Aims Demonstrate the presence of cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT) proteins and mRNAs in isolated human plasma exosomes and evaluate the capacity for exosome‐derived biomarkers to characterize variability in CYP3A4 activity. Methods The presence of CYP and UGT protein and mRNA in exosomes isolated from human plasma and HepaRG cell culture medium was determined by mass spectrometry and reverse transcription–polymerase chain reaction, respectively. The concordance between exosome‐derived CYP3A4 biomarkers and midazolam apparent oral clearance (CL/F) was evaluated in a small proof‐of‐concept study involving six genotyped (CYP3A4 *1/*1 and CYP3A5 *3/*3) Caucasian males. Results Exosomes isolated from human plasma contained peptides and mRNA originating from CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2 J2, 3A4 and 3A5, UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15, and NADPH‐cytochrome P450 reductase. Mean (95% confidence interval) exosome‐derived CYP3A4 protein expression pre‐ and post‐rifampicin dosing was 0.24 (0.2–0.28) and 0.42 (0.21–0.65) ng ml–1 exosome concentrate. Mean (95% confidence interval) exosome CYP3A4 mRNA expression pre‐ and post‐rifampicin dosing was 6.0 (1.1–32.7) and 48.3 (11.3–104) × 10–11 2‐ΔΔCt, respectively. R2 values for correlations of exosome‐derived CYP3A4 protein expression, CYP3A4 mRNA expression, and ex vivo CYP3A4 activity with midazolam CL/F were 0.905, 0.787 and 0.832, respectively. Conclusions Consistent strong concordance was observed between exosome‐derived CYP3A4 biomarkers and midazolam CL/F. The significance of these results is that CYP3A4 is the drug‐metabolizing enzyme of greatest clinical importance and variability in CYP3A4 activity is poorly described by existing precision dosing strategies.

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 posttranscriptional 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 39-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 39-UTRs. This microRNAmediated repression was significantly abrogated by mutating the miR-376c binding site in the 39-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 39-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.

Glucuronidation is an enzymatic process that terminally inactivates steroid hormones, including estrogens and androgens, thereby influencing carcinogenesis in hormone-dependent cancers. While estrogens drive breast carcinogenesis via the estrogen receptor alpha (ERa), androgens play a critical role as prohormones for estrogen biosynthesis and ligands for the androgen receptor (AR). In this study, the expression and regulation of two androgen-inactivating enzymes, the UDPglucuronosyltransferases UGT2B15 and UGT2B17, was assessed in breast cancer. In large clinical cohorts, high UGT2B15 and UGT2B17 levels positively influenced diseasespecific survival in distinct molecular subgroups. Expression of these genes was highest in cases positive for ERa. In cell line models, ERa, AR, and the transcription factor FOXA1 cooperated to increase transcription via tandem binding events at their proximal promoters. ERa activity was dependent on FOXA1, facilitated by AR activation, and potently stimulated by estradiol as well as estrogenic metabolites of 5a-dihydrotestosterone. AR activity was mediated via binding to an estrogen receptor half-site 3 0 to the FOXA1 and ERa-binding sites. Although AR and FOXA1 bound the UGT promoters in ARpositive/ERa-negative breast cancer cell lines, androgen treatment did not influence basal transcription levels. Ex vivo culture of human breast tissue and ERa þ tumors provided evidence for upregulation of UGT2B15 and UGT2B17 by estrogen or androgen treatment. ERa binding was evident at the promoters of these genes in a small cohort of primary tumors and distant metastases. Collectively, these data provide insight into sex steroid receptor-mediated regulation of androgen-inactivating enzymes in ERa þ breast cancer, which may have subtypespecific consequences for disease progression and outcomes.