Prediction of clinically significant prostate cancer through urine metabolomic signatures: A large-scale validated study.

PURPOSE: Currently, there are no accurate markers for predicting potentially lethal prostate cancer (PC) before biopsy. This study aimed to develop urine tests to predict clinically significant PC (sPC) in men at risk. METHODS: Urine samples from 928 men, namely, 660 PC patients and 268 benign subjects, were analyzed by gas chromatography/quadrupole time-of-flight mass spectrophotometry (GC/Q-TOF MS) metabolomic profiling to construct four predictive models. Model I discriminated between PC and benign cases. Models II, III, and GS, respectively, predicted sPC in those classified as having favorable intermediate risk or higher, unfavorable intermediate risk or higher (according to the National Comprehensive Cancer Network risk groupings), and a Gleason sum (GS) of ≥ 7. Multivariable logistic regression was used to evaluate the area under the receiver operating characteristic curves (AUC). RESULTS: In Models I, II, III, and GS, the best AUCs (0.94, 0.85, 0.82, and 0.80, respectively; training cohort, N = 603) involved 26, 24, 26, and 22 metabolites, respectively. The addition of five clinical risk factors (serum prostate-specific antigen, patient age, previous negative biopsy, digital rectal examination, and family history) significantly improved the AUCs of the models (0.95, 0.92, 0.92, and 0.87, respectively). At 90% sensitivity, 48%, 47%, 50%, and 36% of unnecessary biopsies could be avoided. These models were successfully validated against an independent validation cohort (N = 325). Decision curve analysis showed a significant clinical net benefit with each combined model at low threshold probabilities. Models II and III were more robust and clinically relevant than Model GS. CONCLUSION: This urine test, which combines urine metabolic markers and clinical factors, may be used to predict sPC and thereby inform the necessity of biopsy in men with an elevated PC risk.

Aspalathus linearis suppresses cell survival and proliferation of enzalutamide-resistant prostate cancer cells via inhibition of c-Myc and stability of androgen receptor.

Enzalutamide, a nonsteroidal antiandrogen, significantly prolonged the survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patients receiving enzalutamide frequently develop drug resistance. Rooibos (Aspalathus linearis) is a shrub-like leguminous fynbos plant endemic to the Cedarberg Mountains area in South Africa. We evaluated the possibility of using a pharmaceutical-grade green rooibos extract (GRT, containing 12.78% aspalathin) to suppress the proliferation and survival of enzalutamide-resistant prostate cancer (PCa) cells. Treatment with GRT dose-dependently suppressed the proliferation, survival, and colony formation of enzalutamide-resistant C4-2 MDV3100r cells and PC-3 cells. Non-cancerous human cells were more resistant to GRT treatment. GRT suppressed the expression of proteins involved in phosphoinositide 3-kinase (PI3K)-Akt signaling, androgen receptor (AR), phospho-AR (Ser81), cyclin-dependent kinase 1 (Cdk1), c-Myc and Bcl-2 but increased the expression of apoptotic proteins. Overexpression of c-Myc antagonized the suppressive effects of GRT, while knockdown of c-Myc increased the sensitivity of PCa cells to GRT treatment. Expression level of c-Myc correlated to resistance of PCa cells to GRT treatment. Additionally, immunofluorescence microscopy demonstrated that GRT reduced the abundance of AR proteins both in nucleus and cytoplasm. Treatment with cycloheximide revealed that GRT reduced the stability of AR. GRT suppressed protein expression of AR and AR's downstream target prostate specific antigen (PSA) in C4-2 MDV3100r cells. Interestingly, we observed that AR proteins accumulate in nucleus and PSA expression is activated in the AR-positive enzalutamide-resistant PCa cells even in the absence of androgen. Our results suggested that GRT treatment suppressed the cell proliferation and survival of enzalutamide-resistant PCa cells via inhibition of c-Myc, induction of apoptosis, as well as the suppression of expression, signaling and stability of AR. GRT is a potential adjuvant therapeutic agent for enzalutamide-resistant PCa.

