The Mechanism Of Anoikis Resistance Regulated By Transcription Factor Spib In Nsclc Cells

Objects: Anoikis is a type of programmed cell death caused by the detachment of cells from the extracellular matrix(ECM).The acquisition of anoikis resistance is an important mechanism in cancer invasiveness and metastatic behavior.In the advanced stages of cancer,autophagy is thought to promote tumor progression by enhancing the ability of cells to adapt to various environmental stresses.Generally,the autophagy induced by matrix deprivation can enhance anoikis resistance of tumor cells and thus promote tumor metastasis.Our previous study has found that a lymphocyte lineage-specific transcription factor SPIB is ectopically expressed in lung cancer cells and promotes metastasis of lung cancer.It is speculated that SPIB may play a role in anoikis resistance of lung cancer cells,the underlying molecular mechanisms of which are still unclear.Therefore,the current study mainly explores the transcriptional regulation and possible autophagy mechanism mediated by SPIB in the process of non-small cell lung cancer(NSCLC)cells resisting anoikis,aiming to provide theoretical and experimental basis for clinical diagnosis and treatment of NSCLC with SPIB as a potential target.Methods: Part Ⅰ,the function of SPIB in anoikis resistance.The expression of SPIB in lung cancer cell lines was determined by q RT-PCR and western blot.Anoikis was analyzed by cell death assay in five lung cancer cell lines after 5 days of ECM detachment.The expression of SPIB was then examined in both attached and suspended H1703,A549 and H1975 cells.Cell death assay was again performed to analyze anoikis of SPIB down-regulated H1703 and H460 cells and over-expressed H1975 cells,respectively.Meanwhile,the whole cell lysates of H1703 and H460 cells with indicated treatments were used to test the apoptosis markers,including full-length PARP,cleaved-PARP and cleaved-CASP3.Finally,Transwell assay was adopted to examine the effect of SPIB on the invasion of detached lung cancer cells.Immunohistochemical staining against SPIB in 27 primary human lung cancer tissues and their matched lymph node metastases is applied to analyze the role of SPIB in metastasis.Part Ⅱ,autophagy regulation is adopted by SPIB to enhance anoikis resistance.Four cell lines stably expressing GFP-LC3-RFP-LC3ΔG were cultured in attached condition or subjected to suspension for 24 h before analyzing the autophagic flux with fluorescence microscopy.RNA sequencing was then performed to profiling the transcriptional changes upon SPIB silencing in both attached and suspended cultures.To further determining the function of SPIB in autophagy,cells expressing GFP-LC3 were observed under fluorescence microscope.The expression of LC3B-II,SQSTM1,ATG7,ATG5 and BECN1 was quantified by protein immunostaining.Moreover,trehalose,a typical autophagy activator and bafilomycin A1(Baf A1),a classic autophagy inhibitor,were also used to indicate the changes of autophagy flux.Finally,the subcellular structures were examined by transmission electron microscopy of H1703 cells treated as indicated.Part Ⅲ,SPIB performs transcriptional regulation in anoikis resistance.The enrichment of SPIB in genome-wide was analyzed by Ch IP-seq technique.Genes bound to the promoter region were extracted from the total genes enriched by SPIB,and the function of the genes was analyzed by GSEA.The RNA levels of autophagy and anoikis-related genes were examined by q RT-PCR.Subsequently,genes with significant difference in RNA levels were selected for Ch IP-PCR verification.Then the promoter activity of SNAP47 regulated by SPIB was determined by Luciferase assay.Additionally,the protein level of SNAP47 was tested with immunoblot upon SPIB depletion in adherent and suspended cultured lung cancer cells.Finally,Baf A1 was used to confirm the relationship between CLDN2 and autophagic degradation.The function of CLDN2 in anoikis was determined by cell death assay.Results: Part Ⅰ,SPIB enhances anoikis resistance of NSCLC cells.The protein levels of SPIB in lung cancer cell lines were divergent.The level of SPIB was positively correlated with the ability of cells to resist anoikis.Detachment from the ECM induced the expression of SPIB.The loss of SPIB increased the expression of apoptosis protein markers,including cleaved-CASP3 and cleaved-PARP,and promoted anoikis.Therefore,SPIB enhanced the invasion and metastasis potential of lung cancer cells.Part Ⅱ,the down-regulation of SPIB inhibits autophagy.Suspension culture increases the formation of autophagosomes,which can be reduced by SPIB depletion.SPIB knockdown modulated the expression of autophagy-related genes at a transcriptional level.The inhibition of SPIB significantly reduced the expression of LC3B-II,SQSTM1,ATG5,ATG7 and BECN1,and increased the accumulation of swollen autophagic vesicles in suspended lung cancer cells.Part Ⅲ,SPIB enhances NSCLC anoikis resistance by promoting SNAP47 transcription and CLDN2 autophagy degradation.SPIB enriched in the promoter regions of autophagy and anoikis related genes such as BOK,BMF and SQSTM1.A genomic site approximately 0.5 kb upstream of the SNAP47 transcriptional start site(TSS)was occupied by SPIB.The depletion of SPIB in suspension cells inhibited SNAP47 transcription and reduced protein expression.SPIB knockdown promoted CLDN2 expression and inhibited its autophagic degradation.Thereby,accumulated CLDN2 activated anoikis of lung cancer cells.Conclusion: The matrix deprivation induces SPIB expression in NSCLC cells.Highly expressed SPIB activates SNAP47 transcription and accelerates autophagy process.Meanwhile,SPIB inhibits CLDN2 expression and promotes its autophagic degradation.SPIB regulates anoikis resistance through the above two pathways,thereby enhancing the potential of invasion and metastasis.This study has depicted a novel mechanism for NSCLC cells to resist anoikis,providing a promising candidate for the clinical treatment and drug development of lung cancer metastasis.