NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, China
- Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, United States
- Department of Radiation Oncology, University of Miami Miller School of Medicine, United States
- Sylvester Comprehensive Cancer Center, United States
Figures
Figure 1 with 1 supplement
NMNAT is upregulated in Rasv12-induced glial neoplasia in Drosophila.
(A, B) Larval CNS at 100 AEL with glial expression of GFP+ GFP or Rasv12+ GFP was probed for F-actin (white), Repo (red), and DAPI (blue). The yellow dashed lines mark the boundary of glial …
Figure 1—figure supplement 1
Glial neoplasia tissue area in Drosophila larval CNS.
The Drosophila larval CNS with glial expression of Rasv12+ GFP (green) was probed for Repo (red). The glial neoplasia tissue and non-tumor glial area are marked with cyan and white dashed lines, …
Figure 2 with 3 supplements
NMNAT is required for glial neoplasia growth in Drosophila.
(A, B) Larval CNS at 100 AEL with glial expression of Rasv12+lacZ, Rasv12+NmnatRNAi, lacZ+Rasv12, PC+Rasv12, PCWR+Rasv12, and PD+Rasv12 was probed for F-actin (green), Repo (red), DAPI (blue), and …
Figure 2—figure supplement 1
NMNAT expression is lower in NmnatRNAi fly than wild-type control fly.
Quantification of NMNAT intensity in glial cells or glial neoplasia area. Flies were expressing lacZ+lacZ or Rasv12+NmnatRNAi. Data are presented as median ± quartiles, n≥3. Significance level was …
Figure 2—figure supplement 2
NMNAT downregulation in glial cells does not affect brain morphology in adult fly.
Brains of two DAE flies with glial expression of GFP+GFP or GFP+NmnatRNAi were probed for Repo (red), Nmnat (white), and DAPI (blue). The third and fourth rows are high magnification of the boxed …
Figure 2—figure supplement 3
NMNAT-PCWR has no NAD+ synthesis enzyme activity.
(A) Diagram of the continuous coupled enzyme assay where NAD+ synthesized by NMNAT is reduced to NADH by alcohol dehydrogenase (ADH). The production of NADH is measured by absorbance at 340 nm. …
Figure 3 with 3 supplements
NMNAT expression is essential to the proliferation of human GBM cells.
(A, D) The xCELLigence real-time cell analysis assay was used to monitor the growth index of T98G cells after NMNAT knockdown by transfecting siNMNAT1 or siNMNAT2, or after NMNAT overexpression by …
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Figure 3—source data 1
siRNA sequences for NMNAT1/2 knockdown and primer sequences for PCR.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig3-data1-v1.doc
Figure 3—figure supplement 1
T98G cells viability is inhibited after knockdown NMNAT1 or NMNAT2.
(A) The confluency of T98G cells is decreased after transfected with siNMNAT1 or siNMNAT2 96 hr. (B) Cell viability 96 hr after transfection was measured by an MTT assay. Data are presented as mean …
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Figure 3—figure supplement 1—source data 1
T98G cell viability was inhibited after knockdown NMNAT1 or NMNAT2.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig3-figsupp1-data1-v1.doc
Figure 3—figure supplement 2
Knockdown of NMNAT does not affect cell cycle.
(A–E) Cell cycle was detected by flow cytometry after T98G cells was transfected with siRNA Control, siNMNAT1-1, siNMNAT1-2, siNMNAT2-1, and siNMNAT2-2, respectively. (F) Quantification of cells in …
Figure 3—figure supplement 3
NMNAT protein is upregulated in human glioma cells.
SVG p12 is human glial cells. T98G and U87MG are human glioma cells. Proteins were extracted from cells were probed for NMNAT1, NMNAT2, and β-actin and quantification. NMNAT2 overexpression from …
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Figure 3—figure supplement 3—source data 1
NMNAT protein was upregulated in human glioma cells.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig3-figsupp3-data1-v1.doc
Figure 4 with 1 supplement
Overexpression of NMNAT decreases caspase-3 activation in glial neoplasia.
