Modulation of pyruvate kinase alternative splicing by the polypyrimidine-tract binding protein promotes gemcitabine resistance in pancreatic cancer cells

Sara Calabretta 1,2, Pamela Bielli 3, Ilaria Passacantilli 1,2, Emanuela Pilozzi 4, Volker Fendrich 5, Gabriele Capurso 2, Gianfranco Delle Fave 2 and Claudio Sette 1,3

1 Department of biomedicine and prevention, University of Rome Tor Vergata, Rome, Italy

2 Department of science medical/chirurgic and translational medicine, University of Rome La Sapienza, Rome, Italy

3 Laboratory of neuroembriology, Fondazione Santa Lucia, Rome, Italy

4 Department of clinic and molecular medicine, University of Rome La Sapienza, Rome, Italy

5Department of surgery, Philipps-University Marburg, Marburg, Germany

Running title: PKM2 and PTBP1 contribute to drug resistance in PDAC

Keywords: PKM2, PTBP1, gemcitabine, drug resistance, pancreatic adenocarcinoma

Corresponding Authors:Claudio Sette

Dept. of Biomedicine and Prevention

University of Rome “Tor Vergata”

Via Montpellier, 1

00133, Rome, Italy

Telephone: 3906 72596260

Fax: 3906 72596268

Email:

Gianfranco Delle Fave

Dept. of science medical/chirurgic and translational medicine

University of Rome, “La Sapienza”

Via di Grottarossa, 00189 Rome, Italy

Telephone: 3906 33775691

Fax: 3906 33775526

Email:

Supporting Document:Includes 2 supplementary tables and 6 supplementary figures with the corresponding figure legends.

Supplementary Materials and Methods

Cell transfections and extracts preparation.

For RNA interference of PKM2, PANC-1 cells were transfected twice on consecutive days with 60 nM PKM2 siRNAs (see Table 2) using Lipofectamine RNAiMAX and Opti-MEM medium (Life Technologies, California, USA) according to manufacturer’s instructions. Cells were harvested after 24 hours after the last transfection for protein and immunofluorescence analyses.

For PKM2 over-expression experiments, PCL cells were transfected with pEGFPC1 and pEGFPC1-PKM2 (kindly provided by Dr. Axel Ullrich) plasmids using Lipofectamine 2000 Opti-MEM medium (Life Technologies, California, USA) according to manufacturer’s instructions and used for immunofluorescence analysis.

For Western blot analysis, mouse embryo (E 13.5) and adult brain were resuspended in lysis buffer: 150 mM sodium chloride, 50 mM Hepes 7.4, 15 mM magnesium chloride, 20 mM -glicerol-phosphate, 15 mM EGTA, 10% glycerol, 1.0% Triton X-100, 0.5%, 1 mM dithiothreitol, 0.5 mM NaVO4 and protease inhibitor cocktail (Sigma-Aldrich). After 10 min on ice, extracts were centrifuged for 10 min at 12,000 g ,supernatants were collected and used for analysis.

Immunofluorescence analysis

PANC-1 cells transfected with the indicated siRNAs, were seeded at 70% confluence. Cells were then fixed with 4% para-folmaldehyde, permeabilized with 0,1 % Triton X-100 and processed for immunofluorences analisys using anti-PKM2 antibody (1:200, Cell Signaling Technology, MA, USA). For cell death analyses, PCL cells transfected with the indicated vectors were seeded at 70% confluence and treated as described for 72 h. Cells were then processed for immunofluorescence analysis, as described above, using anti-cleaved caspase-3 antibody (1:500; Sigma-Aldrich) and anti-GFP antibody (1:200, Santa Cruz Biotechnology, CA, USA). Positive cells for GFP and cleaved caspase-3 were then counted using fluorescence microscopy. For each treatment, at least 200 cells were counted.

Supplementary Table 1: List of the clinical, histological and pathological features of the patients analysed in Figure 3D-E.

G: grade of cancer cells; T: extent of the tumour; R: residual tumor at the local resection margin; N: extent of spread to regional lymph nodes. Mean age of diagnosis in expressed in years, mean Recurrence Free Survival and Overall Survival are expressed in months. P values are obtained by t test for continuous variables or by Fisher’s exact test for dichotomized groups.

