Activation of Cryptic Donor Splice Sites within the UDP-Glucuronosyltransferase (UGT)1A First-Exon Region Generates Variant Transcripts That Encode UGT1A Proteins with Truncated Aglycone-Binding Domains.

The human UDP-glucuronosyltransferases (UGTs) have crucial roles in metabolizing and clearing numerous small lipophilic compounds. The UGT1A locus generates nine UGT1A mRNAs, 65 spliced transcripts, and 34 circular RNAs. In this study, our analysis of published UGT-RNA capture sequencing (CaptureSeq) datasets identified novel splice junctions that predict 24 variant UGT1A transcripts derived from ligation of exon 2 to unique sequences within the UGT1A first-exon region using cryptic donor splice sites. Of these variants, seven (1A1_n1, 1A3_n3, 1A4_n4, 1A5_n1, 1A8_n2, 1A9_n2, 1A10_n7) are predicted to encode UGT1A proteins with truncated aglycone-binding domains. We assessed their expression profiles and deregulation in cancer using four RNA sequencing (RNA-Seq) datasets of paired normal and cancerous drug-metabolizing tissues from large patient cohorts. Variants were generally coexpressed with their canonical counterparts with a higher relative abundance in tumor than in normal tissues. Variants showed tissue-specific expression with high interindividual variability but overall low abundance. However, 1A8_n2 showed high abundance in normal and cancerous colorectal tissues, with levels that approached or surpassed canonical 1A8 mRNA levels in many samples. We cloned 1A8_n2 and showed expression of the predicted protein (1A8_i3) in human embryonic kidney (HEK)293T cells. Glucuronidation assays with 4-methylumbelliferone (4MU) showed that 1A8_i3 had no activity and was unable to inhibit the activity of 1A8_i1 protein. In summary, the activation of cryptic donor splice sites within the UGT1A first-exon region expands the UGT1A transcriptome and proteome. The 1A8_n2 cryptic donor splice site is highly active in colorectal tissues, representing an important cis-regulatory element that negatively regulates the function of the UGT1A8 gene through pre-mRNA splicing. SIGNIFICANT STATEMENT: The UGT1A locus generates nine canonical mRNAs, 65 alternately spliced transcripts, and 34 different circular RNAs. The present study reports a series of novel UDP-glucuronosyltransferase (UGT)1A variants resulting from use of cryptic donor splice sites in both normal and cancerous tissues, several of which are predicted to encode variant UGT1A proteins with truncated aglycone-binding domains. Of these, 1A8_n2 shows exceptionally high abundance in colorectal tissues, highlighting its potential role in the first-pass metabolism in gut through the glucuronidation pathway.

A Comprehensive Bioinformatic Analysis of RNA-seq Datasets Reveals a Differential and Variable Expression of Wildtype and Variant UGT1A Transcripts in Human Tissues and Their Deregulation in Cancers.

The UGT1A locus generates over 60 different alternatively spliced transcripts and 30 circular RNAs. To date, v2 and v3 transcripts are the only variant UGT1A transcripts that have been functionally characterized. Both v2 and v3 transcripts encode the same inactive variant UGT1A proteins (i2s) that can negatively regulate glucuronidation activity and influence cancer cell metabolism. However, the abundance and interindividual variability in the expression of v2 and v3 transcripts in human tissues and their potential deregulation in cancers have not been comprehensively assessed. To address this knowledge gap, we quantified the expression levels of v1, v2, and v3 transcripts using RNA-seq datasets with large cohorts of normal tissues and paired normal and tumor tissues from patients with six different cancer types (liver, kidney, colon, stomach, esophagus, and bladder cancer). We found that v2 and v3 abundance varied significantly between different tissue types, and that interindividual variation was also high within the same tissue type. Moreover, the ratio of v2 to v3 variants varied between tissues, implying their differential regulation. Our results showed higher v2 abundance in gastrointestinal tissues than liver and kidney tissues, suggesting a more significant negative regulation of glucuronidation by i2 proteins in gastrointestinal tissues than in liver and kidney tissues. We further showed differential deregulation of wildtype (v1) and variant transcripts (v2, v3) in cancers that generally increased the v2/v1 and/or v3/v1 expression ratios in tumors compared to normal tissues, indicating a more significant role of the variants in tumors. Finally, we report ten novel UGT1A transcripts with novel 3' terminal exons, most of which encode variant proteins with a similar structure to UGT1A_i2 proteins. These findings further emphasize the diversity of the UGT1A transcriptome and proteome.

