Mechanism Of SDF-1/CXCR4 Axis In Regulating Lung Cancer Lymphangiogenesis Via Notch1 Pathway

ObjectivesLung cancer always stands first on the list of all cancers regarding the incidence and mortality.Metastasis via lymphatic vessels usually indicates progress of lung cancer,which is also an important factor for patients' poor prognoses.Here the present study was designed to investigate roles of chemokine SDF-1 and its receptor CXCR4 axis and Notchl signaling pathway in lung cancer lymphangiogenesis and to analyze possible mechanisms.MethodsThis study includes three chapters:in the first part,165 patients who received lung resection and were pathologically diagnosed with lung adenocarcinoma(I-IIIA)in Jinling Hospital during January 2010 and December 2012 were selected.Immunohistochemical(IHC)staining for CXCR4 and Notchl was taken with tumor sections,and the correlations between expression of CXCR4/Notchl and patients'clinical characters(such as age,sex,tumor size,lymph node metastasis,etc.),postoperative overall survival or microvessel density(MVD)/lymphatic vessel density(LVD)were carefully analyzed.In the second part,shRNA lentivirus vectors targeting CXCR4 or Notchl were constructed and transfected into lymphatic endothelial cells(LEC).With CXCR4 orNotch1 inhibited in LEC,mRNA levels of some related genes were detected by qPCR,and Fibrin Gel Bead Assay was conducted to find out its impact in lymphangiogenesis in vitro.In the last part,Lewis lung cancer(LLC)cells with stable suppression of CXCR4 or Notchl by lentivirus infection were subcutaneously injected into C57 mice,to make mouse LLC models.With this LLC model,tumor growth and lymphangiogenesis were examined in vivo.ResultsCo-expression of CXCR4 and Notch1 in human lung adenocarcinoma correlated with lymph node metastasis and poor postoperative prognosis,which might be results of its the promotion to higher microvessel density and lymphatic vessel density.After blocking CXCR4 in LEC cells,mRNA levels of Notchl and its downstream genes were significantly down regulated,thereby affecting lymphangiogenesis in FiBA assay.Although VEGFR3 was not significantly affected after CXCR4 or Notchl inhibition,mRNA level of soluble VEGFR3 was significantly up regulated,which might competitively inhibit the function of VEGFR3,thus indirectly suppressing lymphangiogenesis in vitro.What's more,when Notchl and CXCR4 were both blocked,mRNA levels of Notchl downstream genes and lymphangiogenesis in vitro were suppressed much more obviously.Tumor growth and lymphatic vessel density within tumor tissue could be significantly reduced after blocking CXCR4 or Notchl in mouse LLC models.Similarly,when Notchl and CXCR4 were both blocked,decrease in tumor growth and lymphatic vessel density was much more pronounced.ConclusionsOn one hand,CXCR4 could directly regulate expression of Notchl and its downstream genes to modulate lymphangiogenesis in lung cancer;on the other hand,CXCR4 and Notchl could work together to regulate the level of sVEGFR3,a competitive inhibitor of VEGFR3,so as to indirectly modulate lymphangiogenesis in lung cancer.Also,the idea that blocking CXCR4 and Notch1 could inhibit tumor growth and lymphangiogenesis in lung cancer could possibly help the promotion of anti-lymphangiogenesis therapy in future.