Mechanism Of P53^N236S Promoting Tumor-associated Fibroblast Activation

Malignant tumors are prone to metastasis,recurrence and difficult to treat,which seriously affect people's quality of life and threaten life and health.Tumor metastasis is a complex dynamic process involving a variety of factors,including the characteristics of the tumor cells themselves,changes in the host microenvironment.Tumor microenvironment,as a complex microenvironment,is mainly composed of cell components,tumor cells and stromal cells,as well as non-cellular components,extracellular matrix,chemokines and cytokines.Cancer-associated Fibroblasts?CAFs?,as the largest component of the tumor microenvironment,are closely related to the metastasis of malignant tumors.Therefore,in-depth study of the molecular mechanism of tumor-associated fibroblasts affecting tumor metastasis can provide a new method for early diagnosis and treatment of cancer metastasis.p53^N236S(homozygous mutation to p53^S/S,p53^N239S in humans,hereafter referred to as p53S),is a missense point mutation occurring in exon 7 of p53,codon 236 of exon 7mutates from AAT to AGT.This mutation gives it the potential for oncogenes.The p53^N236S236S knock-in mouse model was successfully constructed in the laboratory.It was found that p53^N236S homozygous mutant mice had the characteristics of early tumor onset,high invasive lymphoma and high metastatic sarcoma.Mouse tumor cells were injected subcutaneously into WT,p53^-/-and p53^S/S mice by transplanted tumor experiments,and it was found that tumor cells grew fastest in p53^S/S/S mice,and were immunized with Brdu incorporation and proliferation marker PH3.The blot results confirmed that tumor cells had the highest proliferation level in p53^S/S mice.It is suggested that p53^S/S provides a favorable microenvironment for tumor growth.CAFs are an important component of the tumor microenvironment.qRT-PCR results showed that the tumor tissues of p53^S/S mice highly expressed CAFs-related factors CXCL12 and IGF2.At the same time,we examined the expression of CAFs-related factors in p53^-/-aand p53^S/S/S spontaneous tumors,and found that CAFs-related factors CXCL12,CXCL10,CXCL1,CXCR3,TGFB2 and IL6 were all highly expressed in p53^S/S spontaneous tumors.Because the muscle tissue of mice has the same origin as CAFs,it was found that the expression of CAFs markers FAP,Vimentin and PDGFR?and CAFs-related factors CXCL12,CXCL1and IL6 were also increased in p53^S/S mouse muscle tissues,further indicating that p53S may activate CAFs.Promote early onset and metastasis of tumors.To verify the hypothesis that p53S promotes the early development and metastasis of tumors by activating CAFs,three genotypes of mouse embryonic fibroblasts?MEFs?were prepared:WT,p53^-/-and p53^S/S.qRT-PCR results showed that CXCL12 was highly expressed in p53^-/-and p53^S/S MEFs compared with WT MEFs,and CXCL12 was the highest in p53^S/S MEFs.ELISA experiments also confirmed that CXCL12 is highly expressed in the supernatant of p53^S/S MEFs.In addition,other CAFs-related factors:CXCR4,CXCL1,CXCL2,IGF2,FGF7,FGF10,IL6 and TGFB2 were all up-regulated in p53^S/S MEFs.Tip:p53S may gain new features that promote CAFs activation.Subsequently,the characteristics of CAFs promoting tumor growth and metastasis of p53S/S MEFs were further verified by in vitro and in vivo experiments.The co-culture of PC-3 with WT,p53^-/-and p53^S/S MEFs in Transwell system showed that both p53^-/-and p53^S/S MEFs could promote the migration and invasion of PC-3 cells,while p53^S/S MEFs could promote the migration and invasion of PC-3 cells more significantly.Conditional media derived from WT,p53^-/-and p53^S/S MEFs were prepared.CCK8 was used to detect the effect of PC-3 on the proliferation of PC-3.It was found that PC-3 had the highest proliferation level in p53^S/S MEFs conditioned media.The nude mice xenograft experiments further confirmed that compared with the PC-3 alone injection group and the PC-3+WT MEFs mixed injection group,the PC-3+p53^-/-MEFs mixed injection group had slightly faster tumor growth,while PC-3+p53^S/S MEFs mixed injection group had the fastest growth of tumors.The above data show that p53^S/S MEFs have a stronger ability to promote tumor growth and metastasis than p53^-/-MEFs,and p53S acquires a new function of activating CAFs.The results of HE staining and immunohistochemistry of alpha SMA?CAFs marker?showed that the number of CAFs was higher in p53^S/S MEFs+PC-3 tumor tissues.qRT-PCR data showed that the expression of CAFs markers?alpha SMA,Vimentin and PDGFRA?and related factors?CXCL12,CXCR4 and TGFB2?in p53^S/S MEFs+PC-3tumors was higher than that in p53^S/S MEFs or PC-3 tumors.It is suggested that the tumor cell PC-3 further activates the CAFs characteristics of p53^S/S MEFs.In summary,p53S promotes the activation of CAFs and promotes the early onset and metastasis of tumors.In addition,tumor cell PC-3 further promotes the expression of CAFs-related factors in p53^S/S MEFs,thereby forming a loop that constitutes a tumor-promoting tumor microenvironment.