The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma
- Computational Biology Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, United States
- Basic Sciences Division, Fred Hutchinson Cancer Research Center, United States
- Molecular and Cellular Biology Graduate Program, University of Washington, United States
- David M. Rubenstein Center for Pancreatic Cancer Research, Memorial Sloan Kettering Cancer Center, United States
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, United States
- Department of Pathology, Stony Brook University, United States
- Yale University School of Medicine, United States
- Department of Surgery, Memorial Sloan Kettering Cancer Center, United States
- Dartmouth Norris Cotton Cancer Center, United States
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, United States
Figures
Figure 1 with 1 supplement
UPF1 mutations do not result in the acquisition of squamous histological features or confer a growth advantage to mutant cells in vivo.
(A) Schematic of UPF1 gene structure and corresponding encoded protein domains. Intron 10 (I10) contains the bulk of the mutations reported by Liu et al. Scissors indicate the sites targeted by the …
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Figure 1—source data 1
Source data for mouse tumor volume (Figure 1C).
- https://cdn.elifesciences.org/articles/62209/elife-62209-fig1-data1-v2.xlsx
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Figure 1—source data 2
Source data for mouse survival (Figure 1D).
- https://cdn.elifesciences.org/articles/62209/elife-62209-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Validation experiments in mouse KPC cells.
(A) Titration of adenovirus expressing Cas9 only (AdCas9) or Cas9 and guide RNAs targeting intron 9 and intron 11 of mouse Upf1 (AdUpf1) in KPC cells. Gel corresponds to PCR using genomic DNA with …
Figure 2 with 1 supplement
Mutations in UPF1 intron 10 do not inhibit nonsense-mediated mRNA decay (NMD) or cause exon skipping.
(A) Box plot of NMD efficiency in 293 T cells engineered to contain wild-type (WT) or mutant (P1, P9) UPF1. P1 and P9 correspond to the IVS10+31G>A and IVS10-17G>A mutations reported by Liu et al. …
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Figure 2—source data 1
Source data for qPCR in HEK 293 T cell lines (Figure 2A).
- https://cdn.elifesciences.org/articles/62209/elife-62209-fig2-data1-v2.xlsx
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Figure 2—source data 2
Source data for western blot in HEK 293 T cell lines (Figure 2E).
- https://cdn.elifesciences.org/articles/62209/elife-62209-fig2-data2-v2.xlsx
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Figure 2—source data 3
Source data for RT-PCR in HEK 293 T cell lines (Figure 2F).
- https://cdn.elifesciences.org/articles/62209/elife-62209-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
Validation experiments in human 293 T cells and raw gel images for western blot and RT-PCR.
(A) Sanger sequencing of genomic DNA from engineered 293 T cells verifying introduction of the mutation IVS10+31G>A in a homozygous state, as reported by Liu et al. for patient P1, as well as a …
Figure 3
Many reported UPF1 mutations are identical to genetic variants.
(A) Illustration of the mutations in UPF1 intron 10 (I10) reported by Liu et al. Each row indicates the wild-type (WT) sequence from the reference human genome or mutations reported by Liu et al. …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (H. sapiens) | UPF1 | Ensembl | ENSG00000005007 | |
| Gene (M. musculus) | Upf1 | Ensembl | ENSMUSG00000058301.8 | |
| Strain, strain background (M. musculus) | Mouse | NCI Charles River | Charles River: C57BL/6 albino | Mice for pancreatic injections |
| Strain, strain background (E. coli) | NEB Stable Competent E. coli (High efficiency) | New England Biolabs | C3040 | Chemically competent |
