The Study On The Mechanisms Of IGF-1 Promotes Growth Of Gastric Cancer By Inhibiting Foxo1 Nuclear Retention

Background and ObjectiveGastric cancer (GC) is the fourth most common gastrointestinal malignant human cancer and the second leading cause of death worldwide, with an incidence of approximately one million per year. Nearly half of gastric cancer occurs in East Asia, especially in China. The majority of the subjects of GC were diagnosed at a late stage, resulting in poor prognosis and therapeutic outcome. GC is such a serious public health burden that significantly impairs the quality of patients’ life. The occurrence and development of GC is a complex biological process involving multifacTor, multistep, and multigene. So far, the molecular mechanisms underlying the tumorgenesis of GC are not completely understood.The insulin-like growth factors(IGFs) are a group of protein with high sequence similarity to insulin. The IGF signaling pathway initiates from initiating from binding to cell-surface receptors (IGF-1R and IGF-2R) by either ligands(insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2(IGF-2)). IGF-1R, a receptor tyrosine kinase, appears to be the major functional receptor for IGF-1 and initiates MAPK signaling pathway and PI3K/Akt signaling pathway, which play important role in the cell growth and proliferation, apoptosis, cell differentiation and angiogenesis. Forkhead box protein 01 (FoxO1),a forehead transcription factor family member, has been shown to be a major target protein for the activated P13K/Akt signaling. FoxO1 orchestrates programs of gene expression that regulate cell proliferation, apoptosis and transition. FoxO1 proteins are regulted by phosphorylation, ubiquitination and acetylation.The role of IGF-1/IGF-1 R/Akt/FoxO1 signaling pathway on the pathogenesis of GC has rarely been reported. In this study, we explore a regulatory mechanism and biological functions of IGF-1 signaling pathway in the events of GC to provide reliable theoretical basis on diagnosis and treatment of GC.Materials and methods1.A total of 55 subjects(26 control without GC, and 29 GC patients) were included for the analysis of serum IGF-1 level. All specimens were obtained and pathologically diagnosed at Department of gastroenterology, Shandong Provincal Qianfoshan Hospital of Shandong University from 2008 to 2013. Serum IGF-1 level was measured by using immunoradiometric assays. Phosphorylated IGF-1R protein was detected in 29 GC tissue and adjacent normal tissues by using Western blot.2.Human GC cancer AGS was analyzed for this study. After treatment of IGF-1(100ng/ml), Akt inhibiTor LY294002(20 μ mol/1) and rapamycin(200nmol/1) respectively, western blot was employed to detect the protein expression of phosphorylated IGF-1R、phosphorylated Akt and phosphorylated mTOR. MTT was used to measure growth curve for cell proliferation of GC.3. Constitutively nuclear FoxO1-expressing plasmid was transfected into AGS cells by Liperfectamine 2000. The transfected cells were termed as AGS-nFoxO1. Immnoucytchemistry was used to detect the location of FoxO1. Western bolt was applied to investigate the expression of phosphorylated IGF-1R、phosphorylated Akt and phosphorylated mTOR after treatment of IGF. MTT was used to measure growth curve for cell proliferation of GC after constitutively nuclear FoxO1 expression.Results:1. Significantly higher levels of IGF-1 were detect in the serum of 29 GC patients than 26 control subjects(CTL).(p <0.05), suggesting IGF-1 activity in GC patients. Compared to adjacent normal gastric tissue (NGT), a higher activation of IGF-1R were detected in GC tissues. (p <0.05)2. IGF-1 significantly increased phosphorylation of IGF-1R, the phosphorylation of Akt and mTOR, and significantly increased the cell growth. Moreover, Akt inhibiTor LY294002 completely abolished the effected of IGF-1 on the phosphorylation of Akt ,the phosphorylation of mTOR, and increased in the cell growth, without effect IGF-1R. mTOR inhibiTor rapamycin completely abolished the effect of IGF-1 on the phosphorylation of mTOR without affecting phosphorylation of either IGF-1R or Akt. Besides, rapamycin did not inhibit the IGF-1-induced increase in the cell growth.3. Immunocytochemistry showed IGF-1 induced nuclear exclusion of FoxO1 in AGS cells. AGS-nFoxO1 expressed high levels of nuclear FoxO1,which cannot be phosphorylated due to a defect on phosphorylation site.Sustained nuclear FoxOl completely abolished the effects of IGF-1 on cell growth.Conclusion1. IGF-1R signaling plays a role in the pathogenesis of gastric cancer.2. IGF-1 induced Akt phosphorylation enhances GC cell growth, Akt is downstream of IGF-1 recepTor activation, and is upstream of mTOR signaling pathway.3. IGF-1-induced increase in GC cell growth is mTOR-independent.4. Nuclear exclusion of FoxO1 by IGF-1/Akt is responsible for IGF-1 induced increases in the cell growth of GC.5. IGF-1R/Akt/FoxO1 axis serves as a novel therapeutic target for inhibiting the growth of GC.