Dong Gui Hu

Estrogen Receptor α, Fos-Related Antigen-2, and c-Jun Coordinately Regulate Human UDP Glucuronosyltransferase 2B15 and 2B17 Expression in Response to 17β-Estradiol in MCF-7 Cells

UDP-glucuronosyltransferase 2B15 and 2B17 expression is up-regulated by 17β-estradiol in MCF-7 breast cancer cells, as assessed by quantitative real-time polymerase chain reaction. Using 5′-deletion mapping and site-directed mutagenesis, we demonstrate that 17β-estradiol activation of UGT2B15 gene transcription is mediated by a 282-base pair fragment positioned -454 to -172 nucleotides from the translation start site. This region contains two putative activator protein-1 (AP-1) elements, one imperfect estrogen response element (ERE), and two consensus ERE half-sites. We propose that these five sites act as an estrogen response unit (ERU), because mutation in any site reduces activation of the UGT2B15 promoter by 17β-estradiol. Despite the presence of two AP-1 elements, the UGT2B15 promoter is not responsive to the AP-1 activator phorbol 12-myristate 13-acetate. Although electrophoretic mobility shift assays (EMSA) indicate that the AP-1 proteins c-Jun and Fos-related antigen 2 (Fra-2) bound to the distal AP-1 site, binding of Jun or Fos family members to the proximal AP-1 site was not detected by EMSA. Chromatin immunoprecipitation assays showed a 17β-estradiol-induced recruitment of estrogen receptor (ER) α, c-Jun, and Fra-2 to the 282-bp ERU. The involvement of these three transcription factors in the stimulation of UGT2B15 gene expression by 17β-estradiol was confirmed by siRNA silencing experiments. Mutagenesis and siRNA experiments indicate that UGT2B17 expression is also regulated by 17β-estradiol via the ERU, which is fully conserved in both promoters. Because UGT2B15 and UGT2B17 inactivate steroid hormones by glucuronidation, the regulation of their genes by 17β-estradiol may maintain steroid hormone homeostasis and prevent excessive estrogen signaling activity.

The regulation of UDP-glucuronosyltransferase genes by tissue-specific and ligand-activated transcription factors

Elucidation of the mechanisms regulating UGT genes is of prime importance if the adverse effects of interactions between drugs primarily eliminated by glucuronidation are to be minimized, and if UGT expression is to be manipulated for therapeutic effect. The factors controlling UGT gene expression in the liver include the liver-enriched transcription factors, HNF-1α and HNF-4α, several members of the nuclear-receptor family (CAR, PXR, FXR, LXR, and PPAR), the arylhydrocarbon receptor, and transcription factors involved in stress responses (Nrf2, Maf). HNF-1α, in concert with the intestine-specific transcription factor, Cdx2, and Sp1 regulate UGT gene expression in the gastrointestinal tract, whereas the genes for the major androgen-glucuronidating enzymes, UGT2B15 and UGT2B17, are upregulated by estrogens in breast cell lines and downregulated by androgens in prostate-derived cells. Despite this knowledge, the complex interactions between these transcription factors and their coregulators has not been determined, and the mechanisms regulating UGT gene expression in organs and tissues, other than the liver, gastrointestinal tract, breast, and prostate, remain to be elucidated.

Transcriptional regulation of human UDP-glucuronosyltransferase genes

Abstract Glucuronidation is an important metabolic pathway for many small endogenous and exogenous lipophilic compounds, including bilirubin, steroid hormones, bile acids, carcinogens and therapeutic drugs. Glucuronidation is primarily catalyzed by the UDP-glucuronosyltransferase (UGT) 1A and two subfamilies, including nine functional UGT1A enzymes (1A1, 1A3–1A10) and 10 functional UGT2 enzymes (2A1, 2A2, 2A3, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28). Most UGTs are expressed in the liver and this expression relates to the major role of hepatic glucuronidation in systemic clearance of toxic lipophilic compounds. Hepatic glucuronidation activity protects the body from chemical insults and governs the therapeutic efficacy of drugs that are inactivated by UGTs. UGT mRNAs have also been detected in over 20 extrahepatic tissues with a unique complement of UGT mRNAs seen in almost every tissue. This extrahepatic glucuronidation activity helps to maintain homeostasis and hence regulates biological activity of endogenous molecules that are primarily inactivated by UGTs. Deciphering the molecular mechanisms underlying tissue-specific UGT expression has been the subject of a large number of studies over the last two decades. These studies have shown that the constitutive and inducible expression of UGTs is primarily regulated by tissue-specific and ligand-activated transcription factors (TFs) via their binding to cis-regulatory elements (CREs) in UGT promoters and enhancers. This review first briefly summarizes published UGT gene transcriptional studies and the experimental models and tools utilized in these studies, and then describes in detail the TFs and their respective CREs that have been identified in the promoters and/or enhancers of individual UGT genes.

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.

Identification of androgen receptor splice variant transcripts in breast cancer cell lines and human tissues.

