Targeting sphingolipid metabolism in chronic lymphocytic leukemia.

Elevated levels of circulating C16:0 glucosylceramides (GluCer) and increased mRNA expression of UDP-glucose ceramide glycosyltransferase (UGCG), the enzyme responsible for converting ceramides (Cer) to GluCer, represent unfavorable prognostic markers in chronic lymphocytic leukemia (CLL) patients. To evaluate the therapeutic potential of inhibiting GluCer synthesis, we genetically repressed the UGCG pathway using in vitro models of leukemic B cells, in addition to UGCG pharmacological inhibition with approved drugs such as eliglustat and ibiglustat, both individually and in combination with ibrutinib, assessed in cell models and primary CLL patient cells. Cell viability, apoptosis, and proliferation were evaluated in vitro, and survival and apoptosis were examined ex vivo. UGCG inhibition efficacy was confirmed by quantifying intracellular sphingolipid levels through targeted lipidomics using mass spectrometry. Other inhibitors of sphingolipid biosynthesis pathways were similarly assessed. Blocking UGCG significantly decreased cell viability and proliferation, highlighting the oncogenic role of UGCG in CLL. The efficient inhibition of UGCG was confirmed by a significant reduction in GluCer intracellular levels. The combination of UGCG inhibitors with ibrutinib demonstrated synergistic effect. Inhibitors that target alternative pathways within sphingolipid metabolism, like sphingosine kinases inhibitor SKI-II, also demonstrated promising therapeutic effects both alone and when used in combination with ibrutinib, reinforcing the oncogenic impact of sphingolipids in CLL cells. Targeting sphingolipid metabolism, especially the UGCG pathway, represents a promising therapeutic strategy and as a combination therapy for potential treatment of CLL patients, warranting further investigation.

Menopause and Estrogen Associations With Gut Barrier, Microbial Translocation, and Immune Activation Biomarkers in Women With and Without HIV.

OBJECTIVES: Estrogens may protect the gut barrier and reduce microbial translocation and immune activation, which are prevalent in HIV infection. We investigated relationships of the menopausal transition and estrogens with gut barrier, microbial translocation, and immune activation biomarkers in women with and without HIV. DESIGN: Longitudinal and cross-sectional studies nested in the Women's Interagency HIV Study. METHODS: Intestinal fatty acid binding protein, lipopolysaccharide binding protein, and soluble CD14 (sCD14) levels were measured in serum from 77 women (43 with HIV) before, during, and after the menopausal transition (∼6 measures per woman over ∼13 years). A separate cross-sectional analysis was conducted among 72 postmenopausal women with HIV with these biomarkers and serum estrogens. RESULTS: Women in the longitudinal analysis were a median age of 43 years at baseline. In piecewise, linear, mixed-effects models with cutpoints 2 years before and after the final menstrual period to delineate the menopausal transition, sCD14 levels increased over time during the menopausal transition (Beta [95% CI]: 38 [12 to 64] ng/mL/yr, P = 0.004), followed by a decrease posttransition (-46 [-75 to -18], P = 0.001), with the piecewise model providing a better fit than a linear model (P = 0.0006). In stratified analyses, these results were only apparent in women with HIV. In cross-sectional analyses, among women with HIV, free estradiol inversely correlated with sCD14 levels (r = -0.26, P = 0.03). Lipopolysaccharide binding protein and intestinal fatty acid binding protein levels did not appear related to the menopausal transition and estrogen levels. CONCLUSIONS: Women with HIV may experience heightened innate immune activation during menopause, possibly related to the depletion of estrogens.

The estrogen signaling pathway reprograms prostate cancer cell metabolism and supports proliferation and disease progression.

Just like the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet the incomplete understanding of ERα functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for the treatment of this disease. Using 2 human PCa tissue microarray cohorts, we first demonstrate that nuclear ERα expression was heterogeneous among patients, being detected in only half of the tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single-cell RNA-Seq analyses revealed that estrogens partially mimicked the androgen transcriptional response and activated specific biological pathways linked to proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed the regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprogrammed PCa metabolism, was associated with disease progression, and could be targeted for therapeutic purposes.

