Fibroblasts Promoting Lung Cancer Cells Migration And Invasion Via P53N236S/STAT3/CXCL12 Axis

Lung cancer（LC）is one of the cancers with the highest morbidity and mortality in the world,and its high metastasis rate is the main reason for its recurrence and poor prognosis.Studies have shown that cancer-associated fibroblasts（CAFs）are one of the important causes of lung cancer metastasis.However,the molecular mechanism of CAFs activation and its regulation of lung cancer metastasis has not been fully elucidated.This study mainly revealed the regulation mechanism that p53S/STAT3/CXCL12 signal axis activates the CAFs characteristics of fibroblasts and promotes the migration and invasion of lung cancer cells.Our laboratory previously found that p53^S/S(p53^N236S in human p53^N239S,referred to as p53S)fibroblasts（MEFs）showed CAFs characteristics,such as high expression of CAFS-related proteinsα-SMA,CXCL12,etc.,with unique collagen contraction force of CAFs.p53^S/SMEFs promoted the migration and invasion of human Non-small cell lung cancer cell H1299.Further studies showed that the activated STAT3pathway in p53^S/SMEFs activated its CAFs properties.To verify that p53S mutations in fibroblasts activate the STAT3 pathway to promote the migration and invasion of H1299,RNA interference was first used to knock down p53S in To verify that p53S mutations in fibroblasts activate the STAT3pathway to promote the migration and invasion of H1299,RNA interference was first used to knock down p53S in p53^S/S MEFs.Western Blot results showed that there was no significant change in STAT3 protein expression when p53S protein expression was decreased,while the expression of p-STAT3^Y705,a marker of STAT3 pathway activation,and Vimentin,a marker of CAFs were also decreased.Transwell functional test results also showed that p53^S/S MEFs had a decreased ability to promote the migration and invasion of H1299 and A549 after reduced p53S expression（mobility:11.75±1.37%VS 4.14±0.01%,P<0.005;Invasion rate:3.92±0.68%VS 0.78±0.05%,P<0.05）.Subsequently,STAT3 in p53^S/S MEFs was knocked down by si STAT3 RNA fragments.Western Blot results showed that after STAT3 expression was decreased,p-STAT3^Y705 expression was also decreased,and CAFs marker Vimentin expression was decreased.At the same time,knocking down STAT3 weakened the ability of p53^S/S MEFs to promote migration and invasion of H1299（mobility:11.75±1.37%VS 1.43±0.07%,P<0.005;Invasion rate:3.92±0.68%VS 0.32±0.07%,P<0.05）.It was preliminarily confirmed that p53S activates the STAT3 pathway in fibroblasts to promote lung cancer cell migration and invasion in MEFs.Western Blot results showed that there was no significant change in STAT3 protein expression when p53S protein expression was decreased,while the expression of p-STAT3^Y705,a marker of STAT3 pathway activation,and Vimentin,a marker of CAFs were also decreased.Transwell functional test results also showed that p53^S/SMEFs had a decreased ability to promote the migration and invasion of H1299 and A549 after reduced p53S expression（mobility:11.75±1.37%VS4.14±0.01%,P<0.005;Invasion rate:3.92±0.68%VS 0.78±0.05%,P<0.05）.Subsequently,STAT3 in p53^S/S MEFs was knocked down by si STAT3 RNA fragments.Western Blot results showed that after STAT3 expression was decreased,p-STAT3^Y705 expression was also decreased,and CAFs marker Vimentin expression was decreased.At the same time,knocking down STAT3 weakened the ability of p53^S/S MEFs to promote migration and invasion of H1299（mobility:11.75±1.37%VS1.43±0.07%,P<0.005;Invasion rate:3.92±0.68%VS 0.32±0.07%,P<0.05）.It was preliminarily confirmed that p53S activates the STAT3 pathway in fibroblasts to promote lung cancer cell migration and invasion.Next,we overexpressed p53S in p53^-/-MEFs.In p53^-/-MEFs,high expression of p53S upregulates the protein expression levels of p-STAT3^Y705 and Vimentin,and p53S endows p53^-/-MEFs with stronger ability to promote the migration and invasion of H1299 and A549（H1299 mobility:2.09±0.32%VS 11.75±1.37%,P<0.005;A549mobility:1.67±0.55%VS 12.26±0.39%,P<0.005;H1299 invasion rate:0.58±0.18%VS 5.65±0.11%,P<0.005;A549 invasion rate:0.83±0.01%VS 5.97±0.6%,P<0.005）.In p53^-/-MEFS,p53S is highly expressed,while STAT3 is knocked down,p53S up-regulated p-STAT3^Y705 