“High PKM2” (n=26) / “Low PKM2” (n=16) / p
Mean Age / 65.5 (61.8-69.2) / 70.1 (65.7-74.5) / 0.10
Male Sex / 11 (42%) / 11 (68%) / 0.12
Mean Tumor size (mm) / 37.7 (31.8-43.6) / 36.6 (27.1-46) / 0.8
G3 / 14 (54%) / 8 (50%) / 1
T3 / 20 (31%) / 15 (93%) / 0.2
R1 / 8 (31%) / 6 (37%) / 0.7
N1 / 23 (88%) / 12 (75%) / 0.3
Mean Recurrence Free Survival / 11.6 (11.8-21.3) / 22.1 (12.2-31.9) / 0.04
Mean Overall Survival / 16.6 (11.8-21.3) / 22.1 (12.2-31.9) / 0.2

Supplementary Table 2: List of oligos used in this study

Oligo name / Sequence 5’3’ / Reference
Caspase9 Fw / GCTCTTCCTTTGTTCATCTCC / 1
Caspase9 Rev / CATCTGGCTCGGGGTTACTGC / 1
hCASP2 For ex8 / AACTGCCCAAGCCTACAGAA / 2
hCASP2 Rev ex9 / GCGTGGTTCTTTCCATCTTGTTGGTCA / 2
hCASP2 Rev ex10 / CCTCGTTTGGTGTTCCGCAT / 2
BCL-X ATG Fw / ATGTCTCAGAGCAACCGGGAGCTG / 3
BCL-X TGA Rev / TCATTTCCGACTGAAGAGTGAGCC / 3
BIM UP Fw / AAGCAACCTTCTGATGTAAGTTCT / 4
BIM R2 Rev / ATGGTGGTGGCCATACAAAT / 5
BIM ex2 cost Fw / AAAGACCAAATGGCAAAGCAA / This study
BIM ex2 cost Rev / TGTGGCTCTGTCTGTAGGGA / This study
FAS Fw ex5 / GGAATGCACACTCACCAGCA / This study
FAS Rev ex7 / GTTGGAGATTCATGAGAACCTTGGT / This study
CD44 V5 Fw / TGTAGACAGAAATGGCACCACTG / 6
CD44 V5 Rev / TGGTGCTTGTAGAATGTGGGGTC / 6
CD44 C6 Fw / AGACACATTCCACCCCAGTG / 6
CD44 C7 Rev / CCAGAGGTTGTGTTTGCTCCACC / 6
RON 2507 Fw / CCTGAATATGTGGTCCGAGACCCCCAG / 7
RON 2991 Rev / CTAGCTGCTTCCTCCGCCACCAGTA / 7
cMET Fw XhoI / AGCTCGAGTGGAAGCAAGCAATTTCTT / This study
cMET Rev NotI / AGGCGGCCGCCTCTTCCTATGACTTCA / This study
CCND1 Fw / AGGCGGCCGCATGTGAAGTTCATTTCCAATC / 8
CCND1 D1a Rev bis / AGGATATCGCCTGCCTGGCGCCCTCAGAT / 8
CCND1 D1b Rev / GGGACATCACCCTCACTTAC / 8
USP5/Ex14/XhoI/Fw: / AGCTCGAGAAGAAGTTCACCTTCGGC / 9
USP5/Ex16/HindIII/Rv: / AGAAGCTTATGACCCAGTTCATGGCGG / 9
MNK2 Fw / CCAAGTCCTGCAGCACCCCTG / 10
