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

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.

Figure 1. Chronic treatment with gemcitabine selects DR-PDAC cells

(A) Schematic representation of the protocol used to obtain drug-resistant (DR) PDAC cells from parental PDAC cells (PCL). (B) Representative phase contrast images of PCL- and DR-Pt45P1 (left panels) or PANC-1 (right panels) cells (40× magnification). (C-D) Representative images of the colony assay (upper panels) performed in PCL- and DR-Pt45P1 (C) or PANC-1 cells (D). (C-D). Bar graphs (bottom panels) show the percentage of survival with respect to untreated cells from three experiments (mean ± SD), as assessed by colony formation. Brackets indicate statistical comparison of the indicated samples. Statistical analyses were performed by the paired Student’s t-test. ** p ≤ 0.01.

Figure 2. The PKM2 splice variant is promoted in DR-PDAC cells

(A-E) RT-PCR analysis in PCL- and DR-Pt45P1 or PANC-1 cells of splice variants encoded by the indicated cancer-related genes. Schematic representation of the cancer-related AS events analyzed is shown in the upper panels. Exons (boxes) and introns (lines) are indicated. Black arrows indicate primers used for the RT-PCR analysis (bottom panels). (C) RT-PCRs of PKM gene were followed by PstI digestion in order to distinguish the amplicons. Bar graphs represent the percentage of the indicated AS variants, as assessed by densitometric analysis of the bands. Statistical analyses were performed by the paired Student’s t-test comparing PCL- and DR-PDAC cells values (mean ± SD, n=3, **p<0.01, ns: not significant). (F) HPRT was used as loading control for RT-PCR analyses in panels A-E.

Figure 3. PKM2 protein expression in PDAC cells and PDAC tissues

RT-PCR (A) and Western blot (B) analyses of PKM1 and PKM2 splicing variants in PCL- and DR-PDAC cells. Schematic representation of the PKM gene is shown in the upper panel, black arrows indicate the specific primers used to amplify the PKM1 and PKM2 in PCL- and DR-PDAC cells. HPRT and PKM exon 5-6 region were used as loading control (A). Coomassie staining was used as loading control (B). (C) Western blot analysis of PKM1 and PKM2 protein in PCL-PDAC cells. Coomassie staining was used as loading control. (D) Representative images of PKM2 immunohistochemistry in PDAC tissues (10× magnification). Upper panels show neoplastic glands with weak staining (low PKM2 group; score ≤3), bottom panels show neoplastic glands with strong staining (high PKM2; group score >3). (E) Analysis of recurrence free survival (RFS) of PDAC patients. Low PKM2 group comprised 16 patients (continuous line) while high PKM2 group comprised 26 patients (dotted line); p=0.04 at log-rank test.

Figure 4. Modulation of PKM splicing enhances gemcitabine-induced cell death in DR-PDAC cells

(A-B) RT-PCR (A) and western blot (B) analyses of PKM splicing variants performed in DR-Pt45P1 (left panels) and DR-PANC-1 (right panels) cells transduced with a control ASO (CTRL ASO) or with a specific ASO used to revert PKM splicing (PKM2 ASO) in favour of PKM1 (see Supplementary Table 1). (A) Bar graphs represent the percentage of PKM2 variant, as assessed by densitometric analysis of the bands. Statistical analyses were performed by the paired Student’s t-test comparing DR-PDAC cells values with those obtained in PCL-PDAC cells while brackets indicate statistical comparison of the indicated samples (** p ≤ 0.01; ns: not significant). (B) Coomassie staining was used as protein loading control. (C) Bar graphs show the percentage of cell death from three experiments (mean ± SD) as assessed by immunofluorescence analysis of the cleaved form of caspase-3 in PCL- and DR-PDAC cells transduced with CTRL or PKM2 ASO and treated as indicated. Statistical analyses were performed by the paired Student’s t-test, comparing DR-PDAC cell values with those obtained in PCL-PDAC cells treated with gemcitabine, while brackets indicate statistical comparison of the indicated samples (* p ≤ 0.05, ** p ≤ 0.01, ns: not significant).

Figure 5. PTBP1 is up-regulated in DR-PDAC cells

(A-B) Western blot analysis of PTBP1, PTBP2, hnRNPA1 and hnRNPA2/B1 protein expression in PCL- and DR-Pt45P1 (A) or PANC-1 cells (B). Coomassie staining was used as loading control.

Figure 6. PTPB1 up-regulation is required for PKM2 splicing and gemcitabine resistance in DR-PDAC cells

(A) UV crosslink immunoprecipitation (CLIP) of PTBP1 performed in PCL- or DR-Pt45P1 PDAC cells transfected with either a control (si-ctrl) or PTBP1 (si-PTBP1) siRNAs, in presence of RNaseI (1:1000). Associated PKM pre-mRNA was quantified by qPCR, black arrows indicate primers used to amplify A and B regions (upper panels; see Supplementary Table 1). Data are represented as percentage of input (bottom panels; mean ± SD; n=3). Statistical analyses were performed by the paired Student’s t-test (** p ≤ 0.01, ns: not significant). PTBP1 silencing in DR-Pt45P1 cells and IP efficiency were assessed by western blot analysis. (B) RT-PCR and western blot analyses to evaluate PKM1 and PKM2 expression in DR-PDAC cells transfected with either ctrl or PTBP1 siRNAs. Bar graphs represent the percentage of PKM2 variant, as assessed by densitometric analysis of the bands. Statistical analyses were performed by the paired Student’s t-test comparing the values of DR- PDAC cells transfected with si-ctrl with those obtained in DR-PDAC cell transfected with si-PTBP1 siRNA (** p ≤ 0.01; (upper panels, mean ± SD, n = 3, ** p ≤ 0.01). (B) PTBP1 silencing was assessed by western blot analysis. Coomassie staining was used as loading control. (C-D) Western blot analyses assessing PTBP1 expression levels in PCL- and DR-Pt45P1 or PANC-1 PDAC cells transfected with ctrl or PTBP1 siRNAs. Coomassie staining was used as loading control. (C-D) Bar graphs show the percentage of cell death from three experiments (mean ± SD) as assessed by immunofluorescence analysis of the cleaved form of caspase-3 in PCL-, DR-PDAC cells described in panel C-D and treated as indicated for 72 hours. Statistical analyses were performed by the paired Student’s t-test comparing DR-PDAC cell values with those obtained in PCL-PDAC cells treated with gemcitabine, while brackets indicate statistical comparison of the indicated samples (** p ≤ 0.01, ns: not significant).