Guangzhou, China

Decreased expression of DAB2IP in pancreatic cancer with wild-type KRAS

Yi-Fan Duan, Dong-Feng Li, Yan-Hui Liu, Ping Mei, Yu-Xuan Qin, Liang-Fang Li, Qiu-Xiong Lin and Zi-Jun Li

Guangzhou, China

BACKGROUND:KRAS mutation plays an important role in the pathogenesis of pancreatic cancer. However, the role of wildtype KRAS in the progression of pancreatic cancer remains unknown. The present study was to investigate the expression of the Ras GTPase activating protein (DAB2IP) in pancreatic cancer and its clinical signif i cance.

METHODS:The expression of DAB2IP in pancreatic cancer cell lines and normal human pancreatic ductal epithelial cells was analyzed by Western blotting and real-time quantitative reverse transcription-PCR (qRT-PCR). The KRAS mutational types of pancreatic cancer tissues obtained from pancreatic cancer patients (n=20) were also analyzed. Subsequently, DAB2IP expression was detected in pancreatic cancer tissues, adjacent and normal pancreatic tissues (n=2) by immunohistochemistry, and the relationship between DAB2IP expression and the clinical characteristics of patients was evaluated.

RESULTS:Western blotting and qRT-PCR results showed that DAB2IP expression in pancreatic cancer cells with wildtype KRAS was lower than that in those with mutation-type KRAS and normal human pancreatic ductal epithelial cells (P<0.05). Immunohistochemistry showed that DAB2IP expression was lower in pancreatic cancer tissues than that in adjacent and normal pancreatic tissues (Z=-4.000,P=0.000). DAB2IP expression was lower in pancreatic cancer patients with the wild-type KRAS gene than that in those with KRAS mutations (WilcoxonW=35.000,P=0.042). Furthermore,DAB2IP expression in patients with perineurial invasion was lower than that in those without invasion (WilcoxonW=71.500,P=0.028). DAB2IP expression was lower in patients with more advanced stage than that in those with early clinical stage (WilcoxonW=54.000,P=0.002).

CONCLUSIONS:DAB2IP expression was reduced in patients with pancreatic cancer compared with those with no cancer. DAB2IP expression was correlated with the KRAS gene, perineurial invasion and clinical stage of the disease. Our data indicated that DAP2IP expression can be used as a potential prognostic indicator and a promising molecular target for therapeutic intervention in patients with pancreatic cancer.

(Hepatobiliary Pancreat Dis Int 2013;12:204-209)

DAB2IP;pancreatic cancer; KRAS type; tumor suppressor

Introduction

It is well-known that pancreatic cancer is one of the most malignant tumors. Because of the absence of effective methods for early diagnosis, pancreatic cancer is associated with a high overall mortality (almost 100%), a very low surgical excision rate, and an overall fi ve-year survival rate (<5%). Chemotherapy and radiation treatment are ineffective in most of the patients.[1,2]Therefore, it is essential to fi nd the diagnostic marker and to explore the possible novel therapeutic target(s) in the management of pancreatic cancer.

Many signaling pathways play important roles in the pathogenesis of pancreatic cancer through modulation of proliferation or apoptosis in cancer cells.[3,4]In the present study, we focus on the Ras signaling pathways. In general, Ras alternates between GTP-bound (active) and GDP-bound (inactive) forms in a cycle regulated by Ras guanine nucleotide exchange factors (GEFs)and Ras GTPase activating proteins (GAPs). Ras GEFs release GDP from small G proteins, resulting in the formation of GTP-bound GTPase, whereas Ras GAPs promote the intrinsic GTPase activity of small G proteins, resulting in the conversion of Ras-GTP to Ras-GDP.[5-7]Oncogenic Ras mutants have alterations that affect intrinsic properties of GTPase such as GTP association, GDP dissociation and GTP hydrolysis and are therefore insensitive to the action of Ras GAPs, despite retaining the ability to bind them in the GTP-bound state.[8]KRAS mutations cause unregulated cell proliferation through the RAS-RAF-MEK-ERK kinase pathway and activate a cascade of anti-apoptotic signals through the PI3K-AKT pathway.[9-13]By comparison, the aff i nities of Ras GAPs for wild-type Ras are much higher.[8]Therefore, Ras GAPs only play a role in cancers with wild-type KRAS. Decreased expression of Ras GAP would also activate the RAS signaling pathway and cause abnormal cell biological behavior.[5]

