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Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells

Oncogene volume 35, pages 2031–2039 (2016)Cite this article

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Abstract

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 (AS) 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 favor 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, upregulation 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 upregulation of PTBP1 and modulation of PKM AS 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.

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Acknowledgements

We thank Andrea D’Ascenzo and Enrico Duranti for help with immunohistochemistry; Dr Chiara Naro for helpful suggestions throughout the study; and Dr Vittoria Pagliarini for critical reading of the manuscript. This work was supported by Association for International Cancer Research (AICR) (grant 12-0150), Associazione Italiana Ricerca sul Cancro (AIRC) (grant 14581) and Fondazione Santa Lucia Ricerca Corrente.

Author contributions

SC, PB and IP performed the experiments and analyzed the data. EP and VF performed IHC experiments and score analysis. GC performed statistical analysis of patients; SC, GDF, GC and CS wrote the manuscript. SC and CS designed the study.

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Authors and Affiliations

  1. Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy

    S Calabretta, I Passacantilli & C Sette

  2. Department of Science Medical/Chirurgic and Translational Medicine, University of Rome La Sapienza, Rome, Italy

    S Calabretta, I Passacantilli, G Capurso & G Delle Fave

  3. Laboratory of Neuroembryology, Fondazione Santa Lucia, Rome, Italy

    P Bielli & C Sette

  4. Department of Clinic and Molecular Medicine, University of Rome La Sapienza, Rome, Italy

    E Pilozzi

  5. Department of Surgery, Philipps-University Marburg, Marburg, Germany

    V Fendrich

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  1. S Calabretta
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Correspondence to G Delle Fave or C Sette.

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Calabretta, S., Bielli, P., Passacantilli, I. et al. Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells. Oncogene 35, 2031–2039 (2016). https://doi.org/10.1038/onc.2015.270

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