Studies On Whether Drug Resistance Of P53-Mutant Colorectal Cancer Cells Is Related To RSL1D1 And How To Overcome This

Ribosomal L1 domain containing protein1（RSL1D1）,also called cellular senescence inhibitory gene,a member of the ribosomal Llp/L10e family.To date,numerous studies have demonstrated that RSL.1D1 is an important regulator of rRNA transcriptional processing as well as translational and post-translational modifications of various mRNAs/proteins,and has a function in suppressing cellular senescence.Recently,our group has shown that RSL1D1 plays an oncogenic role in colorectal cancer by interacting with the DNA binding domain of the oncoprotein p53 and recruiting it to the E3 ubiquitinase HDM2,which in turn ubiquitously degrades p53 and downregulates the expression levels of the p53 target genes p21 and PUMA,helping colorectal cancer cells to evade apoptosis and to accelerate cell death through the G1S cell cycle checkpoint,thereby accelerating the progression of colorectal cancer.Numerous studies have shown that the DNA-binding domain of p53（the region where RSL1D1 interacts with p53）loses its oncogenic function after mutation in approximately half of cancer cases,including colorectal cancer,to promote tumour development and lead to drug resistance,while the mutated p53 protein becomes abnormally stable and difficult to degrade by ubiquitination.We hypothesize that the p53 mutation causes RSL1D1 to weaken or even lose its affinity for p53,making it difficult to recruit the p53 mutant protein to HDM2,which in turn leads to the intracellular accumulation of mutant p53 and its pro-cancer effects.The aim of this study is to investigate whether RSL1D1 has difficulty interacting with mutant p53,leading to colorectal tumour progression and drug resistance,and to lay the foundation for a rational targeting strategy for RSL1D1 in p53 mutant/non-mutant cancers in the future.Firstly,we selected two representative mutant proteins R175H（conformational mutation）and R273H（contact mutation）according to the mutation type of p53 as the target of our study,and constructed a series of prokaryotic expression plasmids encoding RSL1D1 and mutant p53 fulllength protein and truncator,and obtained high purity recombinant proteins by IPTG induction and affinity chromatography for GST-pulldown and ELISA assays;to confirm the interacting structural domains of p53 and RSL1D1.The results show that:p53 interacts with RSL1D1-CT domain mainly through the aa 93-224 domain.The mutation of this domain leads to that p53-R175H no longer interacts with RSL1D1,while the p53 protein mutated in R273H（p53-R273H）can still bind to RSL1D1,but showed lower affinity than wild-type p53（p53-WT）.Then,we further validated the above conclusions by bimolecular fluorescence complementation（BiFC）assay and proximity ligation assay（PL A）assay in vivo,by transfecting the constructs encoding RSL1D1,mutant p53（full-length and truncated constructs）into BiFC plasmids and transfecting HCT116^p53+/+cells to examine the protein interactions in vivo.The results showed that RSL1D1 protein was similarly unable to bind to p53-R175H in vivo,but was able to bind to p53-R273H with much lower mutual affinity than RSL1D1-p53-WT.Although both in vitro and in vivo results indicate that aa 93-224 of the p53 protein is the precise domain to bind RSL1D1,aa 225-292 and aa 293-363 also affect the RSL1D1-p53 interaction and are the main reasons for the impaired affinity of rslldl for p53-R273H.To investigated whether RSL1D1 was able to recruit p53-R273H to HDM2,we performed a combined BiFC-IF assay to examine whether the RSL1D1-p53-R273H-HDM2 complex was formed.The results showed that although RSL1D1 still interact with R273H and recruit it to HDM2 to form a triplet complex,its recruitment ability was significantly weaker compared to that of wild type p53.To investigate whether RSL1D1 can ubiquitinate degrade p53-R273H through the recruitment pathway described above,we used a lentivirus system to overexpress the RSL1D1binding domain（RSL1D1-CT）of p53-R273H in human colorectal cancer cell HCT116.The results showed that overexpression of RSL1D1-CT did not lead to an upregulation of p53-R273H protein levels and could no longer be recruited for HDM2 degradation.In HCT116 cells,treatment with HDM2 inhibitor Nutlin-3 significantly increased the level of wild-type p53 protein,but it was difficult to increase the protein levels of p53-R175H and p53-R273H,further confirming that RSL1D1 was difficult to degrade p53 mutant protein through HDM2 dependent pathway.Finally,we investigated the sensitivity of p53 wild type and mutant colorectal cancer cells to chemotherapy drugs through CCK8 and Annexin V/PI experiments.The results showed that the administration of Adriamycin significantly increased the level of p53 protein in HCT116p53+/-cells.thus inducing apoptosis and inhibiting cell proliferation;while in HCT116^p53-R175H and HCT116^p53-R273H cells,the apoptosis rate of Adriamycin-treated cells was significantly lower than that of wildtype p53 cells,indicating that p53 mutation leads to tumor resistance.In summary,this study found that the conformational mutation of p53 lost its binding ability with RSL1D1,while the contact mutation of p53 still retained part of its binding ability with RSL1D1,but the affinity was reduced by about 5 times compared with wild-type p53;due to the weakening or loss of the interaction ability of RSL1D1-p53 mutant protein,RSL1D1 is difficult to degrade mutant p53 in an HDM2 dependent manner.These factors are important reasons for the high stability of p53 mutant proteins in colorectal cancer cells,thus inducing tumor resistance.These findings help to understand the role of RSL1D1 and p53 mutations in tumour development and drug resistance,and are important for the prevention and treatment of malignant tumours.