KDM4 Orchestrates Epigenomic Remodeling of Senescent Cells and Potentiates the Senescence-Associated Secretory Phenotype

Abstract

Cellular senescence restrains the expansion of neoplastic cells through several layers of regulation. We report that the histone H3-specific demethylase KDM4 is expressed as human stromal cells undergo senescence. In clinical oncology, upregulated KDM4 and diminished H3K9/H3K36 methylation correlate with poorer survival of patients with prostate cancer after chemotherapy. Global chromatin accessibility mapping via assay for transposase-accessible chromatin with high-throughput sequencing, and expression profiling through RNA sequencing, reveals global changes of chromatin openness and spatiotemporal reprogramming of the transcriptomic landscape, which underlie the senescence-associated secretory phenotype (SASP). Selective targeting of KDM4 dampens the SASP of senescent stromal cells, promotes cancer cell apoptosis in the treatment-damaged tumor microenvironment, and prolongs survival of experimental animals. Our study supports dynamic changes of H3K9/H3K36 methylation during senescence, identifies an unusually permissive chromatin state, and unmasks KDM4 as a key SASP modulator. KDM4 targeting presents a new therapeutic avenue to manipulate cellular senescence and limit its contribution to age-related pathologies, including cancer.

Keywords: H3K9/H3K36 demethylation; KDM4; age‐related pathology; aging; cellular senescence; epigenetic modification.

FIGURE 1

Histone H3 lysine sites are epigenetically modified upon cellular senescence. (a) Schematic of SILAC‐based identification of intracellular proteins in presenescent (PRE) versus senescent (SEN) cells of the human stromal line PSC27. (b) Column statistics of different categories of protein molecules in output data after SILAC analysis. An asterisk represents identified proteins (732). Data are representative of three independent experiments. (c) Scatterplot of proteins identified by the SILAC procedure. Protein sequence coverage was plotted against protein mass (447 quantifiable). (d) A representative plot derived from characterization of tandem mass spectrometry (MS/MS)‐based quantitative proteomics profiling. For MS scans, the m/z scan range was from 100 to 1100. Intact peptides were detected in the Orbitrap at a resolution of 70,000 with an automatic gain control target value of 1e5. (e) Heatmap depicting genes significantly upregulated in SEN cells after bleomycin (BLEO) treatment. CTRL, control. Genes are ordered by their expression fold change (highest on top) in PRE versus SEN cells after RNA‐seq. (f) Immunoblot analysis of key molecules in DDR, cellular senescence, and the SASP in PSC27 cells induced to senescence by chemotherapeutic agents (TIS), replicative exhaustion (RS) or oncogene‐induced senescence (OIS). PN, passage number. p15, p25, p35 represent different passages in culture. Vector denotes the empty control for human HRasG12V. H3K9me2/3 and H3K36me2/3 are histone H3 methylation markers. CIS, cisplatin; CARB, carboplatin; SAT, satraplatin; DOXO, doxorubicin. (g) IF staining of γH2AX, H3K9me3, and H3K36me3 in PSC27 cells after chemotherapeutic treatment (SAT, BLEO and MIT), replicative exhaustion (REP) or oncogene activation (HRasG12V). Scale bars, 5 μm. Data in (f, g) are representative of three independent biological replicates.

FIGURE 2

KDM4A and KDM4B are expressed in human prostate tumor stroma and correlate with adverse clinical survival. (a) Histological images of KDM4A/B in human PCa tissues. Left, before chemotherapy. Right, after chemotherapy. Scale bars, 100 μm. HE, hematoxylin and eosin. (b) Pathological assessment of stromal KDM4A/B in PCa tissues (42 untreated versus 48 treated). Participants are pathologically assigned into four categories per IHC staining intensity in stroma: 1, negative; 2, weak; 3, moderate; 4, strong expression. p values were determined by two‐way analysis of variance (ANOVA) with Bonferroni's post hoc test. (c) Comparative analysis of KDM4A/B before (B) and after (A) chemotherapy. Each dot represents an individual, with (B and A) data points connected to allow direct profiling per individual (n = 10 participants). p values were determined by two‐sided unpaired t‐test. ^# p > 0.05. *p < 0.05; from left to right, p = 0.6152, p = 0.8451, p = 0.0486, p = 0.5253, p = 0.7660, p = 0.8279, p = 0.0179, and p = 0.4612. (d) Landscape of pathological correlation between KDM4A/B, CXCL8, WNT16B, p16^INK4a, p21^CIP1, Ki67, and PCNA in stroma of individuals with PCa after chemotherapy. Scores derived from histological assessment per factor (48 patients after treatment). (e) Statistical correlation (Pearson analysis) between pathological scores of KDM4A and CXCL8/WNT16B expressed in 48 individuals with PCa. p values were determined by two‐sided unpaired t‐test and adjusted for multiple comparisons. (f) Similar to data in (e) but showing the correlation of KDM4B and CXCL8/WNT16B. (g) Kaplan–Meier analysis of individuals with PCa. DFS stratified according to KDM4A expression (low, average score < 2, green line, n = 20; high, average score ≥ 2, red line, n = 28). (h) Kaplan–Meier analysis of individuals with PCa. DFS stratified according to KDM4B expression (low, average score < 2, yellow line, n = 22; high, average score ≥ 2, pink line, n = 26). Data in (a) are representative of three independent biological replicates. Statistics of (g and h) survival curves derived from Kaplan–Meier analysis, with p values calculated using a two‐sided log‐rank (Mantel‐Cox) test. Data in b are shown as mean ± SD. and representative of three biological replicates. HR, hazard ratio; CI, confidence interval; DFI, disease‐free interval.

