Alcedo Karel P, Guerrero Andres, Basrur Venkatesha, Fu Dong, Richardson Monea L, McLane Joshua S, Tsou Chih-Chiang, Nesvizhskii Alexey I, Welling Theodore H, Lebrilla Carlito B, Otey Carol A, Kim Hong Jin, Omary M Bishr, Snider Natasha T
Department of Cell Biology and Physiology University of North Carolina at Chapel Hill Chapel Hill NC.
Department of Chemistry University of California Davis Davis CA.
Hepatol Commun. 2019 Aug 9;3(10):1400-1414. doi: 10.1002/hep4.1410. eCollection 2019 Oct.
CD73, a cell-surface -linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other -linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal -linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity ( 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity , similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that -linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.
CD73是一种产生细胞外腺苷的细胞表面连接糖蛋白，是癌症免疫治疗的新靶点。尽管抗CD73抗体已进入临床开发阶段，但根据靶细胞和肿瘤类型的不同，CD73具有促肿瘤和抗肿瘤两种功能。本研究的目的是描述人肝细胞癌（HCC）中CD73的调控情况。我们对原发性HCC手术标本进行了分子、生化和细胞分析，并结合HCC细胞的机制研究，检测了CD73的表达、定位和活性。我们分别使用质谱糖组学和RNA测序（RNAseq）分析了CD73聚糖特征以及编码其他连接糖蛋白的转录本的全局变化。CD73在肿瘤肝细胞上表达，在那里它表现出异常的连接糖基化，这与HCC的病因、肿瘤分期或纤维化的存在无关。肿瘤相关CD73的异常糖基化导致5'-核苷酸酶活性降低了3倍（P<0.0001）。从生化角度来看，肿瘤相关CD73在位于C端催化结构域的N311和N333残基上缺乏杂合聚糖和复合聚糖。通过定点诱变阻断N311/N333糖基化产生的CD73，其5'-核苷酸酶活性显著降低，类似于原发性肿瘤。糖基化缺陷的CD73与高尔基体结构蛋白GM130部分共定位，GM130在HCC肿瘤中被强烈诱导。RNAseq分析进一步显示，连接糖蛋白编码基因是HCC肿瘤与相邻组织之间差异表达基因中最大的类别。我们首次详细描述了正常组织和肿瘤组织中CD73的糖基化情况，揭示了一种导致人HCC肿瘤细胞中CD73功能抑制的新机制。本研究结果对于靶向实体瘤中细胞表面CD73的治疗候选抗体以及正在进行HCC临床开发的小分子腺苷受体激动剂具有转化意义。
