Mithramycin demonstrates preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with germline variants in genes involved in bile disposition: ABCB4 (multidrug resistance 3) rs2302387 and ABCB11 [bile salt export pump (BSEP)] rs4668115 reduce transporter expression (P , 0.05) and were associated with $grade 3 transaminitis developing 24 hours after the third infusion of mithramycin (25 mcg/kg, 6 hours/infusion, every day Â7, every 28 days; P , 0.0040). A similar relationship was observed in a pediatric cohort. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, sodium/taurocholate cotransporting polypeptide, organic solute transporter a/b) in several cell lines [Huh7, HepaRG, HepaRG BSEP (2/2)] and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P , 0.0001), and mithramycin inhibited chenodeoxycholic acid-and GW4046-induced FXR-galactose-induced gene 4 luciferase reporter activity (P , 0.001). Mithramycin promoted glycochenodeoxycholic acid-induced cytotoxicity in ABCB11 (2/2) cells and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P , 0.01). Mithramycin is a FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low ABCB11 function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses. SIGNIFICANCE STATEMENT The present study characterizes a novel mechanism of druginduced hepatotoxicity in which mithramycin not only alters farnesoid X receptor (FXR) and small heterodimer partner gene expression but also inhibits bile acid binding to FXR, resulting in deregulation of cellular bile homeostasis. Two novel singlenucleotide polymorphisms in bile flow transporters are associated with mithramycin-induced liver function test elevations, and the present results are the rationale for a genotype-directed clinical trial using mithramycin in patients with thoracic malignancies.
Understanding mechanisms of resistance to abiraterone, one of the primary drugs approved for the treatment of castration resistant prostate cancer, remains a priority. The organic anion polypeptide 1B3 (OATP1B3, encoded by SLCO1B3) transporter has been shown to transport androgens into prostate cancer cells. In this study we observed and investigated the mechanism of induction of SLCO1B3 by abiraterone. Prostate cancer cells (22Rv1, LNCaP, and VCAP) were treated with anti-androgens and assessed for SLCO1B3 expression by qPCR analysis. Abiraterone treatment increased SLCO1B3 expression in 22Rv1 cells in vitro and in the 22Rv1 xenograft model in vivo. MicroRNA profiling of abiraterone-treated 22Rv1 cells was performed using a NanoString nCounter miRNA panel followed by miRNA target prediction. TargetScan and miRanda prediction tools identified hsa-miR-579-3p as binding to the 3′-untranslated region (3′UTR) of the SLCO1B3. Using dual luciferase reporter assays, we verified that hsa-miR-579-3p indeed binds to the SLCO1B3 3′UTR and significantly inhibited SLCO1B3 reporter activity. Treatment with abiraterone significantly downregulated hsa-miR-579-3p, indicating its potential role in upregulating SLCO1B3 expression. In this study, we demonstrated a novel miRNA-mediated mechanism of abiraterone-induced SLCO1B3 expression, a transporter that is also responsible for driving androgen deprivation therapy resistance. Understanding mechanisms of abiraterone resistance mediated via differential miRNA expression will assist in the identification of potential miRNA biomarkers of treatment resistance and the development of future therapeutics.
To ensure accuracy of UGT1A1 (TA)n (rs3064744) genotyping for use in pharmacogenomics-based irinotecan dosing, we tested the concordance of several commonly used genotyping technologies. Heuristic genotype groupings and principal component analysis demonstrated concordance for Illumina sequencing, fragment analysis, and fluorescent PCR. However, Illumina sequencing and fragment analysis returned a range of fragment sizes, likely arising due to PCR “slippage”. Direct sequencing was accurate, but this method led to ambiguous electrophoregrams, hampering interpretation of heterozygotes. Gel sizing, pyrosequencing, and array-based technologies were less concordant. Pharmacoscan genotyping was concordant, but it does not ascertain (TA)8 genotypes that are common in African populations. Method-based genotyping differences were also observed in the publication record (p < 0.0046), although fragment analysis and direct sequencing were concordant (p = 0.11). Genotyping errors can have significant consequences in a clinical setting. At the present time, we recommend that all genotyping for this allele be conducted with fluorescent PCR (fPCR).
