Functional Mechanism Of CBX8 In Cancer Cell Metastasis

Background: Cancer is considersing as one of most deadly diseases,and featured with highly ability of metastasis and invasion as well as poor prognosis.Cancer metastasis is multistep process,including cells morphology changes,introvacular,cell transportion,extravascular cancers,and tumor initiation in new sites.Increasing evidences indicates that several cellular,genetic and epigenetic factors are invovled in this process.Polycomb group(Pc G)of proteins has long been considered to be a paradigmatic model for epigenetic regulation of gene silencing through canonically forming two multimeric protein complexes,Polycomb Repressive Complexes 1 and 2(PRC1 and PRC2)and invovled in a vieriety of biological phenomena such as tumorgenesis.Pc Gs form PRC2 trimethylates the K27 residue of histone 3(H3K27).Then PRC1 via homolog CBXs recognize the trimethylation mark resulting in ubiquitination of H2A119 K which represses transcription.In addition,RAC1 is one of the most important molecular that changes the cells movement through regulating ?-acting.Invasion implies that cancer cells actively get into a tissue by secreting protease to digest the matrix.MMP2(a member of matrix metalloproteinase)plays a pivotal role in cells invasion.It's reported that WNK2 can negatively regulate RAC1 and MMP2 to affect migration and invasion.CBX8,as a member of CBX family,interacts with other Pc G components to form PRC1.Increasing evidences showed that CBX8 level increases in many type cancers,such as glioblastoma,lung cancer,breast cancer,and so on,and indicated that it may play an important role in the process of tumerogenesis and metastasis.However,CBX8 functional mechnism needs elucitive.Methods: 1.CBX8 expression pattern in cancers: systemical analysis of CBX8 expression pattern in GBM,breast cancer and lung cancer in TCGA through c Bio Portal for Cancer Genomics program.2.Generation of CBX8 overexpression and silencing cell lines: Retrovirus plasmids were bought from Origene and transfected into GBM cell lines(U251MG,T98G),breast cancer cell lines(MCF7,MDA-MB-231)and lung cancer cell lines(A549).Selection with G418 for CBX8 overexpressed cell lines and Puromycin for silenced cell lines for three weeks,followed by the confirmation by using q RT-PCR and western blot.3.Migration assay: wounding healing assay was performed in U251 MG,MCF7 and A549 cell lines with CBX8 modulation to evaluate their migration ability by comparing the cells uncovered area,and transwell migration assay in U251 MG,MDA-MB-231 and A549 cell lines with CBX8 modulation as well by measuring with the cell number which passed through the well.Cells morphology was grossly checked after three-step staining.In addintion staining of ?-actin to examine the change of cytoskeleton were also conducted.4.Invasion assay: Cells invasion ablity of U251 MG,MDA-MB-231 and A549 cell lines with CBX8 modulation were mesureed by counting the cell number passing through the wells.NSG Animal model for monitoring metastasiscapacity of CBX8-modulated U251 MG,MDA-MB-231 and A549 cells were employed through tail vein injection.4-6 weeks post injection,bioluminescence imaging wwere caputured to detect tumor cell metastasis.At the end of the experiments,lung tissues were collect for HE staining to check the tumor formation.Mice weight and lung weight also were measured for checking the effect of tumor metastasis on mice and lung growth.5.Regulation relationship between CBX8 and WNK2,RAC1,MMP2: q RT-PCR was performed to measure the expression level of WNK2,RAC1,and MMP2 in U251MG?T98G?MCF7?MDA-MB-231 and A549 with modulated CBX8.Moreover,RAC1 and MMP2 activity in U251 MG,MDA-MB-231 and A549 cell lines and mice serum was also examined to validated the regulation.Chromatin immunoprecipitation assay were performed in CBX8 overexpressed MDA-MB-231 to test the relationship between CBX8 and WNK2 directly.6.Validated the relationship between WNK2 and RAC1,MMP2 using si RNA technique: Transfect wide type U251 MG,MDA-MB-231 and A549 cell lines with three types si RNAs respectively,two days post transfections,q RT-PCR was conducted to check RAC1 and MMP2 expression.Results: 1.TCGA data systemical anlysis presented that CBX8 increased in most cancers,especially in GBM,breast cancer and lung cancer.CBX8 predict a poor prognosis in GBM.However,no significant difference was found in breast cancer and lung cancer.2.Generation of modulated CBX8(overexpression and silencing)cell lines in GBM U251 MG and T98 G cell lines,breast cancer cell lines MCF7 and MDA-MB-231 and lung cancer cell line A549.QRT-PCR and western blot confirmed the modulation of CBX8 in these cell lines.3.Scratch wounding healing presented that overexpression CBX8 increased the migration ability of U251 MG,MCF7 and A549 tumor cells reflecting by the decrease of scratch space.Transwell migration assay obtained the similar result.Moreover,both the three-step staining and ?-acting staining showed that overexpression CBX8 could affect the spindle-shape and ?-actin distribution,which ar consistent with their migration ability.4.Transwell invasion assay showed that overexpression CBX8 increased the invasion ability of U251 MG,MCF7 and A549 tumor cells reflecting by the increased number of cells on the lower side of membrane.Lung metastasis animal model was built through tail vein injection in NSG mice.The data showed that overexpression CBX8 leaded to a higher rate of lung metastasis accompanying by the losing of body weight and lung weight.In addition,HE staining confirmed the conclusion.These data indicated that increased CBX8 could promote a serious lung metastasis.5.CBX8 regulated RAC1 and MMP2 through WNK2 in cancer cells U251MG?T98G?MCF7 ? MDA-MB-231,and A549.The q RT-PCR data showed that WNK2 expression is negatively associated with CBX8 expression,whereas positively associated RAC1 and MMP2 expression.Moreover,increased RAC1 and MMP2 activity in cell lines and mice serum is consistent with this finding.Chromatin immunoprecipitation assay using overexpression MDA-MB-231 cells further confirmed that CBX8 regulated WNK2 expression directly.6.si RNA knockdown WNK2 in U251MG?MDA-MB-231 and A549 further confirmed WNK2 could decrease RAC1 and MMP2 expression,indicating a negatively relationship between WNK2 and RAC1,MMP2.Conclusion: In this study,increased CBX8 has been found in various cancer types.Overexpressing CBX8 can increase the ability of migration and invasion in cancer cell in vitro or in vivo,while silencing CBX8 exibited oppoitie effects.These effect is achieved through the negatively regulation of CBX8 on WNK2 directly,which caused the increase of RAC1 and MMP2 in expression level and activity.