Matriptase-2/NR4A3 axis switches TGF-ß action toward suppression of prostate cancer cell invasion, tumor growth, and metastasis.

Dysregulation of pericellular proteolysis is strongly implicated in cancer metastasis through alteration of cell invasion and the microenvironment. Matriptase-2 (MT-2) is a membrane-anchored serine protease which can suppress prostate cancer (PCa) cell invasion. In this study, we showed that MT-2 was down-regulated in PCa and could suppress PCa cell motility, tumor growth, and metastasis. Using microarray and biochemical analysis, we found that MT-2 shifted TGF-ß action towards its tumor suppressor function by repressing epithelial-to-mesenchymal transition (EMT) and promoting Smad2 phosphorylation and nuclear accumulation to upregulate two TGF-ß1 downstream effectors (p21 and PAI-1), culminating in hindrance of PCa cell motility and malignant growth. Mechanistically, MT-2 could dramatically up-regulate the expression of nuclear receptor NR4A3 via iron metabolism in PCa cells. MT-2-induced NR4A3 further coactivated Smad2 to activate p21 and PAI-1 expression. In addition, NR4A3 functioned as a suppressor of PCa and mediated MT-2 signaling to inhibit PCa tumorigenesis and metastasis. These results together indicate that NR4A3 sustains MT-2 signaling to suppress PCa cell invasion, tumor growth, and metastasis, and serves as a contextual factor for the TGF-ß/Smad2 signaling pathway in favor of tumor suppression via promoting p21 and PAI-1 expression.

Aberrant corticosteroid metabolism in tumor cells enables GR takeover in enzalutamide resistant prostate cancer.

Prostate cancer is driven by androgen stimulation of the androgen receptor (AR). The next-generation AR antagonist, enzalutamide, prolongs survival, but resistance and lethal disease eventually prevail. Emerging data suggest that the glucocorticoid receptor (GR) is upregulated in this context, stimulating expression of AR-target genes that permit continued growth despite AR blockade. However, countering this mechanism by administration of GR antagonists is problematic because GR is essential for life. We show that enzalutamide treatment in human models of prostate cancer and patient tissues is accompanied by a ubiquitin E3-ligase, AMFR, mediating loss of 11ß-hydroxysteroid dehydrogenase-2 (11ß-HSD2), which otherwise inactivates cortisol, sustaining tumor cortisol concentrations to stimulate GR and enzalutamide resistance. Remarkably, reinstatement of 11ß-HSD2 expression, or AMFR loss, reverses enzalutamide resistance in mouse xenograft tumors. Together, these findings reveal a surprising metabolic mechanism of enzalutamide resistance that may be targeted with a strategy that circumvents a requirement for systemic GR ablation.

HSD3B1 and resistance to androgen-deprivation therapy in prostate cancer: a retrospective, multicohort study.