The Somatic Mutation Landscape of UDP-Glycosyltransferase (UGT) Genes in Human Cancers.

The human UDP-glycosyltransferase (UGTs) superfamily has a critical role in the metabolism of anticancer drugs and numerous pro/anti-cancer molecules (e.g., steroids, lipids, fatty acids, bile acids and carcinogens). Recent studies have shown wide and abundant expression of UGT genes in human cancers. However, the extent to which UGT genes acquire somatic mutations within tumors remains to be systematically investigated. In the present study, our comprehensive analysis of the somatic mutation profiles of 10,069 tumors from 33 different TCGA cancer types identified 3427 somatic mutations in UGT genes. Overall, nearly 18% (1802/10,069) of the assessed tumors had mutations in UGT genes with huge variations in mutation frequency across different cancer types, ranging from over 25% in five cancers (COAD, LUAD, LUSC, SKCM and UCSC) to less than 5% in eight cancers (LAML, MESO, PCPG, PAAD, PRAD, TGCT, THYM and UVM). All 22 UGT genes showed somatic mutations in tumors, with UGT2B4, UGT3A1 and UGT3A2 showing the largest number of mutations (289, 307 and 255 mutations, respectively). Nearly 65% (2260/3427) of the mutations were missense, frame-shift and nonsense mutations that have been predicted to code for variant UGT proteins. Furthermore, about 10% (362/3427) of the mutations occurred in non-coding regions (5' UTR, 3' UTR and splice sites) that may be able to alter the efficiency of translation initiation, miRNA regulation or the splicing of UGT transcripts. In conclusion, our data show widespread somatic mutations of UGT genes in human cancers that may affect the capacity of cancer cells to metabolize anticancer drugs and endobiotics that control pro/anti-cancer signaling pathways. This highlights their potential utility as biomarkers for predicting therapeutic efficacy and clinical outcomes.

Regulation of human UDP-glycosyltransferase (UGT) genes by miRNAs.

The human UGT gene superfamily is divided into four subfamilies (UGT1, UGT2, UGT3 and UGT8) that encodes 22 functional enzymes. UGTs are critical for the metabolism and clearance of numerous endogenous and exogenous compounds, including steroid hormones, bile acids, bilirubin, fatty acids, carcinogens, and therapeutic drugs. Therefore, the expression and activities of UGTs are tightly regulated by multiple processes at the transcriptional, post-transcriptional and post-translational levels. During recent years, nearly twenty studies have investigated the post-transcriptional regulation of UGT genes by miRNAs using human cancer cell lines (predominantly liver cancer). Overall, 14 of the 22 UGT mRNAs (1A1, 1A3, 1A4, 1A6, 1A8, 1A9, 1A10, 2A1, 2B4, 2B7, 2B10, 2B15, 2B17, UGT8) have been shown to be regulated by various miRNAs through binding to their respective 3' untranslated regions (3'UTRs). Three 3'UTRs (UGT1A, UGT2B7 and UGT2B15) contain the largest number of functional miRNA target sites; in particular, the UGT1A 3'UTR contains binding sites for 12 miRNAs (548d-5p, 183-5p, 214-5p, 486-3p, 200a-3p, 491-3p, 141-3p, 298, 103b, 376b-3p, 21-3p, 1286). Although all nine UGT1A family members have the same 3'UTR, these miRNA target sites appear to be functional in an isoform-specific and cellular context-dependent manner. Collectively, these observations demonstrate that miRNAs represent important post-transcriptional regulators of the UGT gene superfamily. In this article, we present a comprehensive review of reported UGT/miRNA regulation studies, describe polymorphisms within functional miRNA target sites that may affect their functionalities, and discuss potential cooperative and competitive regulation of UGT mRNAs by miRNAs through adjacently located miRNA target sites.

