Integrin αvβ6 Mediates The Potential For Colon Cancer Cells Of Liver Metastasis And Invasive Growth In Progression

Backgroud and significationColon cancer is one of the commonest malignant tumors affecting men and women together in our society.Invasion growth and distant metastasis are two key factors distinguishing from the benign tumors,which affect the prognosis of patients with colon cancer.How to restrain tumors invasive growth and how to prevent colonic metastasis to liver in patients with colon carcinoma remains one of the most challenging issues in cancer research.Colon cancer progression is closely correlated with the biological feature of cell surface,is thought to be regulated,at least in part,by cell adhesion molecules and matrix-degrading enzymes.Relatively little is known about how these factors might act to enhance invasive growth of colon cancer and mediate the process of colonic metastasis to liver in patients with colon carcinoma.Of all the families of cell adhesion molecules expressed on the surface of cancer cells,the family known as integrins has been best characterized.Integrinαvβ6 expression patterns appear to be directly implicated in the progression of colon cancer in particular.Theαvβ6 integrin is either not expressed or expressed at low levels in normal adult epithelia;however,it becomes highly expressed during tumourigenesis.For example,induction ofαvβ6 expression in oral leukoplakia appears to be a necessary prerequisite for progression to squamous cell cancer and de novo expression ofαvβ6 has been observed in oral squamous and colon cancers. Leading up to this series research,αvβ6 had been reported to enhance proliferation and growth of colon cancer cell in vitro and vivo.In addition,αvβ6 had also been shown to induce secretion of matrix metalloproteinase-9(MMP-9),a matrix-degrading enzymes,in these cell types and enhance the colon cancer cells migration by degeneration of surrounding matrix.This indicated that cancer cells might escape the growth constraints imposed on normal cells when crowded together was by autocrine up-regulation of theαvβ6-mediating MMP-9 secretion.In this study,the major aim of the study was to explore some of the possible mechanisms by which integrinαvβ6 mediates colonic metastasis to liver and invasive growth in colon cancer progression,which provides a new idea and basis for further study.Strategies targeting the integrinαvβ6 can play key roles in the management of patients with advanced colon cancer in the near future.PARTⅠIntegrinαvβ6 mediates the potential for colon cancer cells to colonize in and metastasize to the liverObjectiveColon cell carcinoma is characterized by the propensity for distant metastasis to liver.How to prevent colonic metastasis to liver in patients with colon carcinoma remains one of the most challenging issues in cancer research.Integrin alphavbeta6(αvβ6)is closely correlated with colon cancer progression.However, the link betweenαvβ6 expression in primary colon cancer and colonic metastasis to liver in patients with colon carcinoma remains unknown.Our experimental aims were to detect the effects of integrinαvβ6 on colonization in and colonic metastasis to liver in colon cancer progression.Materials and Methods1.Immunohistochemistry is the most important methodology to detect the effects of integrinαvβ6 expression on both colon cancer and liver metastatic carcinoma specimen in this experimental study.To demonstrate the specificity of the immunohistochemical results,the specificity of againstαvβ6 monoclonal antibody 2G2(obtained from Biogen Idec.)was examined with various colon cancer cells(WiDr cells,SW480 wild cells,SW480β6 transfectants)by immunoprecipitation assay.2.Integrinαvβ6-immunoreactivity(IR)in liver metastasis tissues(63 cases of hepatic metastatic specimens obtained randomly from Jan.1995 to Jan.2005) was performed with Elivision method.Then 358 cases of colon carcinoma specimens obtained randomly from Jan.2001 to Dec.2002 were constructed into two tissue microarrays(200 points / each,constructed by Xi'an Chaoying Biochip Company,Ltd.).Integrinαvβ6 immunoreactivity(IR)was examined according to the Avidin-Biotin Complex protocol.Two groups were divided according to the immunohistochemical results(αvβ6-IR positive orαvβ6-IR negative)to investigate postoperative liver metastases for 3 years.3.To confirm the effect of integrinαvβ6 on enhanced liver colonization and metastatic potential for colon cancer cells in progression,we performed in vivo studies using the liver colonization model(directly liver injection for WiDr cells), and the liver metastatic model(intrasplenic injection for HT29 cell lines).4.To examine the underlying mechanisms associated withαvβ6 regulating colonic metastasis to liver,matrix metalloproteinase-9(MMP-9)levels in the cultures of both HT29 and WiDr transfectants were detected by the Biotrak MMP-9 activity assay system and gelatin zymography assay.The migration capability on fibronectin(FN)for HT29 / WiDr transfectants was examined also by Transwell migration assay in vitro.Results1.The immunoprecipitation assay of various colon cancer cells(WiDr cells, SW480 wild cells,SW480β6 transfectants)for the monoclonal antibody 2G2 showed that integrin avβ6 was specifically recognized by the anti-β6 antibody 2G2.2.Integrinαvβ6 positive rate in liver metastatic tissues(71.4%,45/63)was higher than that in primary colon cancer(34.0%,122/358)(p＜0.01).The results of 3-year follow up for postoperative liver metastasis showed that liver metastasis rates(17%,21/122)for patients withαvβ6 positive was higher than those withαvβ6 negative(only 3%,7/236)(p＜0.01).3.Liver colonization assay(directly liver injection of WiDr transfectants)in nude mice showed that 55%(11 of 20)of the mice injected with WiDr mock transfectants expressingαvβ6,six weeks later,developed liver tumors,and experimental liver metastasis assay in nude mice(intrasplenic injection of HT29 transfectants)showed that 90%(18of 20)of the mice injected intrasplenically with HT29 mock transfectants,four weeks later,developed visible liver metastatic tumors.However,none