Cazet Aurélie, Charest Jonathan, Bennett Daniel C, Sambrooks Cecilia Lopez, Contessa Joseph N
Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut, United States of America.
PLoS One. 2014 Oct 14;9(10):e110345. doi: 10.1371/journal.pone.0110345. eCollection 2014.
Asparagine-linked glycosylation is an endoplasmic reticulum co- and post-translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.
天冬酰胺连接的糖基化是一种内质网共翻译和翻译后修饰，它能使受体酪氨酸激酶（RTK）糖蛋白进行转运并发挥功能。为深入了解糖基化酶对RTK功能的调节作用，我们研究了用短发夹RNA（shRNA）和小干扰RNA（siRNA）敲低磷酸甘露糖异构酶（MPI），该酶是成熟聚糖前体生物合成所必需的。MPI活性丧失降低了U-251和SKMG-3恶性胶质瘤细胞系中FGFR家族受体的磷酸化水平，还导致FRS2、Akt和MAPK信号显著降低。然而，敲低MPI并不影响配体诱导的EGFR或MET RTK的激活或信号传导，这表明FGFRs对MPI抑制更敏感。FGFR信号的降低不是由FGF配体或受体的丧失引起的，而是由受体二聚化的干扰导致的。对敲低MPI的细胞后果的研究表明，由FGFR信号驱动且对恶性胶质瘤临床进展至关重要的细胞程序受到了损害。除了阻断细胞迁移外，敲低MPI还显著降低了电离辐射后胶质瘤细胞的克隆形成存活率。因此，我们的结果表明，对细胞表面受体糖基化所需酶的靶向抑制可被操控，从而对肿瘤细胞表达的关键客户糖蛋白产生离散且有限的影响。此外，这项工作将MPI确定为破坏细胞表面受体依赖性生存信号的潜在酶靶点，以及一种治疗性放射增敏的新方法。
