The Inflammatory Regulation And Mechanism Of P53 Mutation Status In Tumor Microenvironment

Inflammation is the double-edged sword for tumorigenesis,promoting or inhibiting tumor growth depending on the tumor microenvironment.And the inflammatory regulation of tumor microenvironment has become a hot topic in tumor immunology.The macrophages infiltrated in tumor sites play the leading role in tumor-associated inflammation.It has been demonstrated that macrophages in tumor sites are usually educated towards an M2 anti-inflammatory phenotype,and the M2 profile macrophages could further suppress immune response to promote tumor growth,migration and invasion.Reprogramming macrophages in tumor microenvironment and transforming them towards the M1 pro-inflammtory profile to enhance immune response has become an important focus in tumor clinical immunotherapy.The tumor suppressor p53 is commonly mutated in many human tumors,and p53mutations are believed to contribute to the inflammation-associated tumorigenesis.However,the impact of p53 mutants on macrophages in tumor microenvironment remains unclear.In order to understand the effect of p53 mutations on the inflammatory phenotypes of macrophages in tumor microenvironment,we performed the following studies:1、First,we analyzd the inflammatory level among wild-type p53 mice(p53^+/+),p53 null mice(with spontaneously occurred tumor,p53^-/-)and p53^N236S mice(with spontaneously occurred tumor,p53^S/S).Our data revealed the higher expressions of inflammatory factors TNFα,OSMR,c-Myc,Bcl-xl,Cyclin D1,p-STAT3,STAT3 and ERK in liver and spleen from p53^-/-mice and p53^S/S mice than those from p53^+/+mice.These results indicated that the inflammatory level was up-regulated in p53^-/-mice and p53^S/S mice,suggesting the up-regulated inflammatory level during tumorigenesis under p53 dysfunctional status.2、We further compared the inflammatory levels between p53 null mutation and p53point mutation.The results are as follows:In cell levels:Compared with human lung carcinoma cells H1299 with p53 null background,H1299 cells with p53^R175H or p53^R273H showed the higher expressions of PPARγand CSF1,which could induce the macrophage polarization towards the M2profile.Meanwhile,the activation of inflammatory signal pathway IL6/STAT3 in p53^R172H MEFs was also lower than that in p53 null MEFs.These data suggested that the cells with p53^R175H/p53^R273H/p53^R172H mutation maintained the lower inflammation,indicating that p53^R175H/p53^R273H/p53^R172H mutant might have certain impact on regulating the inflammatory phenotype of macrophages in tumor microenvironment.Meanwhile,we overexpressed the oncogene Ras in MEFs with p53 null mutation or p53^R172H mutation,and established the transplanted tumor mouse models to study the impact of p53 mutations on macrophage polarization in solid tumors.Data revealed that compared with the p53 null tumor samples,p53^R172H tumor samples showed the lower expressions of IL6 and M1 macrophage markers CCL3 and i NOS,and the higher expressions of CSF1 and M2 macrophage markers CCL22,CD206 and Arg1.Meanwhile,when compared with p53 null mutation,MEFs with p53^R172Hmutation also formed bigger tumors in vivo.These data suggested that p53^R172H tumor samples maintained the lower level of inflammation and contained more M2phenotype macrophages,indicating that the p53^R172H mutant might promote the M2phenotype polarization of macrophages in tumor sits and promote tumor growth.The results above indicated that compared with the p53 null mutation,p53^R175H/p53^R273H/p53^R172H mutant might induce the anti-inflammatory phenotype of macrophages in tumor microenvironment,maintain the low level of inflammation,and thus obtain the more aggressive tumorgenesis.3、In order to study the impact of different p53 mutation status on macrophages in tumor microenvironment,we further co-cultured the tumor cell-derived conditional media with macrophages to study the regulation of tumor cell-secreted factors in macrophage profiles.We found that the conditional media from H1299 or MEFs with p53 mutations all significantly induced the down-regulated expressions of M1markers TNFα,CCL3,IRF5 and i NOS in macrophages comparing with the control group,and the macrophage phagocytosis was also greatly inhibited.These results suggested that the conditional media from tumor cells with p53 null mutation or p53^R175H/p53^R273H/p53^R172H mutation could induce the M2 phenotype macrophage polarization,indicating that p53 mutations could affect the secretions of factors in tumor cells,thereby