Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion

Abstract

Cellular senescence suppresses cancer by stably arresting the proliferation of damaged cells. Paradoxically, senescent cells also secrete factors that alter tissue microenvironments. The pathways regulating this secretion are unknown. We show that damaged human cells develop persistent chromatin lesions bearing hallmarks of DNA double-strand breaks (DSBs), which initiate increased secretion of inflammatory cytokines such as interleukin-6 (IL-6). Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs). Initiation and maintenance of this cytokine response required the DDR proteins ATM, NBS1 and CHK2, but not the cell-cycle arrest enforcers p53 and pRb. ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence. Furthermore, DDR activity and IL-6 were elevated in human cancers, and ATM-depletion suppressed the ability of senescent cells to stimulate IL-6-dependent cancer cell invasiveness. Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.

Figure 1. Persistent DNA damage signaling correlates with IL-6 secretion

(a) HCA2 fibroblasts were untreated, irradiated with 0.5 Gy, or irradiated with 10 Gy and allowed to recover. Cells were fixed and stained for 53BP1 foci (red) at the indicated intervals after irradiation. (b) Whole cell lysates were prepared at the indicated intervals after irradiation and analyzed by western blotting for the indicated proteins. P-p53, serine 15-phosphorylated p53. (c) HCA2 cells were irradiated with 0.5 or 10 Gy and allowed to recover for the indicated intervals before being fixed and stained for 53BP1 foci. Nuclei with 3 or more foci were quantified. (d) HCA2 cells were irradiated with 10 Gy and analyzed 6 d later for γ-H2AX (red) and the Ser/Thr phosphorylated ATM/ATR substrate motif (p-STK Sub; green) (top panels), or 53BP1 (red) and activated (phosphorylated) ATM (p-ATM; green) (bottom panels). The merged red and green channels display co-localization in yellow. (e) HCA2 cells were irradiated with 0.5 or 10 Gy, and IL-6 secretion was assessed using ELISA over 10 d for the indicated 24 h windows. The data are reported as the fold increase in secretion relative to untreated control cells. (f) Cells were infected with lentiviruses or treated as indicated (expression of p16^INK4A, p16; replicative senescence, SEN-REP; telomere uncapping using a dominant-negative TIN2 protein,TIN2-DN; X-irradiation, X-Ray; hydrogen peroxide, H₂O₂; expression of activated RAS^V12, RAS). Ten days later, cells were analyzed by immunofluorescence to quantify the fraction of cells with 3 or more 53BP1 foci. For representative images of 53BP1 damage foci and for other senescence markers see Supplementary Information, Figs. S1e, S1f-I. (g) Conditioned media from the cell populations described in Fig. 1F were collected over a 24 h interval, and analyzed for IL-6 by ELISA. The data are reported as fold increase in secretion relative to untreated control cells, and are plotted on a log scale (n= number of cell populations analyzed).

Figure 2. Telomeric PDDF promotes increased IL-6 secretion

(a) HCA2 populations were cultured until replicative senescence (PD>71). Top panel: Populations were sampled at the indicated PDs (lower panel X axis). Single cells were analyzed for DNA synthesis (BrdU incorporation over 24 h) and 53BP1 foci (cells with 3 or more foci). Lower panel: Conditioned media were collected over a 24 h interval from the populations analyzed in the top panel. IL-6 was measured by ELISA, and is reported as 10⁻⁶ pg/cell/day (n= number of cell populations analyzed). (b) HCA2 cells at the indicated PDs were fixed and single cells analyzed simultaneously for 53BP1 foci (green) and BrdU incorporation over a 24 h interval (red). Arrows mark nuclei that simultaneously harbor 53BP1 foci and BrdU staining. (c) Cells at PD35 were pulsed with BrdU for 1 h and fixed. Single cells were analyzed simultaneously for 53BP1 foci and BrdU incorporation. BrdU incorporation was scored independent of 53BP1 foci (Total cells) or only in cells with 53BP1 foci (53BP1+). (d) Cells at the indicated PD levels were pulsed with BrdU for 24 h and fixed. Single cells were analyzed by immunofluorescence for intracellular IL-6 (green) and BrdU incorporation (red). (e) Cells were infected with a retrovirus expressing hTERT, selected and cultured for the indicated doublings (PD after hTERT). At the indicated PDs, cells were analyzed for 3 or more 53BP1 foci (top panels) or IL-6 secretion (lower panels), as described for Fig. 2a. (f) Early passage HCA2 or HCA2 cells expressing hTERT were irradiated with 10 Gy, allowed to recover for 8 d, and analyzed for 53BP1 foci (top panel) and IL-6 secretion as described for Fig. 2a. IL-6 secretion is reported as the fold increase compared to unirradiated control cells. There was a non-significant reduction in 53BP1 foci and slight reduction in IL-6 levels (p=0.036, two-tailed student T-test for unpaired samples) in hTERT cells.