and Vimentin protein expression is down-regulated,and the ability of p53S to promote migration and invasion of H1299 and A549 is impaired（H1299 mobility:11.47±0.25%VS 2.14±0.34%,P<0.005;A549 mobility:12.26±0.39%VS 1.52±0.23%,P<0.005;H1299 invasion rate:5.65±0.11%VS 2.04±0.15%,P<0.05;A549 invasion rate:5.97±0.6%VS0.84±0.10%,p<0.005）.These experiments strongly confirmed that p53S activates the STAT3 pathway in fibroblasts to promote the migration and invasion of lung cancer cells.To explore the molecular mechanism of p53S regulation of STAT3 pathway.We detected the expression levels of WT-p53（wild-type p53）/p53S in WT,p53^-/-and p53^S/SMEFs by RT-PCR and Westren Blotting,and found no significant difference in the m RNA expression levels of WT-p53 and p53S.However,the protein expression level of p53S was significantly higher than that of WT-p53,suggesting that the stability of p53S protein was higher than that of WT-p53.After treating p53^S/S MEFs with Ganetespib,the active inhibitor of HSP90,there was no significant change in the expression of HSP90,the protein expression level of p53S decreased significantly,and the p-STAT3^Y705/STAT3 ratio decreased,confirming that p53S increased its protein stability through HSP90.It also suggests that p53S stabilizes p-STAT3 ^Y705and activates the STAT3 pathway through protein level.p-STAT3^Y705is co-regulated by phosphokinase JAK2 and phosphatase SHP2.Co-ip experiment results showed that p53S bound to SHP2 and STAT3,and inhibited the binding of SHP2 and STAT3,thus inhibiting the dephosphorylation of p-STAT3^Y705 and maintaining the continuous activation of STAT3 pathway.CAFs promote tumor metastasis by secreting a variety of cytokines.It was found that the secreted protein concentration from p53^S/SMEFs was significantly higher than WT and p53^-/-MEFS,and the secreted protein concentration in p53^S/SMEFs was decreased by knocking down STAT3 or inhibiting the STAT3 pathway.Conclusion:In p53^S/SMEFs,the activated STAT3 pathway promotes protein secretion.Rt-pcr results showed that CXCL12 was regulated by the STAT3 pathway,suggesting that p53S/SMEFs promoted the migration and invasion of H1299 and A549 by secreting CXCL12.Therefore,hit CXCL12 low in p53^S/SMEFs.Westren Blot results showed that when CXCL12 expression was decreased,the ratio of P-STAT3^Y705/STAT3 in p53^S/S MEFs decreased,suggesting that CXCL12 activated the STAT3 pathway through positive feedback in fibroblasts.Transwell results showed that p53^S/SMEFs reduced the migration and invasion of H1299 and A549 by knocking down CXCL12（mobility:11.75±1.37%VS 1.68±0.17%,P<0.005;Invasion rate:3.92±0.68%VS0.80±0.07%,P<0.005）.CXCL12 normally works on its receptor,CXCR4.In order to specifically inhibit the function of CXCR4 in lung cancer H1299,H1299 cells were cultured on p53^S/SMEFs medium alone or with its receptor inhibitor AMD3100,and the effect of CXCR4 on H1299 migration was detected.It was found that the addition of AMD3100 reduced the ability of p53^S/SMEFs conditioned medium to promote the migration and invasion of H1299 and A549（mobility:9.41±1.25%VS 0.93±0.06%,P<0.05;Invasion rate:5.20±0.68%VS 0.45±0.08%,P<0.05）.The above data suggest that the p53S/STAT3/CXCL12 axis in CAFs promotes lung cancer cell migration and invasion through CXCR4.In conclusion,this study:1.Further confirmed that p53S mutation in fibroblasts can promote the migration and invasion of lung cancer cells H1299 and A549 by activating CAFs properties through the STAT3 pathway.2.The molecular mechanism of p53S activation of STAT3 pathway was preliminarily elucidate:p53S mutant protein increased stability,inhibited the binding of phosphatase SHP2 to STAT3,stabilized p-STAT3,and continuously activated STAT3 pathway.3.CXCL12 is up-regulated by the activated STAT3 pathway in fibroblasts.On the one hand,CXCL12promotes p-STAT3/STAT3 expression through positive feedback and maintains its CAFs characteristics;On the other hand,fibroblast derived CXCL12 promotes lung cancer cell migration and invasion through CXCR4.This study revealed the molecular mechanism by which p53S mutations activate CAFs and promote lung cancer migration and invasion,providing new targets and ideas for targeting CAFs for lung cancer treatment and prognostic analysis.