Non-canonical transcriptional regulation of the poor prognostic factor UGT2B17 in chronic lymphocytic leukemic and normal B cells.

BACKGROUND: High expression of the glycosyltransferase UGT2B17 represents an independent adverse prognostic marker in chronic lymphocytic leukemia (CLL). It also constitutes a predictive marker for therapeutic response and a drug resistance mechanism. The key determinants driving expression of the UGT2B17 gene in normal and leukemic B-cells remain undefined. The UGT2B17 transcriptome is complex and is comprised of at least 10 alternative transcripts, identified by previous RNA-sequencing of liver and intestine. We hypothesized that the transcriptional program regulating UGT2B17 in B-lymphocytes is distinct from the canonical expression previously characterized in the liver. RESULTS: RNA-sequencing and genomics data revealed a specific genomic landscape at the UGT2B17 locus in normal and leukemic B-cells. RNA-sequencing and quantitative PCR data indicated that the UGT2B17 enzyme is solely encoded by alternative transcripts expressed in CLL patient cells and not by the canonical transcript widely expressed in the liver and intestine. Chromatin accessible regions (ATAC-Seq) in CLL cells mapped with alternative promoters and non-coding exons, which may be derived from endogenous retrotransposon elements. By luciferase reporter assays, we identified key cis-regulatory STAT3, RELA and interferon regulatory factor (IRF) binding sequences driving the expression of UGT2B17 in lymphoblastoid and leukemic B-cells. Electrophoretic mobility shift assays and pharmacological inhibition demonstrated key roles for the CLL prosurvival transcription factors STAT3 and NF-κB in the leukemic expression of UGT2B17. CONCLUSIONS: UGT2B17 expression in B-CLL is driven by key regulators of CLL progression. Our data suggest that a NF-κB/STAT3/IRF/UGT2B17 axis may represent a novel B-cell pathway promoting disease progression and drug resistance.

Sex Hormones, the Stool Microbiome, and Subclinical Atherosclerosis in Women With and Without HIV.

CONTEXT: Cardioprotective roles of endogenous estrogens may be particularly important in women with HIV, who have reduced estrogen exposure and elevated cardiovascular disease risk. The gut microbiome metabolically interacts with sex hormones, but little is known regarding possible impact on cardiovascular risk. OBJECTIVE: To analyze potential interplay of sex hormones and gut microbiome in cardiovascular risk. METHODS: Among 197 postmenopausal women in the Women's Interagency HIV Study, we measured 15 sex hormones in serum and assessed the gut microbiome in stool. Presence of carotid artery plaque was determined (B-mode ultrasound) in a subset (n = 134). We examined associations of (i) sex hormones and stool microbiome, (ii) sex hormones and plaque, and (iii) sex hormone-related stool microbiota and plaque, adjusting for potential confounders. RESULTS: Participant median age was 58 years and the majority were living with HIV (81%). Sex hormones (estrogens, androgens, and adrenal precursors) were associated with stool microbiome diversity and specific species, similarly in women with and without HIV. Estrogens were associated with higher diversity, higher abundance of species from Alistipes, Collinsella, Erysipelotrichia, and Clostridia, and higher abundance of microbial ß-glucuronidase and aryl-sulfatase orthologs, which are involved in hormone metabolism. Several hormones were associated with lower odds of carotid artery plaque, including dihydrotestosterone, 3α-diol-17G, estradiol, and estrone. Exploratory mediation analysis suggested that estrone-related species, particularly from Collinsella, may mediate the protective association of estrone with plaque. CONCLUSION: Serum sex hormones are significant predictors of stool microbiome diversity and composition. The gut microbiome may play a role in estrogen-related cardiovascular protection.

Untargeted metabolomics identifies metabolic dysregulation of sphingolipids associated with aggressive chronic lymphocytic leukaemia and poor survival.