FIGURE 3

KDM4 expression is regulated at the posttranslational in senescent cells. (a) Time‐course analysis of KDM4 expression in PSC27 cells after BLEO treatment. Cell lysates were collected at indicated time points after treatment and subject to immunoblot assays. GAPDH, loading control. (b) Time‐course measurement of transcript expression of KDM4 subfamily members and IL‐6, CXCL8, p16^INK4a, and p21^CIP1 in stromal cells after treatment. p values were determined by one‐way ANOVA and adjusted for multiple comparisons. (c) Immunoblot of KDM4 expression in stromal cells treated by BLEO and/or CHX. The upper panel shows a schematic representation of experimental design and timeline. Cell lysates were collected at the indicated time points after CHX addition to media. (d) Immunoblot of KDM4 expression in stromal cells treated by BLEO and/or MG132. Cell lysates were collected after control or BLEO‐induced senescent cells were treated by MG132 for 12 h. (e) Immunoblot appraisal of KDM4 protein levels in PSC27 cells treated by MG132. Cell lysates were collected at time points as indicated by the experimental scheme. (f) Evaluation of KDM4 protein PTM via IP followed by immunoblot assays. Anti‐KDM4A/B was used for IP, with precipitates subjected to immunoblot assay with KDM4A/B antibody. Anti‐ubiquitin was used to probe the ubiquitination profile of KDM4A/B. (g) IF staining of KDM4A/B and p‐53BP1 in stromal cells. Cells were treated by BLEO and subjected to IF staining 7 days later. KDM4A/B, green; p‐53BP1, red. Nuclei (DAPI), blue. Scale bars, 5 μm. (h) Comparative statistics of the average number of foci where KDM4A/B and p‐53BP1 are colocalized in damaged PSC27 per cell. (i) Statistics of stromal cells displaying nuclear colocalization of KDM4A/B and p‐53BP1 in control versus SEN cells. p values were determined by two‐way ANOVA with Bonferroni's post hoc test. (j) Immunoblot assessment of KDM4 and H3K9/H3K36 methylation after chromatin fragmentation. Histone H3, loading control for nuclear lysates. Data in all bar plots are the mean ± SD and representative of three biological replicates. Data in (a, c–e, f, g and j) are representative of three replicates. In (b and h), p values were determined by two‐sided unpaired t‐test and adjusted for multiple comparisons.

