| BackgroundPancreatic cancer is well acknowledged as one of malignancies with extremely dismal prognosis, because the mortality is close to the incidence. A survey of American Cancer Society about cancer incidence and mortality in the United States showed that pancreatic cancer ranked the fourth of all malignant tumors. During the past10years, the incidence of pancreatic cancer increased by about15%, but its5-year survival rate is still below5%. Because of absence of specific clinical manifestations, early diagnosis of pancreatic cancer is difficult, and lymphatic/blood metastases are common. Thus, many patients lost the opportunity of radical treatments. The main reason of death is common advanced disease with invasion of blood vessels and distant metastasis in many patients when diagnosed. The mechanisms of invasion and metastasis of pancreatic cancer is currently unclear. Therefore, investigations concerning invasion and metastasis of pancreatic cancer are of great significance. Chemokines were previously thought to be involved in the inflammatory response. In recent years, they are found to play important roles in initiation and development of cancer, especially in migration and invasion. Our research group identified CXCR7as one of the important differentially expressed genes between pancreatic cancer and normal pancreatic tissues by microarray screening in the DMBA-induced pancreatic cancer model in rats. CXCR7is a recently discovered chemokine receptor, which plays important roles in tumor development, invasion and metastasis. CXCL12, an important ligand of CXCR7, is extensively expressed in many malignant cells, but is rarely expressed in normal cells. Many studies have shown that CXCL12-CXCR7axis promotes tumor growth, invasion and metastasis. However, there is no study referring to its impact on invasion and metastasis of pancreatic cancer, and relative mechanisms. Therefore, exploration of effects of CXCL12-CXCR7axis in the invasion and metastasis in pancreatic cancer is quite important for deepening our understanding for molecular mechanisms of pancreatic cancer. Objectives:1) To observe the effect of CXCL12-CXCR7axis in pancreatic cancer cell proliferation, migration and invasion;2) To explore mechanisms of CXCL12-CXCR7axis in pancreatic cancer cells;3) To validate the impact of CXCR7on growth and metastasis of implanted pancreatic cancer cell in nude mice;4) To analyze the value of CXCL12and CXCR7in predicting the prognosis of pancreatic cancer patients.Methods1) Lentiviral vectors in that CXCR7gene or interfering miRNA of CXCR7was inserted were transferred into pancreatic cancer cell lines, BxPC-3and Capan-1. After antibiotic resistant screening, CXCR7stably silenced or overexpressed pancreatic cancer cell lines were established.2) CCK8assay was used to observe cell proliferation; Transwell migration and invasion models were allied to determine the migration and invasion capabilities of cells; expression and phosphorylation of the proteins in mTOR pathway were detected by Western blot. Through these experiments, influences of CXCR7on phenotypes of pancreatic cancer cells and relative mechanisms were explored.3) Different concentrations of recombinant human CXCL12was used to treat cells (with or without AMD3100, a inhibitor of CXCR4), Transwell model was used to detect migration and invasion, Western blot analysis was adopted to detect expression and phosphorylation of related proteins. All the experiments focused on molecular mechanisms of CXCL12in biological behaviors of pancreatic cancer cells.4) In CXCR7stably silenced or overexpressed cells, the mTOR inhibitor, rapamycin or Torinl, was added, then cell migration and invasion was detected by Transwell assay, expression and phosphorylation of proteins in mTOR pathway was determined by Western blotting. Co-immunoprecipitation was used to show the interaction between CXCR7and mTOR. In cell lines in that CXCR7was stably silenced, Western blot was used to detect expression of proteins in ROCK pathway, downstream of mTOR. In CXCR7stably silenced and parent cells, the mTOR inhibitor, rapamycin or Torinl, was added, then F-actin expression was detected by immunofluorescence staining.5) Orthotopic xenograft liver metastasis model of pancreatic cancer was established in BALB/C nude female mice. Cells were injected orthotopically. Two months later, the mice were sacrificed, the pancreatic tumor volumes of tumor were calculated, and the liver metastatic numbers were counted.6) The prognostic value of CXCL12and CXCR7was explored using immunohistochemical staining.Results1) Based on pancreatic cancer cell lines, BxPC-3and Capan-1, CXCR7stably silenced or overexpressed cell lines were successfully established.2) There was no significant change in proliferation between CXCR7stably silenced or overexpressed cells and control ones (p>0.05). Migration and invasion of CXCR7overexpressed cells were significantly increased compared with control cells, accompanied by enhanced phosphorylation of mTOR,4EBP1and P70S6K; while the opposite trend was observed in CXCR7stably silenced cells, phosphorylation of corresponding proteins was also significantly reduced; regardless of cells in that CXCR7was stably silenced or overexpressed, expression of AKT and p-AKT was not significantly changed.3) After BxPC-3and Capan-1cells were treated with different concentrations of recombinant human CXCL12, migration and invasion of both cell lines were significantly enhanced, CXCR7expression was significantly up-regulated, phosphorylation of mTOR,4EBP1and P70S6K was also significantly increased. Application of AMD3100, the CXCR4inhibitor, in CXCL12-treated cells, did not cause alterations in migration and invasion (p>0.05), CXCR7expression and expression as well as phosphorylation of mTOR,4EBP1and P70S6K.4) After the mTOR inhibitor, rapamycin or Torinl, was added in cells stably overexpressing CXCR7, migration and invasion were significantly reduced, and phosphorylation of mTOR,4EBP1and P70S6K was significantly reduced. Co-immunoprecipitation showed the direct interaction between mTOR and CXCR7. The expression of proteins in ROCK pathway was significantly reduced in CXCR7stably silenced cells. Immunofluorescence staining showed that F-actin was disorganized and depolymerized, when CXCR7was stably silenced, or the mTOR inhibitor, rapamycin or Torinl, was used.5) In pancreatic cancer orthotopic xenograft model in nude mice, growth of orthotopic implanted tumors in the CXCR7stably silenced or overexpressed group was not significantly changed compared with the control groups; the liver metastatic nodule number of CXCR7stably overexpressed group was significantly more than that of the control group (p<0.05), while that of CXCR7stably silenced group was significantly decreased in contrast to the control group (p<0.05).6) Immunohistochemical staining showed that expression of CXCL12and CXCR7was significantly higher in tumor tissues than in para-tumor tissues (p<0.05), and high expression of CXCL12and CXCR7was positively correlated with perineural invasion (p<0.05). Univariate analysis showed that gender, tumor differentiation, lymph node metastasis, CXCL12and CXCR7expression were associated with prognosis (p<0.05), whereas Cox regression analysis identified male gender, poor differentiation, lymph node metastasis, CXCL12and CXCR7co-expression as independent risk factors of unfavorable prognosis (p<0.05).Conclusions1) CXCL12-CXCR7axis had no significant influence on the proliferation of pancreatic cancer cells, but it can extensively accelerate cell migration and invasion.2) Activation of mTOR signaling pathway is involved in the promotion of CXCL12-CXCR7axis for migration and invasion of tumor cells, which is based on the direct interaction between CXCR7and mTOR and its subsequent impact on cell motility through ROCK pathway.3) CXCR7can promote liver metastasis in the pancreatic cancer orthotopic xenograft model, but had no significant effect on the growth of orthotopic tumors.4) CXCL12and CXCR7were highly expressed in pancreatic cancer tissues, and their co-expression was an independent prognostic risk factor. |
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