| BackgroundER is a member of the nuclear receptor superfamily, which plays an important role innormal mammary gland and breast cancer. The pathogenesis of breast cancer is closely involved inestrogen (E2) and estrogen receptor (ER). ER in the nucleus can be directly bound to the estrogenresponse element(ERE) in the promoter region of target genes and regulate the transcription ofthese genes by recuiting coregulatory proteins including coactivators or corepressors, which is socalled ER genomic activity. In addition, recent research have demonstrated that ERon the membrane or in cytoplasm also can mediate rapid non-genomic activity (ER non-genomic activity). The Cross-talk or interaction between ER non-genomic activity andHER2pathways can activate downstream signaling molecules such as MAPK and Akt, leadingto the increased expression of the genes including HER2pathway.These products of genes furtherenhance HER2pathway in turn, resulting in E2-independent cells growth and the resistance toendocrine therapy targeting to E2or ER. Therefore, the ER non-genomic activity is considered asan important mechanism of endocrine therapy resistance. In addition, the ER non-genomicactivity is involved in the regulations of many breast cancer genes. For example, E2-inducedER non-genomic activity regulates the proliferation, migration of breast cancer cells by rapidlyactivating SphK1(Sphingosine kinase) and increasing the activity of Ca2+dependent K+channelconductance.Flap endonuclease1(FEN1) is a member of coregulatory proteins involved inER-mediated transcription and DNA repair. The recent research indicates that FEN1is alsoclosely related to breast cancer. Compared with the corresponding normal breasttissue, FEN1is overexpressed in breast cancer. Our previous research has foundFEN1over-expression in breast cancer cells regulated by E2and the enhanced proliferation of these cells. However, the mechanism on E2–induced up-regulation ofFEN1has not been elucidated. Bioinformatics prediction has found that there are noER binding sites but four potential binding sites of transcription factor Elk-1withinFEN1promoter region. So, we hypothesized that the mechanism thatE2-induced up-regulation of FEN1may be involved in ER-mediated non-genomicactivity but not direct binding of ER to the FEN1promoter region (the genomicactivity). Bsaed on the fact demonstrated by our previous research that E2–inducedup-regulation of FEN1promoter activity depends on the full-length Elk-1in MCF-7celllines, we hypothesized that E2may up-regulate the expression of FEN1throughER-MAPK-Elk-1pathway.Herceptin/Trastuzumab as a humanized monoclonal antibody against HER2caneffectively inhibit the growth and proliferation of breast cancer cells with HER2overexpression, but Herceptin resistance in clinic is very common. The mechanismof Herceptin resistance is not exactly understood HER2pathway as a therapeutictarget of Herceptin may be activated by ER located in the membrane or cytoplasm throughnon-genomic activity, resulting in increased dosage of Herceptin and weaken efficiencyof Herceptin. It has been reported that ER located in the membrane or cytoplasm candirectly interact with the IGF-1R and PI3K/Akt through non-genomic activity in other nonbreast cancer cells with ER-positive, leading to the activation of IGF-1R and Akt,andthe activation of IGF-1R or Akt has been demonstrated as one of the mechanism invovledin Herceptin resistance. All of these evidences indicate that the ER non-genomic activitymay play a potential role in Herceptin resistance of ER positive breast cancer cells.Contents(1)the mechanism on E2–induced FEN1up-regulation through the ER-MAPK signalpathway in breast cancer was explored, using RT-PCR, Western blot, EMSA andChIP assay.(2) the effect and mechanism of ER non genomic activity on herceptin in ERpositive breast cancer cells were observed, using transient transfection and reporter assay，RT-PCR,Western blot, immunofluorescence， confocal and CoIP assay.Results(A) The mechanism on E2-induced FEN1up-regulation in breast cancer throughER non-genomic activity (1) E2can up-regulate FEN1expression in breast cancer cells.(2) E2can activity the phosphorylation of MAPK and Elk through ER non-genomicactivity.(3) E2-induced FEN1up-regulation is caused by Elk-1binding to SRE sites in FEN1promoter region.(4) The expression of ER, FEN1and the phosphorylation of MAPK and Elk in breastcancer tissues was higher than in the corresponding normal breast tissue,which furtherdemonstrated E2-induced FEN1up-regulation through ER-MAPK-Elk-1pathway.(B) The role and mechanism of ER non-genomic activity in Hereceptin resistance(1) Both E2and growth factor, Heregulin can quickly up-regulate the phosphorylationexpression of MAPK and Akt.(2) Herceptin can effectively inhibit the phosphorylation of MAPK and Akt inducedby Heregulin.(3) E2can interfer the inhibitory effect of Herceptin on the phosphorylation of MAPKand Akt induced by Heregulin.(4) ER can interact with IGF-1R and both of them can co-located in the cytoplasm ormembrane, indicating that the IGF-1R may also be invovled in E2-induced ER non-genomic activity.Conclusions(1) E2can up-regulate FEN1expression in breast cancer cells through ER non-genomicactivity or ER-MAPK-Elk-1pathway.(2) E2can interfer the inhibitory effect ofHerceptin on ER-positive breast cancer cells through ER non-genomic activity.Purpose and significance(1) this research provides a new mechanism for E2–induced up-regulation of FEN1, whichis helped to explain the reasons on the observed overexpression of FEN1in clinical breastcancer tissue. At the same time, combined with the previous work, we verify the role ofFEN1as a potential marker in the process of breast cancer, explaining the relationship ofFEN1and E2, ER, which are known breast cancer markers. These studies deepen ourunderstanding of the FEN1expression regulation and the pathogenesis of breast cancer,which provides a basic theoretical evidence for finding a new treatment strategy to enhancethe chemotherapy sensitivity of breast cancer.(2) The clarified mechanism that E2can interfer the efficacy of Herceptin through ER non-genomic activity in ER-positive breastcancer cells may provide a new and important mechanism for Herceptin resistance anda new theoretical basis on the combined strategy for breast cancer treatment withHerceptin and endocrine therapy drugs in clinic. |
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