Isoform-specific disruption of the TP73 gene reveals a critical role for TAp73γ in tumorigenesis via leptin
- Comparative Oncology Laboratory, Schools of Veterinary Medicine and Medicine, University of California, Davis, United States
- Department of Pathology, University of Texas Southwestern Medical Center, United States
- Department of Biochemistry and Molecular Medicine, University of California, Davis, United States
- Department of Urology Surgery, School of Medicine, University of California, Davis, United States
Figures
Figure 1
E11 skipping is detected in a subset of human cancers and dog lymphomas.
(A, B) Ratio of TP73 exon 11 exclusion was examined in lung squamous cell carcinoma (LUSC) and head-neck squamous cell carcinoma (HNSC) along with their normal tissues by using TCGA SpliceSeq …
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Figure 1—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig1-data1-v1.pdf
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Figure 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig1-data2-v1.tif
Figure 2 with 2 supplements
Deletion of exon 11 in the TP73 gene leads to isoform switch from α to γ, resulting in enhanced cell proliferation and migration as well as altered epithelial morphogenesis.
(A) Schematic representation of p73 isoforms and isoform switch resulting from E11 deletion. (B) The level of p73 splicing variants was examined by RT-PCR in isogenic control and E11-KO H1299 cells. …
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Figure 2—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig2-data1-v1.pdf
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Figure 2—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig2-data2-v1.tif
Figure 2—figure supplement 1
Deletion of E11 in TP73 leads to enhanced TAp73gamma expression and subseuqntly, enhanced cell proliferation and migration.
(A) Schematic representation of the strategy to target TP73 exon 11 using two guide RNAs. (B, C) The levels of p73α/γ, p73β/ε, and GAPDH transcripts were measured in isogenic control and E11-KO …
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Figure 2—figure supplement 1—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig2-figsupp1-data2-v1.tif
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Figure 2—figure supplement 1—source data 3
Numerical data for Figure 2—figure supplement 1G.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig2-figsupp1-data3-v1.xlsx
Figure 2—figure supplement 2
Ectopic TAp73gamma promotes cell migration.
(A) H1299 cells were uninduced or induced to express HA-tagged TAp73g for 24 hr, followed by western blot analysis with antibodies against HA or actin. (B) H1299 cells were uninduced or induced to …
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Figure 2—figure supplement 2—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig2-figsupp2-data1-v1.pdf
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Figure 2—figure supplement 2—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig2-figsupp2-data2-v1.tif
Figure 3 with 1 supplement
TAp73γ is primarily responsible for the oncogenic effects observed in E11-KO cells.
(A) Isogenic control and E11-KO H1299 cells transfected with a scrambled siRNA or siRNAs against p73α/γ for 3 d, followed by RT-PCR analysis to measure the levels of p73α, p73γ, and actin …
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Figure 3—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig3-data1-v1.pdf
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Figure 3—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig3-data2-v1.tif
Figure 3—figure supplement 1
TAp73gamma, but not DNp73 is required for the enhanced cell mgiration and proliferation.
(A) Schematic representation of the locations of p73α/γ siRNAs. (B) Isogenic control and E11-KO H1299 cells were transiently transfected with a scrambled siRNA or siRNA against p73α/γ for 3 d, …
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Figure 3—figure supplement 1—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig3-figsupp1-data1-v1.pdf
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Figure 3—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig3-figsupp1-data2-v1.tif
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Figure 3—figure supplement 1—source data 3
Numerical data for Figure 3—figure supplement 1C.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig3-figsupp1-data3-v1.xlsx
Figure 4 with 1 supplement
Deletion of E11 in the Trp73 gene leads to shortened lifespan, increased incidence of spontaneous tumor and chronic inflammation in mice.
(A) The levels of Trp73α, Trp73γ, Trp73ζ and actin transcripts were measured in WT, E11-HET, and E11-KO MEFs by RT-PCR analysis. (B) SA-β-gal staining was performed with WT and E11-KO MEFs. The …
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Figure 4—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig4-data1-v1.pdf
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Figure 4—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig4-data2-v1.tif
Figure 4—figure supplement 1
Mice dificient in TP73 E11 were infertile and prone to diffuse large B cell lymphoma and chronic inflammation.
(A) Schematic representation of the strategy to generate E11-deficient mouse model and the location of four guide RNAs. (B) Genotyping to verify WT, E11-HET, and E11-KO MEFs. (C) Representative …
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Figure 4—figure supplement 1—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig4-figsupp1-data1-v1.pdf
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Figure 4—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig4-figsupp1-data2-v1.tif
Figure 5 with 1 supplement
E11-deficient mice are prone to obesity.
