Biomarker Inference and the Timing of Next-Generation Sequencing in a Multi-Institutional, Cross-Cancer Clinicogenomic Data Set.

PURPOSE: Observational clinicogenomic data sets, consisting of tumor next-generation sequencing (NGS) data linked to clinical records, are commonly used for cancer research. However, in real-world practice, oncologists frequently request NGS in search of treatment options for progressive cancer. The extent and impact of this dynamic on analysis of clinicogenomic research data are not well understood. METHODS: We analyzed clinicogenomic data for patients with non-small cell lung, colorectal, breast, prostate, pancreatic, or urothelial cancers in the American Association for Cancer Research Biopharmaceutical Consortium cohort. Associations between baseline and time-varying clinical characteristics and time from diagnosis to NGS were measured. To explore the impact of informative cohort entry on biomarker inference, statistical interactions between selected biomarkers and time to NGS with respect to overall survival were calculated. RESULTS: Among 7,182 patients, time from diagnosis to NGS varied significantly by clinical factors, including cancer type, calendar year of sequencing, institution, and age and stage at diagnosis. NGS rates also varied significantly by dynamic clinical status variables; in an adjusted model, compared with patients with stable disease at any given time after diagnosis, patients with progressive disease by imaging or oncologist assessment had higher NGS rates (hazard ratio for NGS, 1.61 [95% CI, 1.45 to 1.78] and 2.32 [95% CI, 2.01 to 2.67], respectively). Statistical interactions between selected biomarkers and time to NGS with respect to survival, potentially indicating biased biomarker inference results, were explored. CONCLUSION: To evaluate the appropriateness of a data set for a particular research question, it is crucial to measure associations between dynamic cancer status and the timing of NGS, as well as to evaluate interactions involving biomarkers of interest and NGS timing with respect to survival outcomes.

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.

Pictet-Spengler condensations using 4-(2-aminoethyl)coumarins.

Androgen-deprivation therapy (ADT) is only a palliative measure, and prostate cancer invariably recurs in a lethal, castration-resistant form (CRPC). Prostate cancer resists ADT by metabolizing weak, adrenal androgens to growth-promoting 5α-dihydrotestosterone (DHT), the preferred ligand for the androgen receptor (AR). Developing small-molecule inhibitors for the final steps in androgen metabolic pathways that utilize 17-oxidoreductases required probes that possess fluorescent groups at C-3 and intact, naturally occurring functionality at C-17. Application of the Pictet-Spengler condensation to substituted 4-(2-aminoethyl)coumarins and 5α-androstane-3-ones furnished spirocyclic, fluorescent androgens at the desired C-3 position. Condensations required the presence of activating C-7 amino or N,N-dialkylamino groups in the 4-(2-aminoethyl)coumarin component of these condensation reactions. Successful Pictet-Spengler condensation, for example, of DHT with 9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one led to a spirocyclic androgen, (3R,5S,10S,13S,17S)-17-hydroxy-10,13-dimethyl-1,2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16,17-docosahydro-7'H,11'H-spiro-[cyclopenta[a]phenanthrene-3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)-one. Computational modeling supported the surrogacy of the C-3 fluorescent DHT analog as a tool to study 17-oxidoreductases for intracrine, androgen metabolism.

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.

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.

Mathematical modeling of intracrine androgen metabolism in prostate cancer: Methodological aspects.

