cases of PDAC and 20 cases of normal pancreatic tissue) were used in our immunohistochemical analysis. The paraffin-embedded tissue blocks were cut into 5-lm serial sections onto Superfrost Plus glass slides, incubated at 65 °C for 24 h. Then, the slides were deparaffinized through a graded series of xylene and rehydrated through a graded series of alcohol to distilled water. Subsequently, the slides were processed for antigen retrieval using microwave heating in citrate buffer. Then, 5 % normal goat serum was applied to the slides for 30 min at 37 °C. Next, the sections were incubated overnight with anti-ApoE antibody (diluted 1:200) at 4 °C. Then, biotinylated secondary antibody (1:5,000) was added for 30 min at 37 °C, and the DAB detection kit (Maixin Systems) was used for the detection of the immunostaining. Two pathologists were invited to view the staining of the tissue microarray separately without knowing the clinical patient data. Semiquantitative evaluation of ApoE level was determined by combining the intensity of staining (range, 0–3; 0 absent, 1 weak, 2 moderate, 3 strong) and proportion of staining (range, 0–3; C50 % score 3, 10–49 % score 2, 1–9 % score 1). A total score [3 was considered as positive expression of ApoE.

Expression of ApoE mRNA in pancreatic ductal adenocarcinoma and normal pancreas

Enzyme-linked immunosorbent assay (ELISA) measurement Apolipoprotein E and CA19-9 were measured in sera using commercial ELISA kits (Uscn Life Science Inc.). The tests were performed according to the manufacturer’s protocols. The absorbance values for the two proteins were measured at 450 nm using a Stat Fax 2100 microplate reader (Awareness Technology Inc., USA) within 10 min. Statistical analysis All data were analyzed using IBM SPSS19.0 software. Numerical data were compared using independent Student’s t tests. The relationships between ApoE expression and clinicopathological characteristics were analyzed using chi-square or Fisher’s exact tests, Mann–Whitney U test, and Spearman’s rank correlation coefficients (two-sided). Receiver operating characteristic (ROC) curves methods were used to determine the sensitivity and specificity of markers in distinguishing PDAC cases from NC. Normal cutoff value was defined for ApoE (45.9 lg/ml) as the optimum point at which sensitivity and specificity were maximized, and 37 U/ml was adopted as the CA19-9 cutoff value [17]. A p value \0.05 was considered statistically significant.

Real-time PCR was used to investigate the expression of ApoE transcript levels in 10 PDAC tissues and 10 NC tissues. The results showed an obvious increase in the amount of ApoE mRNA in cancerous tissues compared with normal tissues (Table 1). The relative quantities of ApoE mRNA were 4.48 (2.418, 13.239) and 1 (0.343, 3.042) in PDAC tissues and NC tissues. There were statistically significant differences between the two groups (p \ 0.001). These results confirmed the differential ApoE protein expression of ApoE in PDAC from translational levels. Expression of ApoE protein in pancreatic ductal adenocarcinoma and normal pancreas Then, we examined the expression of ApoE protein in 6 cancerous tissues and 6 normal tissues by western blot. As expected, the results revealed that the ApoE protein was up-expressed in PDAC tissues compared with NC tissues. Representative images of western blot are shown in Fig. 1. Immunohistochemical characteristics of ApoE in pancreatic ductal adenocarcinoma and normal pancreas We also investigated the expression of ApoE in 55 PDAC tissues and 20 NC tissues by immunohistochemical method. High ApoE expression was detected in 43 of 55 (78.2 %) PDAC cases, mainly located in cytoplasm of cancer cells. On the contrast, only 3 of 20 (15 %) cases were positive of ApoE in NC tissues (Fig. 2). Correlation of immunohistochemical variables of ApoE with clinicopathologic parameters According to the immunohistochemical results, patients were divided into ApoE-negative and ApoE-positive groups. The correlation between ApoE and routine clinicopathological characteristics was investigated. We found that Table 1 Quantitative analysis of ApoE mRNA expression in PDAC and NC tissues using real-time PCR Sample

N

NC tissue

10

PDAC tissue

10

Relative value (range) 1 (0.343, 3.042) 4.48 (2.418, 13.239)

DCta

p

12.377 ± 0.744 \0.001 10.214 ± 0.699

a

The difference of cycle number at the threshold level of ApoE expression; mean ± standard deviation

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Fig. 1 Western blot analysis of ApoE in tissues from NC (N) and PDAC (T). The expression of ApoE was significantly higher in PDAC tissues than in NC tissues. b-actin was used as a reference

Fig. 2 Immunohistochemical staining of ApoE in PDAC and NC tissues. ApoE is strongly positive in PDAC tissues but negative or weakly positive in NC tissues (magnification, 9400)