(A) Representative images of H&E-stained visceral adipose tissues from male and female WT and E11-HET mice. (B–, C) Visceral adipose tissue (VAT) mass of male (B) and female (C) WT and E11-HET mice …
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Figure 5—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig5-data1-v1.pdf
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Figure 5—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig5-data2-v1.tif
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Figure 5—source data 3
Numerical data for Figure 5.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig5-data3-v1.xlsx
Figure 5—figure supplement 1
p73gamma maintains the levels of triglycerides and cholesterol in cells.
(A) The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyltransferase 1 (GGT1) mRNA were measured in the livers from WT and E11+/- mice. (B) The levels of …
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Figure 5—figure supplement 1—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig5-figsupp1-data1-v1.pdf
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Figure 5—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig5-figsupp1-data2-v1.tif
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Figure 5—figure supplement 1—source data 3
Numerical data for Figure 5–figure supplement 1C–D.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig5-figsupp1-data3-v1.xlsx
Figure 6 with 1 supplement
E11 deficiency leads to elevated production of Leptin, a novel target of TAp73g.
(A) The levels of TNFα, IL6, IL-1α, Leptin, and actin transcripts were measured in visceral adipose tissue (VAT) and liver from WT and E11-HET mice. (B, C) The levels of serum (B) and VAT (C) Leptin …
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Figure 6—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig6-data1-v1.pdf
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Figure 6—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig6-data2-v1.tif
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Figure 6—source data 3
Numerical data for Figure 6.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig6-data3-v1.xlsx
Figure 6—figure supplement 1
E11 dificiency alters lipid level via increased leptin levels.
(A) Representative images of H&E-stained vesical adipose tissues from male and female WT and E11-HET mice. (B) The levels of Leptin and actin transcripts were measured in WT and E11-KO MEFs. (C) …
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Figure 6—figure supplement 1—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig6-figsupp1-data1-v1.pdf
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Figure 6—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig6-figsupp1-data2-v1.tif
Figure 7 with 1 supplement
Leptin is a critical mediator of TAp73γ-mediated oncogenesis and altered lipid storage.
(A, D) The levels of Leptin and actin transcripts were measured in isogenic control and E11-KO H1299 cells transfected with a scrambled siRNA or siRNAs against Leptin. (B, C) The levels of …
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Figure 7—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig7-data1-v1.pdf
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Figure 7—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig7-data2-v1.zip
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Figure 7—source data 3
Numerical data for Figure 7.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig7-data3-v1.xlsx
Figure 7—figure supplement 1
Leptin is required for p73gamma to enhance cell migration.
(A) The levels of Leptin and actin transcripts were measured in isogenic control and E11-KO Mia-PaCa2 cells transiently transfected with a scrambled siRNA or a siRNA against Leptin for 3 d. (B, C) …
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Figure 7—figure supplement 1—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig7-figsupp1-data1-v1.pdf
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Figure 7—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig7-figsupp1-data2-v1.tif
Figure 8 with 1 supplement
Targeting p73γ or Leptin inhibits tumor growth in vivo.
(A) Xenograft tumor growth in nude mice from isogenic control or E11-KO H1299 cells (error bars represent SEM, * indicates p<0.05 by Student’s t-test). (B) Images of tumors excised from isogenic …
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Figure 8—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig8-data1-v1.pdf
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Figure 8—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig8-data2-v1.tif
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Figure 8—source data 3
Numerical data for Figure 8.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig8-data3-v1.xlsx
Figure 8—figure supplement 1
The levels of p73gamma and Leptin are measured in multiple sets of tumors.
(A) TAp73α, TAp73γ, and actin proteins were measured in tumors from isogenic control and E11-KO groups. (B) TAp73γ and actin proteins were measured in E11-KO tumors with or without p73γ knockdown. (C…
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Figure 8—figure supplement 1—source data 1
Labeled gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig8-figsupp1-data1-v1.pdf
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Figure 8—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/82115/elife-82115-fig8-figsupp1-data2-v1.tif
Additional files
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Supplementary file 1
Survival time, tumor spectrum, steatosis, inflammation, and other abnormalities in WT, Trp73+/- and E11+/- mice.
(a) Wild-type (WT) mice (n = 56) – survival time, tumor spectrum, steatosis, inflammation, and other abnormalities. (b) Trp73+/- mice (n = 30) – survival time, tumor spectrum, steatosis, inflammation, and other abnormalities. (c) E11-HET mice (n = 30) – survival time, tumor spectrum, steatosis, inflammation, and other abnormalities.
- https://cdn.elifesciences.org/articles/82115/elife-82115-supp1-v1.docx
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Supplementary file 2
Primers for generating vectors.
(a) The primers used to generate plasmids’ expression vectors. (b) The primers used for genotyping. (c) The primers used for RT-PCR and ChIP.
- https://cdn.elifesciences.org/articles/82115/elife-82115-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/82115/elife-82115-mdarchecklist1-v1.docx
