Estrogen receptor beta (ER-β) regulates diverse physiological functions in the human body. Current studies are confined to ER-β1, and the functional roles of isoforms 2, 4, and 5 remain unclear. Full-length ER-β4 and -β5 isoforms were obtained from a prostate cell line, and they exhibit differential expression in a wide variety of human tissues/cell lines. Through molecular modeling, we established that only ER-β1 has a full-length helix 11 and a helix 12 that assumes an agonist-directed position. In ER-β2, the shortened C terminus results in a disoriented helix 12 and marked shrinkage in the coactivator binding cleft. ER-β4 and -β5 completely lack helix 12. We further demonstrated that ER-β1 is the only fully functional isoform, whereas ER-β2, -β4, and -β5 do not form homodimers and have no innate activities of their own. However, the isoforms can heterodimerize with ER-β1 and enhance its transactivation in a ligand-dependent manner. ER-β1 tends to form heterodimers with other isoforms under the stimulation of estrogens but not phytoestrogens. Collectively, these data support the premise that ( i ) ER-β1 is the obligatory partner of an ER-β dimer, whereas the other isoforms function as variable dimer partners with enhancer activity, and ( ii ) a single functional helix 12 in a dimer is sufficient for gene transactivation. Thus, ER-β behaves like a noncanonical type-I receptor, and its action may depend on differential amounts of ER-β1 homo- and heterodimers formed upon stimulation by a specific ligand. Our findings have provided previously unrecognized directions for studying ER-β signaling and design of ER-β-based therapies.

SUMMARY High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit diminished estrogen receptor β (ERβ) expression. We report that a key function of ERβ and its specific ligand 5α-androstane-3β,17β-diol (3β-adiol) is to maintain an epithelial phenotype and repress mesenchymal characteristics in prostate carcinoma. Stimuli (TGF-β and hypoxia) that induce an epithelial-mesenchymal transition (EMT) diminish ERβ expression, and loss of ERβ is sufficient to promote an EMT. The mechanism involves ERβ-mediated destabilization of HIF-1α and transcriptional repression of VEGF-A. The VEGF-A receptor neuropilin-1 drives the EMT by promoting Snail1 nuclear localization. Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF-1α and VEGF expression, and Snail1 nuclear localization compared to low Gleason grade cancers.

Autophagy is an important cellular process by which ATG5 initiates the formation of double membrane vesicles (DMVs). Upon infection, DMVs have been shown to harbor the replicase complex of positive-strand RNA viruses such as MHV, poliovirus, and equine arteritis virus. Recently, it has been shown that autophagy proteins are proviral factors that favor initiation of hepatitis C virus (HCV) infection. Here, we identified ATG5 as an interacting protein for the HCV NS5B. ATG5/NS5B interaction was confirmed by co-IP and metabolic labeling studies. Furthermore, ATG5 protein colocalizes with NS4B, a constituent of the membranous web. Importantly, immunofluorescence staining demonstrated a strong colocalization of ATG5 and NS5B within perinuclear regions of infected cells at 2 days postinfection. However, colocalization was completely lacking at 5 DPI, suggesting that HCV utilizes ATG5 as a proviral factor during the onset of viral infection. Finally, inhibition of autophagy through ATG5 silencing blocks HCV replication.

Two flavonoids, genistein and apigenin, have been implicated as chemopreventive agents against prostate and breast cancers. However, the mechanisms behind their respective cancer-protective effects may vary significantly. The goal of this study was to determine whether the antiproliferative action of these flavonoids on prostate (DU-145) and breast (MDA-MB-231) cancer cells expressing only estrogen receptor (ER) beta is mediated by this ER subtype. It was found that both genistein and apigenin, although not 17beta-estradiol, exhibited antiproliferative effects and proapoptotic activities through caspase-3 activation in these two cell lines. In yeast transcription assays, both flavonoids displayed high specificity toward ERbeta transactivation, particularly at lower concentrations. However, in mammalian assay, apigenin was found to be more ERbeta-selective than genistein, which has equal potency in inducing transactivation through ERalpha and ERbeta. Small interfering RNA-mediated downregulation of ERbeta abrogated the antiproliferative effect of apigenin in both cancer cells but did not reverse that of genistein. Our data unveil, for the first time, that the anticancer action of apigenin is mediated, in part, by ERbeta. The differential use of ERalpha and ERbeta signaling for transaction between genistein and apigenin demonstrates the complexity of phytoestrogen action in the context of their anticancer properties.

We have previously demonstrated that Protein Kinase D1 (PKD1) interacts with E-cadherin and is associated with altered cell aggregation and motility in prostate cancer (PC). Because both PKD1 and E-cadherin are known to be dysregulated in PC, in this study we investigated the functional consequences of combined dysregulation of PKD1 and E-cadherin using a panel of human PC cell lines. Gainand loss of function studies were carried out by either transfecting PC cells with fulllength E-cadherin and/or PKD1 cDNA or by protein silencing by siRNAs, respectively. We studied major malignant phenotypic characteristics including cell proliferation, motility, and invasion at the cellular level, which were corroborated with appropriate changes in representative molecular markers. Down regulation or ectopic expression of either E-cadherin or PKD1 significantly increased or decreased cell proliferation, motility, and invasion, respectively, and combined down regulation cumulatively influenced the effects. Loss of PKD1 or E-cadherin expression was associated with increased expression of the pro-survival molecular markers survivin, β-catenin, cyclin-D, and c-myc, whereas overexpression of PKD1 and/or E-cadherin resulted in an increase of caspases. The inhibitory effect of PKD1 and E-cadherin on cell proliferation was rescued by coexpression with β-catenin, suggesting that β-catenin mediates the effect of proliferation by PKD1 and E-cadherin. This study establishes the functional significance of combined dysregulation of PKD1 and E-cadherin in PC and that their effect on cell growth is mediated by β-catenin.