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.

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.

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.

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.

A Rare UGT2B7 Variant Creates a Novel N-Glycosylation Site at Codon 121 with Impaired Enzyme Activity.

The UDP glucuronosyltransferase (UGT) superfamily comprises glycoproteins that reside in the endoplasmic reticulum membranes and that undergo post-translational modifications (PTMs). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and examine only a small fraction of the genetic diversity; however, rare variants (frequency <1%) might have a significant effect because they are predicted to greatly outnumber common variants in the human genome. We discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine instead of an aspartic acid (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin in human embryonic kidney (HEK293) cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease of 49% and 40% in the formation of zidovudine and mycophenolic acid glucuronides, respectively. A systematic survey of the Short Genetic Variations database uncovered 32 rare, naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway resulting from changes in PTMs, such as N-linked glycosylation with consequences on drug metabolism.

ABCC5 and ABCG1 polymorphisms predict irinotecan-induced severe toxicity in metastatic colorectal cancer patients.

OBJECTIVE: Irinotecan is a cytotoxic agent used widely for the treatment of solid tumors, particularly for metastatic colorectal cancers. Treatment with this drug frequently results in severe neutropenia and diarrhea that can markedly impact the course of treatment and patients' quality of life. Pharmacogenomic tailoring of irinotecan-based chemotherapy has been the subject of several investigations, but with limited data on ATP-binding cassette (ABC) and solute carrier (SLC) transporter genes. MATERIALS AND METHODS: In this study, we aimed to discover toxicity-associated markers in seven transporter genes participating in irinotecan pharmacokinetics involving the ABC transporter genes ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, and ABCG2 and the solute carrier organic anion transporter gene SLCO1B1 and using a haplotype-tagging single-nucleotide polymorphisms (n=210 htSNPs) strategy. The profiles of 167 metastatic colorectal cancer Canadian patients treated with FOLFIRI-based regimens were examined and the findings were replicated in an independent cohort of 250 Italian patients. RESULTS: In combined cohorts, a two-marker ABCC5 rs3749438 and rs10937158 haplotype (T-C) predicted lower risk of severe diarrhea [odds ratio (OR) of 0.43; P=0.001]. The co-occurrence of ABCG1 rs225440T and ABCC5 rs2292997A predicted the risk of severe neutropenia (OR=5.93; P=0.0002), which was further improved when incorporating the well-known risk marker UGT1A1*28 rs8175347 (OR=7.68; P<0.0001). In contrast, carriers of one protective marker (UGT1 rs11563250G) but none of these risk alleles experienced significantly less severe neutropenia (8.2 vs. 34.0%; P<0.0001). CONCLUSION: This combination of predictive genetic markers could potentially lead to better risk assessment and may thus improve personalized treatment.

Quantitative profiling of human renal UDP-glucuronosyltransferases and glucuronidation activity: a comparison of normal and tumoral kidney tissues.

Renal metabolism by UDP-glucuronosyltransferase (UGT) enzymes is central to the clearance of many drugs. However, significant discrepancies about the relative abundance and activity of individual UGT enzymes in the normal kidney prevail among reports, whereas glucuronidation in tumoral kidney has not been examined. In this study, we performed an extensive profiling of glucuronidation metabolism in normal (n = 12) and tumor (n = 14) kidneys using targeted mass spectrometry quantification of human UGTs. We then correlated UGT protein concentrations with mRNA levels assessed by quantitative polymerase chain reaction and with conjugation activity for the major renal UGTs. Beyond the wide interindividual variability in expression levels observed among kidney samples, UGT1A9, UGT2B7, and UGT1A6 are the most abundant renal UGTs in both normal and tumoral tissues based on protein quantification. In normal kidney tissues, only UGT1A9 protein levels correlated with mRNA levels, whereas UGT1A6, UGT1A9, and UGT2B7 quantification correlated significantly with their mRNA levels in tumor kidneys. Data support that posttranscriptional regulation of UGT2B7 and UGT1A6 expression is modulating glucuronidation in the kidney. Importantly, our study reveals a significant decreased glucuronidation capacity of neoplastic kidneys versus normal kidneys that is paralleled by drastically reduced UGT1A9 and UGT2B7 mRNA and protein expression. UGT2B7 activity is the most repressed in tumors relative to normal tissues, with a 96-fold decrease in zidovudine metabolism, whereas propofol and sorafenib glucuronidation is decreased by 7.6- and 5.2-fold, respectively. Findings demonstrate that renal drug metabolism is predominantly mediated by UGT1A9 and UGT2B7 and is greatly reduced in kidney tumors.

Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential.

Phase II metabolism is prominently governed by UDP-glucuronosyltransferases (UGTs) in humans. These enzymes regulate the bioactivity of many drugs and endogenous small molecules in many organs, including the liver, a major site of regulation by the glucuronidation pathway. This study determined the expression of hepatic UGTs by targeted proteomics in 48 liver samples and by measuring the glucuronidation activity using probe substrates. It demonstrates the sensitivity and accuracy of nano-ultra-performance liquid chromatography with tandem mass spectrometry to establish the complex expression profiles of 14 hepatic UGTs in a single analysis. UGT2B7 is the most abundant UGT in our collection of livers, expressed at 69 pmol/mg microsomal proteins, whereas UGT1A1, UGT1A4, UGT2B4, and UGT2B15 are similarly abundant, averaging 30-34 pmol/mg proteins. The average relative abundance of these five UGTs represents 81% of the measured hepatic UGTs. Our data further highlight the strong relationships in the expression of several UGTs. Most notably, UGT1A4 correlates with most measured UGTs, and the expression levels of UGT2B4/UGT2B7 displayed the strongest correlation. However, significant interindividual variability is observed for all UGTs, both at the level of enzyme concentrations and activity (coefficient of variation: 45%-184%). The reliability of targeted proteomics quantification is supported by the high correlation between UGT concentration and activity. Collectively, these findings expand our understanding of hepatic UGT profiles by establishing absolute hepatic concentrations of 14 UGTs and further suggest coregulated expression between most abundant hepatic UGTs. Data support the value of multiplexed targeted quantitative proteomics to accurately assess specific UGT concentrations in liver samples and hepatic glucuronidation potential.

The relative protein abundance of UGT1A alternative splice variants as a key determinant of glucuronidation activity in vitro.

Alternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene, which represents one of the best examples of a drug-metabolizing gene regulated by AS. Shorter UGT1A isoforms [isoform 2 (i2)] are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be a determinant of the global transferase activity of cells and tissues. Here we tested whether i2-mediated inhibition increases with greater abundance of the i2 protein relative to the isoform 1 (i1) enzyme, using the extrahepatic UGT1A7 as a model and a series of 23 human embryonic kidney 293 clonal cell lines expressing variable contents of i1 and i2 proteins. Upon normalization for i1, a significant reduction of 7-ethyl-10-hydroxycamptothecin glucuronide formation was observed for i1+i2 clones (mean of 53%) compared with the reference i1 cell line. In these clones, the i2 protein content varied greatly (38-263% relative to i1) and revealed two groups: 17 clones with i2 < i1 (60% ± 3%) and 6 clones with i2 ≥ i1 (153% ± 24%). The inhibition induced by i2 was more substantial for clones displaying i2 ≥ i1 (74.5%; P = 0.001) compared with those with i2 < i1 (45.5%). Coimmunoprecipitation supports a more substantial i1-i2 complex formation when i2 exceeds i1. We conclude that the relative abundance of regulatory i2 proteins has the potential to drastically alter the local drug metabolism in the cells, particularly when i2 surpasses the protein content of i1.

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.

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.

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.

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.

Deferiprone glucuronidation by human tissues and recombinant UDP glucuronosyltransferase 1A6: an in vitro investigation of genetic and splice variants.

Tissue iron overload constitutes a major health problem for people who require regular blood transfusions, such as those with beta-thalassemia major. Deferiprone is a hydroxypyridinone iron chelator used therapeutically to remove this excess iron and prevent tissue damage. Deferiprone is metabolized by UDP-glucuronosyltransferases (UGTs) into deferiprone 3-O-glucuronide (DG), but a systematic evaluation of the contribution of individual human UGTs and the impact of genetic variations of UGTs have not been conducted. Sixteen human UGT1A and UGT2B were studied for deferiprone glucuronidation, and their clearances were compared in human tissue samples. DG was measured by liquid chromatography coupled with mass spectrometry. DG was primarily produced in vitro by UGT1A6, and a second glucuronide metabolite was discovered. UGT1A6, as well as liver and kidney human microsomes, had similar kinetic profiles and clearance (Cl(int) = 1.4-3.0 mul/min/mg), but clearance by intestinal microsomes was much lower (0.04 mul/min/mg). Binding of deferiprone to microsomal preparations was not significant. Genetic variants of UGT1A6 had K(m) values similar to the reference protein (UGT1A6*1), but their V(max) values were reduced by 25 to 70%. The UGT1A6 splice variant isoform 2, detected in the liver and kidney, had no transferase activity for deferiprone. When UGT1A6_i2 was coexpressed with the classic UGT1A6_i1 isoform, velocity was reduced for deferiprone but remained similar for 4-nitrophenol or serotonin glucuronidation. In conclusion, deferiprone glucuronidation seems to depend almost totally on UGT1A6, especially in the liver. Genetic variations and differences in the expression of splice variants represent a potential source of variation in deferiprone metabolism.

