C. Sette

Gemcitabine triggers a pro-survival response in pancreatic cancer cells through activation of the MNK2/eIF4E pathway

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive neoplastic disease. Gemcitabine, the currently used chemotherapeutic drug for PDAC, elicits only minor benefits, because of the development of escape pathways leading to chemoresistance. Herein, we aimed at investigating the involvement of the mitogen activating protein kinase interacting kinase (MNK)/eIF4E pathway in the acquired drug resistance of PDAC cells. Screening of a cohort of PDAC patients by immunohistochemistry showed that eIF4E phosphorylation correlated with disease grade, early onset of disease and worse prognosis. In PDAC cell lines, chemotherapeutic drugs induced MNK-dependent phosphorylation of eIF4E. Importantly, pharmacological inhibition of MNK activity synergistically enhanced the cytostatic effect of gemcitabine, by promoting apoptosis. RNA interference (RNAi) experiments indicated that MNK2 is mainly responsible for eIF4E phosphorylation and gemcitabine resistance in PDAC cells. Furthermore, we found that gemcitabine induced the expression of the oncogenic splicing factor SRSF1 and splicing of MNK2b, a splice variant that overrides upstream regulatory pathways and confers increased resistance to the drug. Silencing of SRSF1 by RNAi abolished this splicing event and recapitulated the effects of MNK pharmacological or genetic inhibition on eIF4E phosphorylation and apoptosis in gemcitabine-treated cells. Our results highlight a novel pro-survival pathway triggered by gemcitabine in PDAC cells, which leads to MNK2-dependent phosphorylation of eIF4E, suggesting that the MNK/eIF4E pathway represents an escape route utilized by PDAC cells to withstand chemotherapeutic treatments.

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

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.

Gemcitabine Treatment Causes Deregulation of Epithelial to Mesenchymal Transition Transcription Factors Transcription and Translation in Pancreatic Ductal Adenocarcinoma Cells

Context Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease, characterised by limited response to chemotherapeutic treatment and early metastasis, leading to very poor prognosis. Epithelial to mesenchymal transition (EMT), a process finely regulated both at transcription and splicing level, contributes to PDAC invasion and affects the response to chemotherapeutic drugs. The expression of the EMT transcription factor ZEB1 is inversely related to sensitivity cells to gemcitabine treatment. Notably, ZEB1 encodes multiple splice variants that mainly differ in the 5’ untranslated region (UTR). However, the biological role of these splice variants in EMT and drug resistance is currently unknown. Objective Characterization of the molecular events involved in the acquisition of gemcitabine resistance in PDAC cells. Methods PCR analysis of EMT genes; Western blot analysis of proteins of the mTOR pathway; 7-mGTP cap assay of cap-dependent translation; polysomal-RNPs fractioning for analysis of mRNA translation. Results PDAC cells exposed to gemcitabine for 72 hours up-regulated mesenchymal genes, including ZEB1, which is known to confer chemoresistance. This response is accompanied by inhibition of mTOR pathway and cap-dependent translation, as confirmed by reduced assembly of the translation initiation complex eIF4F. Conversely, cap-independent translation is not impaired by the drug. In this context, ZEB1 splice variants containing different 5’ UTRs are differentially loaded on polysomes, suggesting that expression of specific variants allows ZEB1 translation during drug treatment. Conclusion Our results show that treatment with gemcitabine alters the expression of EMT genes and that these events are concomitant to important alteration in the translational program. Together, these processes can drive to different translational patterns in presence of gemcitabine, as shown for the ZEB1 variants, which may take part to the mechanisms leading to chemoresistance of PDAC cells.