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BECN1 promotes radiation-induced G2/M arrest through regulation CDK1 activity: a potential role for autophagy in G2/M checkpoint. Huang, R., Gao, S., Han, Y. et al. Cell Death Discov. 6, 70 (2020). https://doi.org/10.1038/s41420-020-00301-2

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Molecules 2017, 22(12), 2045; https://doi.org/10.3390/molecules22122045

Introduction: Cell cycle control: DNA damage checkpoint

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see notes below for details

Cell cycle control: DNA damage checkpoint (modified from Aarts et al. [55]). Reproduced with permission from Nick Turner, Current Opinion in Pharmacology; published by Elsevier, 2013.

In the cell cycle, there are two checkpoint arrests that allow cells to repair damaged DNA in order to maintain genomic integrity. Many cancer cells have defective G1 checkpoint mechanisms, thus depending on the G2 checkpoint far more than normal cells. G2 checkpoint abrogation is therefore a promising concept to preferably damage cancerous cells over normal cells. The main factor influencing the decision to enter mitosis is a complex composed of Cdk1 and cyclin B. Cdk1/CycB is regulated by various feedback mechanisms, in particular inhibitory phosphorylations at Thr14 and Tyr15 of Cdk1. In fact, Cdk1/CycB activity is restricted by the balance between WEE family kinases and Cdc25 phosphatases. The WEE kinase family consists of three proteins: WEE1, PKMYT1, and the less important WEE1B. WEE1 exclusively mediates phosphorylation at Tyr15, whereas PKMYT1 is dual-specific for Tyr15 as well as Thr14. Inhibition by a small molecule inhibitor is therefore proposed to be a promising option since WEE kinases bind Cdk1, altering equilibria and thus affecting G2/M transitio

Intoduction: Autophagy and Beclin 1 (BECN1) phosphorylation.

Front. Cell Dev. Biol., 12 October 2018 | https://doi.org/10.3389/fcell.2018.00137

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(A) The scheme depicts the phases of autophagosome assembly from initiation to nutrient recycling. The steps include the structural transformation from the pre-autophagosomal structure (PAS) to phagophore to autophagosomes, culminating in the fusion of autophagosomes with lysosomes facilitating the degradation of their contents in autolysosomes. The regulatory protein complexes involved are depicted with their components and the presence of regulatory phosphorylation event/events are shown. (B) Primary structure of BECN1 showing the BCL2/BCL-XL binding BH3 motif (residues 105–130), flexible helical domain (F, residues 141–171), and the central coiled coil domain (CCD, residues 175–265). Evolutionary conserved domain (residues 248–337) and β/α-repeated, autophagy-related (BARA) domain (265–450aa) is represented together as ECD-BARA (Mei et al., 2016). The approximate locations of pro-autophagy (green) and inhibitory (red) phosphorylation sites are shown. The contributions of the different domains to complex formation with interactors are also indicated. (C) Phosphorylation-dependent conversion of inactive BECN1 homodimer/BCL2-complex to an active PI3K-III complex is depicted. The STK4-mediated BECN1-BH3 domain phosphorylation (negative regulator of autophagy), triple phosphorylation of BCL2 which releases BCL2 from BECN1, representative phosphorylation events in the N-terminal domain (NTD) promoting BECN1-BCL2 dissociation as well as activating the PI3K-activity are presented (positive regulation).

Materials and Methods

Cell lines and animal models

Reagents and drugs

Major Assays

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Fig. 1 A-G

Assays:

A. qRT-PCR

B. qRT-PCR

C. Western blotting

D. Western blotting

E. Quantification of Fluorescence Confocal Microscopy of LC3 Puncta

F. Fluorescence Confocal Microscopy

G. Fluorescence Confocal Microscopy

Cell lines:

D-G. MDA-MB-231

Abbreviations and Treatments

3MA: ?

Wortamannin? ?

