FEN1 Function And Feasibility Study As An Anti-tumor Target

Flap endonuclease-1(FEN1)is a multifunctional,structure-specific nuclease that has a critical role in maintaining human genome stability.Regulatory mechanisms of FEN1 in cells are crucial to maintaining normal cell growth.We sought FEN l-interacting proteins to further explore the new function of FEN1.We have applied tandem affinity purification methods and mass spectrometry for finding the new FEN 1-interaction partners.We have shown that HSP70 interacts with FEN1 and promotes FEN1 activity and BER efficiency.FEN1 mutations have been detected in human cancer specimens and have been suggested to cause genomic instability and cancer predisposition.However,the exact relationship bet,ween FEN1 deficiency and cancer susceptibility remains unclear.In the current work,we report a novel colorectal cancer-associated FEN 1 mutation,L209P.Here,we elucidated the mechanisms through several levels,including molecular,cellular and mouse model et al.We established point mutations of human FEN1 and then purified those recombination proteins.We found that the FEN1 L209P mutation lacks the FEN,exonuclease(EXO)and gap endonuclease(GEN)activities of FEN1 but retains DNA substrate-binding affinity.The L209P FEN1 variant interferes with the function of the wild-type FEN 1 enzyme in a dominant-negative manner and impairs long-patch base excision repair in vitro and in vivo.We also observed the phenotypes in cellular level.Compared with wild-type(WT)FEN1-containing cells,cells expressing the L209P FEN1 variant were more susceptible to DNA-damaging agents and accumulated more DNA damage lesions in the genome.Consequently,L209P FEN 1-containing cells displayed various chromosomal aberrations including chromosomal breakage and chromosomal aneuploidy.We further show that L209P FEN1-containing cells displayed a higher cellular transformation rate and increased tumor growth in a mouse xenograft model.Thus,our current study demonstrates that FEN1 mutation(L209P)can interfere with base excision repair and induce cellular transformation.These data indicate that human cancer-associated genetic alterations in the FEN1 gene can contribute substantially tocancer development.Based on the above findings,FEN1 is required for genome stability and functional loss of FEN 1 results in the initiation of cancer.However,at the same time,FEN1 expression level in cancer cells is much higher than normal cells.We demonstrated that FEN1 is overexpressed in breast cancers and is essential for rapid proliferation of cancer cells.We showed that manipulating FEN1 levels in cells alters the response of cancer cells to chemotherapeutic drugs.We collected cancer samples and compared the levels of FEN1 protein.We found that high expression of FEN 1 is associated with increased malignancy and cancer patients with high expression of FEN1 have poor prognoses.Suppression of FEN1 in cells leads to the retardation of DNA replication and accumulation of unrepaired DNA intermediates,resulting in DSBs and apoptosis.Therefore,we speculated that inhibiting FEN1 function could suppress cancer cell proliferation.Based on this assumption,we proposed that targeting FEN1 could serve as a potent strategy for cancer therapy.Furthermore,we identified a small molecular compound,SC 13 that specifically inhibits FEN1 activity,thereby interfering with DNA replication and repair in vitro and in cells.SC 13 suppresses cancer cell proliferation and induces chromosome instability and cytotoxicity in cells.Importantly,SC 13 sensitizes cancer cells to DNA damage-inducing therapeutic modalities and impedes cancer progression in a mouse model.These findings could establish a paradigm for the treatment of breast cancer and other cancers as well.