of visible tumors in liver was observed in the mice injected with either WiDr antisenseβ6 or HT29 antisenseβ6 transfectants.These results demonstrated thatαvβ6 contributed to promote metastatic potential and survive in liver environment in colon cancer progression.4.Matrix metalloproteinase-9(MMP-9)levels in the cultures of both HT29 and WiDr cells were detected by the Biotrak MMP-9 activity assay system and gelatin zymography assay,showed that suppression ofαvβ6-IR inhibited MMP-9 activity and secretion(p＜0.01).Transwell migration assay in vitro also showed thatαvβ6 promoted migration on fibronectin(FN)for HT29 / WiDr mock compared with HT29 / WiDr antisenseβ6 transfects(p＜0.01),respectively.Conclusions1.Integrin avβ6 in colon cancer cell surface was specifically recognized by the anti- avβ6 monoclone antibody 2G2.2.There is relatively higherαvβ6 positive rate in liver metastatic tissues than in colon cancer tissues,and there is relatively higher liver metastasis rates for patients withαvβ6 positive compared with those withαvβ6 negative,which indicate that integrinαvβ6 is closely correlated with colonic metastasis to liver in progression.3.The progression of liver metastasis was dependent on the biological features of colon cancer cell surface and the microenvironment of targeting organ(liver). Integrinαvβ6 contributed to survive in liver environment and promote liver metastasis for colon cancer cells in progression.4.Integrinαvβ6 promoted secretion of MMP-9 resulting in extracellular matrix(ECM)degradation,and enhanced migration on fibronectin(FN),which are two prerequisite conditions for colon cancer cells of liver metastasis in progression.Based on this view,strategies by targeting to inhibit the avβ6-IR in colon cancer cells could,at least partly,prevent the potential for colon cancer cells of liver colonization and liver metastasis,which provides the novel theory and basis to control the process of colonic metastasis to liver in progression.PARTⅡIntegrinαvβ6 mediates tumor invasive growth in colon cancer progressionObjectiveInvasive growth is one of the most important features distinguishing from the benign tumors and the key factors affecting the prognosis of patients with colon cancer.Our experimental aims were to detect the effects of cell density on both integrinαvβ6 expression and matrix metalloproteinase-9(MMP-9)secretion in colon cancer cells,and the mechanism by which cancer cells sustain invasive growth via a self-perpetuating manner in colon cancer progression.Methods1.Flow cytometry was applied to analyze avβ6 expression in human colon cancer line,WiDr,SW480 cells and the normal human keratinocyte cell line, HaCaT cells,respectively,at low and high cell density culture.2.To detect the disparity of both distribution and intense for integrin avβ6 expression,an immunohistochemical study of integrin avβ6 was performed on tissue microarrays(TMAs)of 200 points(including 100 cases malignant colon specimens).Two cores were taken from each sample,one obtained from invasive tumor portions(tumor margin,especially cell crowding margin at high cell density);the other obtained from non-invasive portions(centre tumor foci at low cell density).3.The MMP-9 activity levels for various colon cancer lines,WiDr cells,SW480 cells,and the normal human keratinocyte cell line,HaCaT cells,respectively,at high- and low- cell density culture were analyzed using Biotrak MMP-9 activity assay system and gelatin zymography assay system.Results1.High cell density evidently enhances integrin avβ6 expression only for WiDr cells expressing avβ6 compared with low density,but no increase was observed for both SW480 cells(lack avβ6 expression)and HaCaT cells(benign epithelial cells).2.The avβ6 expression rate on immunoreactivity(IR)in overall was 38%(38 in 100 cases).High positive expression for avβ6 on immunoreactivity was observed in invasive tumor edged portions(relatively cell crowding margin and high cell density)in 73.7%(28/38)cases,particularly,preferential localization at the edges of infiltrating tumor edge.In non-invasive centre tumor portions (relatively cell loose and low cell density),low avβ6 positive expressive was frequently observed and high avβ6 expressive only seen in 28.9%(11/38)cases.3.Biotrak MMP-9 activity assay indicated that the amounts of MMP-9 secreted per cell were evidently higher for WiDr and SW480β6 cells at high cell density compared with at low cell density(p＜0.01),but no density-dependent increase observed for SW480 wild cells which lack avβ6(p＞0.05).Gelatin zymography assay also indicated that the levels of MMP-9 secreted for SW480β6 cells which expressing avβ6 was evidently higher at high density than at low density,however no density-dependent increase observed for both SW480 cells (lack avβ6 expression)and HaCaT cells(benign epithelial cells).Conclusions1.Cell crowding and dense induces integrin avβ6 expressed in colon cancer cells,which is the promoter of tumor invasive growth in colon cancer progression.2.The characterization of the role ofαvβ6 in colon cancer progression (integrinαvβ6 expressed in colon cancer cells was in a cell density-dependent way) was profoundly different from normal cells.The MMP-9 secretion increased for colon cancer cells in the high cell density culture was mediated inαvβ6 independent way.Blockage ofαvβ6 expressed in colon cancer cells can inhibit MMP-9 secretion.The mechanisms by which MMP-9 secretion increased for colon cancer cells in a cell density-dependent way was also invalid for normal cells.Based on this view,strategies by targeting to inhibit the avβ6 expressed in colon cancer cells,specifically,at least partly,prevent invasive growth for colon cancer in a self-perpetuating way,which provides the novel theory and basis to develop high-performance and asepsis anticancer medicine.3.High cell density induces integrin avβ6 expression,promotes MMP-9 secretion for colon cancer cells,increases the degradation of extracellular matrix (ECM),which constitutes the molecular biological basis for a self-perpetuating system of tumor invasive growth in colon cancer progression.