BACKGROUND: Metabolic dependencies of chronic lymphocytic leukaemia (CLL) cells may represent new personalized treatment approaches in patients harbouring unfavourable features. METHODS: Here, we used untargeted metabolomics and lipidomics analyses to isolate metabolomic features associated with aggressive CLL and poor survival outcomes. We initially focused on profiles associated with overexpression of the adverse metabolic marker glycosyltransferase (UGT2B17) associated with poor survival and drug resistance. RESULTS: Leukaemic B-cell metabolomes indicated a significant perturbation in lipids, predominantly bio-active sphingolipids. Expression of numerous enzyme-encoding genes of sphingolipid biosynthesis pathways was significantly associated with shorter patient survival. Targeted metabolomics further exposed higher circulating levels of glucosylceramides (C16:0 GluCer) in CLL patients relative to healthy donors and an aggressive cancer biology. In multivariate analyses, C16:0 GluCer and sphinganine were independent prognostic markers and were inversely linked to treatment-free survival. These two sphingolipid species function as antagonistic mediators, with sphinganine being pro-apoptotic and GluCer being pro-proliferative, tested in leukemic B-CLL cell models. Blocking GluCer synthesis using ceramide glucosyltransferase inhibitors induced cell death and reduced the proliferative phenotype, which further sensitized a leukaemic B-cell model to the anti-leukaemics fludarabine and ibrutinib in vitro. CONCLUSIONS: Specific sphingolipids may serve as prognostic markers in CLL, and inhibiting enzymatic pathways involved in their biosynthesis has potential as a therapaeutic approach.

Prediagnostic Hormone Levels and Risk of Testicular Germ Cell Tumors: A Nested Case-Control Study in the Janus Serum Bank.

BACKGROUND: It has been hypothesized that poorly functioning Leydig and/or Sertoli cells of the testes, indicated by higher levels of serum gonadotropins and lower levels of androgens, are related to the development of testicular germ cell tumors (TGCT). To investigate this hypothesis, we conducted a nested case-control study within the Janus Serum Bank cohort. METHODS: Men who developed TGCT (n = 182) were matched to men who did not (n = 364). Sex steroid hormones were measured using LC/MS. Sex hormone binding globulin, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were quantified by direct immunoassay. Multivariable logistic regression was used to calculate ORs and 95% confidence intervals (CI) for associations between hormone levels and TGCT risk. RESULTS: Higher FSH levels [tertile (T) 3 vs. T2: OR = 2.89, 95% CI = 1.83-4.57] were associated with TGCT risk, but higher LH levels were not (OR = 1.26, 95% CI = 0.81-1.96). The only sex steroid hormone associated with risk was androstane-3α, 17ß-diol-3G (3α-diol-3G; OR = 2.37, 95% CI = 1.46-3.83). Analysis by histology found that increased FSH levels were related to seminoma (OR = 3.55, 95% CI = 2.12-5.95) but not nonseminoma (OR = 1.19, 95% CI = 0.38-3.13). Increased levels of 3α-diol-3G were related to seminoma (OR = 2.29, 95% CI = 1.35-3.89) and nonsignificantly related to nonseminoma (OR = 2.71, 95% CI = 0.82-8.92). CONCLUSIONS: Higher FSH levels are consistent with the hypothesis that poorly functioning Sertoli cells are related to the development of TGCT. In contrast, higher levels of 3α-diol-3G do not support the hypothesis that insufficient androgenicity is related to risk of TGCT. IMPACT: Clarifying the role of sex hormones in the development of TGCT may stimulate new research hypotheses.

Endogenous sex steroid hormones and risk of liver cancer among US men: Results from the Liver Cancer Pooling Project.

