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.

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.

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 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.

Urinary oestrogen steroidome as an indicator of the risk of localised prostate cancer progression.

BACKGROUND: Prostate cancer (PCa) is the most common cancer in North American men. Beyond the established contribution of androgens to disease progression, growing evidence suggest that oestrogen-related pathways might also be of clinical importance. The aim of this study was to explore the association of urinary oestrogen levels with clinical outcomes. METHODS: Urine samples from the prospective multi-institutional PROCURE cohort were collected before RP for discovery (n = 259) and validation (n = 253). Urinary total oestrogens (unconjugated + conjugated), including oestrone and oestradiol, their bioactive and inactive catechol and methyl derivatives (n = 15), were measured using mass spectrometry (MS). RESULTS: The median follow-up time for the discovery and replication cohorts was 7.6 and 6.5 years, respectively. Highly significant correlations between urinary oestrogens were observed; however, correlations with circulating oestrogens were modest. Our findings indicate that higher levels of urinary oestriol and 16-ketoestradiol were associated with lower risk of BCR. In contrast, higher levels of 2-methoxyestrone were associated with an increased risk of development of metastasis/deaths. CONCLUSIONS: Our data suggest that urinary levels of oestriol and 16-ketoestradiol metabolites are associated with a more favourable outcome, whereas those of 2-methoxyestrone are associated with an elevated risk of metastasis after RP. Further studies are required to better understand the impact of oestrogens on disease biology and as easily accessible urine-based risk-stratification markers.

Associations Between Prediagnostic Concentrations of Circulating Sex Steroid Hormones and Liver Cancer Among Postmenopausal Women.

BACKGROUND AND AIMS: In almost all countries, incidence rates of liver cancer (LC) are 100%-200% higher in males than in females. However, this difference is predominantly driven by hepatocellular carcinoma (HCC), which accounts for 75% of LC cases. Intrahepatic cholangiocarcinoma (ICC) accounts for 12% of cases and has rates only 30% higher in males. Hormones are hypothesized to underlie observed sex differences. We investigated whether prediagnostic circulating hormone and sex hormone binding globulin (SHBG) levels were associated with LC risk, overall and by histology, by leveraging resources from five prospective cohorts. APPROACH AND RESULTS: Seven sex steroid hormones and SHBG were quantitated using gas chromatography/tandem mass spectrometry and competitive electrochemiluminescence immunoassay, respectively, from baseline serum/plasma samples of 191 postmenopausal female LC cases (HCC, n = 83; ICC, n = 56) and 426 controls, matched on sex, cohort, age, race/ethnicity, and blood collection date. Odds ratios (ORs) and 95% confidence intervals (CIs) for associations between a one-unit increase in log2 hormone value (approximate doubling of circulating concentration) and LC were calculated using multivariable-adjusted conditional logistic regression. A doubling in the concentration of 4-androstenedione (4-dione) was associated with a 50% decreased LC risk (OR = 0.50; 95% CI = 0.30-0.82), whereas SHBG was associated with a 31% increased risk (OR = 1.31; 95% CI = 1.05-1.63). Examining histology, a doubling of estradiol was associated with a 40% increased risk of ICC (OR = 1.40; 95% CI = 1.05-1.89), but not HCC (OR = 1.12; 95% CI = 0.81-1.54). CONCLUSIONS: This study provides evidence that higher levels of 4-dione may be associated with lower, and SHBG with higher, LC risk in women. However, this study does not support the hypothesis that higher estrogen levels decrease LC risk. Indeed, estradiol may be associated with an increased ICC risk.

DNA repair gene polymorphisms, tumor control, and treatment toxicity in prostate cancer patients treated with permanent implant prostate brachytherapy.