FIGURE 4

SASP expression is enhanced but H3K9/H3K36 methylation is attenuated by the histone demethylase KDM4A. (a) Quantitative assessment of SASP expression. PSC27 cells were transduced with a lentiviral construct encoding human KDM4A and/or exposed to BLEO before collection for expression assays. Signals were normalized to control cells (transduced with empty vector and untreated). (b) Immunoblot assay of DDR signaling, H3K9/H3K36 methylation, and SASP expression in cells processed in different ways, as described in (a). GPADH, loading control. Chromatin fractionation was performed to evaluate KDM4A/B in nuclei. Histone H3 was the nuclear lysate loading control. (c) Representative images of SA‐β‐gal and BrdU staining of PSC27 cells subjected to treatment as described in (a). Scale bar, 10 μm. (d) Comparative statistics of SA‐β‐gal and BrdU staining results of stromal cells in individual conditions of (a). (e) Expression profiling of hallmark SASP factors in PSC27 cell sublines transduced with lentiviral constructs encoding KDM4A‐specific shRNAs. Scrambled, shRNA control. Cells were subjected to vehicle or BLEO treatment before collection. (f) Expression curves of hallmark SASP factors in stromal cells treated in conditions as described in (e). Cells lysed at the indicated time points after BLEO damage. (g) Immunoblot assays of stromal cells transduced with lentiviral constructs encoding human SUV39H1 (full length), SETD2M (histone methylase domain SET, tagged with HA) or both. Cells were subjected to BLEO treatment after transduction, and lysates were collected 7–10 days later. GAPDH, loading control. (h) Immunoblot analysis of stromal cells treated with BLEO and/or Bay 11–7082 (BAY). Cell lysates were collected 7–10 days after treatment. Nuclear lysates were also prepared to assess nuclear translocation of representative NF‐κB subunits, p65 (RelA) and p50/p105. GAPDH, loading control. BAY (Bay 11–7082), an NF‐κB inhibitor. (i) Quantitative assessment of SASP expression. Cells were subjected to treatment(s) as described in (h). Signals were normalized to CTRL cells per gene. Data in all bar plots shown as mean ± SD and representative of three biological replicates. In (a, e, f, and i) p values were determined by two‐sided unpaired t‐tests and adjusted for multiple comparisons. Data in (b, c, g, and h) are representative of three biological replicates. GM‐CSF, granulocyte‐macrophage colony‐stimulating factor.

FIGURE 5

Targeting KDM4 with a small molecule inhibitor interferes with the SASP but not cell growth or cell cycle arrest. (a) Heat map depicting the influence of DNA damage and ML324, a selective chemical inhibitor of KDM4, on the transcriptomic expression profile of PSC27 cells. Genes sorted by expression fold change when comparing between cells treated by CTRL versus BLEO (highest on top). Asterisks indicate canonical SASP factors affected by ML324. (b) Graphic visualization of pathways by GO profiling. Significantly enriched genes were those downregulated and sorted according to their fold change when senescent cells were exposed to ML324. GPCR, G‐protein‐coupled receptors; BCR, B cell receptor; PID, pelvic inflammatory disease; FRA, Fos‐related antigen. (c) Venn diagram representation of genes upregulated by BLEO (673, relative to CTRL) and downregulated genes by ML324 (348, in relative to BLEO). (d) Gene‐set enrichment analysis profiling of gene expression with significant enrichment scores showing a SASP‐specific signature in BLEO/ML324 co‐treated cells compared with BLEO only‐treated cells. FDR, false discovery rate; NES, normalized enrichment score. (e) In vitro colony formation assay of cells exposed to BLEO and/or ML324 treatment. The upper images are representative images of crystal violet staining; the graph in the lower panel indicates comparative statistics. Scale bar, 200 μm. p values were determined by two‐sided unpaired t‐tests and adjusted for multiple comparisons. (f) SA‐β‐gal staining of cells after treatment by BLEO and/or ML324. Left, representative images. Scale bar, 10 μm. Right, statistics. (g) BrdU staining of cells treated as described in (e). Scale bar, 10 μm. (h) Time course expression of a subset of SASP factors (CXCL8, CSF2, CXCL1, and IL‐6). Cells were subjected to BLEO and/or ML324 treatment. Data in all bar plots are shown as mean ± SD and representative of three biological replicates. In (d) the statistical significance was calculated using one‐way ANOVA with Tukey's post hoc comparison. In (e) (bottom panel), (f and g (right) and h) p values were determined by two‐sided unpaired t‐tests and adjusted for multiple comparisons.