The Expression Profiles and Deregulation of UDP-Glycosyltransferase (UGT) Genes in Human Cancers and Their Association with Clinical Outcomes.

The human UDP-glycosyltransferase (UGTs) superfamily has 22 functional enzymes that play a critical role in the metabolism of small lipophilic compounds, including carcinogens, drugs, steroids, lipids, fatty acids, and bile acids. The expression profiles of UGT genes in human cancers and their impact on cancer patient survival remains to be systematically investigated. In the present study, a comprehensive analysis of the RNAseq and clinical datasets of 9514 patients from 33 different TCGA (the Genome Cancer Atlas) cancers demonstrated cancer-specific UGT expression profiles with high interindividual variability among and within individual cancers. Notably, cancers derived from drug metabolizing tissues (liver, kidney, gut, pancreas) expressed the largest number of UGT genes (COAD, KIRC, KIRP, LIHC, PAAD); six UGT genes (1A6, 1A9, 1A10, 2A3, 2B7, UGT8) showed high expression in five or more different cancers. Kaplan-Meier plots and logrank tests revealed that six UGT genes were significantly associated with increased overall survival (OS) rates [UGT1A1 (LUSC), UGT1A6 (ACC), UGT1A7 (ACC), UGT2A3 (KIRC), UGT2B15 (BLCA, SKCM)] or decreased OS rates [UGT2B15 (LGG), UGT8 (UVM)] in specific cancers. Finally, differential expression analysis of 611 patients from 12 TCGA cancers identified 16 UGT genes (1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2A3, 2B4, 2B7, 2B11, 2B15, 3A1, 3A2, UGT8) that were up/downregulated in at least one cancer relative to normal tissues. In conclusion, our data show widespread expression of UGT genes in cancers, highlighting the capacity for intratumoural drug metabolism through the UGT conjugation pathway. The data also suggests the potentials for specific UGT genes to serve as prognostic biomarkers or therapeutic targets in cancers.

Divergence of Intracellular Trafficking of Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 3 in MCF-7 Breast Cancer Cells and MCF-7-Derived Stem Cell-Enriched Mammospheres.

Breast cancer MCF-7 cell-line-derived mammospheres were shown to be enriched in cells with a CD44+/CD24- surface profile, consistent with breast cancer stem cells (BCSC). These BCSC were previously reported to express key sphingolipid signaling effectors, including pro-oncogenic sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1P3). In this study, we explored intracellular trafficking and localization of SphK1 and S1P3 in parental MCF-7 cells, and MCF-7 derived BCSC-enriched mammospheres treated with growth- or apoptosis-stimulating agents. Intracellular trafficking and localization were assessed using confocal microscopy and cell fractionation, while CD44+/CD24- marker status was confirmed by flow cytometry. Mammospheres expressed significantly higher levels of S1P3 compared to parental MCF-7 cells (p < 0.01). Growth-promoting agents (S1P and estrogen) induced SphK1 and S1P3 translocation from cytoplasm to nuclei, which may facilitate the involvement of SphK1 and S1P3 in gene regulation. In contrast, pro-apoptotic cytokine tumor necrosis factor α (TNFα)-treated MCF-7 cells demonstrated increased apoptosis and no nuclear localization of SphK1 and S1P3, suggesting that TNFα can inhibit nuclear translocation of SphK1 and S1P3. TNFα inhibited mammosphere formation and induced S1P3 internalization and degradation. No nuclear translocation of S1P3 was detected in TNFα-stimulated mammospheres. Notably, SphK1 and S1P3 expression and localization were highly heterogenous in mammospheres, suggesting the potential for a large variety of responses. The findings provide further insights into the understanding of sphingolipid signaling and intracellular trafficking in BCs. Our data indicates that the inhibition of SphK1 and S1P3 nuclear translocation represents a novel method to prevent BCSCs proliferation.

Circular RNAs of UDP-Glycosyltransferase (UGT) Genes Expand the Complexity and Diversity of the UGT Transcriptome.

