Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 , USA; Watson School of Biological Sciences, Cold Spring Harbor, NY 11724, USA.
Open Biol. 2012 Oct;2(10):120133. doi: 10.1098/rsob.120133.
Alternative splicing of the pyruvate kinase M gene involves a choice between mutually exclusive exons 9 and 10. Use of exon 10 to generate the M2 isoform is crucial for aerobic glycolysis (the Warburg effect) and tumour growth. We previously demonstrated that splicing enhancer elements that activate exon 10 are mainly found in exon 10 itself, and deleting or mutating these elements increases the inclusion of exon 9 in cancer cells. To systematically search for new enhancer elements in exon 10 and develop an effective pharmacological method to force a switch from PK-M2 to PK-M1, we carried out an antisense oligonucleotide (ASO) screen. We found potent ASOs that target a novel enhancer in exon 10 and strongly switch the splicing of endogenous PK-M transcripts to include exon 9. We further show that the ASO-mediated switch in alternative splicing leads to apoptosis in glioblastoma cell lines, and this is caused by the downregulation of PK-M2, and not by the upregulation of PK-M1. These data highlight the potential of ASO-mediated inhibition of PK-M2 splicing as therapy for cancer.
丙酮酸激酶 M 基因的可变剪接涉及到外显子 9 和 10 之间的相互排斥选择。使用外显子 10 生成 M2 同工酶对于有氧糖酵解（瓦堡效应）和肿瘤生长至关重要。我们之前的研究表明，激活外显子 10 的剪接增强子元件主要存在于外显子 10 本身，删除或突变这些元件会增加癌细胞中外显子 9 的包含。为了系统地在外显子 10 中寻找新的增强子元件，并开发一种有效的药理学方法迫使从 PK-M2 切换到 PK-M1，我们进行了反义寡核苷酸 (ASO) 筛选。我们发现了针对外显子 10 中新型增强子的有效 ASO，可强烈切换内源性 PK-M 转录物的剪接以包含外显子 9。我们进一步表明，ASO 介导的剪接方式改变导致神经胶质瘤细胞系发生细胞凋亡，这是由 PK-M2 的下调引起的，而不是由 PK-M1 的上调引起的。这些数据强调了 ASO 介导的 PK-M2 剪接抑制作为癌症治疗的潜力。
