SDF4 Promotes Proliferation,migration And Invasion Of Glioblastoma Cells Through NFAT1/β-catenin Axis

SDF4（Stromal Cell Derived Factor 4）,a member of the CREC family,is a calcium-binding protein that is important in regulating intracellular calcium ion level and cargo sorting.Recently,some studies have showed that SDF4 played a key role in the development of human cancer with multiple biological functions,such as intracellular calcium homeostasis,cell proliferation,endoplasmic reticulum stress and so on.Gliomas are the most common primary intracranial tumour,which is caused by the canceration of glial cells in the central nervous system,accounting for about40%-50%of all intracranial tumors,among which about 50%are malignant glioblastoma（GBM）.Epithelial-mesenchymal Transition（EMT）is referred to the transformation of Epithelial cells to Mesenchymal cells under specific physiological and pathological conditions,which is closely related to the invasion and metastasis of cancer.Changes in intracellular Ca²⁺level modulate versatile downstream signaling pathways and control various physiological and pathological cellular processes,including proliferation,metabolism,and gene transcription.Store-operated Ca²⁺entry（SOCE）,including the sensoring of Ca2+by endoplasmic reticulum and the opening of calcium channels,which is a broad and evolutionarily conserved Ca²⁺influx.Cytoplasmic Ca²⁺plays an important role in many cellular processes and tumor development.The objective of this paper is to explore the role of SDF4 in glioblastoma by using techniques such as molecular biology,cell biology,molecular pathology,and to elucidate the molecular mechanism,of SDF4 in regulating the proliferation and invasion of glioblastoma.The main results are as follows:（1）High SDF4 expression is correlated with poor patient prognosis.We first detected SDF4 expression in three GBM cell lines（LN-229,U-87 MG,A172）and normal astrocytes cell SVGp12.We found that SDF4 was high expression in GBM cell lines.To further determine whether SDF4 expression is implicated in GBM patient prognosis,we analyzed the survival date form the Tumor-Glioma-French-284 database,which is available from the online R2genomics analysis and visualization platform.We found that high SDF4 expression was implicated in poor overall survival.Then,Kaplan-Meier analysis showed that the level of SDF4 expression was increased significantly in malignant glioma compared with normal brain tissues.These results showed that GBM overexpressed SDF4 compared with normal brain tissue,and the high SDF4expression is correlated with poor prognosis.（2）SDF4 is required for GBM cell proliferation and invasion.To investigate the importance of SDF4 in GBM cell proliferation and invasion.We knocked down SDF4 in GBM cell lines（U-87 MG and LN-229）by using lentivirus-mediated sh RNA technique.We examined Brd U incorporation and showed that the positive rate of DNA synthesis decreased after SDF4-knockdow.Flow cytometry showed that SDF4-knockdow could induce cell cycle arrest at S phase.Next,MTT assay showed that the GBM cell growth could be significantly inhibited after SDF4 knockdow.Subsequently,we detection the expression of S cell cycle regulatory proteins.and found that the expressions of CDK4,c-myc and Cyclin D1 were significantly decreased in the SDF4 knockdow cell.In addition,To determine whether SDF4 was involved in the migration and invasion of GBM cells,we performed migration assays and invasion assays.When SDF4 expression was knocked down in U-87 MG and LN-229 cells,these cells migrated much slower than control cells.Wound healing assays showed that the migration and invasion ability of SDF4 cells were significantly reduced after SDF4 knockdow.Furthermore,We used western blot to examine the expression of metastasis related proteins（E-cadherin,β-catenin,MMP3,MMP9,Vimentin）expression.Last we overexpressed SDF4 in SDF4-knockdown cells,in which SDF4overexpression could restore their ability to proliferation,migrate and invade in the SDF4-knockdown cells.The results suggested that SDF4 plays an important role in the proliferation and invasion of GBM cells.