The human UDP-glycosyltransferase (UGT) gene superfamily generates 22 canonical transcripts coding for functional enzymes and also produces nearly 150 variant UGT transcripts through alternative splicing and intergenic splicing. In the present study, our analysis of circRNA databases identified backsplicing events that predicted 85 circRNAs from UGT genes, with 33, 11, and 19 circRNAs from UGT1A, UGT2B4, UGT8, respectively. Most of these UGT circRNAs were reported by one database and had low abundance in cell- or tissue-specific contexts. Using reverse-transcriptase polymerase chain reaction with divergent primers and cDNA samples from human tissues and cell lines, we found 13 circRNAs from four UGT genes: UGT1A (three), UGT2B7 (one), UGT2B10 (one), and UGT8 (eight). Notably, all eight UGT8 circRNAs contain open reading frames that include the canonical start AUG codon and encode variant proteins that all have the common 274-amino acidN-terminal region of wild-type UGT8 protein. We further showed that one UGT8 circRNA (circ_UGT8-1) was broadly expressed in human tissues and cell lines, resistant to RNase R digestion, and predominately present in the cytoplasm. We cloned five UGT8 circRNAs into the Zinc finger with KRAB and SCAN domains 1 vector and transfected them into HEK293T cells. All these vectors produced both circRNAsand linear transcripts with varying circular/linear ratios (0.17-1.14).Western blotting and mass spectrometry assays revealed that only linear transcripts and not circRNAs were translated. In conclusion, our findings of nearly 100 circRNAs greatly expand the complexity and diversity of the UGT transcriptome; however, UGT circRNAs are expressed at a very low level in specific cellular contexts, and their biologic functions remain to be determined. SIGNIFICANCE STATEMENT: The human UGT gene transcriptome comprises 22 canonical transcripts coding for functional enzymes and approximately 150 alternatively spliced and chimeric variant transcripts. The present study identified nearly 100 circRNAs from UGT genes, thus greatly expanding the complexity and diversity of the UGT transcriptome. UGT circRNAs were expressed broadly in human tissues and cell lines; however, most showed very low abundance in tissue- and cell-specific contexts, and therefore their biological functions remain to be investigated.

The Expression Profiles of ADME Genes in Human Cancers and Their Associations with Clinical Outcomes.

ADME genes are a group of genes that are involved in drug absorption, distribution, metabolism, and excretion (ADME). The expression profiles of ADME genes within tumours is proposed to impact on cancer patient survival; however, this has not been systematically examined. In this study, our comprehensive analyses of pan-cancer datasets from the Cancer Genome Atlas (TCGA) revealed differential intratumoral expression profiles for ADME genes in 21 different cancer types. Most genes also showed high interindividual variability within cancer-specific patient cohorts. Using Kaplan-Meier plots and logrank tests, we showed that intratumoral expression levels of twenty of the thirty-two core ADME genes were associated with overall survival (OS) in these cancers. Of these genes, five showed significant association with unfavourable OS in three cancers, including SKCM (ABCC2, GSTP1), KIRC (CYP2D6, CYP2E1), PAAD (UGT2B7); sixteen showed significant associations with favourable OS in twelve cancers, including BLCA (UGT2B15), BRCA (CYP2D6), COAD (NAT1), HNSC (ABCB1), KIRC (ABCG2, CYP3A4, SLC22A2, SLC22A6), KIRP (SLC22A2), LIHC (CYP2C19, CYP2C8, CYP2C9, CYP3A5, SLC22A1), LUAD (SLC15A2), LUSC (UGT1A1), PAAD (ABCB1), SARC (ABCB1), and SKCM (ABCB1, DYPD). Overall, these data provide compelling evidence supporting ADME genes as prognostic biomarkers and potential therapeutic targets. We propose that intratumoral expression of ADME genes may impact cancer patient survival by multiple mechanisms that can include metabolizing/transporting anticancer drugs, activating anticancer drugs, and metabolizing/transporting a variety of endogenous molecules involved in metabolically fuelling cancer cells and/or controlling pro-growth signalling pathways.

The UGTome: The expanding diversity of UDP glycosyltransferases and its impact on small molecule metabolism.

