Roles And Molecular Mechanism Of Integrin αvβ6Trafficking In Regulating The Migration Of Colon Cancer Cells

Background:Colon cancer is one of most common digestive malignant tumors. In recent years, its incidence is increasing continuously as a result of living standard improvement and diet changes. Although its diagnosis and treatment has greatly improved, the mortality rate is still high, just a little lower than lung cancer and liver cancer, and rank3in our country.Integrins are a major family of cell surface-adhesion receptors, which is composed of18a subunit and8β subunit that can assemble into24different heterodimers with non-covalent bindings. They mediate cell-cell and cell-extracellular matrix interactions and play key roles in many essential biological processes, such as proliferation, differentiation, invasion and metastasis.Integrin αvβ6is the only heterodimer that β6can form. It expresses either absent or at very low levels in normal tissues. However, it becomes de novo or highly expressed during times of morphogenesis, tissue repair and especially in malignant epithelia, such as colon, oropharynx, breast, ovarian and pancreatic carcinomas. We have previously reported that heterologous expression of αvβ6in tumour cells promotes cell proliferation, enhances secretion of MMP-9, inhibits cell apoptosis, mediates the potential to colonise and metastasise, and acts as a prognostic indicator for early stage tumours. We could inhibit the malignant behaviour of tumour cells by suppression of av(36expression, and norcantharidin can induce apoptosis of HT29cells through the avP6pathway.Moreover, one of our previous findings is that we have verified the direct binding between αvβ6and ERK2in colon cancer cells. When non-phosphorylated ERK2is bound to β6, ERK2can be more efficiently phosphorylated because of conformational changes, and the presence of β6may also shield ERK2from deactivation by cellular phosphatases. We have also confirmed that integrin αvβ6increases its own level at the cell surface via PKC-mediated signalling as tumour cells become crowded. This may contribute to a self-perpetuating system of colon cancer progression in which the integrin avP6provides a means of sustaining tumour cell proliferation and migration.In past two decades, as a result of internalization and recycling assays pioneered by Mark Bretscher, it became clear that some integrin heterodimers were continually internalized from the plasma membrane into endosomal compartments and then recycled back to the cell surface, thus completing an endo-exocytic cycle. Accumulated evidence indicated that integrin trafficking play a very important role in cell migration during cancer metastasis. The trafficking of integrin αvβ6has also been detected in oral squamous cell carcinoma cells by John F. Mashall in2007.On the basis of all these work, this project was focus on the roles and molecular mechanism of integrin avP6trafficking in regulating malignant behavior of colon cancer cells, especially cell migration. We will study on the relationship between ERK2activation and the endo-exocytic cycle of αvβ6, identify the effect of PKC activation on integrin αvβ6trafficking and cell migration, and examine the effect of high/low cell density culture on integrin αvβ6subcellular distribution in colon cancer cells. We sincerely hope that it would provide some useful ideas and data for the future study in this area. Part I Establishment of internalization and recycling assay of integrin αvβ6in colon cancer cells.ObjectiveTo establish the internalization and recycling assay of integrin αvβ6in colon cancer cells.Methods(1) Internalization assay:Cells were surface labelled with0.2mg/ml EZ-Link Sulf-NHS-SS-Biotin at4℃for1h. Labelled cells were washed in cold PBS and transferred to DMEM at37℃. At the indicated time points, the media was aspirated, and the flasks were transferred to ice rapidly and washed twice with cold PBS. Labelled biotin was removed from proteins remaining at the cell surface by treatment with a MesNa solution for15min at4℃. MesNa was quenched by the addition of20mM IAA for10min at4℃. The cells were lysed, and the amount of biotinylated integrin αvβ6was determined by capture-ELISA.(2) Recycling assay:Following surface labelling with0.2mg/ml EZ-Link Sulf-NHS-SS-Biotin at4℃for1h, cells were transferred to DMEM for30min at37℃. The flasks were then transferred to ice rapidly and incubated in MesNa solution to remove biotin remaining at the cell surface. The internalized integrin αvβ6pool was chased from the cells by returning them to37℃in DMEM for the indicated times. The cells were then returned to ice, and biotin at the cell surface was removed by a second treatment with MesNa. After quenching with20mM IAA, the cells were lysed, and the protein concentrations of the samples were equalised. The levels of biotinylated integrin αvβ6were determined by capture-ELISA.(3) Capture-ELISA:ELISA plates with96wells were coated with5μg/ml αvβ6antibodies (10D5) in Na2CO3(pH9.6) at4℃overnight and blocked with PBS containing0.1%Tween-20(PBST) with5%BSA for1h at room temperature. Integrin αvβ6was captured by overnight incubation of50μl cell lysate at4℃. Unbound material was removed by extensive washing with PBS-T, and the wells were incubated with Streptavidin-HRP in PBS-T containing1%BSA for1h at4℃. Following further washing, biotinylated av(36was detected by a chromogenic reaction with OPD.ResultsInternalization and recycling assay of integrin av(36in colon cancer cells were established successfully, and the trafficking of αvβ6in HT29and WiDr cells were examined. It was found that integrin αvβ6subunits were internalized and recycled with similar kinetics in both cell lines. The internal pools of integrin αvβ6achieved a steady state within30min, and about70%of the internalized integrin avP6subunits were recycled back to the cell membrane with30min.ConclusionIntegrin αvβ6were continually internalized from cell membrane into cytoplasma and recycled back to the cell surface in colon cancer cells, thus completing an endo-exocytic cycle. All the biological process of integrin αvβ6must on the basis of this