regulating the anti-inflammatory phenotype of macrophages in tumor microenvironment.Besides,we further compared the inflammation regulation of tumor microenvironment in macrophages between p53 null mutation and p53 point mutation.The results indicated that the environment under p53 point mutation status,such as p53^R273H/p53^R172H,could acquire the oncogene function and more significantly induce the anti-inflammatory profile macrophages.The results above illustrated that tumor progression under p53 dysfunctional status was accompanied with the up-regualted inflammation.And tumor microenvironments under different p53 mutation status could induce the M2 phenotype macrophages by regulating the secretions of factors in tumor cells.Moreover,p53 point mutation could more significantly induce M2 phenotype macrophages and maintain the lower inflammation than p53 null mutation.Based on these,p53 point mutation such as p53^N236S/p53^R172H showed the more aggressive tumorigenesis in vivo.In order to antagonize the impact of p53 mutations on M2 profile polarization in macrophages,we then screened several natural products.Through comparing the half inhibitory concentration(IC₅₀)of natural products on tumor cells,we removed the natural products with higher toxicity to tumor cells or less effect on tumor cell viability and found that curcumin showed the intermediate IC₅₀ to tumor cells,hence we finally chosed curcumin in the following assays.In order to eliminate the direct antitumor role of curcumin,we further detected the concentrations of curcumin that did not show obvious inhibition on tumor cells.By treating tumor cells with low concentration curcumin,the regulation role of curcumin on the expressions of tumor cell factors in reprogramming macrophages was illustrated in vitro and in vivo.And the data suggested that:1、By treating the tumor cells with low concentration curcumin,we found that curcumin did not directly kill tumor cells,but inhibited the IL6/STAT3 signal and down-regulated the expressions of PPARγand CSF1 in tumor cells.It was proved that STAT3 signal was usually abnormally activated during tumorigenesis and STAT3,PPARγand CSF1 could induce the M2 profile macrophage polarization.These data suggested that curcumin regulated the inflammation in tumor cells and tumor environment.Furthermore,by collecting the tumor cell-derived conditional media to culture macrophages,data revealed that the conditional media from curcumin-treated tumor cells with different p53 mutations all induced the up-regulated expressions of M1 markers TNFα,IRF5,CCL3 and i NOS in macrophages comparing with the ones cultured in the conditional media from tumor cells with different p53 mutations.These results suggested that low concentration curcumin could regulate the tumor cell’s immuno-modulative secretion,re-modulate the tumor microenvironment and shift the p53 mutation educated M2 macrophage polarization back towards M1profile.2、In order to verify the antitumor immuno-modulation of curcumin in vivo,we then subcutaneously injected the curcumin-pretreated MEFs into mice with normal immune capacity.We found that by pretreating these MEFs with curcumin in vitro,the tumor samples with different p53 mutations all showed the inhibited IL6/STAT3signal,the decreased expressions of PPARγand CSF1,the up-regulated expressions of NF-κB subunits RELB and p65 and the down-regulted expressions of M2 markers CCL22,IRF4,CD206 and Arg1.Meanwhile,the tumor growth was greatly inhibited.These results suggested that curcumin application in vitro could re-modulate the tumor inflammatory environment in tumor-bearing mice,inhibit the p53 mutation educated M2 macropahge polarization and ultimately suppress the tumorigenesis of MEFs with p53 null or p53^R172H mutation.In conclusion,our study illustrated that low inflammation was accompanied with tumorigenesis under p53 dysfunctional status,and p53 mutation could regulate the expressions of inflammation factors in tumor cells and induce the M2 macrophages.In addition,our study further demonsrated that p53 point mutation could more significantly induce the M2 macropahge polarization and maintain the lower inflammation than p53 null mutation,thereby exhibiting the more aggressive tumorgenesis.And curcumin application could re-modulate the tumor inflammatory environment,shift the p53 mutation educated M2 macrophage polarizaiton back towards M1 profile and further inhibit the tumor growth.This study might provide a potential in tumor personalized immunotherapy for tumor patients with different p53status.