Expression and sub-cellular localization of the CCAAT/enhancer binding protein Α in relation to postnatal development and malignancy of the prostate

Background C/EBPΑ is a critical mediator of terminal differentiation and a tumor suppressor through its strong antiproliferative actions on cell cycle regulatory proteins. C/EBPΑ also appears to regulate androgen receptor (AR) AR signaling. There, is a paucity of information on the expression and sub-cellular localization of C/EBPΑ in normal mouse and human prostate and in prostate cancer. Methods Immunohistochemistry of tissues including tissue arrays, quantitative polymerase chain reaction and mRNA expression database mining. Results In the mouse prostate epithelium, C/EBPΑ was present at 1 week postnatal localized in the cytosol, began to show nuclear localization at 8 weeks and continued to show prominent nuclear expression at 10 weeks and beyond; C/EBPΑ mRNA was expressed at all ages. In humans, C/EBPΑ showed prominent nuclear localization from peripubescence up to middle age but was sequestered in the cytosol in older individuals; the mRNA level for C/EBPΑ remained essentially unchanged. Most prostate adenocarcinomas expressed a range of levels of C/EBPΑ mRNA and protein that were relatively high in metastatic tumors in a manner that correlated with AR expression; however, most cells showed C/EBPΑ sequestered in the cytosol. Conclusions Temporal changes in sub-cellular localization of C/EBPΑ are consistent with a role in prostate differentiation and as a prostate tumor suppressor; the cytoplasmic sequestration of C/EBPΑ, unique to older human prostates, is arguably a permissive condition for the greater frequency of proliferative disorders of the prostate. In malignant prostate C/EBPΑ may be available to regulate AR signaling through transient changes in its sub-cellular localization. Prostate 68: 1206–1214, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60221/1/20779_ftp.pd

The amino-terminal domain of the androgen receptor co-opts extracellular signal-regulated kinase (ERK) docking sites in ELK1 protein to induce sustained gene activation that supports prostate cancer cell growth

The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8 m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions

Kinases and protein phosphorylation as regulators of steroid hormone action

Although the primary signal for the activation of steroid hormone receptors is binding of hormone, there is increasing evidence that the activities of cell signaling pathways and the phosphorylation status of these transcription factors and their coregulators determine the overall response to the hormone. In some cases, enhanced cell signaling is sufficient to cause activation of receptors in medium depleted of steroids. Steroid receptors are targets for multiple kinases. Many of the phosphorylation sites contain Ser/Thr-Pro motifs implicating proline-directed kinases such as the cyclin-dependent kinases and the mitogen-activated kinases (MAPK) in receptor phosphorylation. Although some sites are constitutively phosphorylated, others are phosphorylated in response to hormone. Still others are only phosphorylated in response to specific cell signaling pathways. Phosphorylation of specific sites has been implicated not only in overall transcriptional activity, but also in nuclear localization, protein stability, and DNA binding. The studies of the roles of phosphorylation in coregulator function are more limited, but it is now well established that many of them are highly phosphorylated and that phosphorylation regulates their function. There is good evidence that some of the phosphorylation sites in the receptors and coregulators are targets of multiple signaling pathways. Individual sites have been associated both with functions that enhance the activity of the receptor, as well as with functions that inhibit activity. Thus, the specific combinations of phosphorylations of the steroid receptor combined with the expression levels and phosphorylation status of coregulators will determine the genes regulated and the biological response

SIRNA-Directed In Vivo Silencing of Androgen Receptor Inhibits the Growth of Castration-Resistant Prostate Carcinomas

BACKGROUND: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools

SOD Mimetics: A Novel Class of Androgen Receptor Inhibitors That Suppresses Castration-Resistant Growth of Prostate Cancer

Advanced prostate cancer (PCa) is the second-leading cause of cancer-related deaths among American men. The androgen receptor (AR) is vital for PCa progression, even in the face of castrate levels of serum testosterone following androgen ablation therapy, a mainstay therapy for advanced PCa. Downregulation of superoxide dismutase 2 (SOD2), a major intracellular antioxidant enzyme, occurs progressively during PCa progression to advanced states, and is known to promote AR activity in PCa. Therefore, this study investigated the effects of SOD mimetics on AR expression and function in AR-dependent LNCaP, CWR22Rv1, and LAPC-4AD PCa cells. Treatment with Tempol, a SOD mimetic, not only lowered cellular superoxide levels, but also concomitantly attenuated AR transcriptional activity and AR target gene expression in a dose- and time-dependent manner, in the presence and absence of dihydrotestosterone, the major endogenous AR agonist. Tempol's inhibition of AR was mediated, in large part, by its ability to decrease AR protein via increased degradation, in the absence of any inhibitory effects on other nuclear receptors. Tempol's inhibitory effects on AR were also reproducible with other SOD mimetics, MnTBAP and MnTMPyP. Importantly, Tempol's effects on AR function were accompanied by significant in vitro and in vivo reduction in castration-resistant PCa survival and growth. Collectively, this study has demonstrated for the first time that SOD mimetics, by virtue of their ability to suppress AR function, may be beneficial in treating the currently incurable castration-resistant PCa in which SOD2 expression is highly suppressed