The Role And Correlated Molecular Mechanism Of Integrin αυβ6 In The Survial And Invasion Of Colon Cancer Cells

BackgroundIntegrins,which are composed of transmembraneαandβsubunits,are a family of transmembrane glycoprotein adhesion receptors that interact with the extracellular matrix(ECM) of basal membranes.These interactions contribute to cell adhesion and cell to cell contacts and let integrins transmit "outside-in signals" that trigger a variety of intracellular signaling events such as calcium flux,synthesis of inositol lipid and cyclins,alteration of intracellular pH,and activation of focal adhesion kinase(FAK),mitogen activated protein kinase(MAPK) and phosphatidylinositol-3 kinase(PI3K). Integrin-mediated signals also regulate a number of cellular functions that have an impact on the initiation as well as the progression of cancers, including proliferation,apoptosis,cell motility,invasion,and angiogenesisIntegrinανβ6 is a receptor for fibronectin,tenascin,vitronectin,and latent tumor growth factor-β.This integrin is restrictedly distributed to epithelial cells. It is primarily expressed during fetal development and wound healing.Down regulation of the integrinβ6 subunit reduces inflammatory responses in the lungs and skin.Up-regulation of the integrinανβ6 is found in epithelial carcinomas including the oral cavity,stomach,lung,breast and pancrea.The integrinανβ6 now emerges as an attractive new candidate as a therapeutic target for metastatic colon carcinoma.In our previous work,we have reported the relationship between integrinανβ6,MMP-9,MMP-2 and ECM degradation in colon cancer.We reported thatανβ6 expression in colon cancer cells lead to a relative increase in secretion of the matrix metaUoproteinase gelatinase B(MMP-9) over its respective inhibitor and that this secretion paralleled the level of cell-surfaceβ6 expression.Theανβ6-mediated gelatinase B secretion was associated with increased proteolysis of denatured collagen at the cell surface,and inactivation of gelatinase B inβ6 -expressing tumour cells inhibited cell spreading and proliferation within 3-dimensional collagen matrices,we previously demonstrated a direct link between the extracellular signal-regulated kinase ERK2 and the growth-promoting cell adhesion molecule,integrinανβ6,in colon cancer cells.Down- regulation ofβ6 integrin subunit expression inhibited tumour growth in vivo and MAP kinase activity in response to serum stimulation.Deletion of the ERK2 binding site on theβ6 cytoplasmic domain also inhibited tumour growth Integrin-mediated MMP-9 secretion was dependent upon direct binding between theβ6 integrin subunit and extracellular signal-regulated kinase which lead to outside-in signalization through the MAPK pathway,and was involved in tumour growth and integrin expression.The most compelling finding is thatανβ6 is defined as a novel,independent prognostic indicator for aggressive colon cancer.Our recent study also shown that patients who wereανβ6-positive had higher rates of liver metastasis than those who wereανβ6-negative.The expression ofανβ6 was detected in 36.7% of gastric carcinomas,and the expression was associated with Lauren type, differentiation,N stage and TNM stage of the tumours.The Kaplane Meier plot showed that patients who wereανβ6 negative had much longer survival times than those who wereανβ6 positive.ανβ6 is also defined as a novel,independent prognostic indicator for gastric carcinoma in humans.Taking all the data together,Integrinανβ6 plays an important role in the invasion and migration of gastrointestinal cancers.Our study here wants to further investigate the role and correlated molecular mechenism ofανβ6 in the survival and progression of colon cancer.Our results can provide molecular therapeutic target for the treatment of colon cancer.PartⅠ:Supression of integrinανβ6 by RNA Interference in colon cancer cells inhibits extracellular matrix degradation through the MAPK pathwayIntegrinανβ6 plays a very important role in the progression of colon cancer cells and is now defined as a novel,independent prognostic indicator for aggressive colon cancer in humans.Herein,we use the RNA interfering technology to downregulate the expression ofανβ6 in colon cancer cells.Our data demonstrate that plasmid vector based shRNA can effectively down-regulateανβ6 expression in protein and mRNA levels.Supression of integrinανβ6 inhibits the phosphorylation and nonphosphorylation level of ERK1/2,the secretion of uPA,pro-MMP-9 and pro-MMP-2 in tumor conditioned medium,and more important,inhibits MAPK-dependent[~3H] labelled collagenⅣdegradation via the plasminogen activation cascade.Our study demonstrates in vitro that supression of integrinανβ6 inhibits extracellular matrix degradation through the MAPK pathway,PartⅡ:Integrinανβ6 Can Regulate eIF4E activity and VEGF Translation through the PI-3K/Akt/mTOR pathway: A Novel Survival Mechanism for Colon Cancer CellsThe integrinανβ6 has been shown to facilitate key functions of colon carcinoma cells,including their ability to migrate,invade,and evade apoptosis.In this study we identified a novel mechanism by which integrinανβ6 regulates VEGF expression,and the survival of colon carcinoma cells.We found that suppression of integrinανβ6 decreases VEGF protein but not mRNA expression,which suggested thatανβ6 may influence the translation level of VEGF.Further investigation show that the mechanism involves the ability of this integrin to stimulate the phosphorylation and inactivation of 4E-binding protein(4E-BP1),a translational repressor that inhibits the function of eukaryotic translation initiation factor 4E(eIF4E).The regulation of 4E-BP1 phosphorylation byανβ6 derives from the ability of this integrin to activate the PI-3K-Akt pathway and,consequently,the rapamycin-sensitive kinase mTOR that can phosphorylate 4E-BP1.Importantly,we show that thisανβ6-dependent regulation of VEGF translation plays an important role in the survival of colon carcinoma cells by sustaining a VEGF autocrine signaling pathway that involves activation of PI-3K and Akt.These findings provide a mechanism that substantiates the reported role ofανβ6 in carcinoma progression.