The Study For The Abnormal Expression Of DAB2IP And Its Significance In Pancreatic Cancer

Background:It is well-known that pancreatic cancer is one of the most malignant tumors. Due to the absence of effective methods for early diagnosis, pancreatic cancer is associated with high overall mortality (almost100%), very low surgical excision rates and an overall five-year survival rate of less than5%. Chemotherapy and radiation treatment are ineffective in most patients. Therefore, investigation of novel diagnosis and therapeutic targets is urgently required.The signaling pathways which correlate with pancreatic cancer include RAS-RAF-MAPK pathways. PI3K-AKT pathways. NF-κB pathways. EGFR pathways. TGF-βpathways. WNT pathways、Hedgehog (Hh) pathways. Notch pathways. STAT pathways and mucin pathways and so on.These signaling pathways play an important role in pathogenesis of pancreatic cancer through influencing the proliferation or apoptosis of cancer cells.Here,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 while, RAS-GAPs promote the intrinsic GTPase activity of the small G proteins, resulting in the conversion of RAS-GTP to RAS-GDP. Oncogenic RAS mutants have alterations that affect intrinsic properties of the 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. KRAS mutations can 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. By comparison, the affinities of Ras-GAPs for wild-type RAS are much higher. 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.There are16kinds of RAS-GAPs which had been reported in recent years:RASAL1, RASAL2, RASAL3, RASA1, RASA2, RASA3, RASA4,IQGAP1, IQGAP2, IQGAP3, DAB2IP, SYNGAP1, GAPVD1, ARHGAP5, G3BP1, NF1. DAB2IP is one of16types 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.Aim:In this study, we sought to define the expression of the RAS-GTPase activating protein, DAB2IP, in pancreatic cancer cells and tissues and explored the potential role and clinical significance of DAB2IP in pancreatic cancer.It will provide a theoretical basis for further elucidation of the molecular mechanisms of pancreatic cancer development process and a new target of pancreatic cancer diagnosis and treatment.Method:1. The cell lines (wild-type (WT) KRAS:Bxpc-3; mutant KRAS: Capan-2,Sw1990,CFPAC-1,Aspc-1,Panc-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 at37℃under an atmosphere containing5%CO2. Total RNA was extracted from five types of pancreatic cancer cells (WT KRAS:Bxpc-3; mutant KRAS:Capan-2,Sw1990,CFPAC-l,Aspc-l,Panc-1) and the normal human pancreatic ductal epithelial cells H6C7using TRIzol reagent (Invitrogen,USA)according to the instructions provided by the manufacturer. Primers for amplification of16kinds of RAS-GAPs (RASAL1, RASAL2, RASAL3, RASA1, RASA2, RASA3, RASA4,IQGAP1, IQGAP2, IQGAP3, DAB2IP,SYNGAP1, GAPVD1, ARHGAP5, G3BP1, NF1) and GAPDH (control) were designed by Shanghai Jierui Company(China). Quantitative real-time PCR analysis (qRT-PCR) was performed using an ABI PRISM7500Quantitative PCR system. Amplification conditions were as follows:95℃for10min followed by50cycles of (95℃for30s,58℃for30s and72℃for30s). 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 values=2-△CT, in which the ACT value for each GAP was calculated by subtracting the average CT value for the target gene from the average CT value for the GAPDH gene.According to the RAS-GAP expression spectrum in pancreatic cancer we examined and RAS-GAP activity table reported in previous literature,we chose DAB2IP which have different expression between pancreatic cancer cells with WT KRAS and those with mutant KRAS and have GAP activity to do further research.2. Proteins were extracted from WT KRAS and mutant KRAS pancreatic cancer cells and normal human pancreatic ductal epithelial cells using RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined with a BCA kit. Samples of the total protein lysates (10μl) were subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes. Polyclonal anti-DAB2IP (1:2000, Abcam) and anti-GAPDH (1:7500,Biowrld) antibodies were used as primary detection reagents and horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000,CST) 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 BandScan5.0.3. Paraffin-embedded specimens of pancreatic cancer tissues paraffin section and related adjacent tissues paraffin section (n=20) were collected during the period from2008to2009in Guangdong General Hospital. Normal pancreatic tissue paraffin section(n=2) were also used as controls. Patients had not received preoperative chemotherapy and radiotherapy or immune/biological intervention. Genomic DNA was extracted using