The Molecular Mechanism Of P53^N236s In Promoting Tumor-Associated Fibroblast Activation By STAY3 Pathway

Cancer-associated Fibroblast（CAFs）,an important component of tumor microenvironment,regulate tumor growth and invasion by secreting various tumor-promoting effect factors in a paracrine manner,which provided a new insight on anti-tumor therapy.At present,the role of p53 and mutant p53 in CAFs activation is still unclear.P53N236S(homozygous mutation is p53^S/S,p53^N239S in human beings,hereinafter referred to as p53S)is a missense point mutation occurring on exon 7 of p53,which shows that codon 236 on exon 7 is mutated from AAT to AGT.Previous study show that p53^S/S mouse embryonic fibroblasts（MEFs）have CAFs characteristics that promote the growth,migration and invasion of prostate cancer cell PC-3.CAFs usually takes a-SMA as a marker,has strong collagen contraction ability and secretes CAFs-related effect factors to perform its tumor-promoting function.WT,p53^-/-and p53^S/S MEFs cells were used as materials to detect the expression level of a-SMA in the cells by Western blotting.It was found that a-SMA was highly expressed in p53^S/SMEFs.The following gel collagen contraction experiments showed that p53^S/SMEFs had the collagen contraction ability peculiar to CAFs.Subsequent ELISA experiments also showed that p53^S/SMEFs increased their ability to secrete CAFs effector CXCL12.Meanwhile,p53^S/S MEFs can promote the migration and invasion of human non-small cell lung cancer cell H1299.The above data suggest that p53S promotes the activation of CAFs characteristics.In order to further study the molecular mechanism of p53S regulating the activation of CAFs characteristics.Microarray chip scanning and GSEA analysis of MEFs cells showed that the IL6-JAK-STAT3 pathway was activated in p53^S/SMEFs.Western blotting results also showed that in p53^S/SMEFs,the expression of STAT3 pathway activated marker p-STAT3^（Y705）increased.After blocking IL6-JAK-STAT3 pathway,with the decrease of p-STAT3^（Y705）protein expression,the protein level ofα-SMA decreases,the collagen contractility of p53^S/S MEF decreases,the ability to secrete CXCL12 decreases,and the ability of p53^S/SMEF to promote the migration and invasion of H1299 also decreases.It indicates that p53S activates CAFs via IL6-JAK-STAT3pathway.So,how does p53S activate the IL6-JAK-STAT3 pathway?ROS accumulation is an activator of IL6-JAK-STAT3.The level of ROS in cells was detected,and it was found that the level of ROS in p53^S/S MEFs was significantly lower than that in WT MEFs.At the same time,by detecting the protein expression of PGC-1a,the expression of PGC-1a in p53^S/S MEFs was also found to be elevated.It is suggested that p53S does not activate IL6-JAK-STAT3 pathway through accumulated ROS.After detecting the expression of the upstream and downstream gene proteins of IL6-JAK-STAT3,it was found that the level of upstream phosphokinase p-JAK2^1007/1008protein increased and the expression of phosphatase SHP2 protein did not change.It was preliminarily speculated that p53S activated the IL6-JAK-STAT3 pathway by up-regulating p-JAK2^1007/1008,thus activating the CAFs characteristic.During the study,it was found that the expression of p-STAT3^（Y705）andα-SMA protein increased during the continuous passage of WT MEFs to senescence,the contractility of collagen increased,and the ability to secrete CXCL12 increased,showing CAFs characteristics similar to p53^S/S MEFs.During aging,p16 expression level increases,DNA damage marker r-H2AX increases and ROS accumulation may activate STAT3 pathway,thus activating its CAFs characteristics.Therefore,the expression levels of p-STAT3^（Y705）andα-SMA in p16^-/-MEFs were detected.it was found that p16^-/-MEFs also expressed p-STAT3^（Y705）andα-SMA at high levels,excluding activation of IL6-JAK-STAT3 pathway caused by p16 protein elevation.However,the high level of r-H2AX in p16^-/-MEFs may promote activation of STAT3 pathway.WT MEFs were treated with Dox,and it was found that p-STAT3 and a-SMA increased with the increase of r-H2AX expression.However,DNA damage can cause ROS accumulation.Therefore,while Dox is treating WT MEFs cells,NAC is used to remove ROS in the cells,and the expression of p-STAT3^（Y705）protein is found to decrease,indicating that ROS accumulation in fibroblasts can activate its CAFS characteristics through L6-JAK-STAT3 pathway.To sum up,this study shows that p53S activates IL6-JAK-STAT3 via up-regulation of p-JAK2^1007/1008,thus activating CAFs characteristics.In addition,ROS accumulation also activates CAFs characteristics through IL6-JAK-STAT3 pathway during fibroblast senescence.