The Role Of FEN1 In Tumor Targeted Therapy And Its Regulatory Mechanism

The number of people with cancer is increasing in the world.Cancer has become one of the major diseases affecting people health.In the 2000 annual meeting of the international tumor gene therapy conference,biological therapy was considered to be one of the methods for comprehensive cancer therapy.Therapies of anti-tumor drugs are changing into the age of precise medical around the world.The most mature area is represented by molecular targeted anti-tumor drugs.Flap Endonuclease 1(FEN1)is a structure specific endonuclease that plays an important role in DNA replication and DNA repair.In the process of DNA replication,FENI can mature Okazaki fragments.FEN1 is involved in the Long Patch Base Excision Repair in the process of Base Excision Repair(BER).In addition,FEN1 can also maintain the stability of gene and telomere.Studies have shown that FEN1 is expressed at low levels in quiescent cells,but is highly expressed in proliferative tissues and cancers including lung,breast,gastric,prostate and pancreatic cancers.We previously observed that FEN1 was over-expressed in different tumors when compared to the normal cells.Inhibition of FEN 1 expression can slow cell growth and proliferation,and also it can enhance the sensitivity to chemotherapeutic drugs.This suggested that targeting FEN1 could be a potential strategy for the treatment of cancer.We had designed FEN1 specific inhibitor SC 13 which can inhibit FEN1 activity.SC 13 can inhibit the growth and proliferation of tumor cells.In vivo and vitro,SC 13 can significantly enhance the sensitivity of cells to low-concentration chemotherapy and improve the chemotherapeutic efficacy.This suggested that the combinative biotherapy targeting FEN1 and chemotherapy provide a new therapeutic strategy for cancer.However,studies on upstream regulatory mechanism of FEN1 are still unknown,so we further explored an in-depth study on this.AKT is also known as protein kinase B(PKB)and its activation can be mediated by the activation of phosphoinositide 3-OH kinase(PI3K).AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways.AKT signaling pathway is closely related to the development of cancer cells and activated AKT can improve DSB repair ability.AKT activity inhibition impairs DNA repair in glioblastoma cells.The activated AKT can phosphorylate IκB kinase,release and activate NFκB/p65.Activated NFκB/p65 can enter into the nucleus and regulate its target genes.Based on this,we hypothesised that AKT pathway may be associated with base excision repair pathway.So,A549 cells was our experimental subject and we had showed that AKT activity inhibition significantly decreases the expression of FEN1 in both mRNA and protein levels.The Co-IP experiment showed that FEN1 can’t interact with AKT.It was reported that phosphorylation modification can lead to instability of FEN1 because of ubiquitination and degradation by proteasome pathway.Regulation of FEN1 by AKT pathway may be mediated by the indirect transcription.We used the software to predict the potential transcription factor binding sites in FEN1 promoter regions and found that there were some transcription factor binding sites including SP-1 and NFκB.We found that activated NFκB/p65 can increase the expression of FEN1 in both mRNA and protein levels.EMSA and ChIP experiments visually showed that NFκB/p65 can bind to FEN1 promoter which is regulated by AKT activity.We found that AKT activity inhibition can sensitize A549 cells to Cisplatin,activate p53 apoptosis signal,accumulate DNA double-stranded breaks,lead to instability chromosome and eventually induce cell apoptosis.In addition,we had established the stable cell lines with overexpression and knockdown of FEN 1 to show that FEN1 expression can affect resistance to Cisplatin.Therefore,it can be concluded that cancer cells control the ability of DNA repair by regulating expression of FEN 1.Our study showed that the sustained activation of AKT can activate nuclear transcription factor NFκB/p65 by phosphorylating IκB kinase to promote expression of FEN1.It can strengthen the ability of DNA repair and improve the resistance of tumor cells to chemotherapeutic drugs.Our study mainly revealed three main results:1 FEN1 is over-expressed in various kinds of tumors and FEN1 expression is closely related to the tumor malignancy grade.Inhibition of FEN1 activity could suppress cancer cell growth and sensitize cancer cells to chemotherapeutic drugs.2 The FEN1 specific inhibitor SC 13 can effectively sensitize cancer cells to chemotherapy and improve the efficacy of chemotherapy.3 In cancer cells,AKT activation can up-regulated FEN1 expression by activating NFκB/p65 which can increase the transcription level of FEN 1.Through this study,we clarified the relationship bewteen FEN1 and tumor development.Inhibition FEN 1 activity plays important role in tumor biological therapy.In addition,we illuminated upstream regulatory mechanism of FEN 1 which provides a better strategy and theoretical basis for the combinative biotherapy and chemotherapy.