Cytosolic glutathione‐S‐transferase A1 (GSTA1) plays a primary role in the metabolic clearance of the aromatase inhibitor, Exemestane

Abstract

Breast Cancer is the second leading cause of cancer death among women in the USA, with estrogen‐receptor positive (ER+) tumors accounting for 75% of breast cancers in postmenopausal women. Endocrine therapy targeting the ER pathway is a major treatment modality for ER+ patients. Exemestane (EXE) is a third generation aromatase inhibitor which blocks the aromatase enzyme in the final step of estrogen biosynthesis. Studies examining the long‐term use of EXE as a chemopreventive agent demonstrated a reduction in breast cancer incidence by more than 65%. EXE represents an improvement in breast cancer therapy, but inter‐individual variability exists in overall patient response and adverse events that may be attributed to variation in EXE metabolism. Preliminary studies have demonstrated that cysteine conjugates of EXE and its active metabolite 17β‐dihydro‐EXE (17β‐DHE) comprise 77% of total EXE metabolites in the urine of subjects taking EXE. The initial step in cysteine conjugate formation is glutathione (GSH) conjugation in a reaction catalyzed by the glutathione‐S‐transferase (GST) family of the enzymes involved in the metabolism of a variety of exogenous and endogenous substances. The goal of the present study was to identify hepatic GSTs active in the metabolism of EXE and 17β‐DHE. In a screening of the Human Protein Atlas, a total of 12 cytosolic and 3 microsomal GSTs were found to be hepatically expressed. Commercially available cytosolic GSTs were purchased, and their activity was verified with a common GST substrate, 1‐chloro‐2.4‐dinitrobenzene (CDNB). Additionally, GSTT1, GSTO1, GSTZ1, GSTA1, GSTM3, and GSTP1 were produced as recombinant histidine‐tagged proteins using an E‐coli expression system and found to be 97–99% pure by silver staining. Microsomal GSTs were cloned with a V5 epitope tag, overexpressed in the HEK293 cell line, and verified by Western Blot analysis using an anti‐V5 antibody. The 12 cytosolic and 3 microsomal GSTs were screened by incubating GSH with EXE or 17β‐DHE (0.125mM) using 0.5 μg of pure recombinant protein or 20 μg of microsomal protein. GS‐conjugates of EXE and DHE were detected using ultra‐performance liquid chromatography combined with tandem mass spectrometry (UPLC‐MS/MS). Results from the individual EXE‐GSH conjugation assays indicate that the cytosolic enzymes GSTA1, GSTM3, GSTP1 and GSTT1 are active against EXE and marginally active against 17β‐DHE, with GSTA1 exhibiting the highest relative activity. The three screened microsomal MGST1, MGST2 and MGST3 all exhibit activity against both EXE and 17β‐DHE, with MGST2 exhibiting the highest activity against 17β‐DHE. These data suggest that both cytosolic and microsomal GSTs are responsible for EXE‐GS and 17β‐DHE‐GS conjugate formation in vitro and that GSH conjugation by GSTs may be an important metabolic pathway contributing to excretion and elimination of EXE in people. Support or Funding Information This work is supported by grants from NIH, National Cancer Institute (grant R01‐ CA164366; to P. Lazarus) and the Health Scien...