DAB2IP is one of the 16 types of Ras GAPs that have been discovered in recent years. Some studies have indicated low expression of DAB2IP in human liver cancer and prostatic carcinoma and DAB2IP has been implicated as a tumor suppressor. However, the role of DAB2IP in the progression of pancreatic cancer remains to be elucidated. In this study, we sought to def i ne the expression of the Ras GTPase activating protein, DAB2IP, in pancreatic cancer cells and tissues and explored the potential role and clinical signif i cance of DAB2IP in pancreatic cancer.

Methods

Human tissue samples and cell lines

The cell lines (wild-type KRAS: Bxpc-3; mutant KRAS: Capan-2, Sw1990, Aspc-1; normal human pancreatic ductal epithelial cells: H6C7) were purchased from the American Type Culture Collection and the Shanghai Cell Bank. All cells were cultured at 37 ℃ under an atmosphere containing 5% CO2. Paraff i n-embedded specimens of pancreatic cancer tissues and related adjacent tissues (n=20) were collected during the period from 2008 to 2009 in Guangdong General Hospital. Normal pancreatic tissue paraff i n sections (n=2) were used as controls. Twenty patients (14 males and 6 females) had not received preoperative chemotherapy and radiotherapy or immune/biological intervention. The patients aged from 37 to 73 years (median 63.5). The Diagnosis and Treatment Standards of Pancreatic Cancer (2011 Edition) formulated by the Ministry of Health of China were used as a reference to determine the staging of pancreatic cancer. The pathological types of these tissues were ductal adenocarcinoma and were moderately differentiated.

Real-time quantitative reverse transcription-PCR (qRTPCR) analysis

Total RNA was extracted from four types of pancreatic cancer cells (wild-type KRAS: Bxpc-3; mutant KRAS: Capan-2, Sw1990, Aspc-1) and the normal human pancreatic ductal epithelial cells H6C7 using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. Primers for amplif i cation of DAB2IP and GAPDH (control) were designed by Shanghai Jierui Co., (Shanghai, China). Primer sequences were as follows: DAB2IP forward: 5'-CCT GGA CGA TGT GCT CTA TG-3'; DAB2IP reverse: 5'-TCT TCT TCT TCT TGT CGG TCT C-3'.

qRT-PCR was performed using an ABI PRISM 7500 Quantitative PCR system (Applied Biosystems, Foster City, CA, USA). Amplif i cation conditions were as follows: 95 ℃ for 10 minutes followed by 50 cycles (95 ℃ for 30 seconds, 58 ℃ for 30 seconds and 72 ℃ for 30 seconds). Each sample was examined in triplicate and the amount of product was normalized relative to that of GAPDH. Quantitative values were calculated according to the formula: Quantitative value=2-?CT, in which the ?CT value for each GAP was calculated by subtracting the average CT (cycle threshold) value for the target gene from the average CT value for the GAPDH gene.

Western blotting analysis

Proteins were extracted from wild-type KRAS and mutant KRAS pancreatic cancer cells and normal human pancreatic ductal epithelial cells using RIPA buffer (Shanghai Biocolor BioScience Technology Company, Shanghai, China) containing protease and phosphatase inhibitors. Protein concentrations were determined with a BCA kit (Guangzhou Weijia Technology Company, Guangzhou, China). Samples of the total protein lysates (10 μL) were subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes (Millipore Company, Shanghai, China). Polyclonal anti-DAB2IP (1:2000, Abcam, Cambridge, UK) and anti-GAPDH (1:7500, Bioworld Technology, St. Louis, USA) antibodies were used as primary detection reagents and horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000, CST, Danvers, USA) was used as the secondary detection reagent. Immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL, Invitrogen, USA) system, and band density was analyzed by BandScan 5.0 software.