BACKGROUND: Radiotherapy and brachytherapy are common treatments for localized prostate cancer (PCa). However, very few studies evaluated the association of variations in DNA damage response genes and treatment outcomes and toxicity in brachytherapy-treated patients. PURPOSE: To evaluate the association of inherited germline variations in DNA repair-associated genes with tumor control and treatment toxicity in patients treated with low-dose-rate prostate brachytherapy (LDRB). MATERIAL AND METHODS: The cohort consists of 475 I-125 LDRB patients with a median follow-up of 51 months after seed implantation. Patients were genotyped for 215 haplotype tagging single nucleotide variations (htSNPs) in 29 candidate genes of DNA damage response and repair pathways. Their association with biochemical recurrence (BCR) was assessed using Cox regression models and Kaplan-Meier survival curves. Linear regressions and analysis of covariance (ANCOVA) between early and late International Prostate Symptom Score (IPSS) with htSNPs were used to evaluate the association with urinary toxicity. RESULTS: After adjustment for the established risk factors, six htSNPs in five genes were found to be significantly associated with an altered risk of BCR, with adjusted hazard ratios (HRadj. ) ranging between 3.6 and 11.1 (P < .05). Compared to carriers of the ERCC3 rs4150499C allele, patients homozygous for the T allele (n = 22) had a significant higher risk of BCR with a HR of 11.13 (IC95 = 3.9-32.0; P < .0001; q < 0.001). The Kaplan-Meier survival curve revealed a mean BCR-free survival time reduced from 213 ± 7 to 99 ± 12 months (log-rank P < .0001) for homozygous T carriers compare to noncarriers. For late IPSS (>6 months after treatment), htSNP rs6544990 from MSH2 showed a statistically significant b-coefficient of 1.85 ± 0.52 (P < .001; q < 0.1). Homozygous carriers of the MSH2 rs6544990C allele (n = 62) had a mean late IPSS 3.6 points higher than patients homozygous for the A allele (n = 132). This difference was significant when tested by ANCOVA using pretreatment IPSS as a covariate (P < .01). CONCLUSIONS: This study suggests an association of the intronic variants of the DNA nucleotide excision repair ERCC3 and DNA mismatch repair MSH2 genes with elevated risk of BCR and late urinary toxicity respectively after LDRB. Further validation is required before translational clinical advances.

Emerging roles for UDP-glucuronosyltransferases in drug resistance and cancer progression.

The best-known role of UDP-glucuronosyltransferase enzymes (UGTs) in cancer is the metabolic inactivation of drug therapies. By conjugating glucuronic acid to lipophilic drugs, UGTs impair the biological activity and enhance the water solubility of these agents, driving their elimination. Multiple clinical observations support an expanding role for UGTs as modulators of the drug response and in mediating drug resistance in numerous cancer types. However, accumulating evidence also suggests an influence of the UGT pathway on cancer progression. Dysregulation of the expression and activity of UGTs has been associated with the progression of several cancers, arguing for UGTs as possible mediators of oncogenic pathways and/or disease accelerators in a drug-naive context. The consequences of altered UGT activity on tumour biology are incompletely understood. They might be associated with perturbed levels of bioactive endogenous metabolites such as steroids and bioactive lipids that are inactivated by UGTs or through non-enzymatic mechanisms, thereby eliciting oncogenic signalling cascades. This review highlights the evidence supporting dual roles for the UGT pathway, affecting cancer progression and drug resistance. Pharmacogenomic testing of UGT profiles in patients and the development of therapeutic options that impair UGT actions could provide useful prognostic and predictive biomarkers and enhance the efficacy of anti-cancer drugs.

UGT2B17 modifies drug response in chronic lymphocytic leukaemia.

BACKGROUND: High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents. METHODS: Functional enzymatic assays and patients' plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels. RESULTS: High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub. CONCLUSIONS: Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.

Alternative promoters control UGT2B17-dependent androgen catabolism in prostate cancer and its influence on progression.

BACKGROUND: Perturbation of the major UGT2B17-dependent androgen catabolism pathway has the potential to affect prostate cancer (PCa) progression. The objective was to evaluate UGT2B17 protein expression in primary tumours in relation to hormone levels, disease characteristics and cancer evolution. METHODS: We conducted an analysis of a high-density prostate tumour tissue microarray consisting of 239 localised PCa cases treated by radical prostatectomy (RP). Cox proportional hazard ratio analysis was used to evaluate biochemical recurrence (BCR), and a linear regression model evaluated variations in circulating hormone levels measured by mass spectrometry. The transcriptome of UGT2B17 in PCa was established by using RNA-sequencing data. RESULTS: UGT2B17 expression in primary tumours was associated with node-positive disease at RP and linked to circulating levels of 3α-diol-17 glucuronide, a major circulating DHT metabolite produced by the UGT2B17 pathway. UGT2B17 was an independent prognostic factor linked to BCR after RP, and its overexpression was associated with development of metastasis. Finally, we demonstrated that distinctive alternative promoters dictate UGT2B17-dependent androgen catabolism in localised and metastatic PCa. CONCLUSIONS: The androgen-inactivating gene UGT2B17 is controlled by overlooked regulatory regions in PCa. UGT2B17 expression in primary tumours influences the steroidome, and is associated with relevant clinical outcomes, such as BCR and metastasis.

