Role And Mechanism Of L1CAM In Pancreatic Cancer Progression&Expression Pattern And Clinical Signiifcance Of IGFBP7in Pancreatic Cancer

Pancreatic cancer (PanCa) is one of the most malignant tumors overall, with anaverage5-year survival rate of less than6%. The reasons given for the poorprognosis in PDAC include rapidly disease progression and metastasis in very earlystage, especially retroperitoneal metastasis and liver metastasis. Recently, studiesfound that many growth factors, angiogenesis factors, and proteinases were involvedin progression of PanCa. Perineural invasion (PNI) is also a special way of invasionand metastasis of PanCa, and is an important reason for retroperitoneal metastasis,and also contributed to abdominal pain, and dismal prognosis in PanCa. However,mechanisms of invasion and metastasis in PanCa are still poorly understood. Furtherstudy of molecular mechanisms of invasion and metastasis in PanCa has a hightheoretical and clinical value, not only can helpful to predict progression of tumor,but also provide potential molecule targets for the treatment of PanCa. Cell adhesionmolecular L1(L1CAM) is a member of the neuronal immunoglobulin superfamily ofcell adhesion molecules, has functions of promoting tumor cell proliferation andtumor growth, inhibiting apoptosis, increasing potentiality of migration and invasionin many cancers. Recently, studies found that L1CAM expressed in PanCa,especially in poorly differentiated tissues, metastasis of lymph node and liver.Interestingly, L1CAM has also been detected in a site of neural invasion, and wasassociated with PNI in PanCa. Thus, researchers supposed that L1CAM might beinvolved in progression of PanCa. However, the mechanism for this procession isstill unclear.In addition, although great progresses have been made in the treatment ofmalignances, until now, no effective methods have been established for PanCa. Thus,defining new molecular prognostic markers, and a better understanding of molecularmechanisms of PanCa progression are greatly needed. Insulin-like growth factorbinding protein7(IGFBP7), also called MAC25or IGFBP-related protein-1, hasfunctions of regulating cell proliferation, differentiation, angiogenesis, cell adhesion,and cellular senescence in many cancer cells, IGFBP7also shows to suppress tumorgrowth by inducing cell apoptosis. Furthermore, down-regulation of IGFBP7is alsoassociated with poor postoperative survival in various types of cancers, includingcancers of colorectum, breast, and liver. However, to our knowledge, little is knownabout the expression pattern and the significance of IGFBP7in PanCa. 1. Construction and identification of lentivirus-mediatedsmall hairpin RNA targeting L1CAMObjective: To construct and identify of lentivirus-mediated small hairpin RNAtargeting L1CAM.Methods: We selected a PanCa cell line with the highest expression of L1CAM byusing real-time PCR; and transfected the cell line with lentivirus-mediated shRNAtargeting L1CAM. Transfection effects were examined by fluorescence microscope;and expression of L1CAM was evaluated by Western Blot method.Results: L1CAM expressed in Capan-2, Panc-1, AsPC-1, BxPC-3, sw1990,Patu-8988and CFPAC-1PanCa cell lines, and the expression of L1CAM in Capan-2was higher than those in other PanCa cell lines. The transfection effect of Capan-2was over85%by using20ul of viral suspension (1×108TU/ml) per5×103cells.The expression of L1CAM in Capan-2infected L1CAM-siRNA was dropped by83%compared with parental cells at96h after transfection.Conclusions: We have successfully constructed lentivirus-mediated shRNA targetingL1CAM; which can knock-down the expression of L1CAM in Capan-2.2. Establishment a vitro mode for PNI in PanCa andidentification of the role of L1CAM in PNIObjective: To establish a vitro model for PNI in PanCa, and further evaluation of therole of L1CAM in PNI by using this co-culture mode.Methods: Mouse dorsal root ganglia (DRG) and human PanCa Capan-2cell linewere co-cultured in Matrigel matrix. On this basis, L1CAM-siRNA/DRG andL1CAM-NC/DRG co-culture model were established respectively by using the samemodel. Neurite outgrowth, cell colony growth, migration, and neurite-colony contactwere quantitated by using Image pro plus software.Results: We observed that Capan-2cell could migrate to DRG, and grow around thenerve fiber. Areas of cell colonies in L1CAM-siRNA/DRG group(Day5:230.31±14.89um2) were less than those in L1CAM-NC/DRG group(Day5:308.24±18.37um2, P=0.005). However, no difference was found on neuriteoutgrowth between L1CAM-siRNA/DRG group and L1CAM-NC/DRG group.Conclusions: We demonstrated that the human PanCa cell line Capan-2was neurotropic, and Capan-2/DRG co-cultrue in vitro system provided a method tostudy specific mechanism of PNI in PanCa. We also found that L1CAM play a rolein PNI in PanCa.3. Effect of L1CAM on proliferation, apoptosis and invasiveability of Capan-2cell line and possible mechanismsObjective: To examine the role of L1CAM in proliferation, cell cycle, apoptosis andinvasion in PaCan in vitro and the involved signaling pathway.Methods: Cell proliferations were determined by the method of CCK-8; cell cyclewas analyzed by flow-cytometry. Apoptosis were analyzed by flow-cytometry, aswell as TUNEL method. Cell invasion were performed by Millicell chambers method.Ras/Raf/Mek/Erk signaling pathway proteins (Erk, p-Erk) were detected by westernblot.Results: knock-down of L1CAM expression led to suppress the proliferation andinvasion potential of Capan-2cells in vitro, without effects on apoptosis. Knockdownof L1CAM also resulted in cellular arrest in the G1phase and a reduction of G2andS phase. In determining the pathway through which L1CAM regulated cellproliferation and invasion, L1CAM was found to regulate Erk phosphorylation.Conclusions: We demonstrated that L1CAM might increase PanCa cell proliferation,invasive potential via Ras/Raf/Mek/Erk signaling pathway.4. Expression Pattern and Clinical Significance of IGFBP7in PDACObjective: To investigate the expression pattern and clinical significance of IGFBP7in human PDAC.Methods: IGFBP7expression was evaluated by immunohistochemistry in190patients with PDAC who underwent surgical tumor resection. Expression of IGFBP7was correlated with that of p53and Ki-67, clinicopathologic features. We alsoevaluated overall survival (OS) according to expression of IGFBP7by Kaplan-meierand Cox regression analyses.Results: IGFBP7expression was significantly down-regulated in pancreatic cancertissues compared to adjacent normal pancreas (p<0.001), and was inversely associated with Ki-67expression (r=-0.284, p<0.001). No significant relationshipswere found for clinicopathologic features, such as diameter of tumor, node status,grade and stage. Importantly, low expression of IGFBP7was associated with poorOS, and this was also significant in multivariate Cox regression analysis (HR,1.38;95%CI,1.00-1.91; P=0.05).Conclusions: We demonstrate for the first time that IGFBP7is down-regulated inpancreatic cancer and low expression of IGFBP7is correlated with increasedproliferation and poor postoperative survival. IGFBP7may be a tumor suppressor inPDAC.