Mechanism And Clinical Significance Of IGFBP2 In Regulation Of Invasion And Metastasis Of Pancreatic Cancer

Objective To determine if IGFBP2 is a potential biomarker for diagnosis and prognosis prediction of pancreatic cancer, we detected the level of IGFBP2 in serum sample from pancreatic cancer patients as well as tumor tissue and performed comprehensive analysis of these data together with clinicopathologic information of patients from our cohort. We also observed the effect of IGFBP2 on invasion and metastasis of pancreatic cancer cell and its related mechanism.Methods 1. Collected serum sample from pancreatic cancer patients, chronic pancreatitis and healthy controls. ELISA assay was adopted to detect the level of IGFBP2 in each group respectively. The comparison of IGFBP2 level in different group was performed. Evaluation of IGFBP2 was carried out by comparing CA19-9 and IGFBP2 level in different sub-groups based on clinicopathologic diameters as well as ROC curve of each marker respectively. Survival analysis was carried out based on followed up survival data stratified by different IGFBP2 level. IGFBP2 expression level was also evaluated in tumor tissue sample by immunohistochemistry assay. Logistic regression was used to reveal to relationship of IGFBP2 expression level and clinicopathologic parameters. At last we used Kaplan-Meier Curve to show the correlation of IGFBP2 expression level and prognosis of patients. 2. Constructed IGFBP2 overexpression plasmids and si RNA and observed the effect of IGFBP2 on morphology and biological behavior of pancreatic cancer cell. Western Blot and Immunofluorescence were used to detect the EMT related genes expression and cellular localization. IGFBP2 overexpression stable cell was used to build orthotopic animal model to evaluate the effect of IGFBP2 on metastasis in our in vivo model. 3. c DNA microarray was used to reveal the differential expression of certain genes followed IGFBP2 overexpression and Ingenuity pathway analysis(IPA) software was used to predict the potential activated pathway. Extracted the cellularnuclear and plasma protein and then used Western Blot to detect the p65---major subunit of NF-κB expression and perform immunofluorescence to confirm the cellular localization of p65. Consecutive tissue slides were adopted to confirm the correlation of IGFBP2,p65 E-cadherin and Vimenin.Combination of IGFBP2 recombinant protein and inhibitor of certain pathway was used to validate which pathway was involved in IGFBP2 activated NF-κB. Genetic modified MEF cell and PTEN plasmids was also adopted for mechanistic study.Results 1. We found IGFBP2 was specifically elevated in pancreatic cancer(435.7 ± 234.2 ng/ml) rather than chronic pancreatitis(233.0 ± 59.80 ng/ml)or healthy control(289.9 ± 148.2 ng/ml) group(both P value <0.05). Adjustment with age and gender would not affect the difference. Significant difference of IGFBP2 rather than CA19-9 was revealed in sub-group stratified by diabetes mellitus, lymph nodes metastasis and nerve invasion. ROC curve indicated the combination use of IGFBP2 and CA19-9 showed a more powerful diagnosis of pancreatic cancer than either marker used alone(AUC =0.921). Moreover IGFBP2 was more powerful than CA19-9 in differential diagnosis of pancreatitis and pancreatic cancer(AUC:0.817 vs 0.770, P<0.05) IGFBP2 is an independent prognostic factor of pancreatic cancer. At the cut off value of which is 333.9 ng/ml, Kaplan-Meier survival curve showed elevated IGFBP2 predicted a poorer prognosis(median survival time 13 months vs 8 months P<0.05). The IHC staining of IGFBP2 in the tumor tissue from the same cohort showed IGFBP2 was positively expressed in 71 cases(94.6 %). Logistic regression showed IGFBP2 expression level was correlated with lymph nodes metastasis and overall survival(P<0.05), survival analysis showed a poorer survival for those patients with IGFBP2 staining scoring more than 6(median survival time 9 months vs 16 months P<0.05). 2. Compare with control group, IGFBP2 overexpression cell showed a mesenchymal like morphology change and an increase of motility and invasion ability while downregulation of IGFBP2 showed a epithelia like morphology and an corresponding decrease of motility and invasion ability. However theproliferation of the cell was not changed followed IGFBP2 change. Followed with increasing IGFBP2 expression, E-cadherin and Vimentin expression showed a corresponding EMT like change. In vivo study showed IGFBP2 overexpression promoted distant metastasis of pancreatic cancer cell in orthotopic nude mouse model. 3. c DNA microarray showed IGFBP2 overexpression lead to an increase of Snail and vimentin expression companied with a decrease of CDH1 expression at transcriptional level. Pathway analysis suggested NF-κB pathway was activated by IGFBP2 overexpression.(P<0.001). Immunofluorescence and Western Blot of cellular nuclear protein showed IGFBP2 overexpression induced nuclear translocation of p65 and its phosphorylated form,which indicated an activation of NF-κB and followed by decrease expression of E-cadherin and increase of Vimentin. In the other hand, the knock down of IGFBP2 exhibited just an opposite effect. Consecutive slide staining confirmed the correlation of IGFBP2, p65, E-cadherin and Vimentin in tissue sample.IGFBP2 would not induce EMT when NF-κB is inhibited. Either the inhibititon of PI3K/Akt pathway or knock out of IKKbeta would lead to a failure of IGFBP promoted p65 nuclear localization which would cause NF-κB activation.Conclusion: 1. IGFBP2 was specifically elevated in pancreatic cancer and positively related with lymph node metastasis and nerve invasion while negatively correlated with prognosis of patients. IGFBP2 is a potential diagnosis and prognosis prediction marker for pancreatic cancer. 2. IGFBP2 induced EMT in pancreatic cancer, which lead to an increase of motility and invasion of pancreatic cancer cell. IGFBP2 induced EMT is NF-κB dependent. 3. IGFBP2 inhibit PTEN expression in pancreatic cancer cell which would lead to activation of PI3K/Akt pathway and phosphorylation of IKKbeta to degrade IκB protein. These would promote the nuclear translocation of p65 to activate NF-κB pathway.