MNK2 ex13a Rev / GATGGGAGGGTCAGGCGTGGTC / 10
MNK2 ex13b Rev / GAGGAGGAAGTGACTGTCCCAC / 10
hPKM Fw ex8 / CTGAAGGCAGTGATGTGGCC / 11
hPKM Rev ex11 / ACCCGGAGGTCCACGTCCTC / 11
hHPRT Fw / TGACCAGTCAACAGGGGACA / 12
hHPRT Rev / TTCGTGGGGTCCTTTTCACC / 12
hPKM1 Fw ex9 / CTTCTTATAAGTGTTTAGCAGCAGCT / This study
hPKM2 Fw ex10 bis / GGGGCCATAATCGTCCTCACCA / This study
hPKM rev ex11RT / CAGGTGGGCCTGACGAGCTG / This study
hPKM cost Fw ex5 / CCTGTGGCTGGACTACAAGA / This study
hPKM cost Rev ex6 / CCATATCAACATCCTGCTCGACC / This study
CLIP Intron 8 E Fw (A region) / TGCATGCTTCCACAGGCATC / This study
CLIP Intron 8 F Rev (A region) / TGGGCTAACTTGTGAGAGGC / This study
CLIP Intron 8 G Fw (B region) / CCCATTCTGTCTTCCCATGTGTT / This study
CLIP Intron 8 H Rev (B region) / TTTAGGGGAAGAGGGGGCAAG / This study
EZH2 Fw / AGAATGGAAACAGCGAAGGAT / 13
EZH2 Rev / AGGGCATTCACCAACTCCACAAA / 13
CTTN Fw / GGGATCACCAGGAGAAATTGCA / 13
CTTN Rev / CTTATCCATCCGATCCTTCTGC / 13
RASSF8 Fw / GGCGCAGTTGCGAAACTGA / 13
RASSF8 Rev / AGGCTATGACAACCTCCTGGCA / 13
MINK1 Fw / GCTCTCCCCTGGGAATAAAG / 13
MINK1 Rev / GAGTCCGCTCTTTCAGCAAC / 13
EIF4G2 Fw / ATCGCAGTTTGGAGAGATGG / 13
EIF4G2 Rev / CTGTCCCAGAGGTGGTGTTT / 13
FAM38A Fw / TGGATGTGTGTGGAGGACAT / 13
FAM38A Rev / ATGATGGCGATGAGGAAGAG / 13
CCDC138 Fw / TCCAGATGACGGTGGAATCT / 13
CCDC138 Rev / ATGCTCTGGGTTTGTTGTCC / 13
TPM1 Fw / CCCGTAAGCTGGTCATCATT / 13
TPM1 Rev1 / TAATTGTTCTTCCAGCTGTCGG / 13
TPM1 Rev2 / CCTGAGCCTCCAGTGACTTC / 13
FGFR2 exIIIb Fw / CACTCGGGGATAAATAGTT / 14
FGFR2 exIIIc Fw / CGGTGTTAACACCACGGAC / 14
FGFR2 Rev / ACTCGGAGACCCCTGCCA / 14
CTRL ASO / TCATTTGCTTCATACAGG / 15
PKM2 ASO(46-60) / GCGGCGGAGTTCCTC / 15
siRNA ctrl / AGACGAACAAGUCACCGAC
siRNA PTBP1 / On target plus human PTBP1 5725 siRNA, Dharmacon
siRNA PKM2 / CCAUAAUCGUCCUCACCAA / 16

