Study Of Anti-apoptotic Mechanisms Of Acquired Drug Resistance Derived From P53^N236S

Malignant tumor is a big threat to human life and health. p53, an important tumor suppressor, could be activated to regulate the expression of target genes with central roles in signaling pathways including cell cycle arrest, apoptosis and DNA repair in response to cellular stresses, and to maintain cell homeostasis and inhibit tumorigenesis. In spontaneous human tumors the average mutation frequency of p53 is about 50%. Unlike the majority of other tumor suppressors, which usually undergo biallelic inactivation during tumorigenesis by truncation or deletion mutations, p53 is mainly inactivation by point mutaition. And about 97% of p53 point mutations are locatedat DNA-binding domain (DBD), above 75% of which are missense mutations. In most cases, mutp53 not only lost the function of original wtp53, which is named "Loss of Function (LOF)", but also possess new activities of its own to contribute to tumorigenesis, which is named "Gain of Function (GOF)". It endued mutp53 various biological phenotypes, including genomic instability, cellulars migration and invasion, enhanced anti-apoptotic ability and drug resistance etc. Besides, it has been shown that mutated p53 protein resulted from different site of p53 point mutations and mutation subtypes of the same p53 mutation site might be different in their biological phenotypes. Thus, study the gain of function and loss of function of different mutp53 will help us better understand the role of mutp53 in tumorigenesis.The the research of the mechanism of mutp53 promoting tumorigenesis studies focused on six hotspots of p53 point mutations, including R172H, G245S, R248Q, R249S, R273H and R282W. In 2016, the research of Matthew T Chang et al. on hotspots analysis of p53 mutations identified multiple hotspots of p53 mutations, including the N239S (in mice as N236S). In previous studies with MEFs (Mouse Embryonic Fibroblasts), we found p53S lost the transcriptional regulation function of target genes, such as p21 and cyclin G following ionizing radiation. p53S could acquire new activity similar to oncogene, and it could collaborate to Ras mutation (H-rasG12V) to accelerate the development process of tumor. At the same time, p53S gained drug-resistance after antineoplastic treatment by doxorubicin. And overexpressed Ras could cooperate with p53S to promote drug resistance. Doxorubicin is an anthracycline drugs and can insert into DNA between adjacent bases and generate active radical, and prompt unwind of the DNA double helix and result into DNA breakages. Doxorubicin also inhibits the activity of topoisomerase I and II, and RNA polymerase. It interfers with DNA replication and RNA transcription, and ultimately lead to cell death.Hence, in this study we try to verify that p53S endue cells resistant to doxorubicin treatment, and to explore the drug-resistance mechanisms derived from p53S. First we used different dosage of doxorubicin to treat cells in different period, three kind of cells with p53S or Ras background were used:wild-type p53(WT), p53s/s+v (introduced empty pBABE vector into p53s/s cells), p53s/s+Ras (introduced H-rasG12V-pBABE vector into p53s/s cells and stably expressed Ras). We used conventional method of drug screening (MTT, SRB) to detect doxorubicin cytotoxicity for these three kinds of cells. The results showed that compared with WT cells, p53S cells (p53s/s+v) did not have higher ICso values, suggesting that p535 cells were not resisted to doxorubicin treatment based on IC50 values. However, the growth curves showed that growth rate of WT cell decreased following the gradient dosage treatment of doxorubicin, whereas p53s/s+v cells were still in a state of rapid proliferation. This suggested that p53S cells were resistant to doxorubicin to a certain extent. Then we used 0.5 μg/mL doxorubicin to treat these cells for 12 hours or 24 hours, the results of Western Blot showed the cleavage of caspase3 and PARP in p53S cells were lower than WT cells under the same conditions of doxorubicin treatment. The flowcytometry assay also showed the percentage of apoptotic p53S cells is lower than WT cells. These data suggested that p53S cells achieved doxorubicin resistance by inhibiting apoptosis signaling. Besides, the growth curves also showed that p53s/s+Ras cells grew faster than p53s/s+v cells, and the percentage of apoptotic p53s/s+Ras cells were significantly lower than p53s/s+v cells (9.6% VS 28.3%). These results suggested the stable expression of Ras further increased p53S cells’ drug-resistance and anti-apoptosis ability.Unexpectedly, in p53-knockout cell (p53-/-) we found the similar tolerance to doxorubicin and anti-apoptosis ability. These data suggested that the loss of function of p53 mutation might contribute to the resistance of p53S cells to doxorubicin-induced apoptosis. However, in addition to anti-apoptotic, there are many drug-toleratance mechanisms, such as enhanced DNA repair capacity and increased drug efflux ability, etc. As our results showed, after treatment of gradient concentration of doxorubicin, the expression of γ-H2AX, one of marker proteins of DNA damage, recovered more rapidly in p53S cells than p53-/- cells.These data suggested that p53S cells might achieve drug-resistance through increased DNA damage repair ability. The DNA damages detected by pulsed-field electrophoresis also demonstrated the DNA repair ability of p53-/- cells were relatively slower than p53s/s+v cells. Given time for repairing, the percentage of apoptosis detected by flow cytometry showed that p53-/- cells were more sensitive to doxorubicin than p53S cells. All these results implied that p53S might acquire doxorubicin-toleratance through the mechanism of inhibiting apoptosis pathway and simultaneously increasing DNA damage repair.In order to study anti-apoptotic mechanisms of acquired drug resistance derived from p53N236S, we analyzed ChIP-on-chip microarray data to screen target genes of p53S. Genes related to apoptosis signaling pathways were detected, such as Apafl, which encodes apoptotic protease activating facter-1, and BircS (also known as survivin), which encodes protein belonging to inhibitors of apoptosis proteins (IAP). In order to verify that Apafl and survivin were transcriptional regulated by p53S, we carried out Chromatin immunoprecipitation (ChIP), Western Blot and realtime-PCR experiments. The results of ChIP desplayed p53S could bind to the promoter region of Apafl and survivin. And results of Western blot and realtine-PCR showed p53S could decrease Apafl expression, and increase survivin expression level both transcription level and protein level. At the same time, the activity of caspase3,7 and 9 also decreased, which were participate in mitochondrial apoptosis pathway. These data prompted p53S might be involved in regulation of endogenous apoptotic signaling pathways through binding and transcriptional regulatin of Apafl and survivin. Further, we used siRNA to decrease survivin expression level to study the correlation between the expression of survivin and durg-resistance in p53S cell. The data showed that inhibition of survivin expression by siRNA, sensitisized p53s/s+v cells to doxorubicin treatment. Altough not as sensitive as wild-type p53 cells. Besides, Flow cytometry assay showed that the percentage of apoptosis of p53s/s+Ras cells were lower than that of p53s/s+v cells following decreasing survivin expression level in response to doxorubicin, which implied stable expression of Ras, as well as survivin, contributed to drug-resistance of p53S cells.In summary, our study found p53S could endow tumor cells anti-apoptosis ability and drive acquired-tolerance for antineoplastic drug treatment. And it was demonstrated that the drug-resistance of p53S was mainly caused by inhibiting apoptosis pathways and simultaneously increasing DNA damage repair. In addition, decreasing survivin espression level could rescue the sensitivity of p53S cells to doxorubicin. These data suggested that the high expression of survivin regulated by p53S promoted p53S cells to resist to doxorubicin treatment. Moreover, besides anti-apoptosis, p53S could also increase the ability of DNA repair and promote cell survival. Our study may provide theoretical basis of the clinical treatment strategy targeting p53 mutation.