Loss Of Acetyltransferase PCAF Increases The Resistance Of Colorectal Cancer To 5-fluorouracil

Objective:5-fluorouracil(5-FU)is one of the first-line chemotherapy agents for colorectal cancer(CRC).However,only 10-20% of CRC patients observed effective responses to 5-FU-based chemo-treatment,and patients who initially responded to 5-FU eventually become resistant.Therefore,it is compelling to reveal the mechanism of5-FU resistance,which is essential for identifying novel tumor markers and developing target therapies.PCAF(p300/CBP-associated factor)was the first discovered mammalian histone acetyltransferase homologous to yeast GCN5 p.Because of the acetylation of histone protein and non-histone protein,PCAF participates in several kinds of cell process.The purpose of this study was to investigate whether PCAF expression is involved in promoting cytotoxicity of 5-FU to CRC cell lines,and discuss the potential mechanism between PCAF and 5-FU resistance.Method:1.Select and establish 5-FU resistant CRC cell lines HCT116/5-FU,SW620/5-FU and SW48/5-FU.To examine the differences of HATs(histone acetyltransferases)and HDACs(histone deacetylases)between normal and resistant cell lines,we performed q PCR and Western bolt analysis,and found only PCAF was downregulated in all three resistant cell lines.2.Examine the level of trimethyl H3K27(H3K27me3)in HCT116 and HCT116/5-FU cells.Ch IP-PCR assay was performed to determine the alteration of H3K27me3 binding to the promoter region of PCAF.3.To investigate the contribution of PCAF to 5-FU resistance,we knocked down / overexpressed PCAF in HCT116 cell line.The capability of in vitro tumorigenesis was determined by clonogenic survival assay,cell viability was tested by CCK-8 assay,the protein levels involved in apoptosis(such as cleaved PARP and cleaved Caspase-3)and cell cycle(such as cyclin D1 and phos-Rb)was examined by western blot,cell cycle alteration was determined by PI staining-based flow cytometry approach.4.HCT116 cells were treated with 5-FU after PCAF knockdown,and we examined the expression of the signaling pathways lead to results mentioned above,such as p53 and p21.Co-IP assay was performed to determine acetylation of p53.5.Ch IP assay was performed to determine the occupancy of p53 at its binding site of p21 promoter and the acetylation changes of p21 promoter.6.Perform a CRC xenograft model to prove the function of PCAF in vivo.HCT116 cells stably expressing Flag-PCAF and normal HCT116 cells were subcutaneously transplanted into nude mice.Measure the size of tumors after 5-FU treatment.Tumor specimens were harvested for immunohistochemial(IHC)staining or western bolt analysis to examine the expression of PCAF,p53 and p21.7.Gene expression database was analyzed to investigate the relationship between PCAF,p21 and the 5-year survival of CRC patients.The expression of PCAF in normal tissues,colorectal adenoma and colorectal cancer was also determined by gene expression database analysis.8.The IHC staining of PCAF in paired CRC tissue array was used to determine the alternation of normal tissues and related colorectal cancer tissues.Result:Here,we demonstrate the significant roles of PCAF in promoting the acquired resistance of colorectal cancer to 5-FU.Initially,our data showed that although some HATs or HDACs were up-or down-regulated in certain resistant cells,only PCAF was decreased in all the three resistant CRC cell lines.In order to investigate the function of PCAF in 5-FU resistance,silencing PCAF with si RNA in HCT116,we observed blocked cytotoxicity of 5-FU.PCAF knockdown increased the cell tumorigenesis capability and viability and inhibited apoptosis,leading to resistance to 5-FU.Cell cycle was also disturbed by PCAF knockdown,pushing cells from G1 to S phase.Similar results were observed when we overexpressed PCAF in HCT116 cells.To investigate the molecular mechanism by which PCAF modulated 5-FU resistance,Western Blot and q PCR were performed to examine the expression of p53 and p21 which plays an essential role in apoptosis and cell cycle.Knockdown of PCAF decreased the 5-FU-induced expression of p21 but not affected the level of p53 in HCT116 cells.Co-IP assay demonstrated the acetylation of p53 was decreased.Therefore,we used Ch IP-q PCR approach to demonstrate that 5-FU treatments increased the binding of p53 to the promoter region of p21 and PCAF knockdown attenuated the binding of p53.In vivo experiment,we observed PCAF overexpression significantly decreased the tumor growth determined by tumor size and tumor weight.The number of tumor cells showing positive immunostaining of p21 increased significantly in the tumors overexpressing Flag-PCAF.Consistently,the protein levels of p21 were also increased in HCT116 tumor xenografts carrying Flag-PCAF plasmid DNA.At last,gene expression database analysis from Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)revealed remarkable downregulation of PCAF in colorectal adenoma,a kind of precancerous lesion,and CRC,respectively.In addition,the expression of p21 is positively correlated with the expression of PCAF,and patients with high PCAF-or p21-expression in tumors deserve a higher 5-year survival probability.The quantitative IHC analysis of the paired samples showed decreased IHC staining of PCAF in adenocarcinoma as compared to their adjacent normal tissues.Conclusions:In this study,we demonstrated that PCAF is downregulated in 5-FU resistant CRC cell lines.This downregulation may depend on the level of trimethyl H3K27.PCAF knockdown could decrease the acetylation of p53 expression,resulting in less occupancy of p53 at its binding site of p21 promoter and decreased p21 expression.Consequently,loss of PCAF transfer the cells from G1 to S phase and attenuated the apoptosis induced by 5-FU,making the cells acquire 5-FU resistance.Our study elucidated PCAF as a potential therapeutic target for colorectal cancer patients to attenuate 5-FU resistance.