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. 2018 Jan 25;37(4):427-438.
doi: 10.1038/onc.2017.340. Epub 2017 Oct 2.

AKT overactivation can suppress DNA repair via p70S6 kinase-dependent downregulation of MRE11

D Piscitello  1 , D Varshney  2 , S Lilla  1 , M G Vizioli  3 , C Reid  3 , V Gorbunova  4 , A Seluanov  4 , D A Gillespie  5 , P D Adams  3
Affiliations

AKT overactivation can suppress DNA repair via p70S6 kinase-dependent downregulation of MRE11

D Piscitello et al. Oncogene. .

Abstract

Deregulated AKT kinase activity due to PTEN deficiency in cancer cells contributes to oncogenesis by incompletely understood mechanisms. Here, we show that PTEN deletion in HCT116 and DLD1 colon carcinoma cells leads to suppression of CHK1 and CHK2 activation in response to irradiation, impaired G2 checkpoint proficiency and radiosensitization. These defects are associated with reduced expression of MRE11, RAD50 and NBS1, components of the apical MRE11/RAD50/NBS1 (MRN) DNA damage response complex. Consistent with reduced MRN complex function, PTEN-deficient cells fail to resect DNA double-strand breaks efficiently after irradiation and show greatly diminished proficiency for DNA repair via the error-free homologous recombination (HR) repair pathway. MRE11 is highly unstable in PTEN-deficient cells but stability can be significantly restored by inhibiting mTORC1 or p70S6 kinase (p70S6K), downstream kinases whose activities are stimulated by AKT, or by mutating a residue in MRE11 that we show is phosphorylated by p70S6K in vitro. In primary human fibroblasts, activated AKT suppresses MRN complex expression to escalate RAS-induced DNA damage and thereby reinforce oncogene-induced senescence. Taken together, our data demonstrate that deregulation of the PI3K-AKT/ mTORC1/ p70S6K pathways, an event frequently observed in cancer, exert profound effects on genome stability via MRE11 with potential implications for tumour initiation and therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PTEN deficiency suppresses DNA damage signalling via MRN complex hypomorphism. (a) Western blots of indicated proteins were performed on whole-cell extracts from HCT116 and DLD1 (WT or PTEN−/−) cells 1h post irradiation with 10 Gy. Actin provides the loading control. (b) Western blot analysis was performed on whole-cell extracts from HCT116 WT cells 48 h post transfection with 200 nm smart pool siRNA against MRE11 or non-targeting siRNA pool and 1 h post irradiation with 10 Gy. Actin provides the loading control. (c) Western blot analysis was performed on whole-cell extracts from HCT116 WT cells, generated 1 h after exposure to 10 Gy irradiation and pre-treatment with 50 and 100 μm Mirin for 40 min. Actin provides the loading control.
Figure 2
Figure 2
Checkpoint proficiency and DNA repair is attenuated in the absence of PTEN. (a) FACS analysis measuring phopho-histone H3 levels and PI incorporation in replicate cultures of HCT116 WT and PTEN−/− cells with or without prior exposure to different doses of IR and incubation with nocodazole for 9 h. FACS data present the mitotic index (ratio of cells in M-phase positive for phosphorylated H3 over total number of cells × 100). The data are normalized to the nocodazole (noc) sample not exposed to IR and average±S.D. of n=3 are plotted. (b) Immunofluorescence analysis of phosphorylated H2Ax (γ-H2AX) and RPA foci in HCT116 WT with or without pre-treatment with Mirin, and in PTEN−/− cells 4 h after exposure to 10 Gy IR. Quantification of the number of RPA foci per nuclei. Error bars show S.D. for three independent biological replicates. (c) Frequency of NHEJ and HR was analysed with two independent NHEJ and HR constructs in HCT116 WT and PTEN−/− cell lines. The WT cell line was either pre-transfected with control siRNA or siRNA pool against MRE11 or pre-treated with 15 μm Mirin 40 min prior to transfection with linearized constructs. The ratio of GFP+/DsRed+ cells was used to measure repair efficiency and is presented as a percentage of the untreated WT control. Error bars show s.e.m. for three independent biological replicates. ** denote P<0.01 as calculated by Student’s t-test. (d) Survival curves plotted with the number of colonies determined by the GelCount (Oxford Optronix, Abingdon, UK). % Survival was calculated relative to the cells untreated with IR. Error bars show s.d. for three independent biological replicates.