References:

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9. Bielli P, Bordi M, Di BiasioV, Sette C. Regulation of BCL-X splicing reveals a role for the Polypyrimidine-tract binding protein (PTBP1/hnRNP I) in alternative 5 ‘splice site selection. Nucleic Acids Res.2014, in press.

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Supplementary Figure legends:

Supplementary Figure 1: Dose-response of PCL and DR-PDAC cells to treatments with gemcitabine

(A) Cell death analysis performed by trypan blue cell count of Pt45P1 and PANC-1 PCL-PDAC cells treated asindicated. Bar graphs represent the percentage of trypan blue positive cells (mean ± SD, n=3).(B-C)Bar graphs show the percentage of cell death from three experiments (mean ± SD) as assessed by trypan blue cell count (B) andimmunofluorescence analysis of the cleaved form of caspase-3 (C) in PCL- and DR-PDAC cells treated as indicated. Statistical analysis was performed by the paired Student’s t-test, brackets indicate statistical comparison of the indicated samples.(** p ≤ 0.01).

Supplementary Figure 2: Analysis of cancer-related AS events in PCL- and DR-PDAC cells

(A-C) 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, left). 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).

Supplementary Figure 3. Specificity of PKM2 antibody in PDAC cells and in neoplastic versus non neoplastic pancreatic glands

(A) Immunofluorescence analysis of PKM2 in PANC-1 cells transfected with either ctrl or PKM2 siRNAs. (B) Western Blot analysis of PKM1 and PKM2 protein expression in PANC-1 cells transfected with either ctrl or PKM2 siRNAs.(C) Western blot analysis of mouse embryo (E13.5) and mouse adult brain (three-month old). (D) Representative images of PKM2 immunohistochemistry in PDAC tissues (10X magnification). Stronger cytoplasmic staining for PKM2 was observed in neoplastic glands (red box, right) than in non neoplastic ductal and acinar cells (black box, left). The antibody was used at increasing dilution (1:800, 1:1600; 1:3000). Immunohistochemistry without antibody for PKM2 was used as negative control.

Supplementary Figure 4. Over-expression of PKM2 in Pt45P1 and PANC-1 PCL promotes resistance to gemcitabine

Cell death analysis performed by immunofluorescence analysis of the cleaved form of caspase-3 of PCL-Pt45P1 (A) and PCL-PANC-1 (B) cells transfected with GFP or GFP-PKM2 and treated as indicated. White arrows indicate cells positive for GFP and cleaved caspase-3, blue arrows indicate cells negative for GFP and positive for cleaved caspase-3. Bar graphs represent the fold increase of treated cells positive to GFP and cleaved caspase-3 respect with the untreated cells (mean ± SD, n=3). Statistical analyses were performed by the paired Student’s t-test, comparing PCL-PDAC cell values without gemcitabine with those obtained in PCL-PDAC cells treated with gemcitabine, while brackets indicate statistical comparison of the indicated samples (* p ≤ 0.05, ns: not significant).

Supplementary Figure 5:Expression of cancer-related splicing factors in PCL- and DR-PDAC cells

(A) Western blot analysis ofPTBP1 protein expression in PCL-Pt45P1 and PCL-PANC1 cells. Coomassie staining was used as loading control.

(B) Western blot analysis of a group of cancer-related splicing factors in PCL- and DR-Pt45P1 (left panels)or PANC-1 cells (right panels). Coomassie staining was used as loading control.

Supplementary Figure 6: Alternative splicing events regulated by PTBP1 in PCL- and DR-PDAC cells

(A-D) RT-PCR analysis of PTBP1 regulated AS in PCL- and DR-PDAC cells of PTBP1-dependent mutually exclusive exons inclusion (A), exon inclusion(B), exon skipping splicing events (C).Schematic representation of exons (boxes) and introns (lines) in the genomic regions analyzed is illustrated above the agarose or acrylamide gelsshowing the PCR-amplified products. Black arrows indicate primers used (see Supplementary Table 1) for the RT-PCR analysis (bottom panels). (D)HPRT was used as loading control.(E)RT-PCR analysis of FGFR2 in DR-PDAC cells transfected either with ctrl or PTBP1 siRNAs. (F) HPRT was used as loading control.

Supplementary Figure 7: The PTBP1/PKM2 axis is required for cisplatin resistance of DR-PDAC cells

(A) Analysis of cisplatin (CDDP)-induced cell death in PCL- and DR-PDAC cells assessed by trypan blue cell count.Bar graphs represent the percentage of trypan blue positive cells treated as indicated,brackets indicate statistical comparison of the indicated samples (mean ± SD, n=3, ** p ≤ 0.01). (B-C)Bar graphs show the percentage of cell death assessed by trypan blue cell count (mean ± SD, n=3) in DR-PDAC cells transfected with control ASO or PKM2 ASO (B) or with ctrl or PTBP1 siRNAsand treated as indicated (C). RT-PCR analyses (B-C) and western blot (C) analyses performed to evaluate PKM1 and PKM2 isoforms and PTBP1 expression levels, respectively, are shown.(C)Coomassie staining was used as loading control. (B-C)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 CDDP, while brackets indicate statistical comparison of the indicated samples (** p ≤ 0.01, ns: not significant).