(A) Glial neoplasia from files expressing lacZ, PC, PCWR, or PD were probed for Repo (red), F-actin (green), DAPI (blue), and caspase-3 (gray). The top row shows the whole glial neoplasia area. The …
Figure 4—figure supplement 1
Blocking caspase pathway in Rasv12 overexpressing fly.
(A) Diagram of caspase pathway in mammalian and Drosophila. (B) Flies with Rasv12+lacZ, Rasv12+Diap1, Rasv12+p35, Rasv12+DCP1 RNAi, and Rasv12+DroncRNAi were probed for Repo (green) and F-actin …
Figure 5 with 2 supplements
NMNAT decreases caspase-3 activation in human glioma cells.
(A) T98G cells were transfected with siNMNAT1 or siNMNAT2 and stained with DAPI (blue) and caspase-3 (white). (B) T98G cells were transfected with DsRed (red), DsRed-NMNAT1 (red), or DsRed-NMNAT2 …
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Figure 5—source data 1
NMNAT decreased caspase-3 activation in human glioma cells.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig5-data1-v1.doc
Figure 5—figure supplement 1
Membrane of Western blot analysis.
Proteins were extracted from T98G cells transfected with siRNA (left), or plasmids and treated with cisplatin for 8 hr (right) for Western blot analysis. P32 was considered as pro-caspase-3. P17/19 …
Figure 5—figure supplement 2
Cleaved caspase-3 is reduced after NMNAT overexpression.
U87MG cells were treated with CDDP and probed for p53, caspase-3, and β-actin. NMNAT, nicotinamide mononucleotide adenylyltransferase.
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Figure 5—figure supplement 2—source data 1
Cleaved Ccaspase-3 was reduced after NMNAT overexpression.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig5-figsupp2-data1-v1.doc
Figure 6
Nmnat-PC inhibits DNA damage-induced p53 activation in glial neoplasia.
(A) Glial neoplasia from flies expressing lacZ, PC, PCWR, or PD were stained with H2Av (red), Repo (green), and F-actin (magenta). The second and third rows are high magnification of the boxed areas …
Figure 7
p53 inhibitor increases glial neoplasia volume in CNS and larvae lethality.
(A) Flies expressing Rasv12 and CD8GFP were treated with DMSO or p53 inhibitor, respectively, stained with cleaved caspase-3 (red) and DAPI (blue). The first column is the whole CNS of flies. White …
Figure 8 with 1 supplement
p53 depletion rescues NMNAT knockdown induced caspase-3 activation in glioma.
(A) Proteins were extracted from U87MG and T98G cells transfected with siNMNAT1/2 after knockdown of p53 or GFP by shRNA lentivirus transduction for Western blot analysis. β-actin was used as an …
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Figure 8—source data 1
p53 depletion rescued NMNAT knockdown-induced caspase-3 activation in glioma.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig8-data1-v1.doc
Figure 8—figure supplement 1
p53 depletion rescues NMNAT knockdown induced caspase-3 activation in glioma.
(A) Proteins were extracted from U87MG and T98G cells transfected with siRNA for Western blot analysis. β-actin was used as an internal control. (B) Quantification of cleaved caspase-3 in Western …
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Figure 8—figure supplement 1—source data 1
p53 depletion rescued NMNAT knockdown-induced caspase-3 activation in glioma.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig8-figsupp1-data1-v1.doc
Figure 9 with 2 supplements
NMNAT interacts with p53 and PARP1 and upregulates PARylation.
(A) Proteins were extracted from T98G cells transfected with plasmids for Western blot and dot blot analyses using anti-PAR antibody. (B) Quantification of PAR in Western blot analysis. Data are …
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Figure 9—source data 1
NMNAT interacts with p53 and PARP1 and upregulates PARylation.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig9-data1-v1.doc
Figure 9—figure supplement 1
PARylation is reduced after NMNAT knockdown.