Background & Aims: Incidence rates of liver cancer in most populations are two to three times higher among men than women. The higher rates among men have led to the suggestion that androgens are related to increased risk whereas oestrogens are related to decreased risk. This hypothesis was investigated in the present study via a nested case-control analysis of pre-diagnostic sex steroid hormone levels among men in five US cohorts. Methods: Concentrations of sex steroid hormones and sex hormone-binding globulin were quantitated using gas chromatography-mass spectrometry and a competitive electrochemiluminescence immunoassay, respectively. Multivariable conditional logistic regression was used to calculate odds ratios (ORs) and 95% CIs for associations between hormones and liver cancer among 275 men who subsequently developed liver cancer and 768 comparison men. Results: Higher concentrations of total testosterone (OR per one-unit increase in log2 = 1.77, 95% CI = 1.38-2.29), dihydrotestosterone (OR = 1.76, 95% CI = 1.21-2.57), oestrone (OR = 1.74, 95% CI = 1.08-2.79), total oestradiol (OR = 1.58, 95% CI=1.22-20.05), and sex hormone-binding globulin (OR = 1.63, 95% CI = 1.27-2.11) were associated with increased risk. Higher concentrations of dehydroepiandrosterone (DHEA), however, were associated with a 53% decreased risk (OR = 0.47, 95% CI = 0.33-0.68). Conclusions: Higher concentrations of both androgens (testosterone, dihydrotestosterone) and their aromatised oestrogenic metabolites (oestrone, oestradiol) were observed among men who subsequently developed liver cancer compared with men who did not. As DHEA is an adrenal precursor of both androgens and oestrogens, these results may suggest that a lower capacity to convert DHEA to androgens, and their subsequent conversion to oestrogens, confers a lower risk of liver cancer, whereas a greater capacity to convert DHEA confers a greater risk. Impact and implications: This study does not fully support the current hormone hypothesis as both androgen and oestrogen levels were associated with increased risk of liver cancer among men. The study also found that higher DHEA levels were associated with lower risk, thus suggesting the hypothesis that greater capacity to convert DHEA could be associated with increased liver cancer risk among men.

Circulating adrenal 11-oxygenated androgens are associated with clinical outcome in endometrial cancer.

Context: Recent evidence support that androgens play an important role in the etiology of endometrial cancer (EC). Adrenal-derived 11-oxygenated androgens are highly potent agonists of the androgen receptor (AR), comparable to testosterone (T) and dihydrotestosterone (DHT) that have not been studied in the context of EC. Methodology: We studied a cohort of 272 newly diagnosed postmenopausal EC cases undergoing surgical treatment. Circulating concentrations of seven 11-oxygenated androgens including precursors, potent androgens and their metabolites were established in serum samples collected before and 1 month after surgery using a validated liquid chromatography tandem mass spectrometry method (LC-MS/MS). Free (unconjugated) and total (free + sulfate and glucuronide conjugates following enzymatic hydrolysis) were analyzed in relation to clinicopathological features, recurrence and disease-free survival (DFS). Results: Levels of 11-oxygenated androgens were weakly correlated to those of canonical androgens such as testosterone (T) and dihydrotestosterone (DHT), with no evidence of their association with clinicopathological features. Levels of 11-oxygenated androgens declined after surgery but remained higher in overweight and obese compared to normal weight cases. Higher levels of preoperative free 11-ketoandrosterone (11KAST) were associated with an increased risk of recurrence (Hazard ratio (HR) of 2.99 (95%CI=1.09-8.18); P=0.03). Postoperative free 11ß-hydroxyandrosterone (11OHAST) levels were adversely associated with recurrence and DFS (HR = 3.23 (1.11-9.40); P=0.03 and 3.27 (1.34-8.00); P=0.009, respectively). Conclusion: 11-oxygenated androgen metabolites emerge as potential prognostic markers of EC.

A Non-Canonical Role for the Glycosyltransferase Enzyme UGT2B17 as a Novel Constituent of the B Cell Receptor Signalosome.