Mithramycin has shown significant preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with inter-individual variation in genes involved in bile disposition: ABCB4(MDR3) rs2302387 and ABCB11(BSEP) rs4668115 variants reduce transporter expression (P<0.05) and were associated with ≥Grade 3 liver function test (LFT) elevations developing 24 hours after the third infusion of mithramycin (25mcg/kg, 6hr/infusion, qdx7, every 28 days;P<0.0040). A similar relationship was observed in a pediatric cohort genotyped for ABCB11. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, NTCP, OSTα/β) in several cell lines (Huh7, HepaRG, HepaRG BSEP (-/-)) and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P<0.0001), and mithramycin inhibited CDCA- and GW4046-induced FXR-GAL4 luciferase reporter activity (P<0.001). Mithramycin promoted GCDC-induced cytotoxicity in cells lacking the BSEP transporter and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P<0.01). Mithramycin is an FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low BSEP function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses. Citation Format: Tristan M. Sissung, Phoebe A. Huang, Ralph Hauke, Edel McCrea, Cody J. Peer, Roberto H. Barbier, Jonathan D. Strope, Ariel M. Ley, Mary Zhang, Julie A. Hong, David Venzon, Jonathan P. Jackson, Kenneth R. Brouwer, Patrick Grohar, John Glod, Brigitte C. Widemann, Theo Heller, David S. Schrump, William D. Figg. Severe hepatotoxicity of mithramycin therapy caused by altering expression of hepatocellular bile transporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2943.
Background: The testosterone uptake transporter OATP1B3 is expressed de novo in prostate tumors and SLCO1B3 gene (encoding OATP1B3) variants are associated with clinical outcomes in patients with prostate cancer receiving androgen deprivation therapy (ADT) and in castration-resistant disease. These findings support the role of OATP1B3 as a major physiologic contributor to androgen distribution and a contributor to resistance to ADT. We have previously shown CBP/p300-mediated SLCO1B3 expression; however, specific transcriptional regulation of de novo expression remains to be elucidated. We characterized the SLCO1B3 promoter in order to better understand the regulatory mechanisms that govern its expression in prostate cancer. Methods: Functional analysis of the SLCO1B3 promoter was conducted using 5’ deletion mutagenesis. Transcriptional activity of the SLCO1B3 gene was measured using the SLCO1B3 promoter-luciferase reporter plasmids, transient transfections and luciferase reporter assays. Promoter activity was modulated by co-transfection with the p300 expression plasmid or treatment with various p300 inhibitor compounds (chetomin, HATi II, or C646). Results: We detected different transcriptional profiles across multiple prostate cancer cells (22Rv1, LNCaP, PC3) and identified a conserved domain responsible for SLCO1B3 transcriptional activity. Variable transcriptional changes in response to p300 inhibitor treatments were observed in both the conserved domain plasmid and the full-length reporter with the HATi II compound showing a consistent 2 to 3-fold increase in luciferase activity. Putative transcription factor binding sites are being evaluated by site-directed mutagenesis to determine potential regulatory elements involved in transcriptional activity of the gene. Conclusions: Our data suggest that several multi-protein transcription factor complexes assemble at distinct regulatory elements in the SLCO1B3 promoter, driving tissue-specific expression of OATP1B3 in prostate cancer. Understanding the underlying regulatory mechanisms of OATP1B3 expression and the transporter’s role in prostate cancer progression will aid in its development as a potential therapeutic target. Citation Format: Roberto H Barbier, Edel M McCrea, Jonathan D Strope, Phoebe A Huang, Tristan M Sissung, Douglas K Price, Cindy H Chau, William D Figg. Mechanisms governing the transcriptional regulation of the liver-specific transporter OATP1B3 in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A069. doi:10.1158/1535-7163.TARG-19-A069
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