The androgen receptor (AR) is widely expressed in human tissues and has biological function in many male and female organs. In particular, the AR plays a critical role in the biology and pathology of the prostate gland. AR activity inhibits breast growth and has pleiotropic actions in breast cancer that are subtype-dependent. Expression of AR splice variants (ARVs) and their role in prostate carcinogenesis has been elucidated in recent studies. We hypothesised that ARVs are also expressed in breast cancers and other hormone sensitive tissues. Herein, the expression of five previously identified ARV transcripts with documented transcriptional capacity (AR-V1, -V3, -V4, -V7, and -V9) was examined in 6 breast (MFM223, MDA-MB-453, MDA-MB-231, ZR75.1, MCF-7, T47D), two prostate (VCaP, LNCaP), and one liver (HepG2) cancer cell lines, a human embryonic kidney cell line (HEK293), and a panel of RNAs representing 21 different human tissues. Four ARVs (V1, V3, V7, V9) were detected to some degree in almost all cell lines and tissues. In addition, four novel ARVs containing a cryptic exon 9 (CE9) were detected in MDA-MB-453 and VCaP cells. Sequencing of ARV amplicons revealed a single nucleotide substitution within CE3 in lung and placental tissue samples that could be translated as an Ile (ATT)>Val (GTT) substitution in the AR-V7 variant protein. Collectively, these data provides insight into the potential complexity of AR transcriptional splicing events in breast cancer cell lines and diverse human tissues, thereby establishing a rationale for further exploration of ARVs in breast cancer and other human pathologies.

A novel polymorphism in a forkhead box A1 (FOXA1) binding site of the human UDP glucuronosyltransferase 2B17 gene modulates promoter activity and is associated with altered levels of circulating androstane-3α,17β-diol glucuronide.

UDP glucuronosyltransferase 2B17 is present in the prostate, where it catalyzes the addition of glucuronic acid to testosterone and dihydrotestosterone and their metabolites androsterone and androstane-3α,17β-diol. Hence, changes in UGT2B17 gene expression may affect the capacity of the prostate to inactivate and eliminate male sex hormones. In this work, we identify a prevalent polymorphism, −155G/A, in the proximal promoter of the UGT2B17 gene. This polymorphism modulates UGT2B17 promoter activity, because luciferase-gene reporter constructs containing the −155A allele were 13-fold more active than those containing the −155G allele in prostate cancer LNCaP cells. The −155G/A polymorphism is contained within a putative binding site for the transcription factor Forkhead Box A1 (FOXA1). Using gene reporter, electromobility shift, and chromatin immunoprecipitation analyses, we show that FOXA1 binds to this site and stimulates the UGT2B17 promoter. Furthermore, down-regulation of FOXA1 in LNCaP cells substantially reduces UGT2B17 mRNA levels. The binding of FOXA1 and subsequent stimulation of the UGT2B17 promoter is greatly reduced in the presence of the −155G allele compared with the −155A allele. Consonant with its capacity to be stimulated by FOXA1, the UGT2B17 −155A allele, compared with the −155G allele, is associated with higher levels of circulating androstane-3α,17β-diol glucuronide. Although the initial phases of prostate cancer are androgen-dependent and UGT2B17 inactivates androgens, there was no association of the UGT2B17 −155G/A polymorphism with prostate cancer risk. In summary, this work identifies FOXA1 as an important regulator of UGT2B17 expression in prostate cancer LNCaP cells and identifies a polymorphism that alters this regulation.

Regulation of Human UGT2B15 and UGT2B17 by miR-376c in Prostate Cancer Cell Lines

Given the prime importance of UDP-glucuronosyltransferase (UGT) 2B15 and UGT2B17 in inactivating testosterone and dihydrotestosterone, control of their expression and activity in the prostate is essential for androgen signaling homeostasis in this organ. Although several studies provide evidence of transcriptional control of UGT2B15 and UGT2B17 by various endogenous and exogenous compounds, potential post-transcriptional regulation of UGT2B15 and UGT2B17 by microRNAs (miRs) in prostate cancer cells has not been examined. The present study identified a putative miR-376c target site in the 3′-untranslated regions (UTRs) of both UGT2B15 and UGT2B17 mRNAs. In accordance with the possibility that this miRNA negatively regulates UGT2B15 and UGT2B17 expression, there is an inverse correlation in the levels of miR-376c and UGT2B15/UGT2B17 mRNAs in prostate cancer cell lines versus normal prostate tissue. In LNCaP cells, transfection of miR-376c mimics inhibited the glucuronidations of testosterone, 4-methylumbelliferone (a substrate of UGT2B15), and androsterone (a substrate of UGT2B17). miR-376c reduced both UGT2B15 and UGT2B17 mRNA and protein levels and the activity of luciferase reporters containing UGT2B15 or UGT2B17 3′-UTRs. This microRNA-mediated repression was significantly abrogated by mutating the miR-376c binding site in the 3′-UTRs of both UGTs. Collectively, these data indicate that the expression of UGT2B15 and UGT2B17 is negatively regulated by the binding of miR-376c to the 3′-UTRs of UGT2B15 and UGT2B17 in prostate cancer cells. This represents the first evidence for post-transcriptional regulation of UGT2B15 and UGT2B17 by miRNAs in prostate cancer cells and may have importance in regulating androgen receptor signaling.

