The Role Of Lgr5in Angiogenesis Of Gastric Adenocarcinoma And Its Molecular Regulation Mechanism

Gastric cancer is one of the leading causes of cancer related death in China. Itsfive-year survival rate is not more than40%. Metastasis and recurrence are the principalreasons for the death of gastric cancer patients. Thus exploring the molecularmechanisms of metastasis and recurrence in gastric cancer is fundamental forunderstanding the mechanism of gastric cancer and crucial of improving the clinicaltreatment efficacy. Currently, a large number of studies have shown that the occurrence,development, recurrence and metastasis of cancer is a multi-stage process withcomposite factor. Angiogenesis is a key role in the growth and recurrence of tumor. Thesuppression of angiogenesis can significantly inhibit tumor growth. It has been thehotspot in cancer research to find new therapeutic targets for suppressing angiogenesis.Lgr5is also known as-GPR49. Recent research found it is more specific than theconventional cell surface markers (e.g. CD133, CD44) as the cell surface marker forgastrointestinal tract, follicles or other tumor stem cells such as colorectal cancer cellsand glioblastoma cells. In the former study, we found that Lgr5protein correlated withangiogenesis related gene expression, indicating that Lgr5may play an important role inthe angiogenesis, but the exact function and mechanism are not clear. Therefore,exploiting the mechanism of Lgr5in the angiogenesis of gastric cancer has significanttheoretical and practical importance for explaining the destructive behaviors of gastriccancer and searching for the new targets to suppress angiogenesis.Aim: Lgr5is taken as a molecular marker for tumor. Analyze the relation betweenLgr5protein expression and prognosis in gastric carcinoma and evaluate the possibilityto be an independent risk factor. Investigate the correlation between Lgr5andangiogenic factors (e.g. VEGF and MVD) and discuss its potential relationship withangiogenesis. Then Lgr5over-expression plasmid is constructed. Observe the ability ofover-expression Lgr5cells in proliferation, migration and apoptosis and so on.Bioinformatics analysis and immunological technology is carried out to find thepotential signaling pathways and its regulation mechanism in the gastric cancer angiogenesis. This study will provide the theoretical basis for searching novel target tosuppress angiogenesis.Methods： Specimens were taken from115patients with gastric cancer confirmedby pathology during2005-2006. Immuno-histochemical staining of Lgr5, CD34, VEGF,p-AKT were performed. Lgr5-GFP recombinant plasmid was constructed andtransiently transfected to BGC823gastric cells. Cell growth, proliferation and migrationcapacity with Lgr5over-expression were investigated by plate-cloning, MTT, Transwel.Flow cytometry and Caspase3activity detection were applied to investigate theanti-apoptotic ability of Lgr5over-expressing cells. Bio-informatical analysistechnology to forcast the angiogenesis-associated protein related to Lgr5, which isconfirmed by Western blot detection. The monoclonal antibody was used to block theVEGF/VEGFR signaling pathway and observe downstream molecular changes. Therelative spatial location between Lgr5and p-AKT was studied throughimmune-fluorescence colocalization. The candidate protein-protein interaction wereconfirmed by co-immunoprecipitation.Results： Lgr5expression significantly related (P<0.05) to prognosis in gastriccancer patients. Lgr5could be considered as an independent indicator for gastric cancerprognosis judgment. Lgr5expression and vascular generate relevant indicators (MVD,VEGF) was positively correlated (P<0.05). In combination with tumor angiogenesisindicators, Lgr5can improve the accuracy of the prognosis of gastric cancer.pEGF-Lgr5over-expression plasmid was successfully constructed. Lgr5over-expression can be significantly enhanced gastric cancer cell growth, proliferation,anti-apoptosis and migration. Via Wnt/β-catenin signaling pathway, Lgr5can promoteTCF/LEF nuclear transcription factor recruitment, enhance VEGF mRNA transcription,expression and angiogenesis. VEGF is able to activate gastric cancer cellVEGF/VEGFR auto-crine pathway, and sustained activation of downstream PI3K/AKTsignaling pathway, VEGF expression up-regulate and promote angiogenesis. Negativeregulation of PTEN/PI3K/AKT signaling pathway is not significantly enhanced andfailed to effectively suppress angiogenesis. Lgr5over-expression can still effectivelyactivate PI3K/AKT pathway to promote angiogenesis even during blocking VEGF/VEGFR signaling pathway. Lgr5can be independent on the VEGF/VEGFR pathway,but directly interacts with p-AKT protein and produce non-PI3K-dependent activationin order to activate PI3K/AKT pathway and promote angiogeanesis. Conclusions： Lgr5is an emerging potential molecular marker for cancer stemcells. It can be used as a crucial independent risk factor for gastric cancer prognosis. Itsmalignant biological behavior closely relates to gastric cancer proliferation, migration,and promotion of tumor angiogenesis. Lgr5regulates angiogenesis via the network ofWnt/β-catenin-VEGF/VEGFR2(autocrine)-PI3K/AKT-VEGF. Lgr5can still effectivelyactivated PI3K/AKT/VEGF pathway even during the interference of VEGF and blockVEGF/VEGFR2signaling pathway. Its potential mechanism is to interact with p-AKTprotein and produce a non-PI3K-dependent activation effect, thereby collaborativelypromote tumor angiogenesis with Lgr5/Wnt/β-catenin signaling pathway.