Possible role of death receptor-mediated apoptosis by the E3 ubiquitin ligases Siah2 and POSH

<p>Abstract</p> <p>Background</p> <p>A functioning ubiquitin proteasome system (UPS) is essential for a number of diverse cellular processes and maintenance of overall cellular homeostasis. The ability of proteasome inhibitors, such as Velcade, to promote extrinsic apoptotic effects illustrates the importance of the ubiquitin proteasome system in the regulation of death receptor signaling. Here, we set out to define the UPS machinery, particularly the E3 ubiquitin ligases, that repress apoptosis through the extrinsic pathway. A cell-based genome-wide E3 ligase siRNA screen was established to monitor caspase-8 activity following the addition of TRAIL.</p> <p>Results</p> <p>Data from the high-throughput screen revealed that targeting the RING-finger containing E3 ligase Siah2 as well as the signaling platform molecule POSH (SH3RF1) conferred robust caspase-8 activation in response to TRAIL stimulus. Silencing Siah2 or POSH in prostate cancer cells led to increased caspase activity and apoptosis in response to both TRAIL and Fas ligand. The E3 activity of Siah2 was responsible for mediating apoptosis resistance; while POSH protein levels were critical for maintaining viability. Further characterization of Siah2 revealed it to function downstream of early death receptor events in the apoptotic pathway. The observed apoptosis resistance provides one biological explanation for the induction of Siah2 and POSH reported in lung and prostate cancer, respectively. Expanding on an initial yeast-two-hybrid screen we have confirmed a physical interaction between E3 ligases Siah2 and POSH. Utilizing a yeast-two-hybrid mapping approach we have defined the spacer region of POSH, more specifically the RPxAxVxP motif encompassing amino acids 601-607, to be the site of Siah2 binding.</p> <p>Conclusions</p> <p>The data presented here define POSH and Siah2 as important mediators of death receptor mediated apoptosis and suggest targeting the interaction of these two E3 ligases is a promising novel cancer therapeutic strategy.</p

Inhibition of Dihydrotestosterone Synthesis in Prostate Cancer by Combined Frontdoor and Backdoor Pathway Blockade

Androgen deprivation therapy (ADT) is palliative and prostate cancer (CaP) recurs as lethal castration-recurrent/resistant CaP (CRPC). One mechanism that provides CaP resistance to ADT is primary backdoor androgen metabolism, which uses up to four 3α-oxidoreductases to convert 5α-androstane-3α,17β-diol (DIOL) to dihydrotestosterone (DHT). The goal was to determine whether inhibition of 3α-oxidoreductase activity decreased conversion of DIOL to DHT. Protein sequence analysis showed that the four 3α-oxidoreductases have identical catalytic amino acid residues. Mass spectrometry data showed combined treatment using catalytically inactive 3α-oxidoreductase mutants and the 5α-reductase inhibitor, dutasteride, decreased DHT levels in CaP cells better than dutasteride alone. Combined blockade of frontdoor and backdoor pathways of DHT synthesis provides a therapeutic strategy to inhibit CRPC development and growth

Potential impact of combined inhibition of 3α-oxidoreductases and 5α-reductases on prostate cancer

Prostate cancer (PCa) growth and progression rely on the interaction between the androgen receptor (AR) and the testicular ligands, testosterone and dihydrotestosterone (DHT). Almost all men with advanced PCa receive androgen deprivation therapy (ADT). ADT lowers circulating testosterone levels, which impairs AR activation and leads to PCa regression. However, ADT is palliative and PCa recurs as castration-recurrent/resistant PCa (CRPC). One mechanism for PCa recurrence relies on intratumoral synthesis of DHT, which can be synthesized using the frontdoor or primary or secondary backdoor pathway. Androgen metabolism inhibitors, such as those targeting 5α-reductase, aldo-keto-reductase family member 3 (AKR1C3), or cytochrome P450 17A1 (CYP17A1) have either failed or produced only modest clinical outcomes. The goal of this review is to describe the therapeutic potential of combined inhibition of 5α-reductase and 3α-oxidoreductase enzymes that facilitate the terminal steps of the frontdoor and primary and secondary backdoor pathways for DHT synthesis. Inhibition of the terminal steps of the androgen metabolism pathways may be a way to overcome the shortcomings of existing androgen metabolism inhibitors and thereby delay PCa recurrence during ADT or enhance the response of CRPC to androgen axis manipulation. Keywords: Androstanediol, Dihydrotestosterone, Dutasteride, 3α-oxidoreductases, Androgen deprivation therapy, Abirateron

Recapitulation of prostate tissue cell type-specific transcriptomes by an in vivo primary prostate tissue xenograft model.

Studies of the normal functions and diseases of the prostate request in vivo models that maintain the tissue architecture and the multiple-cell type compartments of human origin in order to recapitulate reliably the interactions of different cell types. Cell type-specific transcriptomes are critical to reveal the roles of each cell type in the functions and diseases of the prostate. A primary prostate tissue xenograft model was developed using fresh human prostate tissue specimens transplanted onto male mice that were castrated surgically and implanted with a device to maintain circulating testosterone levels comparable to adult human males. Endothelial cells and epithelial cells were isolated from 7 fresh human prostate tissue specimens and from primary tissue xenografts established from 9 fresh human prostate tissue specimens, using antibody-conjugated magnetic beads specific to human CD31 and human EpCAM, respectively. Transcriptomes of endothelial, epithelial and stromal cell fractions were obtained using RNA-Seq. Global and function-specific gene expression profiles were compared in inter-cell type and inter-tissue type manners. Gene expression profiles in the individual cell types isolated from xenografts were similar to those of cells isolated from fresh tissue, demonstrating the value of the primary tissue xenograft model for studies of the inter-relationships between prostatic cell types and the role of such inter-relationships in organ development, disease progression, and response to drug treatments

Proteomic Analysis of Charcoal-Stripped Fetal Bovine Serum Reveals Changes in the Insulin-like Growth Factor Signaling Pathway

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells