Study Of Acquired Drug Resistance Derived From P53^N236S Mutation In Tumor Cells

The proto-oncogene activation and tumor-suppressor gene inactivation together lead to tumorigenesis. p53 is believed to be the most important tumor supressor gene. Missense mutation or deletion of TP53 genes were found in about 50% human cancers. The mutp53 protein might possess activities of its own, often not present in the original wtp53 protein, which could actively contribute to various aspects of tumor progression. Such activities, commonly described as gain-of-function (GOF) of mutp53. p53 GOF mainly showed as promoting cell proliferation, enhancing the cell anti-apoptotic ability cells, promoting cell migration and invasion, as well as inducing cellular changes in energy metabolism, etc. In addition, a large number of clinical data have shown that mutant p53 is involved in anticancer drug resistance of tumor cells to. Understanding the GOF of p53 mutations, especially the mechanism of acquired drug resistance will help us further understand the molecular pathways for p53 mutations promoting tumorigenesis, and provide an important basis for personalized cancer therapy and drug targeting.Our previous work found that p53S lost the wtp53 function, and gained the potential of oncogene and promoted tumorigenesis when cooperates with oncogenic Ras, suggesting the GOF of p53S. These data also imply the potential of drug resistence caused by p53S and Ras cooperation.Therefore, this study aim at p53S gain of function in drug resistence, especially the mechanisms underlying. The results showed that p53S caused anti-apoptotic effect in tumor cells, thus theresistence to antineoplastic treatment. We use doxorubicin to treat four kind of cells with p53S or Ras background:p53S/S +Ras (introduced Ras-pBABE vector into p53S/S cells and stably expressed Ras), p53S/S+vector (introduced empty pBABE vector into the p53S/S cells), p53S/+ +Ras, p53S/+ +vector, and wild-type cells (WT). MTT assay and Annexin-V staining showed that, compared with the wild type cells, p53S/S+ ector cells were significantly tolerant to doxorubicin treatment. Interestingly, stable expression of Ras further enhance this tolerance. Then, we try to study the mechanisms for the drug resistance aquired in p53S/S+Ras and p53S/S cells by analyzing the expression level of p53S downstream genes using Real-time PCR and Western Blot assays.The results showed that upon treatment of DNA synthesis inhibitor doxorubicin, p53S lost its function in regulating downstream factors, such as p21 Cip1/Waf1 and puma, hence, p53S lost its regulatory function in both cell cycle and apoptotic pathway. These data suggest that the apoptotic response induced by doxorubicin is weakened in tumor cells bearing p53S. The stable expression of Ras further promotes anti-apoptotic effect. Meanwhile, we also detected the expression level of p16Ink4a, caspase-3 and PARP in p53S cells and found that, compare to wild type p53, p53S could significantly reduce the cleavage of PARP and caspase-3, the latter is the apoptosis executor. Thus, the function of p53S could effectively prevent cells from apoptosis.To further study the molecular mechanisms of acquired drug resistance driven by p53S, we utilized ChIP-on-Chip technology to analyze the downstream promoters regulated by p53S By comparing to the gene promoters regulated by wild-type p53, we obtained the genes specificly regulated by p53S. We screened and verified genes associated with apoptosis and cell cycle regulation, such as bid, caspase and IAPs family members. These data help us to predict that p53S may participate in the regulation of molecular pathway involved in endogenous mitochondria depended cell apoptosis.In summary, our study found that p53S could endow tumor cells anti-apoptosis ability and drive acquired resistance for antineoplastic drug treatment. We tried to explore the possible molecular mechanisms. The results suggest that, p53S may drive tumor cells' drug resistance by regulating the apoptotic pathway. Our study may provide theoretical basis for the clinical treatment strategy targeting p53 mutation.