Figure 3. Loss of p53 accelerates PPDF formation and IL-6 secretion

(a) Replicatively senescent HCA2 cells were infected with the indicated lentiviruses and IL-6 secretion was analyzed 3−4 PDs following reversal of the senescence arrest. IL-6 secretion is reported as 10⁻⁶ pg/cell/day on a log scale (n= number of cell populations analyzed). (b) Early passage HCA2 cells were infected with a GSE22-expressing retrovirus, selected for 4 d (∼3 PDs) and IL-6 secretion was measured ∼2 PDs later. Cells were cultured for the indicated PDs and assessed for 53BP1 foci (top panel) and IL-6 secretion (lower panel) (10⁻⁶ pg/cell/day on a log scale) (n= number of cell populations analyzed). Because GSE22 prevents p53 tetramerization, which is required for transactivation and rapid degradation, GSE22-expressing cells contained abundant inactive p53 protein (see Supplementary Information, Fig. S3a). (c) Early passage HCA2 cells (PD35) were infected with lentiviruses expressing either shp53 or eGFP, selected and cultured until PD55, and analyzed for IL-6 secretion (10⁻⁶ pg/cell/day on a log scale). (d) Two HCA2-GSE22 populations were infected with a retrovirus expressing hTERT and analyzed for 53BP1 foci and IL-6 secretion as described above.

Figure 4. DDR signaling is required for the cytokine response to PDDF

(a) HCA2 cells were infected with lentiviruses expressing shRNAs against GFP (control, shGFP3) or ATM (shATM-11, −12, or −13) and allowed to recover for 5 d. Whole cell lysates were analyzed by western blotting for the indicated proteins. Ponceau staining shows the total proteins. (b) HCA2 cells were infected as above and irradiated with 10 Gy X-Ray. After 9 d, CM were collected over 24 h and assessed for IL-6 by ELISA. IL-6 secretion is reported as fold change over unirradiated shGFP control. ND=not detected. (c) Replicatively senescent HCA2 were infected as above and allowed to recover for 6 d. Conditioned media CM were collected over 24 h and assessed for IL-6 secretion (reported as fold change over shGFP control). (d) A-T cells were irradiated with 10 Gy and allowed to recover for 2 or 10 d. CM were collected over 24 h, and analyzed for IL-6 by ELISA (reported as fold increase compared to unirradiated A-T cells; numerical values are given above the bars). (e) Seckel syndrome cells were irradiated and analyzed for IL-6 as described above for A-T cells. (f-g) HCA2 cells were infected with lentiviruses expressing shRNAs against GFP (control, shGFP3), NBS1 (shNBS1−1, −2, −6, or −7)) or CHK2 (shCHK2−2, −12, or −13), selected and allowed to recover for 7 d. Whole cell lysates were analyzed by western blotting for the indicated proteins. The arrow indicates CHK2; NS indicates a non-specific band detected by the antibody. (h) HCA2 cells from (Fig4f-g) with the indicated shRNA-expressing lentiviruses were irradiated with 10 Gy X-Ray. After 2 d, CM were collected over 24 h and assessed for IL-6 using ELISA (reported as fold change over unirradiated shGFP control; numerical values are given above the bars). (i) Replicatively senescent HCA2 cells were infected with indicated shRNA-expressing lentiviruses and allowed to recover for 6 d. CM were collected over 24 h and assessed for IL-6(reported as fold change over shGFP control; numerical values are given above the bars).

Figure 5. ATM modulates a SASP subset

(a) Factors secreted by presenescent (PRE) or senescent HCA2 (10 d following 10 Gy) were analyzed using antibody arrays. Signals above the PRE baseline are in yellow, signals below baseline in blue. Factors that differed significantly between PRE and XRA cells are displayed (p<0.05, student unpaired T-Test). (b) SASP factors in (A) were analyzed in X-irradiated ATM-depleted cells (XRA(shATM)) and compared to irradiated-senescent HCA2-shGFP cells (XRA). Changes are reported as XRA(shATM) divided by XRA levels. Signals below the XRA baseline are in blue, factors not substantially modulated by ATM-deficiency in gray. (c) T47D invasion stimulated by presenescent (PRE) HCA2 CM was given a value of 1. Compared to PRE and serum-free (SF) media, invasion was significantly (p<0.05) stimulated by 10% serum (FBS), and CM from irradiated HCA2 (XRA), HCA2-shGFP (XRA-shGFP), and HCA2-shATM plus IL-6 (XRA-shATM+IL-6), but not by CM from HCA2-shATM (XRA-shATM) (n=number of samples analyzed). Recombinant IL-6 was added to levels detected by ELISA in XRA-shGFP (3ng/ml). (d) Human breast tissues were analyzed for ATM/ATR serine threonine kinase substrates (p-STK Sub) or IL-6 by immunofluorescence. Data are reported as arbitrary units, with the average normal signal=1 (Signal Intensity). Significance was determined by two-tailed student T-test for unpaired samples using representative fields from 21 normal and 37 cancer tissues. (e) HCA2 cells were untreated or infected with lentiviruses expressing RAS and shRNAs against GFP (shGFP2) or ATM (shATM2), and pulsed with BrdU for 24 h 9−10 d after infection. (f) HCA2 or A-T cells were untreated or infected as in (e) and photographed 6 d later. (g) HCA2 cells infected as in (e) were fixed 10 d later and analyzed for 53BP1 (red), phospho-ATM (green) and DNA (DAPI, blue). Top panel=merge; bottom panels=grayscale for 53BP1 or p-ATM. (h) A-T fibroblasts were untreated or infected as in (e). CM was collected for 24 h 9−10 d after infection and analyzed for IL-6 (fold increase relative to untreated cells; magnitude is given over the bars). (i) HCA2 cells were untreated or infected as in (e) and IL-6 secretion analyzed as above.