The UDP glycosyltransferase (UGT) superfamily of enzymes is responsible for the metabolism and clearance of thousands of lipophilic chemicals including drugs, toxins and endogenous signaling molecules. They provide a protective interface between the organism and its chemical-rich environment, as well as controlling critical signaling pathways to maintain healthy tissue function. UGTs are associated with drug responses and interactions, as well as a wide range of diseases including cancer. The human genome contains 22 UGT genes; however as befitting their exceptionally diverse substrate ranges and biological activities, the output of these UGT genes is functionally diversified by multiple processes including alternative splicing, post-translational modification, homo- and hetero-oligomerization, and interactions with other proteins. All UGT genes are subject to extensive alternative splicing generating variant/truncated UGT proteins with altered functions including the capacity to dominantly modulate/inhibit cognate full-length forms. Heterotypic oligomerization of different UGTs can alter kinetic properties relative to monotypic complexes, and potentially produce novel substrate specificities. Moreover, the recently profiled interactions of UGTs with non-UGT proteins may facilitate coordination between different metabolic processes, as well as providing opportunities for UGTs to engage in novel 'moonlighting' functions. Herein we provide a detailed and comprehensive review of all known modes of UGT functional diversification and propose a UGTome model to describe the resulting expansion of metabolic capacity and its potential to modulate drug/xenobiotic responses and cell behaviours in normal and disease contexts.

The UDP-Glycosyltransferase (UGT) Superfamily: New Members, New Functions, and Novel Paradigms.

UDP-glycosyltransferases (UGTs) catalyze the covalent addition of sugars to a broad range of lipophilic molecules. This biotransformation plays a critical role in elimination of a broad range of exogenous chemicals and by-products of endogenous metabolism, and also controls the levels and distribution of many endogenous signaling molecules. In mammals, the superfamily comprises four families: UGT1, UGT2, UGT3, and UGT8. UGT1 and UGT2 enzymes have important roles in pharmacology and toxicology including contributing to interindividual differences in drug disposition as well as to cancer risk. These UGTs are highly expressed in organs of detoxification (e.g., liver, kidney, intestine) and can be induced by pathways that sense demand for detoxification and for modulation of endobiotic signaling molecules. The functions of the UGT3 and UGT8 family enzymes have only been characterized relatively recently; these enzymes show different UDP-sugar preferences to that of UGT1 and UGT2 enzymes, and to date, their contributions to drug metabolism appear to be relatively minor. This review summarizes and provides critical analysis of the current state of research into all four families of UGT enzymes. Key areas discussed include the roles of UGTs in drug metabolism, cancer risk, and regulation of signaling, as well as the transcriptional and posttranscriptional control of UGT expression and function. The latter part of this review provides an in-depth analysis of the known and predicted functions of UGT3 and UGT8 enzymes, focused on their likely roles in modulation of levels of endogenous signaling pathways.

Deregulation of the Genes that Are Involved in Drug Absorption, Distribution, Metabolism, and Excretion in Hepatocellular Carcinoma.

Genes involved in drug absorption, distribution, metabolism, and excretion (ADME) are called ADME genes. Currently, 298 genes that encode phase I and II drug metabolizing enzymes, transporters, and modifiers are designated as ADME genes by the PharmaADME Consortium. ADME genes are highly expressed in the liver and their levels can be influenced by liver diseases such as hepatocellular carcinoma (HCC). In this study, we obtained RNA-sequencing and microRNA (miRNA)-sequencing data from 371 HCC patients via The Cancer Genome Atlas liver hepatocellular carcinoma project and performed ADME gene-targeted differential gene expression analysis and expression correlation analysis. Two hundred thirty-three of the 298 ADME genes (78%) were expressed in HCC. Of these genes, almost one-quarter (58 genes) were significantly downregulated, while only 6% (15) were upregulated in HCC relative to healthy liver. Moreover, one-half (14/28) of the core ADME genes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, NAT1, NAT2, UGT2B7, SLC22A1, SLCO1B1, and SLCO1B3) were downregulated. In addition, about one-half of the core ADME genes were positively correlated with each other and were also positively (AHR, ARNT, HNF4A, PXR, CAR, PPARA, and RXRA) or negatively (PPARD and PPARG) correlated with transcription factors known as ADME modifiers. Finally, we show that most miRNAs known to regulate core ADME genes are upregulated in HCC. Collectively, these data reveal 1) an extensive transcription factor-mediated ADME coexpression network in the liver that efficiently coordinates the metabolism and elimination of endogenous and exogenous compounds; and 2) a widespread deregulation of this network in HCC, most likely due to deregulation of both transcriptional and post-transcriptional (miRNA) pathways.

