Increased Chemosensitivity via Targeting Testicular Nuclear Receptor 4 (TR4)-Oct4-Interleukin 1 Receptor Antagonist (IL1Ra) Axis in Prostate Cancer CD133+ Stem/Progenitor Cells to Battle Prostate Cancer

Prostate cancer (PCa) stem/progenitor cells are known to have higher chemoresistance than non-stem/progenitor cells, but the underlying molecular mechanism remains unclear. We found the expression of testicular nuclear receptor 4 (TR4) is significantly higher in PCa CD133⁺ stem/progenitor cells compared with CD133⁻ non-stem/progenitor cells. Knockdown of TR4 levels in the established PCa stem/progenitor cells and the CD133⁺ population of the C4-2 PCa cell line with lentiviral TR4 siRNA led to increased drug sensitivity to the two commonly used chemotherapeutic drugs, docetaxel and etoposide, judging from significantly reduced IC₅₀ values and increased apoptosis in the TR4 knockdown cells. Mechanism dissection studies found that suppression of TR4 in these stem/progenitor cells led to down-regulation of Oct4 expression, which, in turn, down-regulated the IL-1 receptor antagonist (IL1Ra) expression. Neutralization experiments via adding these molecules into the TR4 knockdown PCa stem/progenitor cells reversed the chemoresistance, suggesting that the TR4-Oct4-IL1Ra axis may play a critical role in the development of chemoresistance in the PCa stem/progenitor cells. Together, these studies suggest that targeting TR4 may alter chemoresistance of PCa stem/progenitor cells, and this finding provides the possibility of targeting TR4 as a new and better approach to overcome the chemoresistance problem in PCa therapeutics.     

Steroid receptor co-activator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, methoprene tolerant

Metamorphosis in insects is regulated by juvenile hormone (JH) and ecdysteroids. The mechanism of 20-hydroxyecdysone (20E), but not of JH action, is well understood. A basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family member, methoprene tolerant (Met), plays an important role in JH action. Microarray analysis and RNA interference (RNAi) were used to identify 69 genes that require Met for their hydroprene-regulated expression in the red flour beetle, Tribolium castaneum. Quantitative real time PCR analysis confirmed microarray data for 13 of the 16 hydroprene-response genes tested. The members of the bHLH-PAS family often function as heterodimers to regulate gene expression and Met is a member of this family. To determine whether other members of the bHLH-PAS family are required for the expression of JH-response genes, we employed RNAi to knockdown the expression of all 11 members of the bHLH-PAS family and studied the expression of JH-response genes in RNAi insects. These studies showed that besides Met, another member of this family, steroid receptor co-activator (SRC) is required for the expression of 15 JH-response genes tested. Moreover, studies in JH responsive Aag-2 cells revealed that Aedes aegypti homologues of both Met and SRC are required for the expression of the JH-response gene, kr-h1, and SRC is required for expression of ecdysone-response genes. These data suggest the steroid receptor co-activator plays key roles in both JH and 20E action suggesting that this may be an important molecule that mediates cross-talk between JH and 20E to prevent metamorphosis.     

Cellular processing of the glucocorticoid receptor gene and protein: new mechanisms for generating tissue-specific actions of glucocorticoids

Glucocorticoids regulate numerous physiological processes and are mainstays in the treatment of inflammation, autoimmune disease, and cancer. The traditional view that glucocorticoids act through a single glucocorticoid receptor (GR) protein has changed in recent years with the discovery of a large cohort of receptor subtypes arising from alternative processing of the GR gene. These isoforms differ in their expression, gene regulatory, and functional profiles. Post-translational modification of these proteins further expands GR diversity. Here, we discuss the origin and molecular properties of the GR isoforms and their contribution to the sensitivity and specificity of the glucocorticoid response.     

The Drosophila juvenile hormone receptor candidates methoprene-tolerant (MET) and germ cell-expressed (GCE) utilize a conserved LIXXL motif to bind the FTZ-F1 nuclear receptor

Juvenile hormone (JH) has been implicated in many developmental processes in holometabolous insects, but its mechanism of signaling remains controversial. We previously found that in Drosophila Schneider 2 cells, the nuclear receptor FTZ-F1 is required for activation of the E75A gene by JH. Here, we utilized insect two-hybrid assays to show that FTZ-F1 interacts with two JH receptor candidates, the bHLH-PAS paralogs MET and GCE, in a JH-dependent manner. These interactions are severely reduced when helix 12 of the FTZ-F1 activation function 2 (AF2) is removed, implicating AF2 as an interacting site. Through homology modeling, we found that MET and GCE possess a C-terminal α-helix featuring a conserved motif LIXXL that represents a novel nuclear receptor (NR) box. Docking simulations supported by two-hybrid experiments revealed that FTZ-F1·MET and FTZ-F1·GCE heterodimer formation involves a typical NR box-AF2 interaction but does not require the canonical charge clamp residues of FTZ-F1 and relies primarily on hydrophobic contacts, including a unique interaction with helix 4. Moreover, we identified paralog-specific features, including a secondary interaction site found only in MET. Our findings suggest that a novel NR box enables MET and GCE to interact JH-dependently with the AF2 of FTZ-F1.     

The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein

The cancer/testis antigens (CTAs) are an important group of heterogeneous proteins that are predominantly expressed in the testis in the normal human adult but are aberrantly expressed in several types of cancers. Prostate-associated gene 4 (PAGE4) is a member of the CT-X family of CTAs that in addition to testis, is highly expressed in the fetal prostate, and may also play an important role both in benign and malignant prostate diseases. However, the function of this gene remains poorly understood. Here, we show that PAGE4 is a highly (100%) intrinsically disordered protein (IDP). The primary protein sequence conforms to the features of a typical IDP sequence and the secondary structure prediction algorithm metaPrDOS strongly supported this prediction. Furthermore, SDS-gel electrophoresis and analytical size exclusion chromatography of the recombinant protein revealed an anomalous behavior characteristic of IDPs. UV circular dichroism (CD) and NMR spectroscopy confirmed that PAGE4 is indeed a highly disordered protein. In further bioinformatic analysis, the PredictNLS algorithm uncovered a potential nuclear localization signal, whereas the algorithm DBS-Pred returned a 99.1% probability that PAGE4 is a DNA-binding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GC-rich sequence. Silencing PAGE4 expression induced cell death via apoptosis and in mice carrying PCa xenografts, siRNA-mediated knockdown of the PAGE4 mRNA attenuated tumor growth in vivo. Furthermore, overexpressing PAGE4 protected cells from stress-induced death. To our knowledge, PAGE4 is the first example of a CTA that is an IDP with an anti-apoptotic function.