Table_3_RNA m6A Demethylase ALKBH5 Protects Against Pancreatic Ductal Adenocarcinoma via Targeting Regulators of Iron Metabolism.XLSX

Although RNA m6A regulators have been implicated in the tumorigenesis of several different types of tumors, including pancreatic cancer, their clinical relevance and intrinsic regulatory mechanism remain elusive. This study analyzed eight m6A regulators (METTL3, METTL14, WTAP, FTO, ALKBH5, and YTHDF1-3) in pancreatic ductal adenocarcinoma (PDAC) and found that only RNA m6A demethylase ALKBH5 serves as an independent favorable prognostic marker for this tumor. To better understand the molecular mechanism underlying the protective effect conferred by ALKBH5 against pancreatic tumorigenesis, we performed a transcriptome-wide analysis of m6A methylation, gene expression, and alternative splicing (AS) using the MIA PaCa-2 stable cell line with ALKBH5 overexpression. We demonstrated that ALKBH5 overexpression induced a reduction in RNA m6A levels globally. Furthermore, mRNAs encoding ubiquitin ligase FBXL5, and mitochondrial iron importers SLC25A28 and SLC25A37, were identified as substrates of ALKBH5. Mechanistically, the RNA stabilities of FBXL5 and SLC25A28, and the AS of SLC25A37 were affected, which led to their upregulation in pancreatic cancer cell line. Particularly, we observed that downregulation of FBXL5 in tumor samples correlated with shorter survival time of patients. Owing to FBXL5-mediated degradation, ALKBH5 overexpression incurred a significant reduction in iron-regulatory protein IRP2 and the modulator of epithelial-mesenchymal transition (EMT) SNAI1. Notably, ALKBH5 overexpression led to a significant reduction in intracellular iron levels as well as cell migratory and invasive abilities, which could be rescued by knocking down FBXL5. Overall, our results reveal a previously uncharacterized mechanism of ALKBH5 in protecting against PDAC through modulating regulators of iron metabolism and underscore the multifaceted role of m6A in pancreatic cancer.

尽管RNA m6A调控因子已被证实参与多种肿瘤类型（包括胰腺癌）的肿瘤发生，但其临床相关性及其内在调控机制仍扑朔迷离。本研究分析了八种m6A调控因子（METTL3、METTL14、WTAP、FTO、ALKBH5以及YTHDF1-3）在胰腺导管腺癌（PDAC）中的作用，并发现仅RNA m6A去甲基化酶ALKBH5可作为该肿瘤独立的良好预后标志物。为了深入理解ALKBH5对胰腺癌发生所赋予的保护作用的分子机制，我们利用过表达ALKBH5的MIA PaCa-2稳定细胞系，对m6A甲基化、基因表达和选择性剪接（AS）进行了全转录组分析。研究发现，ALKBH5过表达导致RNA m6A水平整体下降。此外，编码泛素连接酶FBXL5、线粒体铁转运蛋白SLC25A28和SLC25A37的mRNA被鉴定为ALKBH5的底物。从机制上讲，FBXL5和SLC25A28的RNA稳定性以及SLC25A37的选择性剪接受到影响，导致其在胰腺癌细胞系中的上调。特别是，我们观察到肿瘤样本中FBXL5的下调与患者生存时间缩短相关。由于FBXL5介导的降解，ALKBH5过表达显著降低了铁调节蛋白IRP2和上皮间质转化（EMT）调节因子SNAI1的水平。值得注意的是，ALKBH5过表达导致细胞内铁水平以及细胞迁移和侵袭能力显著降低，这一效应可以通过敲低FBXL5得到挽救。总体而言，我们的研究结果揭示了ALKBH5在保护胰腺导管腺癌中通过调节铁代谢调控因子所发挥的先前未描述的机制，并突显了m6A在胰腺癌中的多方面作用。