BACKGROUND: HSD3B1 (1245A>C) has been mechanistically linked to castration-resistant prostate cancer because it encodes an altered enzyme that augments dihydrotestosterone synthesis from non-gonadal precursors. We postulated that men inheriting the HSD3B1 (1245C) allele would exhibit resistance to androgen-deprivation therapy (ADT). METHODS: In this multicohort study, we determined HSD3B1 genotype retrospectively in men treated with ADT for post-prostatectomy biochemical failure and correlated genotype with long-term clinical outcomes. We used data and samples from prospectively maintained prostate cancer registries at the Cleveland Clinic (Cleveland, OH, USA; primary study cohort) and the Mayo Clinic (Rochester, MN, USA; post-prostatectomy and metastatic validation cohorts). In the post-prostatectomy cohorts, patients of any age were eligible if they underwent prostatectomy between Jan 1, 1996, and Dec 31, 2009 (at the Cleveland Clinic; primary cohort), or between Jan 1, 1987, and Dec 31, 2011 (at the Mayo Clinic; post-prostatectomy cohort) and were treated with ADT for biochemical failure or for non-metastatic clinical failure. In the metastatic validation cohort, patients of any age were eligible if they were enrolled at Mayo Clinic between Sept 1, 2009, and July 31, 2013, with metastatic castration-resistant prostate cancer. The primary endpoint was progression-free survival according to HSD3B1 genotype. We did prespecified multivariable analyses to assess the independent predictive value of HSD3B1 genotype on outcomes. FINDINGS: We included and genotyped 443 patients: 118 in the primary cohort (who underwent prostatectomy), 137 in the post-prostatectomy validation cohort, and 188 in the metastatic validation cohort. In the primary study cohort, median progression-free survival diminished as a function of the number of variant alleles inherited: 6·6 years (95% CI 3·8-not reached) in men with homozygous wild-type genotype, 4·1 years (3·0-5·5) in men with heterozygous variant genotype, and 2·5 years (0·7 to not reached) in men with homozygous variant genotype (p=0·011). Relative to the homozygous wild-type genotype, inheritance of two copies of the variant allele was predictive of decreased progression-free survival (hazard ratio [HR] 2·4 [95% CI 1·1-5·3], p=0·029), as was inheritance of one copy of the variant allele (HR 1·7 [1·0-2·9], p=0·041). Findings were similar for distant metastasis-free survival and overall survival. The effect of the HSD3B1 genotype was independently confirmed in the validation cohorts. INTERPRETATION: Inheritance of the HSD3B1 (1245C) allele that enhances dihydrotestosterone synthesis is associated with prostate cancer resistance to ADT. HSD3B1 could therefore potentially be a powerful genetic biomarker capable of distinguishing men who are a priori likely to fare favourably with ADT from those who harbour disease liable to behave more aggressively, and who therefore might warrant early escalated therapy. FUNDING: Prostate Cancer Foundation, National Institutes of Health, US Department of Defense, Howard Hughes Medical Institute, American Cancer Society, Conquer Cancer Foundation of the American Society of Clinical Oncology, Cleveland Clinic Research Programs Committee and Department of Radiation Oncology, Gail and Joseph Gassner Development Funds.

The role of homeostatic regulation between tumor suppressor DAB2IP and oncogenic Skp2 in prostate cancer growth.

Altered DAB2IP gene expression often detected in prostate cancer (PCa) is due to epigenetic silencing. In this study, we unveil a new mechanism leading to the loss of DAB2IP protein; an oncogenic S-phase kinase-associated protein-2 (Skp2) as E3 ubiquitin ligase plays a key regulator in DAB2IP degradation. In order to unveil the role of Skp2 in the turnover of DAB2IP protein, both prostate cell lines and prostate cancer specimens with a variety of molecular and cell biologic techniques were employed. We demonstrated that DAB2IP is regulated by Skp2-mediated proteasome degradation in the prostate cell lines. Further analyses identified the N-terminal DAB2IP containing the ubiquitination site. Immunohistochemical study exhibited an inverse correlation between DAB2IP and Skp2 protein expression in the prostate cancer tissue microarray. In contrast, DAB2IP can suppressSkp2 protein expression is mediated through Akt signaling. The reciprocal regulation between DAB2IP and Skp2 can impact on the growth of PCa cells. This reciprocal regulation between DAB2IP and Skp2 protein represents a unique homeostatic balance between tumor suppressor and oncoprotein in normal prostate epithelia, which is apparently altered in cancer cells. The outcome of this study has identified new potential targets for developing new therapeutic strategy for PCa.

A gain-of-function mutation in DHT synthesis in castration-resistant prostate cancer.

