The Effects Of E2 On The Expression Of FEN1 In Breast Cancer Cells And Its Mechanism

Estrogen receptor (ER) is a member of the nuclear receptor superfamily, which plays an important role in normal mammogenesis and pathogenesis of breast cancer. 17β-estradiol (E2) regulates a diverse array of target genes including many growth factors involved in the development of mammary gland cells by its binding to ER. About two-thirds of breast cancer cells depend on E2 and ER for their growth. The ability of ER to regulate the experssion of target genes is influenced by interaction with many coregulatory proteins. In addition to proteins which are called coactivators or corepressors that modify receptors'activities in transcriptional level for accommodating the expression of estrogen response genes, other coregulatory proteins recruited to ER participate in DNA repair and are required to maintain DNA integrity. There is an intimate relationship between ER mediated transcription and DNA repair. Flap endonuclease 1 (FEN1) is a member of such coregulatory proteins, which was discovered recently. FEN-1 which can remove the 5'flap is required for processing of Okazaki fragments during DNA replication and for long patch base excision repair (BER). Recent researches indicated that FEN1 was overexpression in breast cancer cells, and might be concerned with the level of E2. FEN1 could interact with ER, increased the ER-ERE interaction and influenced endogenous estrogen responsive genes expression. Howener, the correlation between E2 and FEN1 in breast cancer cells is not clear at present. In order to identify the relationship between E2 and FEN1, researches were carried out as follow:1. The expression of FEN1 in breast cancer tissues or MCF-7 breast cancer cells and corresponding normal tissues or MCF-10A mammary epithelial cellsUsing RT-PCR, Real-time PCR and Western blot, the transcription and translation leves of FEN1 in MCF-7 breast cancer cells or breast cancer tissues were found to be higher than that in MCF-10A mammary epithelial cells or corresponding normal tissues.2. The effect of E2 on expression of FEN1 in MCF-7 cells E2 was found to induce the up-regulation of FEN1 expression in MCF-7 cells by RT-PCR and Western blot analysis, which was the most obviously at 6h.3. The effect of E2 on the promoter activity of FEN1 in MCF-7 cells In the transient transfection assay using a FEN1 promotor luciferase reportor plasmid, E2 was detected to up-regulate the expression of FEN1 quickly, which could be inhibited by ER antagonist, ICI182,780 and MAPK inhibitor, U0126. Bioinformatics analysis by TFSEARCH (http://mbs.cbrc.jp/ research/db/ TFSEARCH.html) indicated that there were no estrogen response elements (EREs) but 4 potential serum response elements (SREs) sites or Elk-1 binding sites in the FEN1 promoter region (-458 to +278). Constructed Elk-1 negative dominant plasmid was cotransfected with FEN1 promotor luciferase reportor plasmid into MCF-7 cells and the reportor gene assay indicated that E2 could up-regulate FEN1 promoter activity in MCF-7 cells depending on the function of the full length Elk-1.4. The effect of FEN1 overexpression on the trans-activation of ER and the cell proliferation in MCF-7 cells.The cotransfection assay using the FEN1 expression plasmid and the ERE luciferase reportor plasmid indicated that the overexpression of FEN1 could enhance the trans-activation of ER in MCF-7 cells in dose-dependent way. The proliferation of MCF-7 cells stablely overexpressing FEN1 was detected to be higher than that of contriol cells by MTT assay.In summary, our data show that the expression of FEN1 in MCF-7 cells or breast cancer tissues is higher than that in MCF-10A cells or corresponding normal tissues. The expression of FEN1 in breast cancer cells can be up-regulated by E2, which may be involved in the ER-MAPK-Elk-1 pathway. The overexpression of FEN1 can enhance not only the trans-activation of ER in MCF-7 cells in dose-dependent way but also the proliferation of MCF-7 cells. Our findings have firstly identified the functional relationship between E2 and FEN1 in breast cancer cells, which can provide a fundation for further clarification on the regulative mechanism of E2 on the expression of FEN1 in breast cancer cells and its significance.