| Genetic reagent (M. musculus) | AdUpf1 | This paper | N/A | Adenovirus expressing CRISPR gRNAs targeting mouse Upf1 introns 9-11 |
| Cell line (H. sapiens) | HEK 293T | ATCC | CRL-11268 | Human cell line to model patient mutations |
| Cell line (M. musculus) | KPC | Generated from the PDX-1-Cre; LSL-KrasG12D/+; LSL Trp53R172H/+ | N/A | Murine cell line to model Upf1 exon skipping mutations (Hingorani et al., 2005) Provided by Robert Vonderheide |
| Antibody | Anti-human UPF1 (rabbit monoclonal) | Abcam | Cat No. ab10936 | WB: (1:1000) |
| Antibody | Anti-human GAPDH (rabbit polyclonal) | Abcam | Cat No. ab9485 | WB: (1:1000) |
| Antibody | Anti-rabbit secondary antibody (goat monoclonal) | Abcam | Cat No. ab216777 | WB: (1:10000) IRDye 680RD |
| Antibody | Anti-mouse UPF1 (rabbit monoclonal) | Cell signaling technology | Cat No.9435 | WB: (1:1000) |
| Antibody | Anti-mouse tubulin (mouse monoclonal) | Sigma-Aldrich | Cat No. T9206 | WB: (1:2000) |
| Antibody | Anti-rabbit secondary antibody (from donkey) | Amersham | NA93V | WB: (1:5000) |
| Antibody | Anti-mouse secondary antibody (from sheep) | Amersham | NA931 | WB: (1:5000) |
| Antibody | Anti-mouse p40-ΔNp63 (rabbit polyclonal) | Abcam | Cat No. ab166857 | WB: (1:100) |
| Recombinant DNA reagent | pX459/Cas9 expression plasmid | Addgene | Cat No. 48139 | Ran et al., 2013 |
| Recombinant DNA reagent | phCMV-MUP | PMID:9671053 | Plasmid | Control for transfection efficiency of pmCMV-GI-Norm and pmCMV-GI-39Ter |
| Recombinant DNA reagent | pmCMV-Gl-Norm | PMID:9671053 | Plasmid | Transient transfection construct coding for full-length β-globin |
| Recombinant DNA reagent | pmCMV-Gl-39Ter | PMID:9671053 | Plasmid | Transient transfection construct coding for truncated β-globin with PTC at amino acid 39 |
| Sequence-based reagent | mUpf1_F | This paper | PCR primer | GGTGATGAGATTGCTATTGAGC |
| Sequence-based reagent | mUpf1_R | This paper | PCR primer | TGTTCCTGATCTGGTTGTGC |
| Sequence-based reagent | mUpf1-intron_9-gDNA_F | This paper | Guide DNA Oligo | CACCGTTGTGAGGGCCATACCCTTG |
| Sequence-based reagent | mUpf1-intron_9-gDNA_R | This paper | Guide DNA Oligo | AAACCAAGGGTATGGCCCTCACAAC |
| Sequence-based reagent | mUpf1-intron_11-gDNA_F | This paper | Guide DNA Oligo | CACCGCCGTTGAGCTGATGGTGGCT |
| Sequence-based reagent | mUpf1-intron_11-gDNA_R | This paper | Guide DNA Oligo | AAACAGCCACCATCAGCTCAACGGC |
| Sequence-based reagent | hUPF1_F | This paper | Genomic DNA PCR primer | AAAACGTTTGCCGTGGATGAG |
| Sequence-based reagent | hUPF1_R | This paper | Genomic DNA PCR primer | CACATAGAGAGCGGTAGGCA |
| Sequence-based reagent | hUPF1-gDNA_F | This paper | Guide DNA oligo | GCGCGCGGGGCCTCGCCCAT |
| Sequence-based reagent | hUPF1-patient_1-HDR_R | This paper | DNA HDR ultramer | GCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCCGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGTGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG |
| Sequence-based reagent | hUPF1-patient_9-HDR_R | This paper | DNA HDR ultramer | GCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCTGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGCGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG |
| Sequence-based reagent | hUPF1-PAM-control-HDR_R (wild type) | This paper | DNA HDR ultramer | GCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCCGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGCGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG |
| Sequence-based reagent | hUPF1-RT-PCR_F | This paper | RT-PCR primer | GGATGAGATATGCCTGCGGT |
| Sequence-based reagent | hUPF1-RT-PCR_R | This paper | RT-PCR primer | TTCTCGTCGGCAGACGACAG |