Growth of prostate cancer cells is dependent upon androgen stimulation of the androgen receptor (AR). Dihydrotestosterone (DHT), the most potent androgen, is usually synthesized in the prostate from testosterone secreted by the testis. Following chemical or surgical castration, prostate cancers usually shrink owing to testosterone deprivation. However, tumors often recur, forming castration-resistant prostate cancer (CRPC). Here, we show that CRPC sometimes expresses a gain-of-stability mutation that leads to a gain-of-function in 3ß-hydroxysteroid dehydrogenase type 1 (3ßHSD1), which catalyzes the initial rate-limiting step in conversion of the adrenal-derived steroid dehydroepiandrosterone to DHT. The mutation (N367T) does not affect catalytic function, but it renders the enzyme resistant to ubiquitination and degradation, leading to profound accumulation. Whereas dehydroepiandrosterone conversion to DHT is usually very limited, expression of 367T accelerates this conversion and provides the DHT necessary to activate the AR. We suggest that 3ßHSD1 is a valid target for the treatment of CRPC.

Prostate cancer-from steroid transformations to clinical translation.

The survival benefit conferred by two hormonal agents in phase III trials has clinically validated the long suspected and now widely recognized phenomenon of castration-resistant prostate cancer (CRPC) hormone dependence. Abiraterone inhibits steroid 17α-hydroxylase/17,20-lyase (CYP17A1) and blocks androgen synthesis, whereas enzalutamide directly binds and antagonizes the androgen receptor. Both agents are highly effective against CRPC and significantly prolong survival following docetaxel treatment. However, this clinical validation of the androgen pathway has led to questions regarding the fundamental mechanisms of CRPC, as well as resistance to abiraterone and enzalutamide. Our understanding of the predominant steroid transformation pathways that lead to dihydrotestosterone synthesis in CRPC is evolving. The role of steroidogenesis in the development of resistance to abiraterone and enzalutamide remains uncertain. The specific roles of candidate enzyme targets in the development of resistance to these agents must be defined if we are to identify novel targets for improved pharmacologic therapies.

Abiraterone inhibits 3ß-hydroxysteroid dehydrogenase: a rationale for increasing drug exposure in castration-resistant prostate cancer.

PURPOSE: Treatment with abiraterone (abi) acetate prolongs survival in castration-resistant prostate cancer (CRPC). Resistance to abi invariably occurs, probably due in part to upregulation of steroidogenic enzymes and/or other mechanisms that sustain dihydrotestosterone (DHT) synthesis, which raises the possibility of reversing resistance by concomitant inhibition of other required steroidogenic enzymes. On the basis of the 3ß-hydroxyl, Δ(5)-structure, we hypothesized that abi also inhibits 3ß-hydroxysteroid dehydrogenase/isomerase (3ßHSD), which is absolutely required for DHT synthesis in CRPC, regardless of origins or routes of synthesis. EXPERIMENTAL DESIGN: We tested the effects of abi on 3ßHSD activity, androgen receptor localization, expression of androgen receptor-responsive genes, and CRPC growth in vivo. RESULTS: Abi inhibits recombinant 3ßHSD activity in vitro and endogenous 3ßHSD activity in LNCaP and LAPC4 cells, including conversion of [(3)H]-dehydroepiandrosterone (DHEA) to Δ(4)-androstenedione, androgen receptor nuclear translocation, expression of androgen receptor-responsive genes, and xenograft growth in orchiectomized mice supplemented with DHEA. Abi also blocks conversion of Δ(5)-androstenediol to testosterone by 3ßHSD. Abi inhibits 3ßHSD1 and 3ßHSD2 enzymatic activity in vitro; blocks conversion from DHEA to androstenedione and DHT with an IC(50) value of less than 1 µmol/L in CRPC cell lines; inhibits androgen receptor nuclear translocation; expression of TMPRSS2, prostate-specific antigen, and FKBP5; and decreases CRPC xenograft growth in DHEA-supplemented mice. CONCLUSIONS: We conclude that abi inhibits 3ßHSD-mediated conversion of DHEA to active androgens in CRPC. This second mode of action might be exploited to reverse resistance to CYP17A1 inhibition at the standard abi dose by dose-escalation or simply by administration with food to increase drug exposure.