A novel function for UDP glycosyltransferase 8: galactosidation of bile acids.

The human UDP glycosyltransferase (UGT) superfamily comprises four families of enzymes that catalyze the addition of sugar residues to small lipophilic chemicals. The UGT1 and UGT2 enzymes use UDP-glucuronic acid, and UGT3 enzymes use UDP-N-acetylglucosamine, UDP-glucose, and UDP-xylose to conjugate xenobiotics, including drugs and endobiotics such as metabolic byproducts, hormones, and signaling molecules. This metabolism renders the substrate more polar and more readily excreted from the body and/or functionally inactive. The fourth UGT family, called UGT8, contains only one member that, unlike other UGTs, is considered biosynthetic. UGT8 uses UDP galactose to galactosidate ceramide, a key step in the synthesis of brain sphingolipids. To date other substrates for this UGT have not been identified and there has been no suggestion that UGT8 is involved in metabolism of endo- or xenobiotics. We re-examined the functions of UGT8 and discovered that it efficiently galactosidates bile acids and drug-like bile acid analogs. UGT8 conjugates bile acids ∼60-fold more efficiently than ceramide based on in vitro assays with substrate preference deoxycholic acid > chenodeoxycholic acid > cholic acid > hyodeoxycholic acid > ursodeoxycholic acid. Activities of human and mouse UGT8 are qualitatively similar. UGT8 is expressed at significant levels in kidney and gastrointestinal tract (intestine, colon) where conjugation of bile acids is likely to be metabolically significant. We also investigate the structural determinants of UDP-galactose selectivity. Our novel findings suggest a new role for UGT8 as a modulator of bile acid homeostasis and signaling.

Epirubicin Upregulates UDP Glucuronosyltransferase 2B7 Expression in Liver Cancer Cells via the p53 Pathway

Anthracyclines are effective genotoxic anticancer drugs for treating human malignancies; however, their clinical use is limited by tumor resistance and severe cardiotoxicity (e.g., congestive heart failure). Epirubicin (EPI) is less cardiotoxic compared with other canonical anthracyclines (e.g., doxorubicin). This has been attributed to its unique glucuronidation detoxification pathway. EPI is primarily inactivated by UDP-glucuronosyltransferase 2B7 (UGT2B7) in the liver. Hence, the regulation of hepatic UGT2B7 expression is critical for EPI systemic clearance but remains poorly characterized. We show herein that EPI upregulates UGT2B7 expression in hepatocellular carcinoma (HCC) HepG2 and Huh7 cells. Our analyses of deleted and mutated UGT2B7 promoter constructs identified a p53 response element (p53RE) in the UGT2B7 promoter. EPI stimulated UGT2B7 promoter activity via this p53RE and enhanced in vivo p53 binding at this p53RE in HepG2 cells. Knockdown of p53 expression by small interfering RNA silencing technology significantly repressed the capacity of EPI to stimulate UGT2B7 transcription. Furthermore, the p53 activator nutlin-3α significantly enhanced UGT2B7 expression and recruited the p53 protein to the UGT2B7 p53RE in HepG2 cells. Collectively, our results demonstrated that EPI promotes its own detoxification via the p53-mediated pathway. This regulation may contribute to tumor resistance to EPI-containing HCC chemotherapy and may also provide a new explanation for the reduced cardiotoxicity of EPI compared with other anthracyclines. Our finding also suggests that upon exposure to genotoxic agents, detoxifying genes are activated by the p53-mediated pathway to clear genotoxic agents locally within the tumor site or even systemically through the liver.

Forkhead Box Protein A1 Regulates UDP-Glucuronosyltransferase 2B15 Gene Transcription in LNCaP Prostate Cancer Cells

The UDP-glucuronosyltransferases (UGTs) 2B15 and 2B17 are the major UGTs involved in the inactivation and elimination of the active androgens, dihydrotestosterone and testosterone. Although regulation of these UGT genes by various endogenous and exogenous ligands, including steroid hormones and bile acids, is well documented, the mechanisms controlling their basal gene expression are poorly understood. We recently reported that Forkhead box protein A1 (FOXA1) regulates the basal expression of the UGT2B17 gene in prostate cancer cells. In this study, we show that FOXA1 also regulates basal expression of the UGT2B15 gene in the prostate cell line LNCaP (lymph node carcinoma of the prostate). FOXA1 binds to a site −208 to −217 base pairs relative to the UGT2B15 translation start site, as shown by electromobility shift and chromatin immunoprecipitation assays. Mutation of this site prevents binding and substantially decreases basal UGT2B15 promoter activity. Silencing of FOXA1 expression by small interfering RNA significantly reduced UGT2B15 transcript levels, further confirming a crucial role of FOXA1 in controlling UGT2B15 gene expression. Because local inactivation of active androgens by UGT2B15 and UGT2B17 has been shown to be a major determinant of androgen response and signaling activity, regulation of the UGT2B15 and UGT2B17 genes by FOXA1 may have an important role in the maintenance of androgen homeostasis within prostate cancer cells.