Study On The Mechanism Of P53N236S Mutation Regulating Cholesterol-Hedgehog Pathway Leading To Osteosarcoma Metastasis

Tumor metastasis is the main cause of tumor progression,which seriously affects the clinical prognosis of tumor patients and leads to their death.Abnormal activation of the Hedgehog（HH）signaling pathway can promote the occurrence and development of various tumors.In recent years,studies have found that the HH signaling pathway is involved in regulating tumor invasion and metastasis.Interestingly,studies have shown that cholesterol can covalently modify SHH protein and its downstream Smoothened protein and activate the HH pathway.The tumor suppressor p53 is a very important transcriptional regulator that inhibits tumors’occurrence and development by regulating the cell cycle,promoting apoptosis,and regulating metabolism-related pathways.However,mutated p53 loses the function of suppressing tumors and may also acquire some new functions,such as promoting the synthesis of cholesterol,which further promotes the occurrence and development of tumors.The mutation rate of p53 occurrence in osteosarcoma is as high as 80%,but the mechanism of its regulation of osteosarcoma metastasis is less studied.In this study,the p 53 ^N236S（briefly as p53S）mutant mouse primary osteosarcoma cells were used as a model to explore the mechanism by which the mutation promotes cholesterol synthesis and further activates the HH pathway to promote tumor metastasis.Our study used the primary osteosarcoma cells of p53null and p53S mice as the research objects.First,the migration ability of the two genotypes of osteosarcoma cells was detected by wound healing assay and Transwell assay.The results showed that the migration capacity of p53S osteosarcoma cells was enhanced.We further tested their migration capacity in vivo by xenograft experiment via tail vein injection.It showed that the tumor-bearing mice of p53S mutant osteosarcoma cells had a reduced survival rate,continued weight loss,more severe pulmonary edema,and sporadic tumor nodules found in lung surface and HE staining of sections.The above studies indicate that the metastatic ability of p53S tumor cells was stronger than that of p53null tumor cells.Further,we detected the transcription and protein expression levels of HH signaling pathway-related genes in primary osteosarcoma cells and found that the transcriptional levels of Shh and Gli1（Glioma-associated oncogene1）were significantly increased in p53S tumor cells.Moreover,the content of SHH in the cell culture supernatant increased,and the expression of Gli1 in the cells increased.The above results suggest that the HH pathway is activated in p53S osteosarcoma cells.We further treated p53S tumor cells with the HH signaling pathway inhibitor vismodegib and found that their migratory ability was attenuated.The above data suggest that p53S can promote tumor migration by activating the HH signaling pathway.It has been reported that cholesterol can promote its release by modifying SHH and directly binding SMO to activate the HH signaling pathway.To further study the molecular mechanism of p53S promoting HH signal activation,we detected total cholesterol content in tumor cells by enzymatic hydrolysis and Filipin staining.We found that total cholesterol content in p 53S tumor cells increased.We further detected the transcription and protein expression levels of key genes SREBP2 and HMGCR in the Cholesterol synthesis pathway.We found that the expressions of these two key factors were increased in p53S tumor cells.Cholesterol homeostasis is not only related to its synthesis regulation but also affected by uptake and efflux.Therefore,we further detected the expression levels of cholesterol uptake and efflux-related genes Ldlr and Abca1/Abcg1 in the two genotypes of tumor cells.The transcript level and protein expression level of Abca1/Abcg1 were significantly decreased,but the Ldlr was increased.It suggests that the increased cholesterol in p53S tumor cells is not only related to increased synthesis,but also affected by uptake and decreased efflux.We then treated p53S tumor cells with the cholesterol neutralizer MβCD.It showed that the total cholesterol levels were reduced,the HH pathway was inhibited,and the ability to migrate was reduced.It is indicated that p53S promotes cholesterol accumulation by promoting cholesterol synthesis,uptake and inhibiting its efflux.The cholesterol further activates the HH signaling pathway,ultimately enhancing osteosarcoma metastasis ability.Summly,we tested the metastatic ability of p53S osteosarcoma primary cells in vivo and in vitro and preliminarily confirmed that p53S osteosarcoma cells have abnormal activation of the cholesterol synthesis-HH signaling pathway.This study provides a theoretical basis for further revealing the function and mode of action of p53mutation in osteosarcoma metastasis,thereby providing a new potential target for diagnosing and treating osteosarcoma.