| Estrogen receptor (ER) plays essential roles in breast cancer progression and proliferation. It is an important prognostic factor and a target of endocrine therapy. Breast cancer recurrence after surgery, resistance to the anti-estrogen and metastasis suggest the complexity of cancer. It is of great significantce to discover the co-regulators and understand the mechanisms and cross-talk of signal pathways of cancer development and progression to find new targets of the therapy or prognosis of breast cancer.There are two forms of ER, ERαand ERα, which exhibit similar, yet distinct, structural and functional features. Both ERs have two important transcription activation function regions, AF1 and AF2, which interact with co-regulators. There were a few co-regulators which interact with AF1, compared to AF2. The AF-1 activity is E2-independent, and is involved in resistance of the anti-estrogen and cross-talk of signal pathways. Using ERαAF1 as bait, we isolated a novel ERα-interacting protein, CGI-27, from a human mammary library by yeast two-hybrid screen.Until now, little is known about the function of CGI-27 protein. Bioinformatics analysis provides no information of this protein. But crystal structure of human CGI-27 shows that it is homologous to class III nonheme iron-dependent dioxygenases. CGI-27 interacts specifically with phospho-Tyr 1227 of ERBB2 through the SHC adaptor protein and is required for breast carcinoma cell migration. Since there was no commercial antibody of CGI-27 available, we generated the polyclonal antibody of CGI-27 by purification of GST-CGI-27 protein and immunization of Balb/c mouse. We found the ubiquitous expression of CGI-27 protein in rat tissues and various cancer cells using the antibody of CGI-27. The immunochemistry in breast cancer cells showed that CGI-27 is located mainly in the nucleus and membrane.To confirm the interaction between CGI-27 and ERα, the GST pull-down, co-immunoprecipitation(co-IP) were performed. The results indicated that CGI-27 interacted with ERαboth in vitro and in vivo. In addition, ERααalso interacted with CGI-27 in an E2-indepentent manner. The aa1-146 region of CGI-27 interacted with ERα, and the AF1 and AF2 of ERαinteracted with CGI-27. CGI-27 and ERs were co-localized in the nucleus by confocal microscope experiment.In 293T, MCF-7, ZR-75-1 and SKBR3 cells, the transcriptional activity of ERαand ERαwere increased by CGI-27 over-expression in E2-indepent and dose-dependent manner. We also found that the increase of transcriptional activity depended on the interaction between ERαand CGI-27. The expression level of ERαremains unchanged by CGI-27 over-expression. The transcriptional activity of ERαand ERαwere reduced by knockdown of CGI-27.CGI-27 over-expression increased the expression of estrogen receptor downstream genes, such as c-Fos, c-Myc, CatD and CyclinD1, but not PAI1, pS2 and C3. CGI-27 promoted breast cancer cell growth by cell proliferation and cell cycle assays. The soft agar colony forming ability was increased by CGI-27. By injecting CGI-27 over-expressed MCF-7 cells into nude mouse, we found that CGI-27 provoked liver metastasis.ERBB2 and CGI-27 had synergistic effect on ERαtranscriptional activity, but not ERαtranscriptional activity. The ERBB2 inhibitor and c-SRC inhibitor could reduce the enhancement of ERαtranscriptional activity by CGI-27. Further detection of the phosphorylation level of protein kinases showed that CGI-27 enhanced the phosphorylation of ERK1/2, AKT, ERααS118 and ERααS167. The sites of ERααS104, ERααS106, ERααS118, ERααS167, ERααS305, ERααS518 and ERααY537, but not the ERααS236 and ERααT311, were important for CGI-27 mediated ERαactivation. The mutant at H192 of CGI-27 could not increase the ERαtranscriptional activity.In conclusion, CGI-27 interacts with ERs, increases the phosphorylation levels of ERK1/2, AKT and ERα, enhancs ER transcriptional activation, and promotes cell growth and metastasis, ERBB2 and CGI-27 have synergistic effect on ERαtranscriptional activity. All the results indicated that CGI-27 is not only a co-regulator of ERα, but also a critical factor in the progression of breast cancer and endocrine resistance. CGI-27 might be a new target of the anti-tumor drugs.
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