BACKGROUND: Progression of castration-recurrent/resistant prostate cancer (CRPC) relies in part on dihydrotestosterone derived from intratumoral androgen metabolism. Mathematical modeling provides a valuable tool for studies of androgen metabolism in CRPC. This modeling approach integrates existing knowledge about complex biologic systems and provides a means of interrogating the effects of various interventions. We sought to model a single reaction in the androgen biosynthesis network, namely the oxidation of androsterone (AND) to androstanedione (5α-dione) by four 3α-oxidoreductase enzymes, as an initial effort to establish the feasibility of our modeling approach. METHODS: Models were constructed for two cell culture systems, a non-prostate cancer cell line (CV-1) and a prostate cancer cell line (LAPC-4), using the SimBiology app (version 5.3) in MATLAB (version 8.6). The models included components for substrate (AND), product (5α-dione), each of the four enzymes, and each of the four enzyme-substrate complexes. Each enzymatic reaction consisted of a reversible enzyme-substrate binding step and an irreversible catalysis step. Rates of change for each component were described using ordinary differential equations. RESULTS: Mathematical models were developed with model parameter values derived from literature sources or from existing experimental data, which included gene expression measurements and substrate and product concentrations determined using liquid chromatography-tandem mass spectrometry. The models for both cell lines adequately described substrate and product concentrations observed after 12 h treatment with AND. CONCLUSIONS: This modeling approach represents an adaptable, extensible and mechanistic framework that reflects androgen metabolism. The models can be expanded systematically to describe the complex androgen metabolic pathways important for study of novel therapies for CRPC.

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,17ß-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.

Serum-free complete medium, an alternative medium to mimic androgen deprivation in human prostate cancer cell line models.

BACKGROUND: Almost all men who present with advanced prostate cancer (CaP) and some men who fail therapy for clinically localized CaP are treated with androgen deprivation therapy (ADT). CaP cell lines are used to identify and characterize new agents for ADT or investigate mechanisms of ADT resistance. CaP cell lines are maintained in culture medium that contains fetal bovine serum, which contains testosterone (T). Androgen deprivation experiments are performed using media supplemented with androgen-free serum, such as charcoal stripped fetal bovine serum (CS-FBS). However, CS-FBS composition varies from batch-to-batch and variations may impact experimental reproducibility. Serum free media (SFM) may provide a better defined alternative to media supplemented with CS-FBS (CSM). METHODS: Cell growth of six human CaP cell lines was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Androgen levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: MTT assays showed 5 of 6 CaP cell lines grew after 6 days of culture in androgen- deprived SFM or CSM. LNCaP and VCaP growth was stimulated when cells were cultured in SFM or CSM supplemented with T. LNCaP, C4-2, LAPC-4, and VCaP cell growth was inhibited when cultured in SFM or CSM with T and bicalutamide. LC-MS/MS data showed LAPC-4 cells produced similar DHT levels when cultured in T-supplemented SFM or CSM. Dutasteride impaired T to DHT metabolism in LAPC-4. CONCLUSIONS: Media composition contributed to growth differences observed between CaP cells cultured in SFM or CSM. However, the differences in media composition did not impair CaP cell response to T-stimulated growth, bicalutamide growth inhibition, metabolism of T, or dutasteride efficiency. SFM can be used as a better defined alternative to CSM for androgen deprivation experiments.

Efficient Synthesis of Aurone Mannich Bases and Evaluation of their Antineoplastic Activity in PC-3 Prostate Cancer Cells.

An efficient method for regioselective synthesis of C-7 Mannich bases of 6-hydroxyaurones was accomplished by the N,N-dialkylaminomethylation using aminals prepared from dimethylamine, dipropylamine, bis(2-methoxyethyl)amine, N-methylbutylamine, N-methylbenzylamine, morpholine, piperidine, and 1-methylpiperazine. Further transformation of 7-(N,N-dialkylamino)methyl group in these aurones led to formation of C-7 acetoxymethyl and methoxymethyl derivatives of 6-hydroxyaurones, some of which showed promising inhibition of PC-3 prostate cancer cell proliferation in the high nanomolar to low micromolar range that exceeded that of cisplatin.

Developing antineoplastic agents that target peroxisomal enzymes: cytisine-linked isoflavonoids as inhibitors of hydroxysteroid 17-beta-dehydrogenase-4 (HSD17B4).

Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional peroxisomal enzyme, hydroxysteroid 17ß-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the d-3-hydroxyacyl CoA dehydrogenase activity. HSD17B4 was overexpressed in prostate and colon cancer tissues, knocking down HSD17B4 inhibited cancer cell proliferation, suggesting that HSD17B4 is a potential biomarker and drug target and that CLIFs are potential probes or therapeutic agents for these cancers.