Proteins were extracted from T98G cells transfected with siRNA for dot blot analysis using anti-PAR antibody and quantification. Data are presented as mean ± SD, n=3. Significance level was …
Figure 9—figure supplement 2
NMNAT interacts with p53 in U87MG.
Protein samples extracted from U87MG cells transfected with DsRed, DsRed-NMNAT1, or DsRed-NMNAT2 were immunoprecipitated (IP) with a p53 antibody and subjected to immunoblot (IB) analysis for p53, …
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Figure 9—figure supplement 2—source data 1
NMNAT interacts with p53 in U87MG.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig9-figsupp2-data1-v1.doc
Figure 10 with 1 supplement
NMNAT upregulates SIRT1 and reduces acetylation of p53.
(A) Protein samples extracted from T98G cells transfected with DsRed, DsRed-NMNAT1, or NMNAT2 were immunoprecipitated (IP) with a p53 antibody and probed for acetyl-p53 and SIRT1. Red asterisks (*) …
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Figure 10—source data 1
NMNAT upregulates SIRT1 and reduces acetylation of p53.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig10-data1-v1.doc
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Figure 10—source data 2
NMNAT upregulates SIRT1 and reduces acetylation of p53.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig10-data2-v1.doc
Figure 10—figure supplement 1
Acetyl-p53 is reduced after NMNAT overexpression in U87MG.
U87MG cells were treated with CDDP and probed for p53, acetyl-p53, and tubulin. NMNAT, nicotinamide mononucleotide adenylyltransferase.
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Figure 10—figure supplement 1—source data 1
Acetyl-p53 was reduced in U87MG cells under NMNAT overexpression conditions.
- https://cdn.elifesciences.org/articles/70046/elife-70046-fig10-figsupp1-data1-v1.doc
Figure 11 with 2 supplements
Correlation of tumor NMNAT expression levels with glioma progression.
(A, B) LGG (lower grade glioma) data set (GEPIA). Survival curves for patients with tumors expressing median (top 50% vs. bottom 50%) NMNAT1 levels (A), or top 10% versus bottom 10% levels (B), with …
Figure 11—figure supplement 1
Summary of NMNAT1 and NMNAT2 alteration frequency in cancer types.
Alteration of human NMNAT1 and NMNAT2 was queried in TCGA database (https://www.TCGA.com). 1522 cases with altered NMNAT1 and 931 cases with altered NMNAT2 across 32 projects are shown. NMNAT, …
Figure 11—figure supplement 2
Summary of NMNAT1 and NMNAT2 alteration frequency in cancer types.
Alteration of human NMNAT1 and NMNAT2 was queried in cBioPortal database (https://www.cbioportal.com) separately. 10,967 samples in 35 cancer types are shown. NMNAT, nicotinamide mononucleotide …
Tables
Author response table 1
Gene effect analysis using DepMap.
org dataset
| Manuscript | Cancer | Cell Line | Gene | Gene effect (CERES) | Expression (Log2(TPM+1)) |
|---|---|---|---|---|---|
| Liu et al. (2021). bioRxiv.(this manuscript) | Glioma | T98G | NMNAT1NMNAT2 | -0.427-0.0177 | 2.872.29 |
| U87MG | NMNAT1NMNAT2 | -0.1590.156 | 3.15.74 | ||
| Challa et al. (2021). Cell | Ovarian | OVCAR3 | NMNAT1NMNAT2 | -0.2190.0989 | 3.264.18 |
| Shi et al. (2021).Science Advances | Acute myeloid leukaemia | MOLM13 | NMNAT1NMNAT2 | -0.33-0.00213 | 2.310.189 |
| OCIAML2 | NMNAT1NMNAT2 | 0.0588 0.0624 | 0.8881.52 |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70046/elife-70046-transrepform1-v1.doc
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Source data 1
Original data file.
- https://cdn.elifesciences.org/articles/70046/elife-70046-supp1-v1.xlsx