In chronic lymphocytic leukemia (CLL), an elevated glycosyltransferase UGT2B17 expression (UGT2B17HI) identifies a subgroup of patients with shorter survival and poor drug response. We uncovered a mechanism, possibly independent of its enzymatic function, characterized by an enhanced expression and signaling of the proximal effectors of the pro-survival B cell receptor (BCR) pathway and elevated Bruton tyrosine kinase (BTK) phosphorylation in B-CLL cells from UGT2B17HI patients. A prominent feature of B-CLL cells is the strong correlation of UGT2B17 expression with the adverse marker ZAP70 encoding a tyrosine kinase that promotes B-CLL cell survival. Their combined high expression levels in the treatment of naïve patients further defined a prognostic group with the highest risk of poor survival. In leukemic cells, UGT2B17 knockout and repression of ZAP70 reduced proliferation, suggesting that the function of UGT2B17 might involve ZAP70. Mechanistically, UGT2B17 interacted with several kinases of the BCR pathway, including ZAP70, SYK, and BTK, revealing a potential therapeutic vulnerability. The dual SYK and JAK/STAT6 inhibitor cerdulatinib most effectively compromised the proliferative advantage conferred by UGT2B17 compared to the selective BTK inhibitor ibrutinib. Findings point to an oncogenic role for UGT2B17 as a novel constituent of BCR signalosome also connected with microenvironmental signaling.

UGT2B28 accelerates prostate cancer progression through stabilization of the endocytic adaptor protein HIP1 regulating AR and EGFR pathways.

The androgen inactivating UGT2B28 pathway emerges as a predictor of progression in prostate cancer (PCa). However, the clinical significance of UGT2B28 tumoral expression and its contribution to PCa progression remain unclear. Using the Canadian Prostate Cancer Biomarker Network biobank (CPCBN; n = 1512), we analyzed UGT2B28 tumor expression in relation to clinical outcomes in men with localized PCa. UGT2B28 was overexpressed in tumors compared to paired normal adjacent prostatic tissue and was associated with inferior outcomes. Functional analyses indicated that UGT2B28 promoted cell proliferation, and its expression was regulated by the androgen receptor (AR)/ARv7. Mechanistically, UGT2B28 was shown to be a protein partner of the endocytic adaptor protein huntingtin-interacting protein 1 (HIP1), increasing its stability and priming AR/epidermal growth factor receptor (EGFR) pathways, leading to ERK1/2 activation triggering cell proliferation and epithelial-to-mesenchymal transition (EMT). HIP1 knockdown in UGT2B28 positive cells, and dual pharmacological targeting of AR and EGFR pathways, abolished cell proliferative advantages conferred by UGT2B28. In conclusion, UGT2B28 is a prognosticator of progression in localized PCa, regulates both AR and EGFR oncogenic signaling pathways via HIP1, and therefore can be therapeutically targeted by using combination of existing AR/EGFR inhibitors.

Preoperative Circulating 11-Oxygenated Androgens Are Associated With Metastasis-free Survival in Localized Prostate Cancer.

PURPOSE: Adrenal 11-oxygenated androgens may support cancer progression in men with prostate cancer owing to their abundance and androgenic potential. We hypothesized that preoperative circulating levels of 11-oxygenated androgens influence clinical outcomes in men with newly diagnosed localized prostate cancer. MATERIALS AND METHODS: We studied 1,793 treatment-naïve patients and 155 patients who received preoperative treatment with 5α-reductase inhibitors in the prospective PROCURE cohort, for which preoperative plasma samples were obtained prior to radical prostatectomy. Adrenal 11-oxygenated precursors, potent 11-oxygenated androgens and their metabolites (n=7), were quantified using liquid chromatography-tandem mass spectrometry. Circulating levels were evaluated in relation to prognostic factors, disease-free survival, and metastasis-free survival using multivariable Cox proportional hazards models. RESULTS: At a median follow-up of 93.8 months after surgery, 583 patients experienced biochemical recurrence, 104 developed metastatic disease, and 168 deceased. Higher levels of 11-hydroxytestosterone and 11-ketotestosterone were observed in men with PSA >20 ng/mL and positive nodal status (P < .05). In multivariable analyses, no significant association between 11-oxygenated androgens and disease-free survival was observed. Adrenal 11ß-hydroxyandrostenedione, the predominant androgenic 11-ketotestosterone, and its metabolite 11-ketoandrosterone, modeled as quartiles, were associated with metastasis-free survival (P = .06, P = .03, and P = .008, respectively). Significant accumulation of 11-oxygenated androgen precursors and bioactive androgens, but reduced metabolite levels, was observed in patients on 5α-reductase inhibitors (P < .001). CONCLUSIONS: Preoperative circulating 11-oxygenated androgen levels are associated with metastasis-free survival in men with localized prostate cancer undergoing radical prostatectomy and are affected by 5α-reductase inhibitor treatment.