Characterization of Prostate Cancer in a Functional Eunuch.

BACKGROUND: Eunuchs rarely, if ever, develop prostate cancer (CaP). This article reports on a 62-year-old functional eunuch from prepubertal mumps orchitis who developed clinically localized CaP. METHODS: Serum and CaP and benign prostate tissue androgen levels were measured using a validated liquid chromatography-tandem mass spectrometry assay. The assay measures testosterone; dihydrotestosterone (DHT); the adrenal androgens, androstenedione and dehydroepiandrosterone; and the androgen metabolites, androsterone and androstanedione. Gene and protein expression levels of androgen metabolism enzymes, and androgen receptor and androgen-regulated genes were measured using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry, respectively. RESULTS: Intracrine androgen metabolism produced tissue DHT when serum and tissue testosterone levels were castrate and undetectable, respectively. Androgen receptor, androgen-regulated, and androgen metabolism enzyme genes were expressed but at lower levels in CaP than benign tissues. CONCLUSIONS: DHT was synthesized using the primary backdoor androgen metabolism pathway and not using androstenedione or dehydroepiandrosterone via the frontdoor or secondary backdoor pathways.

Regulators of Androgen Action Resource: a one-stop shop for the comprehensive study of androgen receptor action.

Androgen receptor (AR) is a ligand-activated transcription factor that is the main target for treatment of non-organ-confined prostate cancer (CaP). Failure of life-prolonging AR-targeting androgen deprivation therapy is due to flexibility in steroidogenic pathways that control intracrine androgen levels and variability in the AR transcriptional output. Androgen biosynthesis enzymes, androgen transporters and AR-associated coregulators are attractive novel CaP treatment targets. These proteins, however, are characterized by multiple transcript variants and isoforms, are subject to genomic alterations, and are differentially expressed among CaPs. Determining their therapeutic potential requires evaluation of extensive, diverse datasets that are dispersed over multiple databases, websites and literature reports. Mining and integrating these datasets are cumbersome, time-consuming tasks and provide only snapshots of relevant information. To overcome this impediment to effective, efficient study of AR and potential drug targets, we developed the Regulators of Androgen Action Resource (RAAR), a non-redundant, curated and user-friendly searchable web interface. RAAR centralizes information on gene function, clinical relevance, and resources for 55 genes that encode proteins involved in biosynthesis, metabolism and transport of androgens and for 274 AR-associated coregulator genes. Data in RAAR are organized in two levels: (i) Information pertaining to production of androgens is contained in a 'pre-receptor level' database, and coregulator gene information is provided in a 'post-receptor level' database, and (ii) an 'other resources' database contains links to additional databases that are complementary to and useful to pursue further the information provided in RAAR. For each of its 329 entries, RAAR provides access to more than 20 well-curated publicly available databases, and thus, access to thousands of data points. Hyperlinks provide direct access to gene-specific entries in the respective database(s). RAAR is a novel, freely available resource that provides fast, reliable and easy access to integrated information that is needed to develop alternative CaP therapies. Database URL: http://www.lerner.ccf.org/cancerbio/heemers/RAAR/search/.

Antineoplastic Isoflavonoids Derived from Intermediate ortho-Quinone Methides Generated from Mannich Bases.

The regioselective condensations of various 7-hydroxyisoflavonoids with bis(N,N-dimethylamino)methane in a Mannich reaction provided C-8 N,N-dimethylaminomethyl-substituted isoflavonoids in good yield. Similar condensations of 7-hydroxy-8-methylisoflavonoids led to the C-6-substituted analogs. Thermal eliminations of dimethylamine from these C-6 or C-8 N,N-dimethylaminomethyl-substituted isoflavonoids generated ortho-quinone methide intermediates within isoflavonoid frameworks for the first time. Despite other potential competing outcomes, these ortho-quinone methide intermediates trapped dienophiles including 2,3-dihydrofuran, 3,4-dihydro-2H-pyran, 3-(N,N-dimethylamino)-5,5-dimethyl-2-cyclohexen-1-one, 1-morpholinocyclopentene, and 1-morpholinocyclohexene to give various inverse electron-demand Diels-Alder adducts. Several adducts derived from 8-N,N-dimethylaminomethyl-substituted isoflavonoids displayed good activity in the 1-10 µm concentration range in an in vitro proliferation assay using the PC-3 prostate cancer cell line.