Complete each panel in Fig 1. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

Fig. 1 A-G

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Fig. 1: BECN1 was overexpressed in MDA-MB-231, A549, and mouse tissues post-exposure of IR.

a. qRT-PCR was performed to detect BECN1 mRNA expression and the asterisk (*) indicates a significant increase post 4 Gy IR exposure compared to the non-IR exposure in MDA-MB-231 cells. b qRT-PCR was performed to detect BECN1 mRNA expression and the asterisk (*) indicates a significant increase post 4 Gy IR exposure compared to the non-IR exposure in A549 cells. c Western blot assay was performed to detect theATG3 and BECN1 expression in MDA-MB-231 cells (n = 5) post 4 Gy IR exposure. d The protein levels of LC3 and SQSTM1/P62 in MDA-MB-231 cells were detected by Western blotting analysis. GAPDH was used as the loading control. MDA-MB-231 cells cotreated or not cotreated with 3-MA were harvested at the indicated timepoints after 4-Gy γ-ray-irradiation. e MDA-MB-231 cells cotreated or not cotreated with 3-MA or wortamannin were harvested at the indicated timepoints after 4-Gy γ-ray-irradiation. The number of LC3 puncta per cell. was analyzed and the asterisk (*) indicates a significant change compared with control group. f Autophagic GFP-LC3 puncta in 4-Gy γ-ray-irradiated MDA-MB-231 cells cotreated with or without 3-MA were detected by fluorescence confocal microscopy(bars: ×20 magnification). g Autophagic WIPI2in 4-Gy γ-ray-irradiated MDA-MB-231 cells cotreated with or without 3-MA was detected by fluorescence confocal microscopy(bars: ×20 magnification). The data are presented as the means ± SDs from three independent experiments *p < 0.01 compared with the control group.

Fig. 2 A-G

Complete each panel in Fig 2. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

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Fig. 2 A-G

Fig. 2: Involvement of autophagy in the regulation of IR-induced EMT.

a. The BECN1 expression level was analyzed in 20 paired mice lung fibrosis tissues with 20 Gy radiation and their normal tissues without 20 Gy radiation by calculating the integrated IOD/area using Image-Pro Plus version 6.0. Three independent experiments were performed, the student’s t-test was utilized to determine the p-value and the asterisk (*) indicates a significant change compared with control group. b IHC staining was performed to evaluate BECN1 expression in 20 paired mice lung tissues with 20 Gy radiation and their normal tissues without 20 Gy radiation. The IHC images were captured using the AxioVision Rel.4.6 computerized image system. c An autophagy flux experiment was performed to test the autophagy process. The protein levels of BECN1 and LC3B in MDA-MB-231 cells were detected by western blotting analysis. GAPDH was used as the control. MDA-MB-231 cells cotreated with or without 3-MA were harvested at the indicated timepoints after 4-Gy γ-ray-irradiation. d An autophagy flux experiment was performed to test the autophagy process. The protein levels of BECN1 and LC3B in MDA-MB-231 cells were detected by western blotting analysis. GAPDH was used as the control. MDA-MB-231 cells cotreated with or without 3-MA were harvested at the indicated timepoints after4-Gy γ-ray-irradiation. e The effect of BECN1 knockout on autophagy flux was examined. The cells were transfected with tandem GFP-LC3-mRFP-LC3ΔG plasmids mediated by the adenovirus, and fluorescence laser confocal microscopy was performed(bars: ×20 magnification). f Quantitative measurement of GFP and mRFP. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the WT cells. g Western blot assay was performed to detect the radiation-induced EMT biomarkers’ alteration post 4 Gy radiation in MDA-MB-231 cells at indicated timepoints.

Fig. 3 A-H

Complete each panel in Fig 3. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

Fig. 3 A-H

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Fig. 3: Involvement of autophagy in the regulation of IR-induced cell-cycle progression arrest.

a Representative flow cytometry histogram of cell-cycle progression in the population of 4-Gy γ-ray-irradiated HeLa cells with or without cotreatment with 3-MA. b Effect of 3-MA on the G2 and M phase distribution in the population of irradiated cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the control group. c Representative flow cytometry histogram of cell-cycle progression in the population of 4-Gy γ-ray-irradiated BECN1-WT and BECN1-KO MDA-MB-231 cells. d Western blotting analysis of BECN1 in BECN1-WT MDA-MB-231 cells (control) and BECN1-KO cells generated by CRISPR/Cas9. GAPDH served as the internal loading control. e proportions of 4-Gy γ-ray-irradiated BECN1-KO and control cells in the G2 and M phases at the indicated times after IR. The data are presented as the means ±Quantitative measurement of the SDs from three independent experiments; *p < 0.05 compared with the control group. f Representative flow cytometry histogram of cell-cycle progression in the population of 4-Gy γ-ray-irradiated ATG7-WT and AG7-KO MDA-MB-231 cells. g Western blotting analysis of ATG7 in ATG7-WT MDA-MB-231 cells (control) and ATG7-KO cells generated by CRISPR/Cas9. GAPDH served as the internal loading control. h Quantitative measurement of the proportion of 4-Gy γ-ray-irradiated ATG7-KO and control cells in the G2 and M phases at the indicated times after IR. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the control group.

