Tryptophan metabolism and its role in fibroid pathogenesis

摘要 在我们对肌瘤中非编码RNA进行分析的过程中，我们发现了高度异常的过度表达 肌瘤中的色氨酸2，3双加氧酶(TDO2)和吲哚胺2，3双加氧酶(IDO1)。我们证实了这一点 QRT-PCR和Western印迹分析发现，TDO2和TDO2的表达持续升高 IDO1在我们的组织标本中有更多的变量过表达。TDO2在子宫肌瘤中的表达增加 与IDO1相比有较高的表达，且几乎未检测到IDO2的表达。与发病机制有关 肌瘤的增加是TDO2的增加，而不是IDO1的增加是种族依赖的，增加的是 与高加索人相比，非裔美国人明显更高。此外，TDO2的表达 在携带MED12突变的子宫肌瘤中显著增加，并被药物阻断抑制 在体外导致细胞外基质相关基因的增殖和表达减少，炎症和 肌瘤细胞(LSMC)球体中的细胞生长。这些酶在细胞中的异常表达 肌瘤导致犬尿氨酸(Kyn)水平升高，KYN是色氨酸降解的代谢副产物， 已知的芳香烃受体(AhR)的内源性配体。基于这个初步数据，我们假设 以TDO2显著过度表达为特征的色氨酸代谢失调是 肌瘤发病机制及抑制纠正色氨酸代谢紊乱的基础 TDO2和犬尿氨酸水平的正常化将抑制肌瘤的生长和进展 潜在的肿瘤形成。我们在三个目标中提出了这一假说。在AIM 1中，我们将描述TRP 子宫肌层和子宫肌瘤及外植体的代谢，并确定其机制(S)。 用体外方法研究TDO2在子宫肌瘤中的显著过表达。在目标2中，我们将研究 犬尿氨酸及其对调节细胞外基质(ECM)、炎症的下游基因AhR的激活作用 和细胞增殖。AIM 3旨在确定一种TDO2的药理抑制剂的用途，并与 携带shRNA的慢病毒敲除TDO2对小鼠肌瘤建立和发展的影响 子宫肌瘤。这种新的代谢机制对肌瘤的发病机制具有重大的翻译意义，因为它 为旨在纠正肌瘤中色氨酸代谢的新疗法开辟了道路。

Abstract In the course of our profiling for non-coding RNAs in fibroids we discovered highly aberrant overexpression of Tryptophan 2,3 dioxygenase (TDO2) and Indoleamine 2,3-dioxygenase (IDO1) in fibroids. We confirmed this finding by both qRT-PCR and Western blot analysis, and found a consistently elevated expression of TDO2 and more variable overexpression of IDO1 in our tissue specimens. The increment in TDO2 expression in fibroids was higher as compared to IDO1, and the expression of IDO2 was barely detected. Relevant to the pathogenesis of fibroids was that the increment in TDO2 but not IDO1 was race dependent, with the increment being significantly higher in African Americans as compared with Caucasians. Furthermore, the expression of TDO2 was significantly increased in MED12 mutation bearing leiomyomas, and its inhibition by pharmacologic blockade in vitro led to decreased proliferation and expression of genes related to extracellular matrix, inflammation and cell growth in leiomyoma smooth muscle cells (LSMC) spheroids. Aberrant expression of these enzymes in fibroids resulted in increased levels of kynurenine (Kyn), a metabolic byproduct of tryptophan degradation and a known endogenous ligand for Aryl hydrocarbon receptor (AhR). Based on this preliminary data we hypothesized that dysregulation of Trp metabolism as characterized by marked overexpression of TDO2 is fundamental to the pathogenesis of fibroids and correction of Trp metabolic dysregulation by inhibition of TDO2 and normalization of kynurenine levels will inhibit fibroid growth and progression and potentially tumor establishment. We propose to this hypothesis in 3 aims. In aim1 we will characterize Trp metabolism in myometrium and fibroid tumors and explants, and determine the mechanism(s) underlying the marked overexpression of TDO2 in fibroids using an in vitro approach. In Aim 2 we will examine the impact of kynurenine and its activation of AhR on downstream genes regulating extracellular matrix (ECM), inflammation and cell proliferation. Aim 3 is designed to determine the utility of a pharmacological inhibitor of TDO2 and with lentivirus bearing shRNA to knock down TDO2 on fibroid establishment and progression in in vivo mouse models of fibroids. This novel metabolic mechanism for fibroid pathogenesis has great translational significance as it opens the way for novel therapies aimed at correction of tryptophan metabolism in fibroids.