Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells.
Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells.
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DOI：
10.1038/onc.2015.270
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发表时间：
2016-04-21
期刊：
影响因子：
8
通讯作者：
Sette C
中科院分区：
文献类型：
--
作者：
Calabretta S;Bielli P;Passacantilli I;Pilozzi E;Fendrich V;Capurso G;Fave GD;Sette C
胰腺导管腺癌（PDAC）是一种侵袭性和不可治愈的疾病。预后不良是由于多种原因，包括对一线化疗方法吉西他滨的耐药性的获得。因此，迫切需要更直接靶向这种疾病的分子畸变的新疗法。我们发现，PDAC细胞长期暴露于吉西他滨选择了耐药的细胞亚群（DR-PDAC细胞）。重要的是，丙酮酸激酶基因（PKM）的选择性剪接在DR-PDAC细胞中受到不同的调节，导致癌症相关PKM 2亚型的促进，其高表达也与PDAC患者较短的无复发生存期相关。通过反义寡核苷酸切换PKM剪接以有利于替代PKM 1变体挽救了DR-PDAC细胞对吉西他滨和顺铂的敏感性，表明PKM 2表达是耐受药物诱导的遗传毒性应激所必需的。从机制上讲，上调多嘧啶束结合蛋白（PTBP 1），PKM剪接的关键调节剂，与DR-PDAC细胞系中PKM 2的表达相关。PTBP 1在DR-中比在亲本PDAC细胞中更有效地被募集到PKM前体mRNA。因此，DR-PDAC细胞中PTBP 1的敲低减少了其向PKM前mRNA的募集，促进了PKM 1变体的剪接并消除了耐药性。因此，长期暴露于吉西他滨导致PDAC细胞中PTBP 1的上调和PKM选择性剪接的调节，从而赋予对药物的抗性。这些发现指出PKM 2和PTBP 1是改善PDAC对化疗的反应的新的潜在治疗靶点。
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and incurable disease. Poor prognosis is due to multiple reasons, including acquisition of resistance to gemcitabine, the first line chemotherapeutic approach. Thus, there is a strong need for novel therapies, targeting more directly the molecular aberrations of this disease. We found that chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that are drug-resistant (DR-PDAC cells). Importantly, alternative splicing of the pyruvate kinase gene (PKM) was differentially modulated in DR-PDAC cells, resulting in promotion of the cancer-related PKM2 isoform, whose high expression also correlated with shorter recurrence free survival in PDAC patients. Switching PKM splicing by antisense oligonucleotides to favour the alternative PKM1 variant rescued sensitivity of DR-PDAC cells to gemcitabine and cisplatin, suggesting that PKM2 expression is required to withstand drug-induced genotoxic stress. Mechanistically, up-regulation of the polypyrimidine-tract binding protein (PTBP1), a key modulator of PKM splicing, correlated with PKM2 expression in DR-PDAC cell lines. PTBP1 was recruited more efficiently to PKM pre-mRNA in DR- than in parental PDAC cells. Accordingly, knockdown of PTBP1 in DR-PDAC cells reduced its recruitment to the PKM pre-mRNA, promoted splicing of the PKM1 variant and abolished drug resistance. Thus, chronic exposure to gemcitabine leads to up-regulation of PTBP1 and modulation of PKM alternative splicing in PDAC cells, conferring resistance to the drug. These findings point to PKM2 and PTBP1 as new potential therapeutic targets to improve response of PDAC to chemotherapy.