Human RAP2A homolog of the Drosophila asymmetric cell division regulator Rap2l targets the stemness of glioblastoma stem cells
- Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas/Universidad Miguel Hernández de Elche, Spain
- Instituto de Investigación Biomédicas I+12 and Pathology and Neurooncology Unit, Hospital Universitario 12 de Octubre, Spain
- Unidad de Investigación, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Hospital General Universitario de Elche, Spain
- Unidad de Genética Molecular, Hospital General Universitario de Elche, Spain
Figures
Figure 1 with 2 supplements
RAP2A is highly downregulated in glioblastoma (GBM) patients.
(A, B) Analysis of GBM patient versus control samples focused on the level of expression of human homologs of Drosophila asymmetric cell division (ACD) regulators by hierarchical clustering (A) and the Gene Distance Matrix (B). Color-coded scale bar indicates the fold level of expression. (C) Kaplan–Meier survival curves corresponding to patients in The Cancer Genome Atlas (TCGA) (IDHwt) and Gravendeel (IDHwt) cohort in GBM datasets. Low RAP2A expression levels in human GBM are associated with a poor prognosis.
Figure 1—figure supplement 1
Statistical analysis of asymmetric cell division expression levels in glioblastoma (GBM) patient samples.
A hierarchical clustering of genes showing significant or nonsignificant changes in their expression levels is shown. A t-test was applied to the microarray values of the asymmetric cell division regulator genes shown in Figure 1. The statistical values are presented in separate tables corresponding to the significant (A) and nonsignificant (B) changes in the indicated genes. The overall threshold p-value was 0.01. p-Values were based on t distribution. Significance was determined using just Alpha.
Figure 1—figure supplement 2
RAP2A expression levels in the different GBM subtypes and their associated survival rates.
(A) RAP2A expression levels analysis according to IDHwt glioblastoma (GBM) subtypes in The Cancer Genome Atlas (TCGA) cohort. (B–D) Kaplan–Meier survival curves for each GBM subtype, proneural (B), mesenchymal (C), and classical (D), based on high and low RAP2A expression levels.
Figure 2 with 1 supplement
Drosophila RAP2A homolog Rap2l regulates asymmetric cell division (ACD).
(A) Drosophila type II neuroblast (NBII) lineage; the only NB per lineage expresses the transcription factor Dpn, while mature intermediate neural progenitors (INPs) express both Dpn and Ase; GMC, ganglion mother cell; iINP, immature INP; mINP, mature INP. (B) Confocal micrographs showing a control larval brain NBII lineage with one NB (Dpn+ Ase-) and an NBII lineage in which Rap2l has been downregulated displaying an ectopic NB (eNB). (C) Rap2l downregulation in NBII lineages by the wor-Gal4 ase-Gal80 (an NBII-specific driver) causes Cno and Numb localization failures (green arrows) in dividing progenitors (NB or INPs), while aPKC localization is not significantly altered (red arrows). Data shown in the scaled bar graphs was analyzed with a Mann–Whitney U test (B) or a chi-square test (C), *p<0.05, **p<0.01, ns, not significant, n=number of NB lineages analyzed (in B) or number of dividing cells analyzed (in C); scale bar: 10 µm.
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Figure 2—source data 1
Source data of Figure 2B analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig2-data1-v1.xlsx
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Figure 2—source data 2
Source data of Figure 2C (aPKC) analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig2-data2-v1.xlsx
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Figure 2—source data 3
Source data of Figure 2C (cno) analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig2-data3-v1.xlsx
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Figure 2—source data 4
Source data of Figure 2C (Numb) analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig2-data4-v1.xlsx
Figure 2—figure supplement 1
Drosophila RAP2A homolog Rap2l regulates asymmetric cell division (ACD).
Confocal micrographs showing a control larval brain type II neuroblast (NBII) lineage with one NB (Dpn+ Ase-) and NBII lineages in which Rap2l has been downregulated displaying an ectopic NB (eNB). The phenotype of three different, independent Rap2lRNAi lines (KK—as in Figure 2—GD and BDSC) is shown. Data represented in the bar graph was analyzed with a Kruskal–Wallis test (**p<0.01), followed by post hoc Dunn’s test; *p<0.05, **p<0.01, ***p<0.001; n=number of NB lineages analyzed; the number of different brains analyzed per genotype is also indicated in brackets.