A three-lead, programmable, and microcontroller-based electrocardiogram generator with frequency domain characteristics of heart rate variability.

The objective of this study is to design and develop a programmable electrocardiogram (ECG) generator with frequency domain characteristics of heart rate variability (HRV) which can be used to test the efficiency of ECG algorithms and to calibrate and maintain ECG equipment. We simplified and modified the three coupled ordinary differential equations in McSharry's model to a single differential equation to obtain the ECG signal. This system not only allows the signal amplitude, heart rate, QRS-complex slopes, and P- and T-wave position parameters to be adjusted, but can also be used to adjust the very low frequency, low frequency, and high frequency components of HRV frequency domain characteristics. The system can be tuned to function with HRV or not. When the HRV function is on, the average heart rate can be set to a value ranging from 20 to 122 beats per minute (BPM) with an adjustable variation of 1 BPM. When the HRV function is off, the heart rate can be set to a value ranging from 20 to 139 BPM with an adjustable variation of 1 BPM. The amplitude of the ECG signal can be set from 0.0 to 330 mV at a resolution of 0.005 mV. These parameters can be adjusted either via input through a keyboard or through a graphical user interface (GUI) control panel that was developed using LABVIEW. The GUI control panel depicts a preview of the ECG signal such that the user can adjust the parameters to establish a desired ECG morphology. A complete set of parameters can be stored in the flash memory of the system via a USB 2.0 interface. Our system can generate three different types of synthetic ECG signals for testing the efficiency of an ECG algorithm or calibrating and maintaining ECG equipment.

Dihydrotestosterone synthesis bypasses testosterone to drive castration-resistant prostate cancer.

In the majority of cases, advanced prostate cancer responds initially to androgen deprivation therapy by depletion of gonadal testosterone. The response is usually transient, and metastatic tumors almost invariably eventually progress as castration-resistant prostate cancer (CRPC). The development of CRPC is dependent upon the intratumoral generation of the potent androgen, dihydrotestosterone (DHT), from adrenal precursor steroids. Progression to CRPC is accompanied by increased expression of steroid-5α-reductase isoenzyme-1 (SRD5A1) over SRD5A2, which is otherwise the dominant isoenzyme expressed in the prostate. DHT synthesis in CRPC is widely assumed to require 5α-reduction of testosterone as the obligate precursor, and the increased expression of SRD5A1 is thought to reflect its role in converting testosterone to DHT. Here, we show that the dominant route of DHT synthesis in CRPC bypasses testosterone, and instead requires 5α-reduction of androstenedione by SRD5A1 to 5α-androstanedione, which is then converted to DHT. This alternative pathway is operational and dominant in both human CRPC cell lines and fresh tissue obtained from human tumor metastases. Moreover, CRPC growth in mouse xenograft models is dependent upon this pathway, as well as expression of SRD5A1. These findings reframe the fundamental metabolic pathway that drives CRPC progression, and shed light on the development of new therapeutic strategies.

A birth-to-death view of mRNA from the RNA recognition motif perspective.

RNA binding proteins are a large and varied group of factors that are the driving force behind post-transcriptional gene regulation. By analogy with transcription factors, RNA binding proteins bind to various regions of the mRNAs that they regulate, usually upstream or downstream from the coding region, and modulate one of the five major processes in mRNA metabolism: splicing, polyadenylation, export, translation and decay. The most abundant RNA binding protein domain is called the RNA Recognition Motif (RRM)1. It is probably safe to say that an RRM-containing protein is making some contact with an mRNA throughout its existence. The transcriptional counterpart would likely be the histones, yet the multitude of specific functions that are results of RRM based interactions belies the universality of the motif. This complex and diverse application of a single protein motif was used as the basis to develop an advanced graduate level seminar course in RNA:protein interactions. The course, utilizing a learner-centered empowerment model, was developed to dissect each step in RNA metabolism from the perspective of an RRM containing protein. This provided a framework to discuss the development of specificity for the RRM for each required process.