Extensive metabolic consequences of human glycosyltransferase gene knockouts in prostate cancer.

BACKGROUND: Naturally occurring germline gene deletions (KO) represent a unique setting to interrogate gene functions. Complete deletions and differential expression of the human glycosyltransferase UGT2B17 and UGT2B28 genes are linked to prostate cancer (PCa) risk and progression, leukaemia, autoimmune and other diseases. METHODS: The systemic metabolic consequences of UGT deficiencies were examined using untargeted and targeted mass spectrometry-based metabolomics profiling of carefully matched, treatment-naive PCa cases. RESULTS: Each UGT KO differentially affected over 5% of the 1545 measured metabolites, with divergent metabolic perturbations influencing the same pathways. Several of the perturbed metabolites are known to promote PCa growth, invasion and metastasis, including steroids, ceramides and kynurenine. In UGT2B17 KO, reduced levels of inactive steroid-glucuronides were compensated by sulfated derivatives that constitute circulating steroid reservoirs. UGT2B28 KO presented remarkably lower levels of oxylipins paralleled by reduced inflammatory mediators, but higher ceramides unveiled as substrates of the enzyme in PCa cells. CONCLUSION: The distinctive and broad metabolic rewiring caused by UGT KO reinforces the need to examine their unique and divergent functions in PCa biology.

The Glycosyltransferase Pathway: An Integrated Analysis of the Cell Metabolome.

Nucleotide sugar-dependent glycosyltransferases (UGTs) are critical to the homeostasis of endogenous metabolites and the detoxification of xenobiotics. Their impact on the cell metabolome remains unknown. Cellular metabolic changes resulting from human UGT expression were profiled by untargeted metabolomics. The abundant UGT1A1 and UGT2B7 were studied as UGT prototypes along with their alternative (alt.) splicing-derived isoforms displaying structural differences. Nineteen biochemical routes were modified, beyond known UGT substrates. Significant variations in glycolysis and pyrimidine pathways, and precursors of the co-substrate UDP-glucuronic acid were observed. Bioactive lipids such as arachidonic acid and endocannabinoids were highly enriched by up to 13.3-fold (p < 0.01) in cells expressing the canonical enzymes. Alt. UGT2B7 induced drastic and unique metabolic perturbations, including higher glucose (18-fold) levels and tricarboxylic acid cycle (TCA) cycle metabolites and abrogated the effects of the UGT2B7 canonical enzyme when co-expressed. UGT1A1 proteins promoted the accumulation of branched-chain amino acids (BCAA) and TCA metabolites upstream of the mitochondrial oxoglutarate dehydrogenase complex (OGDC). Alt. UGT1A1 exacerbated these changes, likely through its interaction with the OGDC component oxoglutarate dehydrogenase-like (OGDHL). This study expands the breadth of biochemical pathways associated with UGT expression and establishes extensive connectivity between UGT enzymes, alt. proteins and other metabolic processes.

Extensive Alteration of Androgen Precursor Levels After Castration in Prostate Cancer Patients and Their Association With Active Androgen Level.

