Proteome of renal cell line caki-2 with AFMID overexpression and potential biomarker discovery in urine

Aromatic caninurine formamase (AFMID) is an enzyme involved in tryptophan pathway, metabolizing N-formylkynurenine to kynurenine. AFMID had been found significantly down regulated in clear cell renal cell carcinoma (ccRCC) in both tissue and urine samples. Although ccRCC is characterized by a typical Warburg-like phenotype, mitochondrial dysfunction and elevated fat deposition, it is unknown whether AFMID plays a role in tumorigenesis and the development of ccRCC. In present study, AFMID overexpression had inhibitory effects for ccRCC cells, decreasing the rate of cell proliferation. Quantitative proteomics showed that AFMID overexpression altered cellular signaling pathways involved in cell growth, and cellular metabolism pathways, including lipid metabolism and inositol phosphate metabolism. Further urine proteomic analysis indicated that cellular function dysfunction with AFMID overexpression could be reflected in urine. The activity of predicted up-regulators, DDX58, TREX1, TGFB1, SMARCA4, and TNF in ccRCC cells and urine showed opposing change trends. Potential urinary biomarkers were tentatively discovered and further validated using an independent cohort. Protein panel of APOC3, UMOD and LEAP2 achieved an AUC value of 0.862 for the training cohort, and 0.883 for validation cohort. Present study is of significance in terms of highlighting various aspects of pathway changes associated with AFMID enzymes, discovering potential specific biomarkers for potential patient diagnosis and therapeutic targeting.

芳香族犬尿氨酸甲酰胺酶（Aromatic caninurine formamase, AFMID）是一种参与色氨酸代谢通路的酶，可将N-甲酰犬尿氨酸代谢为犬尿氨酸。研究证实，透明细胞肾细胞癌（ccRCC）的组织与尿液样本中，AFMID均呈显著下调表达。尽管ccRCC以典型的瓦伯格样表型、线粒体功能障碍及脂肪沉积增加为特征，但目前尚不明确AFMID是否在ccRCC的发生与发展中发挥作用。本研究发现，AFMID过表达可抑制ccRCC细胞增殖，降低细胞增殖速率。定量蛋白质组学分析显示，AFMID过表达会改变参与细胞生长的细胞信号通路，以及脂质代谢、肌醇磷酸代谢等细胞代谢通路。进一步的尿液蛋白质组学分析表明，AFMID过表达引发的细胞功能异常可在尿液样本中得到反映。预测得到的上调调控因子DDX58、TREX1、转化生长因子β1（TGFB1）、SMARCA4及肿瘤坏死因子（TNF）在ccRCC细胞与尿液中的活性呈现相反的变化趋势。本研究初步筛选得到潜在尿液生物标志物，并通过独立队列完成验证。由载脂蛋白C3（APOC3）、尿调蛋白（UMOD）及肝脏表达抗菌肽2（LEAP2）组成的蛋白标志物组，在训练队列中的曲线下面积（AUC）达0.862，在验证队列中达0.883。本研究阐明了与AFMID相关的多条通路变化特征，为发掘ccRCC的潜在诊断特异性生物标志物及治疗靶点提供了重要依据。