Fig. 4. A-G

Complete each panel in Fig 4. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

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Fig. 4. A-G

Fig. 4: Autophagy-related BECN1 deficiency inactivates the G2/M checkpoint in response to IR.

a Representative flow cytometry histograms of phosphorylated histone H3 (pHH3)-positive mitotic cells in the population of γ-ray-irradiated HeLa cells with or without 3-MA cotreatment. The cells were immunostained with pHH3(Ser10) antibody to detect the proportion of pHH3-positive mitotic cells at the indicated times after IR. b Quantitative measurement of pHH3(Ser10)-positive cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.01 compared with the control group. c Representative flow cytometry histogram of the population of γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB-231 cells. The cells were immunostained with pHH3(Ser10) antibody to detect the proportion of pHH3-positive cells at the indicated times after IR. A rescue experiment was conducted by transfecting BECN1-KO MDA-MB-231 cells with lentiviral vectors expressing BECN1. d Quantitative measurement of pHH3(Ser10)-positive cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the WT cells. e Representative image of mitotic protein monoclonal-2 (MPM2)-positive cells obtained through immunofluorescence staining (bars: ×20 magnification). f Quantification of MPM2 antibody-stained mitotic cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the WT cells. g pHH3 was detected by Western blotting analysis. β-actin served as the internal control.

Fig. 5 A- F

Complete each panel in Fig 5. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

Fig. 5 A- F

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Fig. 5: Autophagy inhibition and BECN1 deficiency disturb the response of G2/M checkpoint-related proteins to IR.

a Western blotting analysis of G2/M checkpoint-regulating proteins (pATM, pCHK2, and CHK2) in the γ-ray-irradiated A549 cells that were cotreated or not cotreated with the autophagy inhibitor 3-MA. The proteins were detected at the indicated timepoints after IR, and GAPDH served as the internal control. b Western blotting analysis of the phosphorylation of G2/M checkpoint-regulating proteins (pCDC25C, pCDK1, and CDK1) in the γ-ray-irradiated A549 cells with or without cotreatment with the autophagy inhibitor 3-MA. The phosphorylation analysis was performed at the indicated timepoints after IR, and GAPDH served as the internal control. c Western blotting analysis of G2/M checkpoint-associated phosphorylated proteins (pCHK1, CHK1, pWEE1, and pMYT1) in the γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB-231 cells. GAPDH served as the internal control. d Western blotting analysis of G2/M checkpoint-associated phosphorylated proteins (pCHK2, CHK2, pCDK1, and CDK1) in the γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB-231 cells. GAPDH served as the internal control. e Quantitative measurement of the levels of pATM, pCHK2, pCDC25C, and pCDK1 in A549 cells with or without 3-MA cotreatment detected at the indicated time point after IR. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 between different groups. f Quantitative measurement of phosphorylated protein levels of pCHK1, pWEE1, pMYT1, and pCDK1 in BECN1-WT and BECN1-KO MDA-MB-231 cells at the indicated timepoints after IR. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 between different groups.

Fig. 6 A-D

Complete each panel in Fig 6. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

Fig. 6 A-D

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Fig. 6: BECN1 deficiency and autophagy inhibition inhibits the dissociation of CDC25C from the CDK1 complex after irradiation.

a The levels of phosphorylated CDK1-pY15 protein in BECN1-KO and BECN1-WT MDA-MB-231 cells were detected 1 and 2 h after 4-Gy irradiation by western blot analysis. β-actin served as the internal loading control. b Effects of 3-MA treatment on the protein–protein interactions of the CDK1 complex in MDA-MB-231 cells after irradiation. Cell lysates were collected from DMSO-treated MDA-MB-231 cells and 3-MA-treated cells at 2 h after 4-Gy irradiation, and immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blot analysis was performed using anti-CDK1, anti-CDC25C, and anti-WEEl antibodies. β-actin was used as the internal loading control. c Effects of BECN1 deficiency on the protein–protein interactions of the CDK1 complex after irradiation. Cell lysates were collected from BECN1-WT and BECN1-KO MDA-MB-231 cells at the indicated timepoints after 4-Gy irradiation, and immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blotting analysis was performed using anti-CDK1, anti-CDC25C, and anti-WEEl antibodies. β-actin was used as the internal loading control. d Effects of BECN1 deficiency and overexpression on the protein–protein interactions of the CDK1 complex in MDA-MB-231 cells with or without 4-Gy irradiation. Cell lysates were collected from BECN1-WT cells, BECN1-KO cells, and BECN1-KO cells transfected with exogenous BECN1 mediated by lentiviral vectors with or without 4-Gy irradiation, and immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blotting analysis was performed using the indicated antibodies. β-actin was used as the internal loading control.