QIAamp DNA FFPE Tissue kit (Qiagen company, China) and used as a template for amplification of the KRAS common mutations point (KRAS12、13、61codon) by PCR. The PCR product was rinsed, pre-denatured and transferred to96-well plates for direct sequencing using an ABI3100Genetic Analyzer to detect KRAS type of pancreatic cancer tissues.4. Paraffin-embedded tissue specimens were sectioned (4μm thick) and slides prepared using standard techniques. Mounted tissue sections were baked at65℃for3h, deparaffinized in xylene and rehydrated through graded alcohols. Antigens were retrieved by heating in1μM sodium citrate (pH6.0) in a pressure cooker at100℃for3min, followed by incubation in3%H2O2for10minutes at room temperature to destroy endogenous peroxidase activity. Non-specific staining was blocked by incubation in10%sheep serum for10min. Tissue sections were incubated with anti-DAB2IP (1:200,Abcam) primary antibodies overnight at4℃, followed by HRP-conjugated incubation with a EnVision goat anti-rabbit antibody secondary antibody (Shanghai gene company, China) for half an hour at room temperature. 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),1(weak staining, light yellow),2(moderate staining, brown yellow) and3(brown). Positive staining cell count (B) was scored as follows:1(<1/3),2(1/3-2/3),3(≥2/3). The degree of positive staining (AxB) was represented as follows:AxB=0:(-),AxB=1-2:(+),AxB=3-4(++), AxB=6-9:(+++). Data were obtained for five to ten randomly selected high magnification fields.5. All statistical analyses were performed using the SPSS13.0statistical software package. Differences in DAB2IP mRNA and protein levels between the pancreatic cells and normal human pancreatic ductal epithelial cells were assessed by the one-way ANOVA and the differences between groups were assessed using the Student-Newman-Kuels (SNK) test. Differences in DAB2IP protein levels between pancreatic cancer tissues and adiacent tissues were assessed by Independent-samples T test.α=0.05(two-sided) as the difference level. Differences in DAB2IP protein expression levels between pancreatic cancer paraffin-embedded tissues and adjacent paraffin-embedded tissues were assessed using two related samples Wilcoxon tests. The relationship between DAB2IP protein expression and clinicopathologic parameters was analyzed using two independent samples Wilcoxon tests. The change of DAB2IP、AKT、P-AKT、ERK、P-ERK、JNK and P-JNK expression levels、 RAS activity and cell viability in48hours after transfecting DAB2IP siRNA were assessed by the one-way ANOVA. P<0.05was considered to be indicative of significance.Result:1.Both the DAB2IP expression levels of mRNA and protein decreased in pancreatic cancer cells comparing with that in the normal human pancreatic ductal epithelial cells (P<0.05). Reduced DAB2IP expression were detected in pancreatic cancer cells with WT KRAS expressing than those those with mutant KRAS (P<0.05)2. Sequencing of pancreatic cancer tissues (n=20) revealed15cases with KRAS gene mutations (8cases:codon12GGT to GAT(G-D);5cases:codon12GGT to GTT(G-V);2cases:codon61CAA to CTA(Q-L), and five cases with WT KRAS. Therefore, the mutation rate was75%.DAB2IP expression was lower in pancreatic cancer tissues than in adjacent tissues (Z=-4.000,P=0.000). DAB2IP expression was lower in pancreatic cancer patients with the wild-type KRAS gene than in those with KRAS mutations (WilcoxonW=35.000, P=0.042). Furthermore, DAB2IP expression in patients with perineurial invasion (PNI) was lower than those without (WilcoxonW=71.500, P=0.028). DAB2IP expression was lower in patients with more advanced stage than in those with early clinical stage (WilcoxonW=54.000, P=0.002). Furthermore, DAB2IP expression was not influenced by gender, age, maximum tumor diameter, infiltration depth and lymph node metastasis (P>0.05).3. Silencing DAB2IP in wild-type KRAS pancreatic cancer cell by DAB2IP siRNA increased the expression of P-Akt、P-ERK and RAS activity,coupled with decreased expression of P-JNK.(P<0.05).It can also increased the proliferation of wild-type KRAS pancreatic cancer cell Bxpc-3(P<0.05)Conclusion:The expression of DAB2IP decreased in pancreatic cancer. This effect correlated with the type of KRAS gene, the presence of PNI and clinical stage of the disease. Selective suppression of DAB2IP results in unrestrained activation of RAS signal pathway in pancreatic cancer cell with wild-type KRAS gene,and increases its proliferation. Thus indicating that DAP2IP expression levels serve as a potential prognostic indicator and promising molecular target for therapeutic intervention in pancreatic cancer patients.