PURPOSE: Dihydrotestosterone and testosterone are thought to be major contributors of prostate cancer progression and resistance. We studied the modulation of 15 circulating steroids by castration and their association with dihydrotestosterone and testosterone levels. MATERIALS METHODS: A total of 116 serum samples were collected from 99 prostate cancer patients and categorized as eugonadal, castration-sensitive prostate cancer, castration-resistant prostate cancer, or castration-resistant prostate cancer under abiraterone acetate. Serum levels of 15 steroids were measured using mass spectrometry and compared between groups using analysis of variance. Intrapatient association of steroid levels and the androgens testosterone and dihydrotestosterone were assessed using Pearson correlation and linear regression. RESULTS: Testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androsterone, androstenediol, estrone, estrone-sulfate, estradiol, and androsterone/3α-diol-3/3α-diol-17-glucuronide levels were significantly decreased in castration-sensitive prostate cancer (castrated) compared to eugonadal patients. Testosterone levels were strongly associated with multiple steroids under eugonadal conditions, whereas they were weakly affected by precursor steroids in castrated patients. By contrast, dihydrotestosterone levels under androgen deprivation therapy were associated with testosterone and the backdoor pathway metabolite androsterone. In castration-resistant prostate cancer patients, levels of androstenedione were significantly associated with testosterone level, while testosterone was the only steroid associated with dihydrotestosterone levels. CONCLUSIONS: Androgen deprivation therapy significantly reduces the levels of 13 circulating steroids. Upon androgen deprivation therapy initiation, the backdoor pathway metabolite androsterone are strongly associated with dihydrotestosterone levels. Under castration-resistant prostate cancer conditions, androstenedione was significantly associated with testosterone levels, suggesting the presence of tumor-related circulating androgens in these patients. These results provide further rationale to intensify treatments with androgen receptor axis signaling pathway inhibitors in patients with prostate cancer.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

A liquid chromatography-mass spectrometry assay for the quantification of nucleotide sugars in human plasma and urine specimens and its clinical application.

Nucleotide sugars and more specifically UDP-sugars, represent a major source of energy, key components of extracellular matrix, glycosylation and glucuronidation reactions, and emerge as important signaling molecules through P2Y14 purinergic receptor. Despite their pivotal role in a variety of physiological and pathological processes and their potential as biomarkers, UDP-sugar composition of biological fluids remains poorly studied. We developed a liquid chromatography electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode for the simultaneous quantification of UDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine in human blood and urine. Relative to existing methods, UDP-sugar recovery was enhanced with perchloric acid and ammonium formate during sample preparation that also significantly improved chromatographic stability. Performance of the assay was validated and allowed the absolute quantification of UDP-sugars with a wide dynamic range (0.1 to 200 ng/mL) using stable deuterated isotopes as internal standards. We report a fast (13 min run) and sensitive method (limit of detection: 10-30 pg/mL; lower limit of quantification ≤ 0.2 ng/ml) to simultaneously quantify five UDP-sugars in a low volume (100 µL) of plasma and urine. Findings identified sex-specific profiles in both plasma and urine of healthy subjects. Applicability was also successfully demonstrated in specimens collected from individuals displaying a variety of medical conditions. This validated method was optimized for a high-throughput assessment of UDP-sugars in specimens of clinical importance and enabled an accurate and reliable absolute quantification of important UDP-sugars in diverse clinical contexts.

Sex steroid modulation of macrophages within the prostate tumor microenvironment.

BACKGROUND: The role of androgens and other sex steroids is known to influence the prognosis and progression of prostate cancer through different disease states. While androgens are generally regarded as immunosuppressive and estrogens as inflammatory, the specific influence of sex steroids on the immune microenvironment of prostate tumors remains incompletely understood. MATERIAL AND METHODS: In this study, we evaluate the link between sex steroids and prostate cancer immune cells, particularly macrophages. Using in vitro and in vivo models, as well as ex vivo culture of patient prostate tissue, we evaluated the influence of androgen, estrogen, and progesterone on immune cells of the prostate microenvironment. RESULTS: In vitro, we observed sex steroids induced indirect changes on prostate cancer cell proliferation via THP-1 derived macrophages, but no clear changes were induced using human monocyte derived macrophages. Comparing immunohistochemistry for immunosuppressive macrophage marker CD163 with concomitant circulating sex steroids from the same patients, we observed a correlation with higher dehydroepiandrosterone (DHEA)-sulfate and estrone-sulfate levels associated with higher prostate CD163 expression. Similar relationships between DHEA and CD163 levels were observed in ex vivo cultured prostate biopsies. Finally, in a murine prostate cancer model of long-term sex steroids we observed significant differences in tumor growth in mice implanted with estrogen and DHEA diffusion tubes. CONCLUSIONS: Our results highlight the complex influence of sex steroids on the immune cell composition of prostate tumors. Understanding this biology may help to further personalized therapy and improve patient outcomes.

