HPEBP4 Promotes ERa-mediated Transactivation In Cancer Cells

hPEBP4 promotes ERα-mediated transactivation in cancer cells by inhibiting proteasome-dependent degradation of ERαHuman phosphatidylethanolamine-binding protein-4 (hPEBP4), a novel molecule identified by us in 2004, is highly expressed in breast cancer, ovary cancer and prostate cancer. Our previous studies about this molecule focused on the role of hPEBP4 in the apoptosis resistance of tumor cells, and we has demonstrated that hPEBP4 promotes cancer cells to resist TNFα/TRAIL-mediated apoptosis by inhibiting the activation of ERK1/2,JNK but enhancing activation of Akt. In addition to the role of this molecule in cellular apoptosis, we are wondering whether there are any other functions of this molecule relatively specifically expressed in tumor cells. We predict that high expression of hPEBP4 in tumor cells is probably the result of constant evolution of tumor cells in response to microenvironment around them. Considering that hPEBP4 is highly expressed in the restricted type of tumors including ovary, breast and prostate cancer,, we suppose that hPEBP4 may participate the cellular response to sex-steroid hormone in tumorigenesis under microenvironment. Therefore, the primary aim of this study is to investigate the role of hPEBP4 in the estrogen recpeptor alpha (ERα)-mediated transacvivation of cancer cells.In order to address the role of hPEBP4 in ERα-mediated transactivation, we firstly used reporter gene assay to determine whether hPEBP4 could affect ERα-mediated transactivation. After silencing hPEBP4 with smll RNA interferance(siRNA) in human breast cancer cells MCF-7, we observed that the luciferase activity significantly decreased, indicating hPEBP4 could promote ERα-mediated transactivation. Real-time PCR and Western blot analysis showed that the activation of endogeneous target genes of ERα, pS2 and Cyclin D1, was also greatly suppressed when hPEBP4 was silenced , further confirming that hPEBP4 could promote ERα-mediated transactivation. The transactivation activity of ERα, as a transcriptional factor, is the major component of ERα-mediated signal pathway. So, our data suggest that hPEBP4 positively regulates ERα-mediated signal pathway and silencing hPEBP4 can significantly suppress the transactivational activity of ERαin breast cancer cells.Then we want to know how hPEBP4 can affect ERα-mediated transcriptional activity, we used chromatin immunoprecipitaion(ChIP) to analyze the binding of ERαwith estrogen responsive element(ERE) after MCF-7 cells were stimulated with E2. We found that silencing hPEBP4 led to less ERαrecruited to ERE, thus suggesting that the reduced ERα-mediated tranactivation may be due to the less amount of ERαbound with ERE.To reveal the mechanism of how hPEBP4 leads to less ERαrecruited to ERE, we obsterved the ERαprotein level in MCF-7 cells 2 h and 6 h after stimulated with E2. We found that E2 stimulation induced an increase of ERαprotein and silencing hPEBP4 dramatically suppressed E2-induced increase of ERα. Given the importance of proteasome-dependent degradation in ERαturnover, we observed the effect of proteasome inhibitor MG132 on the increase of ERαprotein by hPEBP4. We found that MG132 leads to dramatic accumulation of ERαin MCF-7 cells stimulated with E2, and more importantly, the difference of ERαin MCF-7 cells between siRNA and non-siRNA control disappeared after MG132 was added to block proteasome activity. Then we ask one question: whether MG132 has the same effect on ERα-mediated transactivation accordingly? Through reporter gene assay, we showed that MG132 almost completely abolished the inhibitor effect of silencing hPEBP4 on ERα-mediated transactivation. Furthermore, we successfully reversed the decrease of ERα-mediated transactivation by transfecting pSG5-ERαplasmid into hPEBP4-silenced MCF-7 cells. These data demonstrate that hPEBP4 promotes ERα-mediated transactivation by inhibiting proteasome-dependent degradation of ERα.Our previous studies has shown that hPEBP4 can translocate from lysosome to membrane to regulate MAPK and Akt pathway, thereby resulting in the resistance of tumor cells to apoptosis induction. We wonder whether the regulation of signal pathways by hPEBP4 is also invovled in the promotion of ERα--mediated transactivation by hPEBP4. We found that E2 could activate ERK1/2 and Akt pathway in MCF-7 cells, and silencing hPEBP4 enhanced the activation of ERK1/2 but inhibited Akt activation. In addition, we confirmed the interaction between hPEBP4 and membrane-bound ERαwith immunoprecipitation using cytoplasmic protein. Since hPEBP4 could regulate ERα-mediated membrane signal pathway(non-classical pathway), we went further to confirm whether the activity is involved in the effect of hPEBP4 on ERα-mediated transactivation. To test this hypothesis, we used U0126 and LY294002 to block activation of ERK1/2 and PI-3K respectively and then we observed the transcriptional activity with reporter gene assay in MCF-7 cells. We found that both U0126 and LY294002 didn't affect the inhibitory effect of silencing hPEBP4 on ERα-mediated transactivation. And also we didn't observe any effect of the inhibitors on ERαprotein level as demonstrated by Western blot. The data indicate that the effect of hPEBP4 on ERα-mediated transactivation is independent of its regulation of ERK and Akt pathways.Finally, we confirmed the above effect of hPEBP4 by using HeLa cells as cell model. We established stable cell clones overexpressing hPEBP4 by transfecting HeLa cells with pcDNA3.1/His-Myc-hPEBP4 plasmids. We also transiently transfered pSG5-ERαto HeLa cells to acquire an estrogen responsiveness. We found Overexpression of hPEBP4 led to a higher