The Relevant Studies On How Transcription Factor YY1 Inhibits FEN1 Expression And Causes Drug Resistance In Breast Cancer Cells

Drug resistance is a major challenge in cancer therapeutics. Abundant evidence indicates that DNA repair systems are enhanced after repetitive chemotherapeutic treatments, rendering cancers cells drug-resistant. However, the precise molecular mechanism of such a transformation is not known. The effectiveness of most current chemotherapeutic drugs for cancer depends on the ability to induce DNA damage in hyper-proliferating cancer cells, which have inadequate DNA repair capacity. However, the development of multidrug resistance (MDR) in cancer cells poses a major challenge to chemotherapy and greatly limits the anti-cancer efficacy of chemotherapeutic drugs.Flag endonuclease 1 (FEN1) is an important member of this family, playing a pivotal role in DNA replication and repair. Although FEN1 was once widely considered a tumor suppresser based on its role in the maintenance of genomic stability through Okazaki fragment maturation, long-patch base excision repair, rescue of the stalled replication fork, and telomere maintenance, accumulated evidences now indicate that FENl is required for tumor progression. Transcription factor Ying Yang 1(YY1) plays an important role in divergent biologic processes such as embryogenesis, differentiation, cellular proliferation and cancer progression. YY1 is well known for its dual roles in regulating gene expression, either as activator or repressor, depending upon the context in which it binds to.Using the computer programs TRANSFAC, Match 1.0-public, TESS, and TFSEARCH, nearly 200 transcription factors including NF-B and YY-1 were found might bind to the FEN1 promoter region. To validate whether YY1 indeed binds to the predicted YY1 binding site on the FEN1 promoter region, we performed the electrophoretic mobility shift assay (EMSA) using the purified recombinant YY1 protein and the DNA probe, a 29 base pair oligonucleotide covering the predicted YY1 binding site. We found that YY1 effectively binds to the wild type probe, forming the YY1/DNA complex. We then investigated whether YY1 bound to the FEN1 promoter region in the cell by conducting a chromatin immune-precipitation-PCR (ChIP-PCR) and showed that the FEN1 promoter was specifically pulled-down by an YY1-specific antibody but not the control antibody. The results all suggest that transcriptional factor YY1 binds to the FEN1 promoter.We exogenously overexpressed YY1 in 293T and evaluated the FEN1 protein level. We found that the protein level of endogenous FEN1 gradually decreased as the amounts of the plasmid DNA transfected into 293T cells increased. We next examined whether YY1 bound to the FEN1 promoter region and suppressed the gene expression. We subcloned the FEN1 promoter into the pGL4.0 plasmid, so that the expression of the EGFP reporter gene was only driven by the FEN1 promoter. The YY1 expression vector and the pGL4.0-FENl promoter-driven EGFP vector were co-transfected into 293T cells. We then measured the EGFP mRNA level by qPCR and the EGFP protein by flow cytometry. Our data indicated that the ectopic overexpression of YY1 in 293T cells considerably reduced EGFP mRNA and protein levels.We found that in response to treatments with MMC and Taxol, the mRNA level of YY1 was down-regulated by more than 2 folds, while the mRNA level of FEN1 was up-regulated by 3 to 6 folds. We consistently observed that the YY1 protein level was reduced by approximate 2 folds, while the protein level of FEN1 increased by more than 2 folds. Our ChIP analyses indicated that the amount of YY1 bound to the FEN1 promoter reduced by 2 folds upon the MMC treatment. Furthermore, when we expressed the YY1 gene in 293T cells, we observed that the cells harboring this expression plasmid became more sensitive to both MMC and Taxol treatment.We employed an expression array of 26 cancer cell lines in 13 major categories that have been treated with 25 different DNA-damaging agents and therapeutic drugs. The Northern dot blotting results showed that FEN1 expression levels in breast cancer cell lines, MDA-MB-4355 and MDA-MB-231, increased significantly after the treatment with DNA-damaging agents, such as camptothecin, cytochalasin D, MMC, and gamma irradiation.More than 80% of patients with low expression of FEN1 can survive, disease-free, for more than 10 years; however, only less than 55% of patients with high expression can do so. Patients with FEN1-high and YYl-low expression had both statistically significantly poor overall and disease-free postoperative survivals, a fact suggesting that FEN1 and YY1 might have inverse impact on the survival of breast cancer patients. This is consistent with the results that we have obtained from molecular studies using cultured cell lines and clinical drugs. Overall, the FEN1/YY1 interaction and regulatory mechanism might be of clinical importance and should be further investigated.