Intergenic Splicing between Four Adjacent UGT Genes (2B15, 2B29P2, 2B17, 2B29P1) Gives Rise to Variant UGT Proteins That Inhibit Glucuronidation via Protein-Protein Interactions.

Recent studies have investigated alternative splicing profiles of UDP-glucuronosyltransferase (UGT) genes and identified over 130 different alternatively spliced UGT transcripts. Although UGT genes are highly clustered, the formation of chimeric transcripts by intergenic splicing between two or more UGT genes has not yet been reported. This study identified 12 chimeric transcripts (chimeras A-L) containing exons from two or three genes of the four neighboring UGT genes (UGT2B15, UGT2B29P2, UGT2B17, and UGT2B29P1) in human liver and prostate cancer cells. These chimeras typically contain the first five exons of UGT2B15 or UGT2B17 (exons 1-5) spliced to a terminal exon (exon 6) from a downstream UGT gene. Hence they encode truncated UGTs with novel C-terminal peptides. Functional assays of representative chimeric UGT proteins (termed chimeric UGT2B15 and chimeric UGT2B17) showed that they are inactive and can repress the activity of wild-type UGTs. Coimmunoprecipitation assays demonstrated heterotypic interactions between chimeric UGT2B15 (or chimeric UGT2B17) and the UGT2B7 protein. Thus oligomerization of the chimeric UGTs with wild-type UGTs may explain their inhibitory activity. Studies in breast and prostate cancer cells showed that both wild-type and chimeric UGT2B15 and UGT2B17 transcripts are regulated in a similar way at the transcriptional level by sex hormones through their canonical promoters but are differentially regulated at the post-transcriptional level by micro-RNA 376c via their unique 3'-untranslated regions. In conclusion, the formation of chimeric transcripts by intergenic splicing among UGT genes represents a novel mechanism contributing to the diversity of the human UGT transcriptome and proteome. The differential post-transcriptional regulation of wild-type and variant transcripts by micro-RNAs may contribute to their deregulated expression in cancer.

Cooperative Regulation of Intestinal UDP-Glucuronosyltransferases 1A8, -1A9, and 1A10 by CDX2 and HNF4α Is Mediated by a Novel Composite Regulatory Element.

The gastrointestinal tract expresses several UDP-glucuronosyltransferases (UGTs) that act as a first line of defense against dietary toxins and contribute to the metabolism of orally administered drugs. The expression of UGT1A8, UGT1A9, and UGT1A10 in gastrointestinal tissues is known to be at least partly directed by the caudal homeodomain transcription factor, CDX2. We sought to further define the factors involved in regulation of the UGT1A8-1A10 genes and identified a novel composite element located within the proximal promoters of these three genes that binds to both CDX2 and the hepatocyte nuclear factor (HNF) 4α, and mediates synergistic activation by these factors. We also show that HNF4α and CDX2 are required for the expression of these UGT genes in colon cancer cell lines, and show robust correlation of UGT expression with CDX2 and HNF4α levels in normal human colon. Finally, we show that these factors are involved in the differential expression pattern of UGT1A8 and UGT1A10, which are intestinal specific, and that of UGT1A9, which is expressed in both intestine and liver. These studies lead to a model for the developmental patterning of UGT1A8, UGT1A9, and UGT1A10 in hepatic and/or extrahepatic tissues involving discrete regulatory modules that may function (independently and cooperatively) in a context-dependent manner.

Regulation of UDP-Glucuronosyltransferase 2B15 by miR-331-5p in Prostate Cancer Cells Involves Canonical and Noncanonical Target Sites.