Inactivation of Prostaglandin E2 as a Mechanism for UGT2B17-Mediated Adverse Effects in Chronic Lymphocytic Leukemia.

High expression of the metabolic enzyme UDP-glucuronosyltransferase UGT2B17 in chronic lymphocytic leukemia (CLL) cells was associated with poor prognosis in two independent studies. However, the underlying mechanism remains unknown. We hypothesized that UGT2B17 impacts intracellular levels of hormone-like signaling molecules involved in the regulation of gene expression in leukemic cells. We initially confirmed in a third cohort of 291 CLL patients that those with high UGT2B17 displayed poor prognosis (hazard ratio of 2.31, P = 0.015). Consistent with the unfavorable prognostic significance of elevated UGT2B17 expression in CLL patients, high UGT2B17 expression was associated with enhanced proliferation of MEC1 and JVM2 malignant B-cell models. Transcriptomic analyses revealed that high UGT2B17 was linked to a significant alteration of genes related to prostaglandin E2 (PGE2) and to its precursor arachidonic acid, both in cell models and a cohort of 448 CLL patients. In functional assays, PGE2 emerged as a negative regulator of apoptosis in CLL patients and proliferation in cells models, whereas its effect was partially abrogated by high UGT2B17 expression in MEC1 and JVM2 cells. Enzymatic assays and mass-spectrometry analyses established that the UGT2B17 enzyme inactivates PGE2 by its conjugation to glucuronic acid (GlcA) leading to the formation of two glucuronide (G) derivatives. High UGT2B17 expression was further associated with a proficient inactivation of PGE2 to PGE2-G in CLL patient cells and cell models. We conclude that UGT2B17-dependent PGE2 glucuronidation impairs anti-oncogenic PGE2 effects in leukemic cells, thereby partially contributing to disease progression in high UGT2B17 CLL patients.

Combination of germline variations associated with survival of folinic acid, fluorouracil and irinotecan-treated metastatic colorectal cancer patients.

Aim: Germline variants could modify survival of metastatic colorectal cancer patients (mCRC). Patients & methods: The association of 285 haplotype-tagging SNPs in 11 candidate genes and overall survival (OS) was tested in two cohorts totalizing 417 FOLFIRI-treated mCRC. Gene expression was investigated in vitro and in public datasets. Results: In the combined cohort, CES1 rs9921399T>C was associated with prolonged OS (hazard ratio [HR] = 0.40) whereas ABCC1 rs17501011G>A (HR = 2.08) and UGT1 rs1113193G>A (HR = 2.12) were associated with shorter OS (p ≤ 0.005). A combined effect of these polymorphisms was observed with HR of 1.98-2.97 (p < 0.05). The ABCC1 rs17501011A variant reduced reporter-gene activity (p < 0.05) whereas ABCC1 tumor expression was associated with shorter survival (p ≤ 0.013). Conclusion: We identified a combination of genetic determinants that could predict mCRC survival.

Germline variability and tumor expression level of ribosomal protein gene RPL28 are associated with survival of metastatic colorectal cancer patients.

This study investigated the potential of single nucleotide polymorphisms as predictors of survival in two cohorts comprising 417 metastatic colorectal cancer (mCRC) patients treated with the FOLFIRI (folinic acid, 5-fluorouracil and irinotecan) regimen. The rs4806668G > T of the ribosomal protein gene RPL28 was associated with shorter progression-free survival and overall survival by 5 and 9 months (P = 0.002), with hazard ratios of 3.36 (P < 0.001) and 3.07 (P = 0.002), respectively. The rs4806668T allele was associated with an increased RPL28 expression in transverse normal colon tissues (n = 246, P = 0.007). RPL28 expression was higher in colorectal tumors compared to paired normal tissues by up to 124% (P < 0.001) in three independent datasets. Metastatic cases with highest RPL28 tumor expression had a reduced survival in two datasets (n = 88, P = 0.009 and n = 56, P = 0.009). High RPL28 was further associated with changes in immunoglobulin and extracellular matrix pathways. Repression of RPL28 reduced proliferation by 1.4-fold to 5.6-fold (P < 0.05) in colon cancer HCT116 and HT-29 cells. Our findings suggest that the ribosomal RPL28 protein may influence mCRC outcome.