MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas
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
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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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 6

TDO2-mediated Trp metabolism stimulates cell growth more significantly in mut-MED12 versus WT-MED12 LM cells.

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TDO2-mediated Trp metabolism stimulates cell growth more significantly i...
(A and B) MyoF or LM cells were treated with Kyn (A) or the AHR-specific antagonist CH223191 (B) at different doses for 48 hours and analyzed with Caspase-Glo 3/7 and CCK-8 assays for cell apoptosis and viability, respectively (n = 5 for each tissue type). (C) LM cells were transfected with AHR siRNA for 72 hours, and the protein levels of AHR and Cyclin D1 were assessed by immunoblot (n = 4). Since Cyclin D1 protein levels variate between different patient samples, the Cyclin D1 blots were exposed at different lengths (indicated by black line) for different subjects to make the difference between AHR siRNA and control siRNA more apparent. See complete unedited blots in the supplemental material. (D and E) MyoF, WT-MED12 LM, and mut-MED12 LM cells were treated with Trp (D) or the selective TDO2 inhibitor 680C91 (E) at different doses for 48 hours and analyzed for apoptosis and viability (n = 5 for each tissue type). (F) WT- and mut-MED12 LM cells were transfected with TDO2 siRNA for 72 hours, and the protein levels of TDO2 and Cyclin D1 were assessed by immunoblot (n = 3–5). Samples for WT-MED12 LM of patient no. 2 (PT#2) were run on a different gel and sliced together (indicated by black line). The full unedited gel images are shown in Supplemental Data. N.D., not detected. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 by 2-way ANOVA with Tukey’s multiple-comparison test (A, B, D, and E) or paired t test (C and F).