Endogenous estradiol and inflammation biomarkers: potential interacting mechanisms of obesity-related disease.

PURPOSE: Disentangling the effects of endogenous estrogens and inflammation on obesity-related diseases requires a clearer understanding of how the two biological mechanisms relate to each other. METHODS: We studied 155 healthy postmenopausal women not taking menopausal hormone therapy enrolled in the Prostate Lung Colorectal and Ovarian (PLCO) screening cancer trial. From a baseline blood draw, we measured endogenous estradiol and 69 inflammation biomarkers: cytokines, chemokines, adipokines, angiogenic factors, growth factors, acute phase proteins, and soluble receptors. We evaluated the estradiol-inflammation relationship by assessing associations across different models (linear, ordinal logistic, and binary logistic) using a variety of estradiol classifications. We additionally investigated the estradiol-inflammation relationship stratified by baseline obesity status (BMI < 30 stratum and BMI > 30 stratum). RESULTS: Associations of estradiol with 7 inflammation biomarkers met p < 0.05 statistical significance in linear and ordinal models: C-reactive protein (CRP), adiponectin, chemokine (C-X-C motif) ligand-6, thymus activation-regulated chemokine, eosinophil chemotactic protein, plasminogen activator inhibitor-1, and serum amyloid A. The positive association between estradiol and CRP was robust to model changes. Each standard deviation increase in endogenous estradiol doubled a woman's odds of having CRP levels higher than the study median (odds ratio 2.29; 95% confidence interval 1.28, 4.09). Estradiol was consistently inversely associated with adiponectin. Other estradiol-inflammation biomarker associations were not robust to model changes. CONCLUSIONS: Endogenous estradiol appears to be associated with CRP and adiponectin; the evidence is limited for other inflammation biomarkers.

Extragonadal Steroids Contribute Significantly to Androgen Receptor Activity and Development of Castration Resistance in Recurrent Prostate Cancer after Primary Therapy.

PURPOSE: Beyond testosterone, several steroids contribute to the activation of the androgen receptor pathway, but their relative contributions to the activation of the androgen receptor signaling axis in patients with castrated prostate cancer remain unknown. MATERIALS AND METHODS: Serum levels of 9 steroids were measured by mass spectrometry from continuously castrated patients of the PR.7 study (219) and from the PCA24 cohort (116). For each steroid standard curves for dose dependent prostate specific antigen promoter activation were built in castration sensitive (LAPC4) and resistant (VCaP) prostate cancer models. Standard curves were used to determine the androgen receptor activation potency for each steroid measurement from patients in these trials. RESULTS: In LAPC4 and VCaP cells testosterone, dihydrotestosterone and androstenedione induced androgen receptor transcriptional activity, while dehydroepiandrosterone, 5alpha-androstan-3beta,17beta-diol, androstenediol and androsterone stimulated androgen receptor only in VCaP cells. Extragonadal steroids were responsible for 34% (LAPC4) and 88% (VCaP) of the serum total androgen receptor transcriptional activity found in castrated cases. The total androgen receptor transcriptional activity secondary to testosterone, dihydrotestosterone and androstenedione was associated with time to castration resistance in patients from the PR.7 study (HR 2.17, 95% CI 1.12-4.23, p=0.02) in multivariate analysis using the castration sensitive model (LAPC4). Androgen receptor transcriptional activity of extragonadal androstenedione was the only steroid statistically associated with time to castration resistance in univariate analysis (HR 1.89, 95% CI 1.04-3.44, p=0.036). CONCLUSIONS: Extragonadal steroids contribute significantly to the androgen receptor axis activation at castration levels of testosterone in recurrent nonmetastatic prostate cancer and these sustain the development of castration resistance after primary local treatment.

Glucuronidation of Abiraterone and Its Pharmacologically Active Metabolites by UGT1A4, Influence of Polymorphic Variants and Their Potential as Inhibitors of Steroid Glucuronidation.