| Sequence-based reagent | Positive control gBlock to detect UPF1 splice variant (ΔE10-11) | This paper | DNA gBlock | ACATGCGGCTCATGCAGGGGGATGAGATATGCCTGCGGTACAAAGGGGACCTTGCGCCCCTGTGGAAAGGGATCGGCCACGTCATCAAGGTCCCTGATAATTATGGCGATGAGATCGCCATTGAGCTGCGGAGCAGCGTGGGTGCACCTGTGGAGGTGACTCACAACTTCCAGGTGGATTTTGTGTGGAAGTCGACCTCCTTTGACAGGCCGGTGCTGGTGTGTGCTCCGAGCAACATCGCCGTGGACCAGCTAACGGAGAAGATCCACCAGACGGGGCTAAAGGTCGTGCGCCTCTGCGCCAAGAGCCGTGAGGCCATCGACTCCCCGGTGTCTTTTCTGGCCCTGCACAACCAGATCAGGAACATGGACAGCATGCCTGAGCTGCAGAAGCTGCAGCAGCTGAAAGACGAGACTGGGGAGCTGTCGTCTGCCGACGAGAAGCGGTACCGGGCCTTGAAGCGCACCGCAGAGAGAGAGCTGCTGATG |
| Sequence-based reagent | mMup1-qPCR_F | PMID:25564732 | qPCR primer | GACCTATCCAATGCCAATCG (exon 5/6 junction) |
| Sequence-based reagent | mMup1-qPCR_R | PMID:25564732 | qPCR primer | GATGATGGTGGAGTCCTGGT (exon 7) |
| Sequence-based reagent | hβ-globin-qPCR_F | PMID:25564732 | qPCR primer | GCTCGGTGCCTTTAGTGATG (exon 2) |
| Sequence-based reagent | mβ-globin-qPCR_R | PMID:25564732 | qPCR primer | CCCAGCACAATCACGATCATA (exon 3, mouse specific) |
| Commercial assay or kit | RNeasy Plus Mini Kit | Qiagen | Cat No. 79654 | |
| Commercial assay or kit | Zero Blunt TOPO PCR cloning system | ThermoFisher | Cat No. K280020 | |
| Chemical compound, drug | Phosphatase inhibitor | ThermoFisher | Cat No. A32959 | |
| Chemical compound, drug | Protease inhibitor | Thermofisher | Cat No. A32963 | |
| Chemical compound, drug | Penicillin/Streptomycin | GIBCO | Cat No. 15070063 | |
| Chemical compound, drug | Lipofectamine 2000 | ThermoFisher | Cat No. 11668030 | |
| Chemical compound, drug | Lipofectamine 3000 | Invitrogen | Cat No. L3000001 | |
| Chemical compound, drug | Puromycin | ThermoFisher | Cat No. A1113803 | |
| Software, algorithm | GuideScan v1.0 | PMID:28263296 | http://www.guidescan.com/ | |
| Software, algorithm | Fiji v2.0.0 | ImageJ | https://imagej.net/Fiji | |
| Software, algorithm | RSEM v1.2.4 | PMID:21816040 | deweylab.github.io/RSEM/RRID:SCR_013027 | |
| Software, algorithm | Bowtie v1.0.0 | PMID:19261174 | github.com/BenLangmead/bowtie/; RRID:SCR_005476 | |
| Software, algorithm | TopHat v2.0.8b | PMID:19289445 | ccb.jhu.edu/software/tophat/index.shtml RRID:SCR_013035 | |
| Software, algorithm | MISO v2.0 | PMID:21057496 | genes.mit.edu/burgelab/miso/ RRID:SCR_003124 | |
| Software, algorithm | IGV v2.3.90 | Thorvaldsdottir | software.broadinstitute.org/software/igv/ RRID:SCR_011793 | |
| Software, algorithm | Prism v7.0 | GraphPad Prism v7.0 | http://www.graphpad.com/ RRID:SCR_002798 | |
| Other | SuperScript IV Reverse Transcriptase | ThermoFisher | Cat No. 18090010 | |
| Other | Q5 High-Fidelity DNA Polymerase | New England Biolabs | Cat No. NEB #M0491 | |
| Other | PowerUp SYBER Green Master Mix | ThermoFisher | Cat No. A25742 | |
| Other | RNase-Free DNase Set | Qiagen | Cat No. 79254 |
Additional files
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Supplementary file 1
(a) Histopathological classification of pancreatic tumors derived from orthotopic injection of control or Upf1-targeted KPC cells. Tumors displaying >25% squamous differentiation were classified as positive for squamous features. (b) Differentially spliced isoforms identified in 293 T cells engineered to have the ‘patient 1’ UPF1 genotype relative to WT control cells. (c) Differentially spliced isoforms identified in 293 T cells engineered to have the ‘patient 9’ UPF1 genotype relative to WT control cells. (d) Overlap between UPF1 mutations reported by Liu et al. and genetic variants present in the 1000 Genomes, NHLBI Exome Sequencing Project, ExAC, and gnomAD databases. Genomic coordinates are specified with respect to the GRCh37/hg19 genome assembly. (e) Summary statistics of somatic mutations in UPF1 identified in our reanalysis of whole-exome and whole-genome sequencing data from Fang et al. (f) All genetic differences in UPF1 and KRAS from the reference genome, including both somatic mutations and inherited genetic variants, that we identified in our reanalysis of all samples from Fang et al.’s cohorts.
- https://cdn.elifesciences.org/articles/62209/elife-62209-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62209/elife-62209-transrepform-v2.docx
