| Background and objection: Pancreatic cancer(PC)is an aggressive malignancy with an exceedingly low 5-year survival rate,its incidence is increasing year on year and the mortality of PC almost equal to the incidence.Despite decades of research,the basic and clinical diagnosis and treatment technology research of PC has made great progress and breakthrough,however,there is still no significant improvement in survival time of patients with PC.The main reason is that,on the one hand,the lack of effective screening methods for early detection,on the other hands,drug-resistant and relapse remain key challenges for PC.Therefore,potential biomarkers and effect therapeutic targets for PC are urgently required.Ceramide-1-phosphate transfer protein(CPTP)is member of the glycolipid transfer protein(GLTP)family,it can selectively transport the bioactive sphingolipids(SLs)ceramide-1-phosphate(C1P)but other SLs in mammals.CPTP is involved in SLs metabolism and played a significant role in maintaining homeostasis of SLs and associated with autophagy and inflammation.To date,the roles and mechanism of CPTP in PC have not been systematically studied,it worth to study in-depth.On this study,we systematically investigated the roles,potential mechanism and transcriptional regulation of CPTP.the work described here provided a theoretical basis for CPTP as a promising therapeutic target of PC.Methods: The Linked Omics and GEPIA website tool were used to analyze the relationships between CPTP expression level and tumor grade,overall survival(OS)and disease-free survival(DFS)times of PC patients.RT-q PCR and immunohistochemistry analysis were used to measure the m RNA and protein expression levels of CPTP from PC tissue and nontumor normal tissue,respectively,next the correction between CPTP expression and clinicopathological characteristics of PC patients was analyzed according IHC results.Stable human PC cell lines overexpressing CPTP or knocked down were established.The effects of CPTP overexpression and knockdown on proliferation and metastasis of PC cells were examined in vitro and in vivo using Cell Counting Kit-8,colony formation,transwell and matrigel assays,as well as xenograft models.The expression levels of proteins-associated with cell metastasis were analyzed using western blot analysis.RNA sequencing,lipidomic and proteomic analysis were performed to investigate the mechanism of CPTP.Dualluciferase,electrophoretic mobility shift and chromatin immunoprecipitation assays were performed to analyze the transcriptional regulation of CPTP in the PC cell lines.Result: CPTP is highly expressed in PC and is associated with tumor grade and poor prognosis in patients with PC.Overexpression of CPTP indicated that CPTP promotes proliferation,migration and invasion of PC cells in vitro and in vivo,while CPTP koockdown are reverse.Mechanistically,Enrichment analysis of differential genes obtained by transcriptome sequencing found that CPTP was related to abnormal activation of PI3K/AKT signaling.Proteomic analysis showed that CPTP was co-upregulation with seronine kinase and phosphatidylinositol signaling pathway members SH3BP1,KDM5 B and PI4 KA,And PI4 KA is an upstream molecule of the PI3K/AKT signaling.On the other hand,the result of lipidomic analysis demonstrated that alternation of CPTP could affect SLs level in PC cells,including Cer,C1 P,Car,SM,Sph and LPC.C6-cer treatment induced the increase of endogenous Cer level in PC cells,up-regulation of Cer inhibits the cells proliferation,migration and invasion in knockdown CPTP PANC-1 cell.For transcriptional regulation,transcription factors Sp1/Sp3 can bind to the proximal regulatory region of CPTP promoter and regulate its transcription.Overexpression of Sp3 partially reverses the reduction in cell proliferation,migration and invasion by CPTP knockdown.These results indicated that the pro-cancer effect of CPTP on PC was regulated by transcription factors Sp1/Sp3.Conclusion: CPTP is upregulated in PC tumor and related to poor prognosis of PC patients.CPTP promotes the proliferation,migration/invasion and tumor growth and metastasis of PC cells,thus plays a pro-tumor role in PC,and the expression of CPTP is regulated by transcription factors Sp1/Sp3.Mechanistically,CPTP may activates serine/threonine kinase and PI4KA/AKT signaling pathway,in addition,CPTP is involved in SLs metabolism and the change of SLs level may be one of the cancer-promoting mechanisms of CPTP.Taken together,multiple factors contribute to the pro-tumor effect to PC induced by CPTP. |