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Figure 2—figure supplement 1—source data 1
Source data of Figure 2—figure supplement 1 analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig2-figsupp1-data1-v1.xlsx
Figure 3
RAP2A expression in glioblastoma (GBM) neurosphere cultures reduces the stem cell population.
(A) Different GBM cell lines show similar RAP2A mRNA levels and significantly lower levels than in control Astros. (B) RAP2A mRNA levels are significantly higher in the GB5 line after infecting this line with RAP2A (GB5-RAP2A). Data shown in the scaled bar graphs in (A) and (B) was analyzed with an ANOVA and a t-test, respectively; error bars show the SD; n=2 (in A) and 3 (in B) different experiments. (C) Immunofluorescences of the glioblastoma stem cell (GSC) stem cell markers CD133, SOX2, and Nestin reveal a significant reduction in the protein intensity in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the box plots was analyzed with a Mann–Whitney U for CD133 and Nestin and with a t-test for Sox; the central lines represent the median and the box limits the lower and upper quartiles, as determined using R software; crosses represent sample means; error bars indicate the SEM; n=number of sample points; *p<0.05, **p<0.01, ***p<0.001, ns, not significant; scale bar: 10 µm.
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Figure 3—source data 1
Source data of Figure 3C (CD133) analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig3-data1-v1.xlsx
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Figure 3—source data 2
Source data of Figure 3C (SOX2) analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig3-data2-v1.xlsx
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Figure 3—source data 3
Source data of Figure 3C (Nestin) analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig3-data3-v1.xlsx
Figure 4
RAP2A expression in glioblastoma (GBM) neurosphere cultures reduces the stem cell population.
RT-PCRs and western blots show a significant decrease in the mRNA and protein expression levels, respectively, of the glioblastoma stem cell (GSC) markers CD133, SOX2, and Nestin in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student‘s t-test; error bars show the SD; n=3 different experiments; *p<0.05, **p<0.01.
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Figure 4—source data 1
Original files for the blots displayed in Figure 4.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for the blots displayed in Figure 4, labeling the relevant bands.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig4-data2-v1.zip
Figure 5
RAP2A expression in glioblastoma (GBM) neurosphere cultures decreases cell proliferation and sphere size.
(A) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significantly lower number of Ki67-expressing cells per neurosphere than control GB5 neurospheres. Data shown in the box plots was analyzed with a t-test; the central lines represent the median and the box limits the lower and upper quartiles, as determined using R software; crosses represent sample means; error bars indicate the SEM; n=number of sample points. (B) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significant decrease in their size compared to control GB5 neurospheres of the same stage. Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student’s t-test; error bars show the SD; n=total number of neurospheres of three different experiments; *p<0.05; scale bars: 10 µm (in A) and 100 µm (in B).
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Figure 5—source data 1
Source data of Figure 5A analyisis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig5-data1-v1.xlsx
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Figure 5—source data 2
Source data of Figure 5B analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig5-data2-v1.xlsx
Figure 6
RAP2A expression in glioblastoma (GBM) neurosphere cultures fosters asymmetric cell division (ACD) in glioblastoma stem cells (GSCs).
(A) Early-stage GB5 neurospheres expressing RAP2A (GB5-RAP2A) show an odd number of cells (Caussinus and Gonzalez, 2005; Gateff, 1978; Albertson and Doe, 2003) significantly more frequently than control GB5 neurospheres, which show more frequently an even number of cells (Gateff, 1978; Albertson and Doe, 2003; Bello et al., 2006). (B) GB5-RAP2A dividing cells show a significant increase in the number of asymmetric NUMB localization in the progeny compared to control GB5 dividing cells. Data shown in the bar graphs was analyzed with a chi-square test with Yates correction; n=number of neurosphere cell clusters analyzed. **p<0.01; ***p<0.001, scale bar: 20 µm.
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Figure 6—source data 1
Source data of Figure 6A analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig6-data1-v1.xlsx
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Figure 6—source data 2
Source data of Figure 6B analysis.
- https://cdn.elifesciences.org/articles/105690/elife-105690-fig6-data2-v1.xlsx
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Human RAP2A homolog of the Drosophila asymmetric cell division regulator Rap2l targets the stemness of glioblastoma stem cells
eLife 14:RP105690.
https://doi.org/10.7554/eLife.105690.3