（3）SDF4 promotes cell proliferation and invasion through NFAT1 signaling pathway.NFAT is a Ca²⁺-dependent transcription factors.and it activation accelerates important cellular processes,such as cell proliferation,survival and metastasis.To determine whether the proliferative effect and metastatic effects of SDF4 on GBM cells were NFAT dependent,we treated SDF4-knockdown GBM cells with a dephosphorylation inhibitor of NFAT1（FK-506）.MTT assays and Brd U incorporation showed that the proliferation and DNA synthesis ability of cells was significantly inhibited,and Transwell assays and wound healing assays showed that the invasion ability of cells was also inhibited.Consistently,we found that the expression of the protein related to proliferation and invasion（CDK4,Cyclin D1 and MMP3,Snail1）was recovered by Western blot.Last,we performed chromatin immunoprecipitation（CHIP）and double luciferase assay to examine whether NFAT1 could regulated the expression of MMP3 at the transcriptional level,and found that NFAT1 could enrichment at the promoter of MMP3 and activated MMP3 transcription.（4）SDF4 regulates theβ-catenin signal pathway by reducing the ubiquitination ofβ-catenin.To further confirm that SDF4 has a biological impact onβ-catenin,we performed Real-time fluorescence quantitative PCR analysis and found that theβ-catenin levels were not affected in SDF4-knockdown U-87 MG and LN-229 cells.The result suggesting that SDF4 may regulateβ-catenin levels post-transcriptionally.Subsequently,We used the MG132 to treat GBM cells.Western blot results showed that the degradation ofβ-catenin was inhibited after MG132 treating the SDF4-knockdown cells.Indeed SDF4 knockdown could increase the turnover rate ofβ-catenin in the presence of the de novo protein synthesis inhibitor cycloheximide（CHX）.Last.we examined the ubiquitination ofβ-catenin and found that the ubiquitination level ofβ-catenin increase in SDF4-knockdown cells.Taken together,these results indicated that SDF4 regulates theβ-catenin signal pathway by reducing the levels ofβ-catenin ubiquitination.（5）SDF4 affects the ubiquitination ofβ-catenin through Ca²⁺-NFAT1 signaling pathway.We measured the nuclear transfer of NFAT1 by nucleolabel-cytoplasmic separation,and found that NFAT1 is exclusively localized in the cytoplasm when SDF4-knockdown.Consistently,Similar results were also obtained by immunofluorescence.To investigate the physical interaction between NFAT1 and theseβ-catenin,we performed co-immunoprecipitation（Co-IP）experiments and found that NFAT1 associated withβ-catenin in vivo.To confirm whether NFAT1 regulate the ubiquitination ofβ-catenin,we treated GBM ells with a dephosphorylation inhibitor of NFAT1（FK-506）,and found that NFAT1 is almost localized in the cytoplasm andβ-catenin was reduced.Next,FK-506induced the reduction ofβ-catenin was inhibited by MG132.The results indicated that SDF4 could mediate the ubiquitination degradation ofβ-catenin through increased the p-NFAT1 in the cytoplasm.Last,we treated LN-229 cells with a number of calcium channel inhibitors and activators,and found that the ubiquitylation level ofβ-catenin was regulated by intracellular calcium levels.Take together,these results suggested that SDF4 affects the ubiquitination ofβ-catenin through Ca²⁺-NFAT1signaling pathway.In conclusion,in this study,we found that the high expression of SDF4 was associated with poor prognosis in glioblastoma patients,and we first reported that SDF4 could changed the intracellular Ca²⁺and activated the Ca²⁺-NFAT1 signaling pathway.In addition,the activation of SDF4-induced dephosphorylation of NFAT1 in the cytoplasm can demodulate the ubiquitination degradation ofβ-catenin and induced cell proliferation and migration.Our results suggested that SDF4-induced Ca²⁺-NFAT1/β-catenin axis disorders promote GBM cells proliferation and invasion.Thus,our studies provide insights into the applicability of employing the SDF4 as a potential therapeutic target in cancer.