UGT2B15 is an important androgen-metabolizing UDP-glucuronosyltransferase (UGT) and the mechanisms controlling its expression are of considerable interest. Recent studies showed that miR-376c regulates UGT2B15 in prostate cancer cells via a canonical target site in the 3' untranslated region (3'UTR). The UGT2B15 3'UTR also contains a canonical miR-331-5p target site; previous work indicated that deleting this site reduced, but did not abolish, the ability of miR-331-5p to repress a luciferase reporter carrying the UGT2B15 3'UTR We report here the discovery and characterization of a second, noncanonical miR-331-5p target site in the UGT2B15 3'UTR miR-331-5p-mediated repression of a UGT2B15 3'UTR-reporter was partly inhibited by mutating either of the two miR-331-5p target sites separately, but completely abolished by mutating the two sites simultaneously, indicating that the two sites act cooperatively. miR-331-5p mimics significantly reduced both UGT2B15 mRNA levels and glucuronidation activity in prostate cancer cells, confirming that the native transcript is a miR-331-5p target. Transfection of either miR-331-5p or miR-376c mimics repressed the activity of the UGT2B15 3'UTR-reporter; however, cotransfection of both microRNAs (miRNAs) further reduced activity, indicating cooperative regulation by these two miRNAs. A significant negative correlation between miR-331 and UGT2B15 mRNA levels was observed in a tissue RNA panel, and analysis of The Cancer Genome Atlas (TCGA) hepatocellular carcinoma data set provided further evidence that miR-331 may play an important role in regulation of UGT2B15 in vivo. There was no significant correlation between miR-331 and UGT2B15 mRNA levels in the TCGA prostate adenocarcinoma cohort, which may reflect the complexity of androgen-mediated regulation in determining UGT2B15 levels in prostate cancer. Finally, we show that miR-331-5p does not regulate UGT2B17, providing the first evidence for a post-transcriptional mechanism that differentially regulates these two important androgen-metabolizing UGTs.

MiR-193b regulates breast cancer cell migration and vasculogenic mimicry by targeting dimethylarginine dimethylaminohydrolase 1.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is responsible for metabolism of an endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), which plays a key role in modulating angiogenesis. In addition to angiogenesis, tumours can establish a vascular network by forming vessel-like structures from tumour cells; a process termed vasculogenic mimicry (VM). Here, we identified over-expression of DDAH1 in aggressive MDA-MB-231, MDA-MB-453 and BT549 breast cancer cell lines when compared to normal mammary epithelial cells. DDAH1 expression was inversely correlated with the microRNA miR-193b. In DDAH1+ MDA-MB-231 cells, ectopic expression of miR-193b reduced DDAH1 expression and the conversion of ADMA to citrulline. In DDAH1- MCF7 cells, inhibition of miR-193b elevated DDAH1 expression. Luciferase reporter assays demonstrated DDAH1 as a direct target of miR-193b. MDA-MB-231 cells organised into tube structures in an in vitro assay of VM, which was significantly inhibited by DDAH1 knockdown or miR-193b expression. Mechanistically, we found miR-193b regulates cell proliferation and migration of MDA-MB-231 cells, whilst DDAH1 knockdown inhibited cell migration. These studies represent the first evidence for DDAH1 expression, regulation and function in breast cancer cells, and highlights that targeting DDAH1 expression and/or enzymatic activity may be a valid option in the treatment of aggressive breast cancers.

Exemestane and Its Active Metabolite 17-Hydroexemestane Induce UDP-Glucuronosyltransferase (UGT) 2B17 Expression in Breast Cancer Cells.

Exemestane (EXE) is an aromatase inhibitor indicated for endocrine therapy of breast cancer in postmenopausal women. The primary active metabolite of EXE, 17-hydroexemestane (17-HE), is inactivated via glucuronidation, mainly by UDP-glucuronosyltransferase 2B17 (UGT2B17). UGT2B17 also has a primary role in inactivation of endogenous androgens testosterone and dihydrotestosterone and may play an important role in regulation of breast and prostate tumor intracrinology. We recently reported that UGT2B17 could be induced by both estrogenic and androgenic ligands in breast cancer cells via binding of the estrogen receptor α (ERα) or the androgen receptor (AR) to a complex regulatory unit in the proximal UGT2B17 promoter. In this study we show that both EXE and 17-HE increase UGT2B17 mRNA levels in breast cancer MCF-7 and MDA-MB-453 cells, and increase glucuronidation of UGT2B17 substrates, including 17-HE and androsterone. Using antagonists of ERα and AR as well as inhibition mediated by small interfering RNA (siRNA) we demonstrate that EXE and 17-HE induce UGT2B17 expression primarily via the AR. This result is consistent with previous reports that 17-HE can act as an AR ligand. In vitro studies suggest that multiple steroid-responsive DNA elements within the proximal promoter are involved in the response to 17-HE-liganded AR. The up-regulation of UGT2B17 by EXE and 17-HE in breast cancer cells might enhance the local metabolism of 17-HE as well as that of endogenous androgens, hence impacting potentially on treatment outcomes.