FIGURE 6

Accessible chromatin landscape in senescent cells and ML324‐mediated suppression. (a) The average level of ATAC‐seq enrichment (normalized) for the top 1000 most active genes between proliferating cells, SEN cells (bleomycin‐induced) and bleomycin/ML324 co‐treated cells (marked as CTRL, BLEO and BLEO/ML324, respectively). (b) Heat maps showing the expression in fragments per kilobase of transcript per million mapped reads (FPKM) of the SASP hallmark genes (RNA‐seq) and the assessable chromatin enrichment in reads per kilobase of bin per million reads sequenced (RPKM; ATAC–seq) at their promoters (TSS ± 2.5 kb). Example genes are listed alongside each type of heat map. (c) GO analysis results for gene classes of significant expression fold change in proliferating versus senescent cells, and inhibited substantially upon ML324 treatment. Percentage of gene number among all upregulated genes in SEN cells and log10 of p value per class presented, with p < 0.05 (two‐sided unpaired test) as the threshold for significance. (d) Heat maps showing ATAC‐seq enrichment of peaks near the accessible promoters (3.0‐kb upstream of the TSS and downstream of the TES per gene) in each of the assayed samples. Enrichment signals were collected for all active TSSs and TESs, which were assorted by cap analysis of gene expression values, with peaks defined by hierarchical clustering. The top 1000 genes of enrichment signals were selected for analysis. (e) Heat maps depicting ATAC‐seq enrichment of peaks near the accessible promoters (3.0‐kb upstream of the TSS and downstream of the TES per gene) present in each of the assayed samples. The whole genomic range was evaluated for each sample. (f) TF motifs identified from distal ATAC‐seq peaks in each of the group's samples. Only TFs with detectable expression (FPKM ≥ 5) and a motif enrichment p value < 1 × 10⁻¹⁰ in each sample were included. (g) The UCSC browser views show enrichment of ATAC‐seq signals at the promoters of several SASP‐unrelated genes (COMMD9, PRR5L, TRAF6, RAG1 and IFTAP) in contrast to those at the promotes of representative SASP factors (CXCL1, IL‐1β and IL‐6).

FIGURE 7

Therapeutically targeting KDM4 in the damaged TME diminishes cancer resistance conferred by senescent stroma. (a) Illustrative diagram for preclinical treatment of NOD/SCID mice. Two weeks after subcutaneous implantation and in vivo uptake of tissue recombinants, animals received either single (mono) or combinational (dual) agents administered as metronomic treatments. (b) Statistical profiling of tumor end volumes. PC3 cells were xenografted alone or together with PSC27 cells. MIT was administered to induce tumor regression, alone or together with ML324. Right, representative images of tumors. c, Representative BLI of PC3/PSC27 tumor‐bearing animals. Digital signals were proportional to in vivo luciferase activities measured by an IVIS device. (d) Comparative imaging of in vivo senescence of tumor tissues by SA‐β‐gal staining. Scale bars, 200 μm. Right, violin plot of positivity statistics. (e), Transcript assessment of in vivo expression of several canonical SASP factors in stromal cells isolated from tumors of NOD/SCID mice. Statistics were performed with a two‐sided Mann–Whitney U‐test, with upper and lower hinges representing the 25th and 75th percentiles. Horizontal bars show the median value, and whiskers extend to the values no further than 1.5 times the interquartile range from either the upper or lower hinge. (f) Quantitative appraisal of SASP factor expression and senescence marker expression in stromal cells isolated from tumor tissues of animals. Signals for each factor were normalized to the vehicle‐treated group. (g) Statistical assessment of DNA damage and apoptosis in preclinical biospecimens. Values are the percentage of cells positively stained with antibodies to γ‐H2AX or caspase 3 (cleaved). (h) Representative histological images of caspase 3 (cleaved) in tumors at the end of therapeutic regimes. Scale bar, 100 μm. (i) Survival appraisal of mice killed upon development of advanced bulky diseases. Survival duration was calculated from the time of tissue recombinant injection until the day of death. Data were analyzed by a two‐sided log‐rank (Mantel‐Cox) test. Data in all bar plots are the mean ± SD and representative of three biological replicates. In (b (left), d (right), f and g) p values were from a two‐sided unpaired t‐test and adjusted for multiple comparisons. Data in (h) are representative of three biological replicates.

FIGURE 8

Working model depicting epigenomic reprogramming and KDM4‐mediated histone demethylation (H3K9/H3K36), events enabling SASP expression in senescent cells. In genotoxic settings, senescent cells undergo irreversible DNA damage, which triggers enhanced DDR signaling. The chromatin accessibility landscape is remodeled, with a set of activated TFs physically binding to the enhancers and promoters of senescence‐associated genes, including those encoding SASP factors. There is a strong concordance of clustering schemes and a close functional linkage between chromatin accessibility and transcriptional output. Future efforts to combine genome and epigenome sequencing, as well as to generate maps of chromosome conformation, will pave the way to tackle the non‐coding genome in senescent cells. Importantly, technological pipelines demonstrating three‐dimensional epigenomes to allow identification of distinct modes of epigenetic regulation, and studies revealing dynamic engagement of key molecules including, but not limited to, KDM4 upon the onset of global reconfiguration of chromatin and transcription machineries for genome activation, hold the potential to precisely define the epigenetic landscape of senescent cells.