transcriptional activity of ERαand increase of ERαprotein level as well. MG132 blocked the promoting effect of hPEBP4 on ERα-mediated transactivation in HeLa cells. The results suggested that promoting effect of hPEBP4 on ERα-mediated transactivation also exists in other kinds of human cancer cells.In summary, we have demonstrated that hPEBP4 can promote ERα-mediated transactivation in cancer cells by inhibiting proteasome-dependent degradation of ERα. The present study, for the first time, links hPEBP4 with estrogen-induced activity in cancer cells, thus representing a new regulatory mechanism for ERα-mediated signal pathway. Identification of A Small Molecule Inhibitor for hPEBP4 and Its Antitumor EffectHuman phosphatidylethanolamine-binding protein-4 (hPEBP4) is a novel gene identified by our lab a few years ago. Highly expressed in breast cancer cells, it has been determined to play a role in resisting apoptosis. Previous work has reported that MCF-7 and LnCap cells become more sensitive to TNF-α/TRAIL-mediated apoptosis respectively if hPEBP4 was targetedly reduced with siRNA. However, siRNA has its bottleneck when applied to in vivo. Comparatively, development of small molecule inhibitors is more mature in technology and characterized as good absorbance and bioavailability. Therefore, based on the understanding of its effect and mechanism in apoptosis resistance, we intended to discovery a specific chemical inhibitor for hPEBP4.1,Identification of small molecule inhibitor for hPEBP4Due to a lack of X-ray result of hPEBP4 protein crystal, we adopted homology modeling to Figureure out a 3D structure of hPEBP4. To do that we found out the homology proteins by searching protein data bank(http://www.ncbi.nih.gov/BLAST/). Based on those homology proteins, we used two simutation softwares of InsightII and Powell to determine the structure of hPEBP4. Then we tried to seek suitable candidates samL1 molecules from the SPECS molecule library , which contains a pool of about 27,000,0 small molecules, by applying virtual screening method. The following softwares were used consecutively during that process: Dock4.0, Flex and AutoDock3.05. As a result, the cancdidate molecules were finally reduced to 83 after virtual screening.For the remaining 83 candidate molecules, we further screened them with surface plasmon resonance(SPR). As a useful technology in drug discovery, SPR is designed to detect any interaction between two proteins or protein-small molecules based on the local tiny change of electronical flow caused by that interaction. RU is usually used to show how strong the interaction is. In this experiment, we put the recombinant GST-hPEBP4 fusion protein on CM5 Chip and made the small molecules flow past the chip. Interaction between the fusion protein and passing small molecule was measured. With SPR, we selected out 7 small moleccules which could bind with hPEBP4 fusion protein most tightly. These molecules were further subject to MTT assay to examine their effect on proliferation of MCF-7 cells. When added together with TNF-α, two candidates—DC240042, DC240016, show a synergestic effect on inhibiting MCF-7 cell proliferation. But DC240016 alone caused a dramatic cytotoxic effect on MCF-7 cells, so we performed following functional screen only with DC240042.2,Antitumor effect of DC240042We have known that hPEBP4 promotes apoptosis through interaction with Raf-1 and MEK1 and inhibiting the phosphoralation of ERK1/2 and JNK1/2, which provides us a convinent way to rule out off-target effect of DC240042. Western blot was used to detect the activation of JNK1/2 and ERK1/2. We found that incubation with 20ng/mL TNF-αfor 30 minutes could lead to obvious phosphoralation of JNK and ERK1/2. DC240042 pretreatment significantly enhanced that phosphoralation. Co-immunoprecipitation assay suggested that DC240042 could also render the hPEBP4 bind with Raf-1/MEK1 to a less extent.Then we used FACS to investigate whether DC240042 could really promote TNF-α-mediated apoptosis. Probing with R123/PI revealed that DC240042 increased the apoptosis percent caused by 20ng/mL TNF-α. Apoptosis assay with another pair of probes, Annexin V/PI, show DC240042 was synergestically effective in promoting apoptosis when combined with 10, 20 and 50ng/mL TNF-α. FACS also suggested that the effect of DC240042 was dose and time dependent.Next, we wanted to see whether the effect of DC240042 still exist in another cell line L929 that doesn't express hPEBP4 and sensitive to TNF-α. We found that DC240042 hadn't any effect on L929 cells. To exclude the possibility that difference other than hPEBP4 between MCF-7 and L929 lead to the different response to DC240042, we directly silenced hPEBP4 in MCF-7 cells and tested apoptosis. Consistant with previous report, hPEBP4 silence dramatically sensitized MCF-7 cells to TNF-α(almost 3 folds). However, as observed in L929 cells, DC240042 didn't promote TNF-α-mediated apoptosis after hPEBP4 was silenced. These data imply that the effect of DC240042 is totally dependent on hPEBP4. Therefore, DC240042, as purposely designed, targets hPEBP4 to exert its effect.Since there is no flexible and widely accepted in vivo model to perform therapheutic experiments with TNF-α, we used soft-agorase clone forming assay to examine the effect of DC240042 on the anchorage-independent proliferation of MCF-7 cells. Pretreatment of DC240042 significantly enhanced the inhibitory effect of TNF-αon the growth of MCF-7 cells.Because it has been reported that hPEBP4 also plays a role in TRAIL-resistance of prostate cancer. We tested whether DC240042 was effective in LnCap cells. FACS result show that DC240042 could also sensitized LnCap cells to TRAIL-induced apoptosis.