ApoE-positive status was observed more frequently in PDAC patients with advanced T status and TNM stages (p = 0.021 and p = 0.018, respectively). Further, Spearman’s correlations coefficients of ApoE to T status and TNM stages were 0.36 (p = 0.007) and 0.32 (p = 0.016). Because the p values of the clinical differences are of marginal significance, further studies should be performed to address these correlations. There was no significant correlation between ApoE expression and other clinicopathologic parameters, such as age, gender, tumor size, tumor location, histological grade, or perineural invasion in PDAC tissues. The results are summarized in Tables 2 and 3. Serum levels of ApoE and CA19-9 in pancreatic ductal adenocarcinoma Enzyme-linked immunosorbent assay was used to confirm the levels of ApoE and CA19-9 in serum samples from 40 PDAC patients and 40 NC. Serum ApoE and CA19-9 were higher in the PDAC group than in the NC group (median level, 40.7 vs. 55.1 lg/ml, p = 0.003 for ApoE; median level, 18.4 vs. 152.2 U/ml, p = 0.001 for CA19-9). According to the ROC curves analysis, the sensitivity and specificity for distinguishing PDAC from NC were 76.2 and 71.4 %, respectively, for ApoE, 66.7 and 85.7 %, respectively, for CA19-9, and 76.2 and 90.5 %, respectively, for their combination. Table 4 and Fig. 3 show the

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complementary performance of ApoE and CA19-9 in detecting PDAC.

Discussion In the present study, we investigated the expression level of ApoE in human PDAC using real-time PCR, western blot, immunohistochemical and ELISA methods. We found that ApoE was overexpressed in PDAC patients at mRNA and protein levels, mainly located in cytoplasm of PDAC tissues, which further confirmed our proteomic findings [7]. Compared with normal pancreatic tissues, ApoE levels in PDAC patients were significantly higher. In addition, our immunohistochemical analysis indicated that a high level of ApoE was more frequently detected in PDAC patients with advanced T status and TNM stages. The gene encoding ApoE has been mapped to human chromosome 19q13.2; its expressed product includes three distinct isoforms of the protein, namely E1, E2, and E3. ApoE is mainly synthesized by the liver and is involved in lipid metabolism and cholesterol transport. Besides, it also has been the subject of great interest in many other functions such as immune response and regulation, anti-oxidant effects, tissue repair, and cell growth and differentiation [10, 11, 18]. Although the roles of ApoE in the molecular mechanism of malignancies remain unclear, many studies

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Table 2 Correlations between ApoE expression and clinicopathologic parameters in PDAC Parameter

Number of cases (%)

ApoEpositive cases (%)

Overall

55

43 (78.2)

\60

16

14

]60

39

29

Age

Gender

p value

0.507

0.476

0.422

0.516

Clinicopathological parameters

0.36

0.007

0.32

0.016

Table 4 The performance of ApoE and CA19-9 in differentiating PDAC patients from NC Marker candidate

AUC

Cutoff value

Sensitivity (%)

Specificity (%)

ApoE

0.79

45.9 lg/ml

76.2

71.4

0.83

37.0 U/ml

66.7

85.7

0.89

51.9 lg/ml, 41.7 U/ml

76.2

90.5

26

Female

23

17

\20 mm

15

9

CA19-9

]20 mm

40

34

ApoE ? CA19-9

Location Head

42

31

Body/tail

13

12

Histological grade Well

14

8

Moderate–poor

41

35

Perineural invasion Yes

45

37

No

10

6

T1, T2

12

6

T3, T4

43

37

N0

25

17

N1

30

26

T status

N status

M status M0

53

42

M1

2

1

TNM stages I

3

1

II

32

23

III

18

18

IV

2

1

Preoperative CA19-9 (U/ml) B37 [37 Fisher’s exact test; cant (p \ 0.05)

18

11

37

32

b

0.103

1.055

0.304

3.359

0.067

1.245

0.265

5.189

0.023*

2.786

0.095

a

0.392

b

0.018*

3.204

0.073

Mann–Whitney U test; * statistically signifi-

are exploring the relationship between the expression of ApoE and different types of malignancies. There are published reports that have identified ApoE as differentially expressed in some malignant tumors. For instance, Chen et al. [19] found that the level of ApoE was significantly higher in ovarian cancer than in normal ovarian tissues.

p value

TNM stages

32

2.667

ApoE expression Spearman’s correlation

T status

Male Tumor size

a

v2 value

Table 3 Spearman’s correlation analysis of the factors related to ApoE expression in PDAC patients