Figure 3
Figure 3
MRE11 is rapidly degraded in PTEN-deficient HCT116 cells. (a) RT–qPCR analysis of MRE11, RAD50 and NBS1 mRNAs in HCT116 WT and PTEN−/− cells. Data were normalized to ACTIN mRNA and are presented relative to the WT signal. The error bars represent the s.d. of three independent biological replicates. (b) Western blot analysis of HCT116 WT and PTEN−/− cells following treatment with cycloheximide for 7 and 24 h. p53 provides the positive control and actin provides the loading control. The graph represents the quantification of the MRE11 western blot analysis using ImageJ. (c) Western blot of HCT116 WT and PTEN null cells 32 h post transfection of ectopic MRE11A with or without cycloheximide treatment for 8 and 24 h. The graph represents the quantification of the MRE11A-Myc-Flag western blot signal by ImageJ.
Figure 4
Figure 4
MRE11 suppression is stimulated by mTORC1 and S6 kinase signalling. (a) Western blot analysis of HCT116 WT cells transiently transfected with GAG-AKT and lysed at the indicated time-points post transfection. Actin provides the loading control. (b) Western blot analysis of HCT116 PTEN−/− cells treated for 72 h with different inhibitors targeting various kinases within the AKT/mTOR/S6K pathway. Seventy-two hours post treatment, cells were exposed to IR and 1 h after were analysed. Actin provides the loading control.
Figure 5
Figure 5
S6K1 phosphorylates MRE11A in vitro. (a) Sequence analysis of MRE11A reveals the presence of an S6K1 consensus sequence. The analysis identified a site on T597. (b) SDS–PAGE performed on recombinant Myc-MRE11A incubated with γ32P-ATP and varying amounts of recombinant S6K1. The lower panel shows Coomassie staining. (c) Scintillation counts of MRE11 bands cut from the SDS–PAGE gel quantifying 32P incorporation into MRE11A.
Figure 6
Figure 6
MRE11 degradation is mediated by S6K1 phosphorylation on T597. (a) Positive ion MS/MS spectra, of tryptic phosphopeptides (593–604) and (617–625) of MRE11. The b and y ion series are indicated in blue and red, respectively. Dashed lines indicates b and y ion fragments used to infer phosphorylation positions. (b) Summary of a. (c) Western blot of HCT116 PTEN null cells expressing ectopic MRE11A WT or MRE11A T597A or MRE11A T597D 24 h post transfection. Actin provides the loading control.
Figure 7
Figure 7
Activated AKT pathway enhances RAS-induced senescence by inhibition of MRN complex. (a, c) Western blot analysis on IMR90 cells 12 days post infection with retroviruses expressing HRASG12V, Myr-AKT, HRASG12V/Myr-AKT or control virus (CTR). Actin provides a loading control. (b, top) Immunofluorescence images obtained following an alkaline comet assay with IMR90 cells 12 days post infection with CTR, HRASG12V, Myr-AKT or HRASG12V and Myr-AKT retroviruses. (b, bottom) Total DNA damage (sum of single and double DNA breaks) was measured as percentage of DNA migrated in the tail of the comet over the total signal from the head and tail. Error bars show S.D. for three independent biological replicates. *** denotes P<0.001 as calculated by Student’s t-test. Data were quantified by using ImageJ OpenComet, and it was derived from three independent biological replicates. (c) Western blot analysis on IMR90 cells 12 days post infection with retroviruses expressing HRASG12V, Myr-AKT, HRASG12V/Myr-AKT or control virus (CTR). Actin provides a loading control. (d) Quantification of SA β-galactosidase-positive cells plotted as a percentage of total cells. The quantification was performed on the average of 100 cells per sample.
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