Regulation of UDP-Glucuronosyltransferases UGT2B4 and UGT2B7 by MicroRNAs in Liver Cancer Cells.

The transcriptional regulation of UDP-glucuronosyltransferases UGT2B4 and UGT2B7 has been well studied using liver cancer cell lines, and post-transcriptional regulation of these two UGTs by microRNA (miRNA/miR) miR-216b-5p was recently reported. This study describes novel miRNA-mediated regulation of UGT2B4 and UGT2B7 in liver cancer cells. Bioinformatic analyses identified a putative miR-3664-3p binding site in the UGT2B7 3'-untranslated region (UTR) and binding sites for both miR-135a-5p and miR-410-3p in the UGT2B4 3'-UTR. These sites were functionally characterized using miRNA mimics and reporter constructs. A miR-3664-3p mimic induced repression of a luciferase reporter carrying the UGT2B7 3'-UTR in liver cancer cell lines; mutation of the miR-3664-3p site abrogated the response of the reporter to the mimic. Similarly, mutation of the miR-135a-5p site or miR-410-3p site in a luciferase reporter bearing UGT2B4 3'-UTR abrogated the ability of miR-135a-5p or miR-410-3p mimics to reduce reporter activity. Transfection of miR-3664-3p mimics in HepG2 liver cancer cells significantly reduced mRNA and protein levels of UGT2B7, and this led to reduced enzymatic activity. Transfection of miR-135a-5p or miR-410-3p mimics significantly decreased UGT2B4 mRNA levels in Huh7 liver cancer cells. The expression levels of miR-410-3p were inversely correlated with UGT2B4 mRNA levels in The Cancer Genome Atlas cohort of liver hepatocellular carcinoma (371 specimens) and a panel of ten normal human tissues. Similarly, there was an inverse correlation between miR-135a and UGT2B4 mRNA levels in a panel of 18 normal human liver tissues. Together, these data suggest that miR-135a and miR-410 control UGT2B4 and that miR-3664 controls UGT2B7 expression in liver cancer and/or normal liver cells.

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE).

Genetic polymorphisms of human UDP-glucuronosyltransferase (UGT) genes and cancer risk.

Identification of genetic polymorphisms that contribute to the risk of developing cancers is important for cancer prevention. The most recent human genome GRCh38/hg38 assembly (2013) reveals thousands of genetic polymorphisms in human uridine diphosphoglucuronosyltransferase (UGT) genes. Among these, a large number of polymorphisms at the UGT1A and UGT2B genes have been shown to modulate UGT gene promoter activity or enzymatic activity. Glucuronidation plays an important role in the metabolism and clearance of endogenous and exogenous carcinogenic compounds, and this reaction is primarily catalyzed by the UGT1A and UGT2B enzymes. Therefore, it has long been hypothesized that UGT polymorphisms that reduce the capacity to glucuronidate carcinogens and other types of cancer-promoting molecules (e.g. sex hormones) are associated with an increased risk of developing cancers. A large number of case-control studies have investigated this hypothesis and these studies identified numerous UGT polymorphisms in UGT1A and UGT2B genes as genetic risk factors for a wide variety of cancers, including bladder, breast, colorectal, endometrial, esophageal, head and neck, liver, lung, prostate, and thyroid. These UGT polymorphisms may be cancer causative polymorphisms, or be linked to as yet undefined causative polymorphisms, either in UGT genes or neighboring genes. This article presents a comprehensive review of these case-control studies, discusses current areas of uncertainty, and highlights future research directions in this field.

Expression of androgen receptor splice variants in clinical breast cancers.

The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer.