doi: 10.1038/s41586-021-03422-5.
Epub 2021 Apr 14.
AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity
Affiliations
- PMID: 33854232
- PMCID: PMC8864551
- DOI: 10.1038/s41586-021-03422-5
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AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity
Nature.
2021 Apr.
Abstract
Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.
Conflict of interest statement
Figures
a, Schematic for production of the conditional knockout mouse model. b, c, Images of wild-type and Ambra1 cKO P21 mice (b) and brains (c). c, bottom, representative image of PCR amplification of Tm1c, Ambra1 and Cre. d, Wild-type and Ambra1 cKO olfactory bulbs in sagittal sections of E18.5 embryos, stained for Ki67 antibody and Hoechst (n = 3). e, Quantification of Ki67+ cell area in the whole brain of wild-type and Ambra1 cKO E13.5 embryos (sagittal sections shown in Fig. 1b) (n = 5). P value by two-tailed unpaired t-test. f, Representative scheme of NSCs extraction and cell culturing from mouse embryo medial ganglionic eminences (MGE). LGE, lateral ganglionic eminences. g, Densitometry quantification of normalized protein levels in wild-type and Ambra1 cKO NSCs shown in Fig. 1c (n = 4). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. h, Left, representative images of NSCs extracted from mouse embryo medial ganglionic eminences. Right, violin plot of clonal neurosphere diameters in wild-type and Ambra1 cKO NSCs (n = 3; total of 128 neurospheres analysed for each condition). P value by two-tailed unpaired t-test. i, Whole-brain quantification of E13.5 wild-type and Ambra1 cKO cyclin D1 staining normalized over DAPI, represented in Fig. 1e. P value by two-tailed unpaired t-test (n = 5). j, Wild-type and Ambra1 cKO olfactory bulbs in sagittal sections of E18.5 embryos, stained for cyclin D1 antibody and Hoechst (n = 3). k, Sagittal sections of wild-type and Ambra1 cKO E13.5 embryos, stained for cyclin D2 (n = 3). l, Left, representative images of sagittal sections of the mesencephalic ventricular zone in wild-type and Ambra1 cKO E13.5 embryos, stained for RB(pS807/811) (n = 5). Right, quantification of RB(pS807/811-positive area in the mesencephalic ventricular zone of E13.5 wild-type and Ambra1 cKO embryos (n = 5). P value by two-tailed unpaired t-test. m, Left, representative images of RB(pS807/811) in sagittal sections of the olfactory bulb in wild-type and Ambra1 cKO E18.5 embryos. Right, quantification of the number of RB(pS807/811)-positive cells (n = 3). P value by two-tailed unpaired t-test. n, Immunoblot of N-MYC after cycloheximide treatment in wild-type and Ambra1 cKO NSCs (n = 3). o, Quantitative PCR with reverse transcription (qRT‒PCR) of NSCs; the investigated genes are at the bottom of the graph (n = 5). P values by two-tailed unpaired t-test. p, Immunoblot of control and AMBRA1silenced SH-SY5Y cells (n = 3). q, Immunoblot of AMBRA1 immunoprecipitation in SH-SY5Y cells. r, Immunoblot for AMBRA1, PP2AC and N-MYC in SH-SY5Y cells silenced for the indicated genes (n = 3). Unless otherwise stated, n refers to biologically independent samples. For immunoblots, actin was used as loading control. Data are mean ± s.e.m. Scale bars, 250 μm.
a, Densitometric quantification of cyclin D1 and D2 protein levels in the cycloheximide time course normalized over actin (n = 3). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. b, Immunoblot of wild-type or Ambra1 cKO NSCs treated with cycloheximide and/or MG132 for the indicated times. (n = 3). c, Distribution of cell-cycle phases in NSCs after release from nocodazole treatment (n = 3). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. d, Six-hour BrdU incorporation of passage-2 wild-type and Ambra1 cKO NSCs with or without abemaciclib treatment (n = 3). P values by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. e, Percentage of apoptotic cells in wild-type and Ambra1 cKO NSCs (n = 3). EA, early apoptotic; LA, late apoptotic. P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. f, Left, immunoblot of the indicated proteins in NSCs after abemaciclib treatment. Right, densitometry quantification of the indicated proteins (n = 3). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. g, Left, representative images of sagittal sections from wild-type and Ambra1 cKO E13.5 embryos, stained for SOX2 and TBR2. Right, quantification of immunostained positive area (SOX, n = 6; TBR2, n = 4). P values by two-tailed unpaired t-test. h, Left, representative images of sagittal sections of wild-type and Ambra1 cKO E18.5 embryos, stained for TBR2. Right, quantification of immunostained positive area (n = 6). P value by two-tailed unpaired t-test. Arrows indicate TBR2+ cells in the subventricular zone. i, j, Representative images of sagittal sections of wild-type and Ambra1 cKO E18.5 embryos, stained for the neuronal marker NeuN. i, Left, higher magnification of the mesencephalic alar plate. Right, quantification of immunostained positive cells (n = 3). P value by two-tailed unpaired t-test. j, Lower magnification to better appreciate the uncropped quantified area (n = 3). Scale bar, 500 μm. Unless otherwise stated, n refers to biologically independent samples. For immunoblots, actin was used as loading control. Data are mean ± s.e.m. Unless otherwise noted, scale bars represent 250 μm.
a, Immunoblot of control or AMBRA1-silenced U87-MG cells for the indicated proteins (n = 3). b, Immunoblot of cyclin D1 in control or AMBRA1-silenced U87-MG cells treated with cycloheximide and/or MG132 for the indicated times (n = 3). c, Analysis of densitometry for the cyclin D immunoblot in U87-MG cells, silenced for the indicated genes, shown in Fig. 1g (n = 4). P values by one-way ANOVA followed by Dunnett’s multiple comparisons test. d, Left, immunoblot of cyclin D1 in U87-MG cells silenced for AMBRA1 expression and overexpressing empty vector (pcDNA), wild-type AMBRA1 or AMBRA1(ΔWD40). Right, analysis from densitometry (n = 3). P values by one-way ANOVA followed by Tukey’s multiple comparisons test. e, Immunoblot analysis of cyclin D1 immunoprecipitation from protein extracts of control and AMBRA1-silenced U87-MG cells (n = 3). f, Co-immunoprecipitation of AMBRA1 in U87-MG cells transiently overexpressing empty vector, cyclin D1–Flag or cyclin D1(T286A)–Flag. Cells were treated with MG132 for 3 h before lysis (n = 3). g, Fold change in the number of cells in control or AMBRA1-silenced U87-MG cells (n = 11). P value by two-tailed unpaired t-test. h, Immunoblot of the indicated proteins of U87MG, BJ-hTERT and U2OS cells that were untreated or treated with MLN4924 for 4 h (n = 3). i, j, Cells immunostained with cyclin D1, EdU antibody and counterstained with Hoechst. i, Scatter plots reporting single-cell total nuclear intensities of EdU versus Hoechst (cells examined over three independent experiments: siSCR, n = 3,279; siAMBRA1, n = 3,608 cells). j, Box plots (centre line, median; box limits, 25th and 75th percentile) indicating total cyclin D1 nuclear intensities (siSCR, n = 3,279; siAMBRA1, n = 3,608 cells. median siSCR = 169,654; siAMBRA1 = 429,623). k, l, Immunoblot of cell-cycle markers in control and AMBRA1-silenced BJ-hTERT cells (k) and cell-cycle-sorted U2OS-FUCCI cells (l) (n = 3). m, Immunoblot of the indicated proteins in AMBRA1-silenced BJ-hTERT cells synchronized by 24-h serum starvation. Cells were collected after the indicated starvation recovery time points (n = 3). n, Representative images of live-cell imaging of control and AMBRA1-silenced U2OS-FUCCI cells from 0 to 14 h with a 2-h interval between different images. The length of the G1 phase is shown in Fig. 1k (n = 3). Scale bar, 5 μm. o, Cell proliferation in control or AMBRA1-silenced BJ-hTERT cells (24 h and 48 h n = 6; 72 h siSCR n = 6, siAMBRA1 n = 5). p, q, Control or AMBRA1-silenced U2OS-FUCCI cells. p, Representative contour plot. q, Fold increase of cells present in S–G2 phase in AMBRA1-downregulated cells with respect to control cells (n = 10). r, Box plots (centre line, median; box limits, 25th and 75th percentile; whiskers, minimum and maximum) showing the cell-cycle length of siSCR (n = 65; median = 13) or siAMBRA1 (n = 65; median = 8.5) U2OS-FUCCI cells examined over three independent experiments. Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. Data were analysed using a two-tailed unpaired t-test (g, o, q) or two-tailed Mann–Whitney test (j, r). For immunoblots, actin or β-tubulin were used as loading control.
a, Total γH2AX nuclear intensity in the different cell-cycle phases of BJ-hTERT cells (n = 3). Data are mean ± s.d. b, Average number of γH2AX foci in control or AMBRA1-silenced U2OS cells (n = 3). c, Alkaline comet assay of control, AMBRA1- and ATG7-silenced U2OS cells (n = 3). d, Scatter plots showing γH2AX versus Hoechst total nuclear intensities from immunostainings of control, AMBRA1- and ATG7-silenced BJ-hTERT cells. The proportion of γH2AX-positive cells (red, arbitrary cut-off) is indicated (siSCR, n = 721; siAMBRA1, n = 725; siATG7, n = 733 cells examined over 3 independent experiments). e, Immunoblot of γH2AX in control, AMBRA1- and ATG7-silenced BJ-hTERT cells (n = 3). f, Homologous recombination (HR) efficiency in control, AMBRA1- and ATG7-silenced U2OS cells (n = 3). Data are mean ± s.d. g, Number of BRCA1 foci per nucleus in control and AMBRA1-silenced U2OS cells either untreated or treated with 3-Gy irradiation, stained against BRCA1 (n = 500 cells examined over 3 independent experiments, centre indicates the mean). h, Time in mitosis in control (n = 91 cells examined over 3 independent experiments) or AMBRA1-silenced (n = 72 cells examined over 3 independent experiments) cells. Bars represent median and interquartile range. i, Dying cells upon mitotic exit as evaluated by time-lapse imaging (n = 2 independent experiments; more than 60 cells per condition). j, Distribution of 53BP1 nuclear foci in G1 U2OS cells (n = 3). k, Total γH2AX versus Hoechst intensity in control and AMBRA1-silenced BJ-hTERT cells that were untreated or treated with 2 mM hydroxyurea (HU) for 2 h (siSCR, n = 2,481; siAMBRA1, n = 2,237; siSCR + HU, n = 2,484; siAMBRA1 + HU, n = 2,281 cells; scatter plots are representative of n = 3 independent experiments). l, Quantification of normalized protein levels of CHK1 represented in Fig. 2e (n = 3). m, n, BJ-hTERT cells as in Extended Data Fig. 4k treated with cycloheximide or with cycloheximide and 2 mM hydroxyurea. m, Immunoblot analysis of the indicated proteins in total cell lysates. n, Quantification of normalized CHK1 protein expression levels (n = 4). o, p, qRT–PCR analyses of the indicated genes in control or AMBRA1-silenced BJ-hTERT (o) and U2OS (p) cells, respectively (CCNA2, E2F1 and RAD51 n = 5; BRCA1 n = 4; CHEK1 n = 3). q, r, Immunoblot analysis of the indicated proteins in control or AMBRA1-silenced U2OS (q) and BJ-hTERT (r) cells (n = 3 in both conditions). s, Gene ontology (GO) biological processes (2018) from enrichment analysis of DEA (Differential Expression Analysis) genes from RNA sequencing (RNA-seq) experiments. DEA originating from three RNA-seq independent experiments was used as input for the web-based software EnrichR,. P values computed using Fisher’s exact test; clearer bars show a smaller P value. Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. Data were analysed using a two-tailed unpaired t-test (a, b, c, f, j, l, n, o, p) or two-tailed Mann–Whitney test (g, h). For immunoblots, β-tubulin, SOD1 or GADPH were used as loading control.
a, Analysis of DEA genes (from n = 3 independent RNA-seq experiments) predicting the transcription factor activated after depletion of AMBRA1. b, qRT–PCR analyses of the indicated genes in control or AMBRA1-silenced U2OS-FUCCI cells sorted for the different cell-cycle phases (n = 3) c, Immunoblot for the indicated proteins in U2OS cells interfered for the indicated autophagy regulators (n = 3). d, Left, violin plot of γH2AX nuclear mean intensity in control and AMBRA1-silenced BJ-hTERT cells that were untreated or treated with 0.1 μM abemaciclib for 48 h. Right, representative scatter plot of single-cell γH2AX nuclear mean intensity versus Hoechst, and cell cycle phase gating strategies from control and AMBRA1silenced BJ-hTERT cells treated with abemaciclib (n = 643 cells). e, Cell count of control U87-MG cells or U87-MG cells with inducible cyclin D1 expression, three days after stimulation with dox (n = 3). f, Cell count of control BJ-hTERT cells or BJ-hTERT cells with inducible cyclin D1 expression at the indicated time points after stimulation with dox, normalized over non-induced cells (1-d V15+, n = 6; 3-d V15+, 3-d E30+, n = 4; 1-d E30+, 4-d V15+, 4-d E30+, 6-d V15+ and 6-d E30+, n = 5). V15+: dox-treated control cells; E30+: dox-treated cyclin D1-inducible cells. g, h, Percentage of cells in each cell-cycle phase in U87-MG (g) and BJ-hTERT (h) cells, control or with inducible cyclin D1 expression, untreated or 48 h after doxycycline stimulation (n = 3). i, Immunoblot for the indicated proteins in control U87-MG cells or U87-MG cells with inducible cyclin D1 expression at the indicated time points with or without dox stimulation (n = 3). j, k, Mean fork speed (j) (kb min−1) and fork symmetry analysis (k) of DNA fibres from control BJ-hTERT cells and BJ-hTERT cells with inducible cyclin D1 expression treated as in Fig. 2d (scored forks: − dox, n = 312; 3-d dox, n = 449; 4-d dox, n = 429; 6-d dox, n = 426). Data are mean ± s.d. l, Quantification of immunohistochemistry staining in Fig. 2g (n = 3 mice). m, Immunoblot for the indicated proteins in wild-type or Ambra1 cKO NSCs (n = 3). Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. Data were analysed using a two-tailed unpaired t-test (b, l), two-tailed Mann–Whitney test (d, j, k), two-way ANOVA followed by Sidak’s multiple comparisons test (e, g, h) or one-way ANOVA followed by Sidak’s multiple comparisons test (f). Exact P values are provided in the ‘Statistical analysis and data reproducibility’ section of the Supplementary Methods.
a, Bioinformatics analysis of expression data from the TCGA database. Pie charts show the percentage of AMBRA1-low cancers (light blue) with respect to the total (grey) in the indicated datasets. BLCA, bladder urothelial carcinoma; COAD, colon adenocarcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; UCEC, uterine corpus endometrial carcinoma. b, Xena correlation analysis of AMBRA1 mRNA expression and stemness score. The shaded area in the plot indicates the confidence interval (95%). c, Lolliplots showing the distribution of AMBRA1 mutations annotated in TCGA Pan-Cancer Atlas Studies datasets. d, Frequency of AMBRA1 mutations (expressed as a percentage) in TCGA Pan-Cancer Atlas Studies datasets. The cut-off was selected at 2%. e, Oncoprint of AMBRA1 alterations (homodeletions, shallow deletions, mutations), and TP53 and EGFR mutations from TCGA Pan-Lung Cancer datasets. f, Mutual exclusivity and co-occurrence analysis of the indicated genes from TCGA Pan-Lung Cancer datasets. P values derived from one-sided Fisher’s exact test. g, Kaplan–Meier analysis of patients in the Pan-Cancer Atlas Studies database was generated based on the expression level of AMBRA1 (low, below 20%; high, above 80%). Plot was downloaded from the online database GEPIA (http://gepia2.cancer-pku.cn/#analysis ). P values derived from one-sided log-rank Mantel–Cox test). h, Kaplan–Meier analysis of overall survival based on RNA-seq analysis of AMBRA1 mRNA levels using the KM-plotter lung adenocarcinoma database.
a, Schematic representations of the mouse model and initial testing of the system. The KrasG12D transgenic mouse is mated with the conditional Ambra1flox/flox mouse to produce the Ambra1+/+::KrasG12D/+ and the Ambra1flox/flox::KrasG12D/+ genotypes. Lung-specific expression of oncogenic KrasG12D and deletion of Ambra1 is induced by intranasal inoculation with defective adenoviral particles carrying the Cre recombinase. b, Immunoblot analysis of AMBRA1 immunoprecipitation from tissue lung samples from Ambra1flox/flox::KrasG12D/+ mice 16 weeks after administration of AdenoCre (n = 3). c, The expression of the Ambra1 floxed allele after Cre administration was verified by RT–PCR performed in lung tissue samples as in c (n = 3). Primers were designed to distinguish wild-type and floxed alleles. d, Representative examples of H&E images of fixed lungs. Bottom, Magnification of the bronchus, highlighting the tumour initiation site. Scale bar, 1 mm. e, Quantification of immunohistochemistry staining in Fig. 3b (Ki67, n = 3; γH2AX, n = 3; RPA(pS4/8), n = 3; cyclin D1, n = 4; c-Myc(pS62), n = 3 in two independent tumours for each condition). Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. P values for γH2AX and cyclin D1 by two-tailed Welch t-test; P values for Ki67, c-MYC(pS62), RPA(pS4/8) by two-tailed unpaired t-test.
a, Ratio between CHEK1 expression in the AMBRA1-low subpopulation of cancers with respect to normal tissue. b, Gating strategy for Fig. 3c. Bottom, scatter plots of total nuclear DNA intensity versus γH2AX intensity (siSCR DMSO, n = 1,850; siSCR AZD, n = 1,716; siAMBRA1 DMSO, n = 1,866; siAMBRA1 AZD, n = 1,731 cells; representative of three independent experiments). The γH2AX-positive cells (arbitrary cut-off) are indicated in red. Top, Hoechst nuclear intensity versus counts. γH2AX-positive cells are indicated by the red line. c, Immunoblot of AMBRA1, RPA(pS4/8) and β-tubulin in control or AMBRA1-silenced BJ-hTERT cells that were untreated or treated with AZD7762. d, Left, gating strategy for the quantification on the right. Top, Hoechst nuclear intensity versus counts. Bottom, scatter plots reporting single-cell total nuclear DNA intensity versus TUNEL intensity. Right, TUNEL-positive cells in the different cell phases calculated based on Hoechst intensity (n = 3). P values by two-tailed unpaired t-test. e, Viability analysis of control and AMBRA1-silenced BJ-hTERT cells treated with the indicated concentrations of LY2603618 for 24 h (n = 3). P values by two-stage step-up (Benjamini, Krieger and Yekutieli). f, Cell viability in control, AMBRA1- and ATG7-silenced BJ-hTERT cells that were untreated or treated with AZD7762 for 24 h (n = 4 for Control and treatments with 100 nM AZD7762). P value by two-tailed unpaired t-test. g, Cell viability in control and AMBRA1-silenced BJ-hTERT cells that were untreated or treated with olaparib for 24 h (n = 3). Analysis by two-tailed unpaired t-test. h, Fork symmetry analysis from BJ-hTERT cells treated for 24 h with 100 nM AZD7762 or 5 μM LY2603618 (scored forks: siSCR DMSO, n = 533; siSCR AZD, n = 560; siSCR LY, n = 548; siAMBRA1 DMSO, n = 601; siAMBRA1 AZD, n = 543; siAMBRA1 LY, n = 548). P values by two-tailed Mann–Whitney test. Data are mean ± s.d. i, Cell viability analysis of control and AMBRA1-silenced A549, H11299 and HCC827 lung cancer cell lines treated with the indicated concentrations of AZD7762 and LY2603618 (n = 3) for 24 h. P values by two-stage step-up (Benjamini, Krieger and Yekutieli). Data are mean ± s.d. j, Immunoblot of the indicated proteins in control or AMBRA1-silenced A549, HCC827 and H1299 cells (n = 3). k, Immunoblot of sarcoma cell lines (n = 3). l, Immunoblot of SKUT-1B cells treated with the inhibitor MLN4924 for 4 h (n = 3). m, Left, immunoblot of SKUT-1B cells reconstituted with wild-type AMBRA1 or mutant AMBRA1(ΔWD40) or AMBRA1(PXP). Right, densitometry quantification of the indicated normalized protein levels (n = 3). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. n, Late apoptosis analysis in SKUT-1B cells reconstituted with wild-type AMBRA1, AMBRA1(ΔWD40) or AMBRA1(PXP) and treated with 200 nM AZD7762 for 24 h (n = 3). P values by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values are provided in the ‘Statistical analysis and data reproducibility’ section of the Supplementary Methods.
a, Cell viability of Ambra1+/+
and Ambra1gt/gt MEFs treated with AZD7762 or vehicle for 24 h (n = 4 independent experiments). P values by two-tailed unpaired t-test. b, Box plots (centre line, median; box limits, 25th and 75th percentile; whiskers, minimum and maximum) indicating weight of Ambra1+/+
and Ambra1gt/gt MEF xenografts referred to in Fig. 3f (Ambra1+/+ + vehicle, n = 8; Ambra1+/+ + AZD7762, n = 8; Ambra1gt/gt + vehicle, n = 10; Ambra1gt/gt + AZD7762, n = 11 mice). P values by two-tailed unpaired t-test. c, Cell death percentage in control U87-MG cells or overexpressing cyclin D1, either untreated or treated with AZD7762 for 24 h; mean ± s.e.m. (n = 3 independent experiments). P values by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. Unless otherwise stated, data are mean ± s.d.
a, Haematoxylin and eosin (H&E)-stained wild-type (WT; Ambra1flox/flox) and Ambra1 cKO (Ambra1flox/flox:: Nestin-cre) brain sections. DPall, dorsal pallium; HPC, hippocampus; LV, lateral ventricle; OB, olfactory bulb; pVZ, pallium ventricular zone. P4, postnatal day 4. Scale bars, 400 μm (top); 1 mm (middle, bottom). b, Sagittal sections from wild-type and Ambra1 cKO E13.5 embryos, stained for Ki67 and Hoechst. Boxed regions are magnified to the right (8× magnification) (n = 5). mVZ, mesencephalic ventricular zone; vDPall, ventricular dorsal pallium; vIC, ventricular inferior colliculus; vSC, ventricular superior colliculus. Scale bar, 250 μm. c, Immunoblot of the indicated proteins from extracts of wild-type and Ambra1 cKO NSCs (n = 4). d, BrdU incorporation in wild-type and Ambra1 cKO NSCs; time points of BrdU administration are indicated (n = 3). e, Sagittal sections of wild-type and Ambra1 cKO E13.5 embryos, stained for cyclin D1 and Hoechst. Boxed regions are magnified below (6× magnification) (n = 5). Scale bar, 250 μm. f, Immunoblot of cyclin D1 and cyclin D2 after treating wild-type and Ambra1 cKO NSCs with cycloheximide (CHX) (n = 3). g, Immunoblot of cyclin D1 in U87-MG cells in which expression of the indicated E3 ubiquitin ligases was knocked down using small interfering RNA (siRNA) (n = 4). h, Top, co-immunoprecipitation of cyclin D1 and AMBRA1 in AMBRA1-overexpressing U87-MG cells (n = 3). IP, immunoprecipitation. Bottom, model of the regulation of cyclin D1 stability by AMBRA1. i, Immunoblot of cyclin D1 and cyclin D3 in U2OS cells in which expression of the indicated cullin proteins was knocked down using siRNA (n = 3). j, Percentage of cells in the indicated phase of the cell cycle (n = 3). Cells were immunostained for cyclin D1, and counterstained with EdU and Hoechst upon AMBRA1 mRNA interference. k, Box plots (centre line, median; box limits, 25th and 75th percentiles; whiskers, minimum and maximum) showing the length of the G1 phase in siAMBRA1 (n = 59 cells) and control siSCR (n = 63 cells) U2OS cells across three independent experiments. Unless otherwise stated, data are mean ± s.e.m.; n refers to biologically independent samples. Data were analysed using a two-sided one-way ANOVA followed by Sidak’s multiple comparisons test (d), one-way ANOVA followed by Sidak’s multiple comparisons test (j) or two-tailed unpaired t-test (k). NS, not significant. Quantifications of immunoblots are shown in Extended Data Figs. 1g, 2a, 3c.
a, Scatter plots reporting single-cell γH2AX, EdU and Hoechst total nuclear intensities from AMBRA1-silenced (siAMBRA1) and control (siSCR) BJ-hTERT cells (siSCR, n = 716 cells; siAMBRA1, n = 715 cells; representative of three independent experiments). b, Left, AMBRA1-silenced and control U2OS cells stained for γ-tubulin (red), histone H3 phosphorylated at Ser10 (H3(pS10); green) and Hoechst (blue). Scale bars, 10 μm. Right, quantification of mitotic cells showing anaphase bridges and lagging chromosomes (chr) (n = 3). c, Left, AMBRA1-silenced and control BJ-hTERT cells immunostained for RPA (siSCR, n = 704 cells; siAMBRA1, n = 720 cells). Scale bars, 10 μm. Right, quantification of the average number of RPA foci and scatter plot of Hoechst intensity versus RPA intensity (n = 3). d, Top, DNA fibres from control and AMBRA1-silenced BJ-hTERT cells. Scale bars, 10 μm. Bottom, quantification of mean fork speed (kb min−1) and of fork symmetry analysis after incorporation and staining of 5-iodo-2’-deoxyuridine (IdU) and 5-chloro-2’-deoxyuridine (CldU). Scored forks: siSCR, n = 301; siAMBRA1, n = 233. Data are mean ± s.d. e, Immunoblot analysis of the indicated proteins in control or AMBRA1-silenced BJ-hTERT cells. f, Immunoblot analysis of cyclin D1 and γH2AX in control BJ-hTERT cells or cells in which cyclin D1 expression was induced by doxycyclin (dox) treatment for the indicated number of days. The prefixes i- and e- indicate the induced and the endogenous form of cyclin D1, respectively (n = 3). g, Sagittal sections of wild-type or Ambra1 cKO E13.5 embryos, stained for γH2AX antibody (n = 3). Scale bar, 40 μm. Quantification of immunohistochemistry is shown in Extended Data Fig. 5l. Actin or β-tubulin were used as loading controls. Unless otherwise stated, data are mean ± s.e.m.; n refers to biologically independent samples. Data were analysed using a two-tailed unpaired t-test (b, c) or two-tailed Mann–Whitney test (d).
a, H&E-stained mouse lung sections showing neoplastic lesions in Ambra1-proficient (Ambra1+/+::KrasG12D/+) and Ambra1-deficient (Ambra1flox/flox::KrasG12D/+) lung tissue 20 weeks after adenoviral infection. Four different samples are shown and are representative of four mice. Scale bar, 1 mm. b, Immunohistochemistry analyses of Ki67, γH2AX, RPA(pS4/8), cyclin D1 and c-MYC(pS62) at 20 weeks after infection. Scale bar, 40 μm. c, Left, AMBRA1-silenced and control BJ-hTERT cells were treated with 100 nM AZD7762 for 24 h and immunostained for γH2AX and Hoechst. Scale bars, 5 μm. Right, quantification of pan-γH2AX-positive cells after inhibition of CHK1 by treatment with AZD7762 for 24 h (n = 3 independent experiments). d, Left, the indicated BJ-hTERT cells were treated with AZD7762 for 24 h and stained with TUNEL and Hoechst. Scale bars, 5 μm. Right, quantification of the average percentage of TUNEL-positive cells (n = 3 independent experiments). e, Survival curves of mice xenotransplanted with control sarcoma SKUT-1B cells or SKUT-1B cells reconstituted with AMBRA1 (SKUT-1B AMB). Mice were treated with vehicle or AZD7762 (n = 4 mice). f, Assessment of the volume of Ambra1+/+ and Ambra1gt/gt MEF xenografts in mice treated with AZD7762 or vehicle (Ambra1+/+ + vehicle, n = 8 for days 17–21, n = 7 from day 24; Ambra1+/+ + AZD7762, n = 8; Ambra1gt/gt + vehicle, n = 10; Ambra1gt/gt + AZD7762, n = 11 mice). Data are mean ± s.d. g, AMBRA1 regulates the G1–S-phase transition by mediating the degradation of cyclin D proteins and c-MYC. A defective AMBRA1–cyclin D axis causes a premature entry into S phase, leading to replication stress and genome instability. The increased DNA damage causes faster tumour growth and neurodevelopmental defects. pRB, phosphorylated RB. Quantification of immunohistochemistry is shown in Extended Data Fig. 7e. Unless otherwise stated, data are mean ± s.e.m. Data were analysed using a two-tailed unpaired t-test (c, d, f) or log-rank Mantel–Cox test (e). In f, day 23,*P = 0.0348; day 24, P = 0.0353; day 25, *P = 0.0228; ****P < 0.0001.
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