Mutational analysis

Genomic DNA was extracted using QIAamp DNA FFPE Tissue kit (Qiagen company, Dusseldorf, Germany) and used as a template for amplif i cation of the KRAS common mutations point by PCR with the Premix Ex TaqTMHot Start Version kit (TaKaRa company, Dalian, China). Primer sequences were as follows: KRAS 1 exon sense: 5'-CTT AAG CGT CGA TGG AGG AG-3'; KRAS 1 exon antisense: 5'-GTA TCA AAG AAT GGT CCT GC-3'; KRAS 2 exon sense: 5'-CTT TGG AGC AGG AAC AAT GTC-3'; KRAS 2 exon antisense: 5'-TGC ATG GCA TTA GCA AAG ACT C-3'.

The amplif i cation conditions were as follows: 95 ℃ for 5 minutes followed by 35 cycles (95 ℃ for 45 seconds, 58 ℃ for 45 seconds, 72 ℃ 1 minute) and a fi nal extension at 72 ℃ for 5 minutes. Reactions were then held at 12 ℃. PCR products were purif i ed and sequenced. PCR analysis was performed using the ABI Big Dye Terminator v3.1 kit (Applied Biosystems, Foster City, CA, USA) under the following conditions: 96 ℃ for 1 minute followed by 25 cycles of (96 ℃ for 10 seconds, 55 ℃ for 5 seconds, 60 ℃ for 4 minutes). Reactions were then held at 4 ℃. The PCR product was rinsed, pre-denatured and transferred to 96-well plates for direct sequencing using an ABI 3100 Genetic Analyzer.

Immunohistochemistry

Paraf fi n-embedded tissue specimens were sectioned (4 μm) and slides prepared using standard techniques. Mounted tissue sections were baked at 65 ℃ for 3 hours, deparaf fi nized in xylene and rehydrated through graded alcohols. Antigens were retrieved by heating in 1 μmol/L sodium citrate (pH 6.0) in a pressure cooker at 100 ℃for 3 minutes, followed by incubation in 3% H2O2for 10 minutes at room temperature to destroy endogenous peroxidase activity. Non-speci fi c staining was blocked by incubation in 10% goat serum for 10 minutes. Tissue sections were incubated with anti-DAB2IP (1:200, Abcam) primary antibodies overnight at 4 ℃, followed by incubation with HRP-conjugated goat anti-rabbit antibody (Shanghai Gene Company, China) for half an hour at room temperature. The sections were stained with DAB and hematoxylin, dehydrated through graded alcohols and xylene and then mounted. Human breast cancer specimens were used as the positive control and PBS was substituted for the primary antibody as the negative control. Positively stained cells exhibited clear cell structure, accurate positioning of positive granules and a distinct contrast to the background staining. Positive staining intensity (A) was graded as follows: 0 (no staining), l (weak staining, light yellow), 2 (moderate staining, brown yellow) and 3 (brown). Positive staining cell count (B) was as follows: 1 (<1/3), 2 (1/3-2/3), 3 (≥2/3). The degree of positive staining (A × B) was as follows: A×B=0: (-), A×B=1-2: (+), A×B= 3-4: (++), A×B=6-9: (+++). Data were obtained from fi ve to ten randomly selected high magnif i cation fi elds (magnif i cation ×400).

Statistical analysis

Statistical analyses were made using the SPSS13.0 statistical software package. Differences in DAB2IP mRNA and protein levels between pancreatic cells and normal human pancreatic ductal epithelial cells were assessed by one-way ANOVA and the differences between groups were assessed using the Student-Newman-Kuels (SNK) procedure. α=0.05 (two-sided) served as the difference level. Differences in DAB2IP protein expression levels in pancreatic cancer tissues, adjacent tissues and normal pancreatic tissues were assessed using two related Wilcoxon's tests for samples. The relationship between DAB2IP protein expression and clinicopathologic parameters was analyzed using two independent Wilcoxon's tests for samples.P<0.05 was considered to be statistically signif i cant.