When the expression of ApoE was inhibited using ApoEspecific siRNA, cell cycle arrest and apoptosis were observed in an ApoE-expressing ovarian cancer cell line. Based on in vivo and in vitro studies [20], the overexpression of ApoE may promote cancer migration and proliferation, which has contributed to an aggressive clinical course in patients with lung adenocarcinoma. Moreover, a high concentration of ApoE may lead to increased resistance to the chemotherapy drug cisplatin, and is closely associated with poor survival in lung adenocarcinoma patients. Thus, ApoE may play important roles in the pathogenesis and progress of such tumors. By binding to the LDL receptor, and then serving as a trigger to activate certain downstream signal transductions, cancer-derived ApoE may promote cell proliferation and prevent cell apoptosis. In addition, ApoE may modify the expression of some cytokines to maintain tumor proliferation and survival. Support for this comes from a study showing that ApoE suppressed the lipopolysaccharide and poly (I–C)-inducted secretion of tumor necrosis factor-a, interleukin (IL)-6, and IL-1b [21], which have been shown to play important roles in the pathogenesis and development of tumors [22], in the murine monocyte macrophage cell line. By promoting cholesterol efflux, ApoE may alter the membrane microenvironment and disrupt toll-like receptor signaling pathways, which are needed for the activation of cytokine production. The investigators also found that a tandem repeat peptide of ApoE lacking in the lipid-binding domain was also able to inhibit such inflammatory cytokine production in macrophages. Hence, ApoE could reduce the production of inflammatory factors in a mechanism independent of lipid transport. Evidence from an animal experiment also indicated that proinflammatory and profibrogenic genes were up-regulated, necroinflammation and macrophage infiltration were exacerbated, and fibrosis was advanced in

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Fig. 3 Receiver operating characteristic curves (ROC) of serum levels of ApoE, CA19-9, and ApoE/CA19-9 combined for the detection of PDAC. The areas under the curve (AUC) for ApoE and CA19-9 were 0.79 and 0.83, respectively, whereas their combination was 0.89

hyperlipidemic-prone ApoE-deficient (ApoE-/-) mice with hepatic inflammation [23]. Regarding PDAC, studies by Grønborg et al. [24] using quantitative proteomics methods revealed that the level of ApoE was elevated approximately 20-fold in a pancreatic cancer cell line (Panc1) compared with NC. Their subsequent immunohistochemical analysis further confirmed the overexpression of ApoE in PDAC tissues. Interestingly, a recent report has shown that pancreatic cancer cells utilized lipid in preference to carbohydrate for energy, which may increase the turnover of ApoE [25]. Our study showed that the ApoE protein was significantly elevated in PDAC, which indicated that the ApoE overexpression might be a potential characteristic of PDAC. Our data also revealed a possible relationship between ApoE expression and clinical characteristics of the PDAC patients. The high expression of ApoE seemed to be positively correlated with later T stages and advanced TNM stages. Although the detailed mechanisms are poorly understood, the fact that ApoE upregulation results in aggressive behavior has been observed in many tumors. In gastric cancer, a high level of ApoE was related with advanced T status, N status, TNM stages, and poor survival [26, 27]. Studies of prostate cancer revealed that ApoE overexpression was associated significantly with Gleason score as well as local and distant invasiveness [28]. Some studies have drawn opposing conclusions. Specific peptide sequences of ApoE have been reported to have antiinfective and anti-inflammatory properties as well as an effect against tumor angiogenesis [29]. Another study showed that the ApoE 133–162 peptide had anti-tumor activity against all tested human cancer cell lines in a dosedependent manner using the MTT cytotoxic assay and the

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assay of calcein leakage from artificial liposomes. The tested cell lines included two gastric cancer cell lines (MKN-7, MNN-1), two pancreatic cancer cell lines (PANC-1, PaCa-2), and one colon cancer cell line (COLO201) [30]. From this perspective, the up-regulation of ApoE in PDAC may simply be part of a protective physiological response. Further studies focusing on the possible mechanism of ApoE in the development of PDAC are required. Apolipoprotein E is a PDAC-related marker rather than a specific marker, as evidenced by several reports mentioned above showing altered levels of ApoE in the sera of patients with other diseases. However, our ELISA results showed that the measurement of ApoE was useful for the detection of PDAC in patients, with sensitivity and specificity of 76.2 and 71.4 %, respectively. When used in combination with other markers such as CA19-9, ApoE could improve the specificity or sensitivity and provide additional information for the discrimination of PDAC patients from normal individuals. ApoE has three different isoforms in serum (E1, E2, and E3), and each isoform possibly contributes differently to the development of PDAC. The specificity or sensitivity of ApoE for the diagnosis of PDAC may be improved by measuring the levels of its different isoforms. Further studies are needed to address this issue. In conclusion, this study, together with our previous proteomic findings, demonstrates that ApoE expression is significantly up-regulated in PDAC. ApoE alone or in combination with other markers may provide additional information for the diagnosis and clinical management of PDAC. Acknowledgments This work was supported by Anhui Province Science and Technology Projects Foundation (No. 11010402172). Conflict of interest

None.

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