Variability in testosterone measurement between radioimmunoassay (RIA), chemiluminescence assay (CLIA) and liquid chromatography-tandem mass spectrometry (MS) among prostate cancer patients on androgen deprivation therapy (ADT).

INTRODUCTION & AIMS: Monitoring testosterone (T) levels is recommended to assess the effectiveness of androgen deprivation therapy (ADT) in advanced prostate cancer. T levels below 20 ng/dL have been associated with better outcomes. Three main measures for T exist including radioimmunoassay (RIA), chemiluminescence assay (CLIA) and mass spectrometry (MS). While CLIA and RIA are ubiquitous, MS is regarded as the reference standard. We set out to determine the discordance of T measurements amongst men on ADT. METHODS: A retrospective review of men with prostate cancer on ADT for ≥3 months was conducted. Serum samples were split in triplicate. Observational data was reported and T measurements were compared analyzing for variability looking for categorical concordance. Over and under-estimation rates were calculated. RESULTS: Ninety-five patients were included with a mean age of 70 (50-92) years. Mean ADT duration was 24.1 (3-144) months. Ninety-five percent of patients had T ≤20ng/dL by MS and CLIA as compared to only 80% by RIA. After subdividing into T categories of ≤20, 20 to 50 and ≥50 ng/dL concordance analysis showed that 4.3% and 18.9% of T measured by MS would have a different category result when remeasured by CLIA (Kappa 0.84) or RIA (Kappa 0.50) respectively. CLIA and RIA overestimated T in 66.7% of patients with T <20 ng/dL measured by MS. Conversely CLIA and RIA underestimated T in only 4.4% of cases with T >20 ng/dL measured by MS. CONCLUSIONS: There is significant variability in T measured with RIA, CLIA and MS. CLIA and RIA overestimated T levels in majority of patients leaving a concern of misdiagnosing truly castrate patients as being inadequately treated.

Circulating estradiol and its biologically active metabolites in endometriosis and in relation to pain symptoms.

Objectives: Endometriosis (EM) is an estrogen-dominant inflammatory disease linked to infertility that affects women of reproductive age. EM lesions respond to hormonal signals that regulate uterine tissue growth and trigger inflammation and pain. The objective of this study was to evaluate whether estradiol (E2) and its biologically active metabolites are differentially associated with EM given their estrogenic and non-estrogenic actions including proliferative and inflammatory properties. Design: We performed a retrospective study of 209 EM cases and 115 women without EM. Methods: Pain-related outcomes were assessed using surveys with validated scales. Preoperative serum levels of estradiol (E2) and estrone (E1), their 2-, 4- and 16- hydroxylated (OH) and methylated (MeO) derivatives (n=16) were measured by mass spectrometry. We evaluated the associations between estrogen levels and EM anatomic sites, surgical stage, risk of EM, and symptoms reported by women. Spearman correlations established the relationships between circulating steroids. Results: Of the sixteen estrogens profiled, eleven were detected above quantification limits in most individuals. Steroids were positively correlated, except 2-hydroxy 3MeO-E1 (2OH-3MeO-E1). Higher 2OH-3MeO-E1 was linked to an increased risk of EM (Odd ratio (OR)=1.91 (95%CI 1.09-3.34); P=0.025). Ovarian EM cases displayed enhanced 2-hydroxylation with higher 2MeO-E1 and 2OH-E1 levels (P< 0.009). Abdominal, pelvic and back pain symptoms were also linked to higher 2OH-3MeO-E1 levels (OR=1.86; 95%CI 1.06-3.27; P=0.032). Conclusions: The 2-hydroxylation pathway emerges as an unfavorable feature of EM, and is associated with ovarian EM and pain related outcomes.