Results

Reduced DAB2IP mRNA and DAB2IP protein expression in pancreatic cancer cells with wild-type KRAS

qRT-PCR was used to analyze DAB2IP mRNA in four types of pancreatic cancer cells and normal human pancreatic ductal epithelial cells. DAB2IP mRNA was reduced in pancreatic cancer cells with wild-type KRAS, but this was rarely observed in cells with mutant KRAS and in normal pancreatic ductal cells (P<0.05) (Fig. 1).

Fig. 1.qRT-PCR used to analyze DAB2IP mRNA levels in four types of pancreatic cancer cells and pancreatic ductal cells.

DAB2IP protein expression was detected in pancreatic cancer cells with mutant and wild-type KRAS and normal human pancreatic ductal epithelialcells by Western blotting. In accordance with mRNA levels, DAB2IP protein expression in pancreatic cancer cells with wild-type KRAS were lower than that in those with mutant KRAS and normal pancreatic ductal cells (P<0.05) (Fig. 2).

Expression of DAB2IP in pancreatic cancer and adjacent tissues

Immunohistochemistry showed that DAB2IP expression was signif i cantly lower in pancreatic cancer tissues than that in adjacent tissues and normal pancreatic tissues (Z=-4.000,P=0.000) (Table 1, Fig. 3).

Relationship between DAB2IP expression and KRAS type in pancreatic cancer tissues

Sequencing of pancreatic cancer tissues (n=20) revealed 15 cases with KRAS gene mutations (8 cases: codon 12 GGT to GAT (G-D); 5 cases: codon 12 GGT to GTT (G-V); 2 cases: codon 61 CAA to CTA (Q-L), and 5 cases with wild-type KRAS (Fig. 4). Therefore, the mutation rate was 75%. DAB2IP expression was lower in pancreatic cancer patients with the wild-type KRAS gene than that in patients with KRAS mutations (WilcoxonW=35.000,P=0.042) (Table 2).

Fig. 2.DAB2IP protein expression levels were detected in pancreatic cancer cells with different types of KRAS and pancreatic ductal cell by Western blotting.

Fig. 3.Expression of DAB2IP was investigated by immunohistochemistry (original magnification ×400).A: positive control (breast cancer);B: negative control (pancreatic cancer, PBS was substituted for the primary antibody);C: normal pancreatic tissue;D: pancreatic cancer tissue with wild-type KRAS;E: pancreatic cancer tissue with mutant KRAS;F: adjacent tissue.

Relationship between DAB2IP protein expression and clinicopathologic parameters of pancreatic cancer patients

The relationship between DAB2IP protein expression and clinicopathologic parameters of pancreatic cancer patients was investigated to assess clinical signif i cance. DAB2IP expression in patients with perineurial invasion (PNI) was lower than that in those without PNI (WilcoxonW=71.500,P=0.028). DAB2IP expression was lower in patients with more advanced clinical stage than that in those with early clinical stage (WilcoxonW=54.000,P=0.002). DAB2IP expression was not inf l uenced by gender, age, maximum tumor diameter, inf i ltration depth and lymph node metastasis (P>0.05) (Table 3).

Table 1.Differences in DAB2IP protein expression between pancreatic cancer and adjacent tissues

Table 2.Relationship between DAB2IP expression and KRAS type in pancreatic cancer tissues (n=20)

Fig. 4.Mutational analysis of pancreatic cancer tissues.A: KRAS 12 codon wild-type;B: KRAS 12 codon GGT to GTT (G-V);C: KRAS 12 codon GGT to GAT (G-D);D: KRAS 61 codon wild-type;E: KRAS 61 codon CAA to CTA (Q-L).

Table 3.Relationship between the DAB2IP protein expression and clinicopathologic parameters of pancreatic cancer patients

Discussion

A relatively high proportion of human pancreatic cancers (60%-90%) are associated with oncogenic mutations of KRAS. However, the role of wild-type KRAS and the Ras signaling pathway in the progression of pancreatic cancer remains to be elucidated. DAB2IP is one of 16 types of Ras GAPs that have been discovered over recent years. DAB2IP was identif i ed as a protein that interacts with the tumor suppressor DAB2/DOC2 or the apoptosis signal-regulating kinase 1 (ASK1) in tumor necrosis factor (TNF)-mediated JNK/P38 MAPK pathways.[14,15]Analysis of the genomic organization of DAB2IP revealed that DAB2IP contains a common structural region called the GAP-related domain (GRD), which is the catalytic unit of this protein and activates the GTPase activity of Ras protein. DAB2IP also has Ras GAP activityin vivoandin vitro.[16]Recent studies demonstrated that DAB2IP was frequently downregulated in metastatic prostate cancer and was therefore associated with poor prognosis. Furthermore, DAB2IP knockdown cells were found to be resistant to radiation-induced apoptosis.[17-21]Some studies[22-24]demonstrated that DAB2IP is reduced in human liver cancer, thus indicating that DAB2IP acts as a tumor suppressor.

In this study, qRT-PCR and Western blotting analyses showed that DAB2IP expression was reduced at the mRNA and protein levels in pancreatic cancer cells with wild-type KRAS, although this effect was rarely observed in those with mutant KRAS genes and pancreatic ductal cells. In accordance with these results, immunohistochemical analysis indicated that DAB2IP expression was lower in pancreatic cancer compared with adjacent and normal pancreatic tissues. DAB2IP expression was lower in pancreatic cancer patients with wild-type KRAS gene compared with that in the KRAS mutant group. These results suggested that wild-type KRAS tumors also possess abnormal Ras signaling pathways.

The pancreas is a highly innervated organ. Pancreatic cancer easily invades the perineural space from the weakened areas of the perineurium. It is generally accepted that PNI is responsible for high recurrence and pain and is an independent prognostic factor in pancreatic cancer whereby more serious neural inf i ltration is associated with poor prognosis.[25,26]Immunohistochemical analysis revealed a correlation between the expression of DAB2IP and the presence of PNI and clinical stage. Expression of DAB2IP was lower in patients with PNI compared with those without PNI. DAB2IP expression was lower in advanced clinical stage patients compared with that in early clinical stage patients. These observations suggest that decreased DAB2IP expression is associated with the development and inf i ltration of pancreatic cancer and that DAB2IP is a potential prognostic indicator of this disease.

In conclusion, our data indicate that DAB2IP acts as a pancreatic cancer suppressor. Furthermore, DAB2IP expressions are correlated with the type of the KRAS gene, the presence of PNI, and the clinical stage of pancreatic cancer. DAB2IP is implicated as a potential prognostic indicator and a promising molecular target for pancreatic cancer therapy. However, the specif i c mechanism by which DAB2IP inf l uences the development and progression of this disease remains to be fully elucidated. Further studies to explore some potential mechanisms by which DAB2IP expression inf l uences the pathogenesis or natural progression of pancreatic cancer are required.

Contributors:LZJ proposed the study. DYF and LZJ performed research and wrote the fi rst draft. DYF and LDF collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. LZJ is the guarantor.

Funding:This work is supported by grants from the Project of International Cooperation in Guangzhou Province (2010B050700014) and the Science and Technology Planning Project of Guangzhou (2011J4100006).

Ethical approval:Not needed.

Competing interest:No benef i ts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received September 26, 2012

Accepted after revision December 19, 2012

AuthorAff i liations:Southern Medical University, Guangzhou 510515, China (Duan YF); Department of Gastroenterology (Duan YF, Qin YX, Li LF and Li ZJ), Medical Research Center (Li DF and Lin QX), and Department of Pathology (Liu YH and Mei P), Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China

Zi-Jun Li, PhD, Department of Gastroenterology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China (Tel: 86-20-83827812-61921; Fax: 86-20-64085875; Email: zijunli2005@yahoo.com.cn)

? 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(13)60032-6