Abiraterone (Abi) acetate (AA) is a prodrug of Abi, a CYP17A1 inhibitor used to treat patients with advanced prostate cancer. Abi is a selective steroidal inhibitor that blocks the biosynthesis of androgens. It undergoes extensive biotransformation by steroid pathways, leading to the formation of pharmacologically active Δ4-Abi (D4A) and 5α-Abi. This study aimed to characterize the glucuronidation pathway of Abi and its two active metabolites. We show that Abi, its metabolites, and another steroidal inhibitor galeterone (Gal) undergo secondary metabolism to form glucuronides (G) in human liver microsomes with minor formation by intestine and kidney microsomal preparations. The potential clinical relevance of this pathway is supported by the detection by liquid chromatography-tandem mass spectrometry of Abi-G, D4A-G, and 5α-Abi-G in patients under AA therapy. A screening of UGT enzymes reveals that UGT1A4 is the main enzyme involved. This is supported by inhibition experiments using a selective UGT1A4 inhibitor hecogenin. A number of common and rare nonsynonymous variants significantly abrogate the UGT1A4-mediated formation of Abi-G, D4A-G, and 5α-Abi-G in vitro. We also identify Gal, Abi, and its metabolites as highly potent inhibitors of steroid inactivation by the UGT pathway with submicromolar inhibitor constant values. They reduce the glucuronidation of both the adrenal precursors and potent androgens in human liver, prostate cancer cells, and by recombinant UGTs involved in their inactivation. In conclusion, tested CYP17A1 inhibitors are metabolized through UGT1A4, and germline variations affecting this metabolic pathway may also influence drug metabolism. SIGNIFICANCE STATEMENT: The antiandrogen abiraterone (Abi) is a selective steroidal inhibitor of the cytochrome P450 17α-hydroxy/17,20-lyase, an enzyme involved in the biosynthesis of androgens. Abi is metabolized to pharmacologically active metabolites by steroidogenic enzymes. We demonstrate that Abi and its metabolites are glucuronidated in the liver and that their glucuronide derivatives are detected at variable levels in circulation of treated prostate cancer patients. UDP-glucuronosyltransferase (UGT)1A4 is the primary enzyme involved, and nonsynonymous germline variations affect this metabolic pathway in vitro, suggesting a potential influence of drug metabolism and action in patients. Their inhibitory effect on drug and steroid glucuronidation raises the possibility that these pharmacological compounds might affect the UGT-associated drug-metabolizing system and pre-receptor control of androgen metabolism in patients.

Factors Affecting Interindividual Variability of Hepatic UGT2B17 Protein Expression Examined Using a Novel Specific Monoclonal Antibody.

Accurate quantification of the metabolic enzyme uridine diphospho-glucuronosyltransferase (UGT) UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers revealed strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification (r = 0.93), and three major expression patterns (absent, low, or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, located in the binding site for the transcription factor forkhead box A1 (FOXA1) of the UGT2B17 promoter. The highest level of expression was observed for individuals carrying at least one rs59678213 A allele. Multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213, reaching 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize the UGT2B17 level in various disease states and establish more precisely the contribution of the UGT2B17 enzyme to drug and hormone metabolism.

Post-transcriptional Regulation of UGT2B10 Hepatic Expression and Activity by Alternative Splicing.

The detoxification enzyme UDP-glucuronosyltransferase UGT2B10 is specialized in the N-linked glucuronidation of many drugs and xenobiotics. Preferred substrates possess tertiary aliphatic amines and heterocyclic amines, such as tobacco carcinogens and several antidepressants and antipsychotics. We hypothesized that alternative splicing (AS) constitutes a means to regulate steady-state levels of UGT2B10 and enzyme activity. We established the transcriptome of UGT2B10 in normal and tumoral tissues of multiple individuals. The highest expression was in the liver, where 10 AS transcripts represented 50% of the UGT2B10 transcriptome in 50 normal livers and 44 hepatocellular carcinomas. One abundant class of transcripts involves a novel exonic sequence and leads to two alternative (alt.) variants with novel in-frame C termini of 10 or 65 amino acids. Their hepatic expression was highly variable among individuals, correlated with canonical transcript levels, and was 3.5-fold higher in tumors. Evidence for their translation in liver tissues was acquired by mass spectrometry. In cell models, they colocalized with the enzyme and influenced the conjugation of amitriptyline and levomedetomidine by repressing or activating the enzyme (40%-70%; P < 0.01) in a cell context-specific manner. A high turnover rate for the alt. proteins, regulated by the proteasome, was observed in contrast to the more stable UGT2B10 enzyme. Moreover, a drug-induced remodeling of UGT2B10 splicing was demonstrated in the HepaRG hepatic cell model, which favored alt. variants expression over the canonical transcript. Our findings support a significant contribution of AS in the regulation of UGT2B10 expression in the liver with an impact on enzyme activity.