trafficking.SignificanceEstablishment of internalization and recycling assay is the basis of this project. Part II The effect of integrin αvβ6-ERK2direct binding on integrin αvβ6trafficking.ObjectiveTo identify the effect of integrin αvβ6-ERK2direct binding on integrin αvβ6trafficking. MethodsTo determine whether the activation of ERK2could affect integrin αvβ6trafficking, we performed internalization and recycling assays in the presence of PD98059, an inhibitor of MEK, which can block the phosphorylation of ERK2. To determine whether the interaction between β6and ERK2is essential for the process of integrin αvβ6trafficking, we compared internalization and recycling of integrin avP6in SW480cells stably transfected with wild-type β6or Del. Mutant β6.ResultsIn HT29cells, addition of PD98059inhibited the internalization rate of integrin avP6but did not alter its recycling. In SW480cells, the Del. Mutant β6exhibited impaired internalization compared with wild-type β6, but the deletion of the ERK2binding site had no effect on the recycling. This was consistent with the effect of PD98059. However, it was found that PD98059treatment had no effect on the initial internalization of integrin αvβ6, but the deletion of ERK2binding site was able to slow the αvβ6internalization at the beginning of the incubation time.ConclusionIntegrin αvβ6was able to facilitate the phosphorylation of ERK2and vice versa; the activation of ERK2was an essential step for the internalization of integrin αvβ6. There could be mutual activation effects between integrin avP6and ERK2.SignificanceIt was clarified that the relationship of integrin avp6-ERK2direct binding and integrin αvβ6trafficking. Part III The effect of integrin αvβ6trafficking on the migration of colon cancer cells. ObjectiveTo identify the effect of integrin αvβ6trafficking on the migration of colon cancer cells.MethodsCompare the effects of10D5(a functional blocking antibody of integrin αvβ6), PD98059or primaquine (a drug that inhibits receptor recycling and vesicular transport) on the migration of HT29cells towards fibronectin, which is the ligand of integrin αvβ6. Examine the migrating ability of SW480wild-type β6, SW480Del. Mutant β6and SW480mock cells towards fibronectin. Compare the differences and identify the effects of10D5, PD98059or primaquine treatment on them.ResultsCompared to HT29cells without any treatment, the application of10D5or PD98059both greatly inhibited the migration of HT29cells towards fibronectin. Similar results were seen following the addition of primaquine. Unlike the wild-type β6transfectant, SW480Del. Mutant β6cells have an impaired ability to migrate towards fibronectin. PD98059was able to inhibit the migration of wild-type β6transfected cells, but it had no inhibitory effect on the other two cell lines. Application of10D5or primaquine caused a similar level of migration for the three different cells.ConclusionThe migration of HT29cells or SW480wild-type β6cells towards fibronectin was in an αvβ6-dependent manner, and the expression of integrin αvβ6, integrin αvβ6-ERK2binding, integrin αvβ6trafficking are all crucial factors in this process. The disturbance of any component would have a negative effect on it.SignificanceThese results suggest that the internalization and recycling of αvβ6integrins plays a large role in the migration of colon cancer cells and that direct binding between αvβ6and ERK2may be a crucial part of the cycle. Part IV The effect of PKC on integrin αvβ6trafficking and cell migration.ObjectiveTo identify the effect of PKC on integrin αvβ6trafficking and cell migration.MethodsTo determine whether the activation of PKC could affect integrin avP6trafficking, we performed internalization and recycling assays in the presence of PMA, an activator of PKC. Combined application of PMA and PD98059was also completed. Then we examined the effect of PKC activation on cell migration and whether the application of10D5, PD98059or primaquine was able to reverse this effect.ResultsPKC was able to accelerate both the internalization and recycling of integrin avP6in HT29cells. The increase in the internalization rate by PMA could be attenuated by PD98059, but the recycling rate of integrin αvβ6was still increased. The promotion effect of PMA on cell migration was obvious, and the combined application of10D5, PD98059or primaquine with PMA was able to reverse this effect. Moreover, PKC activation caused a significant increase in the migration of SW480cells with wild-type (36, but little effect was observed on the other two transfectants.ConclusionThese results suggest that PKC was able to promote integrin av(36trafficking, and the positive effect of PKC on cell migration is through an αvβ6-dependent mechanism.SignificanceIt was clarified that the effect of PKC activation on integrin αvβ6trafficking and cell migration. PartⅤ The effect of high/low cell density culture on integrin avp6subcellular distribution in colon cancer cells.ObjectiveTo identify the effect of high/low cell density culture on integrin avP6subcellular distribution in colon cancer cells.MethodsIdentical suspensions of HT29cells were seeded into different dishes to achieve high or low-density cultures. The αvβ6trafficking was detected by internalization and recycling assay, and the expression of integrin αvβ6on cell membrane was examined by FACScan analyses. Then the cells were lysed and the total expression of integrin αvβ6in lysates were examined by western blot.ResultsIt was observed that the internalization and recycling rate of integrin avP6were both accelerated while cells became crowded, and the levels of integrin αvβ6at the cell surface increased significantly. However, it was proved that the total cellular content of αvβ6integrin was constant.ConclusionIt indicated that the changes in expression of integrin αvβ6at the cell surface were due to the redistribution of αvβ6in cells by the regulation of internalization and recycling of the heterodimer.SignificanceIt suggested a self-perpetuating system of colon cancer progression in which the integrin avP6provides a means of sustaining tumour cell proliferation and migration.