MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas
Azna Zuberi, … , Serdar E. Bulun, Ping Yin
Azna Zuberi, … , Serdar E. Bulun, Ping Yin
Published August 22, 2023
Citation Information: JCI Insight. 2023;8(18):e171305. https://doi.org/10.1172/jci.insight.171305.
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Research Article Metabolism Reproductive biology

MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas

Abstract

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

Authors

Azna Zuberi, Yongchao Huang, Ariel J. Dotts, Helen Wei, John S. Coon V, Shimeng Liu, Takashi Iizuka, Olivia Wu, Olivia Sotos, Priyanka Saini, Debabrata Chakravarti, Thomas G. Boyer, Yang Dai, Serdar E. Bulun, Ping Yin

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Figure 3

mut-MED12 stimulates TDO2 expression and Kyn production in primary LM cells.

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mut-MED12 stimulates TDO2 expression and Kyn production in primary LM ce...
(A) Bar graph showing normalized RNA-Seq counts of TDO2, IDO1, IDO2, and CYP1B1 in WT-MED12 LM (n = 4), mut-MED12 LM (n = 6), and matched MyoF (n = 10) tissues. The default settings of DESeq2 package were used to normalize the counts. (B) Genome browser track view of ChIP-Seq data showing enrichment of MED12 and H3K4me3 in the TDO2 gene locus in mut- versus WT-MED12 LM (n = 3 of each genotype). (C) qPCR quantification of mRNA levels of TDO2 and IDO1 in WT-MED12 LM (n = 7), mut-MED12 LM (n = 9), and matched MyoF (n = 16). (D and E) Representative western blot images (D) and ImageJ quantification (E) showing higher TDO2 protein levels in mut-MED12 LM (n = 15) than in WT-MED12 LM (n = 13) and matched MyoF (n = 28). Some of the matched tissues for which we had both RNA and protein available were used for qPCR analysis in C. The protein isolated from primary mut-MED12 LM cells transfected with control siRNA or TDO2 siRNA, and from G44N mut-MED12 and the control WT-MED12 cell lines (see ref. 83 for detailed information of the cell lines), was used to show TDO2 band specificity. The images of other samples are shown in Supplemental Figure 3C; see complete unedited blots in the supplemental material. N.D., not detected; L, LM; M, MyoF. (F) Kyn and Trp levels in WT-MED12 LM (n = 8), mut-MED12 LM (n = 24), and matched MyoF (n=32) tissues were quantified by HPLC-MS/MS. Data are normalized to the metabolite levels in MyoF (red dotted line). Scattered dot plots adjacent to the bar graphs show the mean levels in LM in each genotype. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0005, #Padj = 1.83 × 10–10, ##Padj = 1.4 × 10–62 by DESeq2 (A), 2-way ANOVA with Tukey’s multiple-comparison test (C and E), or unpaired t test (F).