Sex-dependent association of circulating sex steroids and pituitary hormones with treatment-free survival in chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is not considered a hormone-regulated cancer although sex is a recognized risk factor with men more frequently diagnosed and developing progressive disease. We hypothesized that variable hormonal exposure may have a sexually dimorphic influence on treatment-free survival (TFS). In 156 CLL cases, we quantitatively profiled 29 circulating steroids (progesterone, adrenal precursors, androgens, estrogens, and catechol estrogens) as well as luteinizing hormone (LH) and follicle-stimulating hormone. Median TFS was shorter for men than that for women (80.7 vs. 135.0 months, P = 0.033). Circulating hormone profiles in CLL patients were significantly different from those of healthy donors. In male CLL cases, higher LH levels were associated with shorter TFS (adjusted hazard ratio (HRadj) 2.11; P = 0.004). In female CLL cases, high levels of the potent androgens testosterone and dihydrotestosterone and the sum of methoxy estrogens were associated with an improved TFS with HRadj values of 0.24 (P = 0.007), 0.54 (P = 0.023), and 0.31 (P = 0.034), respectively. Reduced TFS was observed for women with CLL exhibiting high expression of the steroid-inactivating UGT2B17 enzyme. This study is the first to establish a link between the outcome of CLL patients, sex steroids, and pituitary hormones, revealing a sex-specific hormonal imbalance associated with disease progression.

Cross-Talk between Alternatively Spliced UGT1A Isoforms and Colon Cancer Cell Metabolism.

Alternative splicing at the human glucuronosyltransferase 1 gene locus (UGT1) produces alternate isoforms UGT1A_i2s that control glucuronidation activity through protein-protein interactions. Here, we hypothesized that UGT1A_i2s function as a complex protein network connecting other metabolic pathways with an influence on cancer cell metabolism. This is based on a pathway enrichment analysis of proteomic data that identified several high-confidence candidate interaction proteins of UGT1A_i2 proteins in human tissues-namely, the rate-limiting enzyme of glycolysis pyruvate kinase (PKM), which plays a critical role in cancer cell metabolism and tumor growth. The partnership of UGT1A_i2 and PKM2 was confirmed by coimmunoprecipitation in the HT115 colon cancer cells and was supported by a partial colocalization of these two proteins. In support of a functional role for this partnership, depletion of UGT1A_i2 proteins in HT115 cells enforced the Warburg effect, with a higher glycolytic rate at the expense of mitochondrial respiration, and led to lactate accumulation. Untargeted metabolomics further revealed a significantly altered cellular content of 58 metabolites, including many intermediates derived from the glycolysis and tricarboxylic acid cycle pathways. These metabolic changes were associated with a greater migration potential. The potential relevance of our observations is supported by the down-regulation of UGT1A_i2 mRNA in colon tumors compared with normal tissues. Alternate UGT1A variants may thus be part of the expanding compendium of metabolic pathways involved in cancer biology directly contributing to the oncogenic phenotype of colon cancer cells. Findings uncover new aspects of UGT functions diverging from their transferase activity.

Prediagnostic circulating inflammation markers and endometrial cancer risk in the prostate, lung, colorectal and ovarian cancer (PLCO) screening trial.

