Tryptophan metabolism and its role in fibroid pathogenesis

摘要 在我们对子宫肌瘤中的非编码RNA进行分析的过程中，我们发现了以下基因的高度异常过表达： 纤维瘤中色氨酸2，3-双加氧酶（TDO 2）和吲哚胺2，3-双加氧酶（IDO 1）。我们证实了这一点 通过qRT-PCR和Western印迹分析发现，TDO 2和TDO 3的表达持续升高， IDO 1在我们的组织标本中的过度表达。TDO 2在子宫肌瘤中的表达增加 与IDO 1相比，IDO 2的表达更高，并且几乎未检测到IDO 2的表达。与发病机制有关 TDO 2而不是IDO 1的增加是种族依赖性的， 与白人相比，非洲裔美国人明显更高。此外，TDO 2的表达 在携带MED 12突变的平滑肌瘤中， 导致细胞外基质、炎症和炎症相关基因的增殖和表达降低， 平滑肌细胞（LSMC）球体中的细胞生长。这些酶的异常表达， 子宫肌瘤导致犬尿氨酸（Kyn）水平增加，这是色氨酸降解的代谢副产物， 已知的芳烃受体（AhR）内源性配体。基于这些初步数据，我们假设 以TDO 2显著过表达为特征的Trp代谢失调， 纤维瘤发病机制和通过抑制纠正Trp代谢失调的基础 TDO 2的降低和犬尿氨酸水平的正常化将抑制纤维瘤的生长和进展， 潜在的肿瘤形成。我们从三个方面提出了这一假设。在aim 1中，我们将描述Trp 子宫肌层和纤维瘤肿瘤和外植体中的代谢，并确定其潜在的机制。 使用体外方法在肌瘤中显著过表达TDO 2。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 和细胞增殖。目的3旨在确定TDO 2的药理学抑制剂的效用， 携带shRNA的慢病毒敲低TDO 2对体内小鼠模型中纤维瘤建立和进展的影响 纤维瘤这种新的代谢机制对纤维瘤的发病机制具有重大的转化意义，因为它 开辟了新的治疗方法，旨在纠正纤维瘤中的色氨酸代谢。

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.