Application of Mannich bases to the synthesis of hydroxymethylated isoflavonoids as potential antineoplastic agents.

The regiospecific Mannich aminomethylation of 7-hydroxyisoflavonoids using bis(N,N-dimethylamino)methane afforded C-8 substituted N,N-dimethylaminomethyl adducts, and the regioselective aminomethylation of 5-hydroxy-7-methoxyisoflavonoids afforded predominantly the C-6 substituted N,N-dimethylaminomethyl adducts. Acetylation of these C-6 or C-8 Mannich bases with potassium acetate in acetic anhydride provided access to the corresponding acetoxymethyl derivatives that were subsequently converted to hydroxymethyl- and methoxymethyl-substituted 5-hydroxy- or 7-hydroxyisoflavonoids related to naturally occurring flavonoids. The C-8 acetoxymethyl, hydroxymethyl or methoxymethyl-substituted isoflavonoids possessed promising inhibitory potency in the low micromolar range in a prostate cancer PC-3 cell proliferation assay.

Roles for the backdoor pathway of androgen metabolism in prostate cancer response to castration and drug treatment.

Almost all men who present with advanced prostate cancer (CaP) and many men who fail potentially curative therapy are treated with androgen deprivation therapy (ADT). ADT is not curative and CaP recurs as the lethal phenotype. The goal of this review is to describe the evolution of adrenal androgen blockade, how new androgen measurement methods have furthered understanding of androgen metabolism, and how further understanding of the backdoor pathway of androgen metabolism may lead to interventions that extend survival even more.

The role of intracrine androgen metabolism, androgen receptor and apoptosis in the survival and recurrence of prostate cancer during androgen deprivation therapy.

Prostate cancer (CaP) is the most frequently diagnosed cancer and leading cause of cancer death in American men. Almost all men present with advanced CaP and some men who fail potentially curative therapy are treated with androgen deprivation therapy (ADT). ADT is not curative and CaP recurs as the lethal phenotype. The goal of this review is to apply our current understanding of CaP and castration-recurrent CaP (CR-CaP) to earlier studies that characterized ADT and the molecular mechanisms that facilitate the transition from androgen-stimulated CaP to CR-CaP. Reexamination of earlier studies also may provide a better understanding of how more newly recognized mechanisms, such as intracrine metabolism, may be involved with the early events that allow CaP survival after initiation of ADT and subsequent development of CR-CaP.

Proteasomal regulation of caspase-8 in cancer cell apoptosis.

Previous studies demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of caspase-8. The present study investigated the impact of proteasome inhibition on caspase-8 stability, ubiquitination, trafficking, and activation in cancer cells. Using caspase-8 deficient neuroblastoma (NB7) cells for reconstituting non-cleavable mutant forms of caspase-8, we demonstrated that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8, in response to proteasome inhibitor and GST-TRAIL. Moreover in the LNCaP human prostate cancer cells, caspase-8 polyubiquitination occurs after TRAIL stimulation and caspase-8 processing. Subcellular fractionation analysis revealed caspase-8 activity in both cytosol and plasma membrane fractions in both NB7 reconstituted caspase-8 cell lines, as well the LNCaP prostate cancer cells. The present results suggest that caspase-8 stabilization through proteasome inhibition leads to reactivation of the extrinsic pathway of apoptosis and identify E3 ligase mediating caspase-8 polyubiquitination, as a novel molecular target. Inhibition of this E3 ligase in combination with TRAIL towards restoring apoptosis signaling activation may have potential therapeutic significance in resistant tumors.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.