Inflammation is proposed to increase risk of developing endometrial cancer, but few prospective epidemiologic studies have investigated the relationship between circulating inflammation markers and endometrial cancer risk. In a nested case-control study within the PLCO Screening Trial we measured serum levels of 64 inflammation-related biomarkers in 284 incident endometrial cancer cases and 284 matched controls. Using multivariable logistic regression inflammation markers were evaluated individually and combined into a cross-validated inflammation score. Of 64 markers, 22 were associated with endometrial cancer risk at p < 0.05 and 17 of 22 markers remained associated after multiple testing corrections. After adjusting for BMI and estradiol, SERPINE1 [quartile(Q)4 vs. Q1 odds ratio (OR) (95% confidence interval (CI)), p trend = 2.43 (0.94-6.29), 0.03] and VEGFA [2.56 (1.52-4.30), 0.0002] were positively associated with endometrial cancer risk, while CCL3 [0.46 (0.27-0.77), 0.01], IL13 [0.55 (0.33-0.93), 0.01], IL21 [0.52 (0.31-0.87), 0.01], IL1B [0.51 (0.30-0.86), 0.01] and IL23 [0.60 (0.35-1.03), 0.02] were inversely associated with risk. We observed large differences in ORs across BMI-inflammation score categories. Endometrial cancer risk was most pronounced among obese women with the highest inflammation score tertile (T) [10.25 (3.56-29.55) vs. normal BMI/T1]. Several inflammation markers were prospectively associated with endometrial cancer, including adipokines, pro- and anti-inflammatory cytokines, angiogenic factors and acute phase proteins. Inverse associations with anti-inflammatory markers (IL13, IL21), other inflammation markers/mediators (CCL3, IL1B, IL23), and a robust positive association between VEGFA and endometrial cancer risk were independent of BMI and estradiol, suggesting that these factors may influence risk through other mechanisms.

Correlation between prostate volume and single nucleotide polymorphisms implicated in the steroid pathway.

OBJECTIVES: A few preliminary studies have suggested a link between some genetics variants and benign prostatic hyperplasia (BPH). Our goal was to study the link between a set of single nucleotide polymorphisms (SNPs) implicated in the steroid pathway and accurate measurement of prostate volume in a cohort of men who underwent radical prostatectomy. METHODS: Clinical and pathological data including prostate weight were obtained from 611 Caucasian patients with small volume, localized prostate cancer treated by radical prostatectomy. Patients were genotyped for 90 SNPs located inside or nearby genes implicated in the steroid pathway (Sequenom iPLEX). Correlation between prostate weight and genotypes from each SNP was studied by analysis of covariance, adjusted on age and tumor stage. A Bonferroni correction was applied, and the SNPs implicated were then incorporated in a multivariable model. RESULTS AND LIMITATIONS: Seven SNPs located in or nearby genes implicated in steroid hormone metabolism were significantly associated with prostate volume: HSD17B2 (rs1119933), ESR2 (rs8006145), SULT2B1 (rs279451), NQO1 (rs2917670), ESR1 (rs1569788), GSTP1 (rs1138272), and CYP19A1 (rs17523880). Significant association was maintained after multivariate analysis for four SNPs, indicating their independent association with prostate volume. The power of the association of each SNP with prostate volume was comparable to the effect of age. The strongest associations were found with variants in ESR1, ESR2, HSD17B2, and CYP19A1 genes, indicating a potential role of the estrogen signaling pathway in genesis of BPH. CONCLUSIONS: Our results are in favor of an implication of estrogen biotransformation and signaling pathways in the pathophysiology of BPH.

Evidences of Biological Functions of Biliverdin Reductase A in the Bovine Epididymis.

Epididymal sperm binding protein 1 (ELSPBP1) is secreted by the epididymal epithelium via epididymosomes and is specifically transferred to dead spermatozoa during epididymal transit. We identified biliverdin reductase A (BLVRA) as a partner of ELSPBP1 by immunoprecipitation followed by tandem mass spectrometry. Pull down assays showed that these two proteins interact in the presence of zinc ions. The BLVRA enzyme is known to convert biliverdin to bilirubin, both of which possess antioxidant activity. Assessment by real-time RT-PCR showed that BLVRA is highly expressed in the caput and the corpus epididymis, but is expressed at lower levels in the testis and the cauda epididymis. It is primarily found in the soluble fraction of the caput epididymal fluid, is barely detectable in the cauda fluid, and is detectable to a lesser extent in the epididymosome fraction of both caput and cauda fluids. Immunocytometry on epididymal sperm showed that BLVRA is found on all sperm recovered from the caput region, whereas it is undetectable on cauda sperm. Biliverdin and bilirubin are found in higher concentrations in the caput epididymal fluid, as measured by mass spectrometry. Lipid peroxidation was limited by 1 µM of biliverdin, but not bilirubin when caput spermatozoa were challenged with 500 µM H2O2. Since immature spermatozoa are a source of reactive oxygen species, BLVRA may be involved in the protection of maturing spermatozoa. It is also plausible that BLVRA is implicated in haemic protein catabolism in the epididymal luminal environment.