Fig. 7 A-F

Complete each panel in Fig 7. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

Fig. 7 A-F

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Fig. 7: The radiation-induced cytoplasmic translocation of CDC25C is blocked under BECN1-deficient conditions.

a The levels of CDC25C and BECN1 proteins in the cytoplasm and nucleus, respectively, in BECN1-KO and BECN1-WT MDA-MB-231 cells after 4-Gy irradiation were detected by Western blot analysis. β-actin served as the internal loading control. Tubulin and LAMIN A/C proteins were detected as the control cytoplasmic and nuclear proteins, respectively. Exogenous BECN1-expressing vectors were transfected into BECN1-KO cells for the rescue experiment. b CDC25C and CDK1 in BECN1-WT and BECN1-KO MDA-MB-231 cells with or without 4-Gy γ-ray-irradiation were detected by immunofluorescence confocal microscopy(bars: ×20 magnification). c CDC25C and CDK1 in BECN1-WT and BECN1-KO MDA-MB-231 cells with or without 4-Gy γ-ray-irradiation were detected by immunofluorescence confocal microscopy. Exogenous BECN1-expressing vectors were transfected into BECN1-KO cells for the rescue experiment(bars: ×20 magnification). d. The BECN1 levels after 4-Gy γ-ray-irradiation with or without 3-MA treatment were assayed by immunofluorescence microscopy (bars: ×20 magnification). e The levels of BECN1, tubulin and LAMIN A/C proteins in the cytoplasm and nucleusafter 4-Gy irradiation with or without cotreatment with 3-MA were detected by Western blot analysis. β-actin served as the internal loading control. f Effects of siRNA-mediated knockdown of ATG5 and ATG7 on the radiation-induced nuclear translocation of BECN1 (cells???)

Fig. 8. A-F

Complete each panel in Fig 8. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

Fig. 8. A-F

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Fig. 8: BECN1 mediates the interaction of CDC25C with CHK2.

a The interactions of BECN1 with CDC25C, CHK2, CHK1, 13-3-3α, WEE1, CDK1, CCNB1, and BCL2in MDA-MB-231 cells with or without 4-Gy irradiation were assayed, and immunoprecipitates were prepared with anti-BECN1 or anti-IgG antibodies. Western blot analyses were performed using the indicated antibodies. b The interactions of BECN1 with CDC25C and CHK2 in either the cytoplasm or nucleusof MDA-MB-231 cells with or without 4-Gy irradiation were assayed, and immunoprecipitates were prepared with anti-BECN1, anti-CHK2, anti-CDC25C, or anti-IgG antibodies. Western blot analysis was performed using anti-CDC25C and anti-CHK2 antibodies. c BECN1-truncated mutants were constructed according to the listed illustrator. d Flag-BECN1, flag-BECN1-A, flag-BECN1-B, flag-BECN1-C, flag-BECN1-D, flag-BECN1-E, and empty vector were overexpressed in MDA-MB-231 cells. Immunoprecipitation was performed using a flag antibody. e Flag-BECN1, flag-BECN1-A, flag-BECN1-B, flag-BECN1-C, flag-BECN1-D, flag-BECN1-E, and empty vector were overexpressed in BECN1-KO MDA-MB-231 cells. Immunoprecipitation was performed using anti-CDC25 antibody, and western blot analysis was performed using anti-CDC25C and anti-CHK2 antibodies. f The cells (??????) were transfected with the indicated Flag- or HA-tagged expression vectors, and the immunoprecipitations were performed using anti-HA, anti-Flag, or anti-IgG antibodies. Western blot analyses were performed using anti-Flag, anti-HA, anti-CDK1, and anti-CCNB1 antibodies. β-actin served as the internal loading contro