Metformin Reduces Histone H3K4me3&nbsp;at the Promoter Regions of Positive Cell Cycle Regulatory Genes in Lung Cancer Cells

doi:10.21203/rs.3.rs-125949/v1

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Metformin Reduces Histone H3K4me3 at the Promoter Regions of Positive Cell Cycle Regulatory Genes in Lung Cancer Cells

https://doi.org/10.21203/rs.3.rs-125949/v1

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published 09 Feb, 2021

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Background

This study aimed at understanding the effect of metformin on histone H3 modifications at the promoters of cell cycle regulatory genes in lung cancer cells.

Methods

Histone H3 modifications were analyzed using ChIP-seq, and changes in DNA methylation and chromatin accessibility were evaluated using the SureSelect Methyl-Seq Target Enrichment System and ATAC-seq, respectively. MLL2 overexpression was analyzed in tumor and matched normal tissues from 42 non-small cell lung cancer (NSCLC) patients.

Results

Metformin showed little effect on DNA methylation or chromatin accessibility at the promoter regions of cell cycle regulatory genes in lung cancer cells but significantly downregulated histone H3K4 methyltransferase MLL2 and reduced H3K4me3 levels at the promoters of positive cell cycle regulatory genes such as CDK1, CDK6, and E2F8. Eighty-eight genes involved in cell cycle showed reduced H3K4me3 levels in response to metformin, and 27% of them showed mRNA downregulation. The siRNA-mediated knockdown of MLL2 significantly reduced the H3K4me3 levels at the promoters of positive cell cycle regulatory genes. MLL2 overexpression was found in 14 (33%) of 42 NSCLC patients, with a higher prevalence in females (P = 0.04). A Cox proportional hazards analysis showed that recurrence-free survival of adenocarcinoma patients with MLL2 overexpression was approximately 1.32 (95% CI = 1.08–4.72; p = 0.02) times poorer than in those without it after adjusting for sex and pathologic stage.

Conclusions

The present study suggests that metformin might reduce H3K4me3 levels at the promoters of positive cell cycle regulatory genes through MLL2 downregulation in lung cancer cells. And, MLL2 may be a potential therapeutic target for reducing the recurrence of lung adenocarcinoma.

Cancer Biology

Oncology

Lung cancer

Metformin

H3K4 methylation

MLL2

Cell cycle

Lung cancer is the leading cause of cancer-related death worldwide. Despite the development of new therapeutic agents, the overall five-year survival rate of non-small cell lung cancer (NSCLC) is still less than 20% [1]. Epigenetic and genetic changes play an important role in the development and progression of lung cancer. Epigenetic modifications affect transcriptional initiation by altering the binding affinity between transcription factors and promoter regions or by regulating transcriptional elongation [2]. Unlike oncogenic mutations, epigenetic alterations can be reversible, making them potential therapeutic targets. Some DNA methyltransferase inhibitors and histone deacetylase inhibitors have been approved by the US Food and Drug Administration to treat certain hematologic malignancies [3, 4]. However, single-agent epigenetic therapy has shown limited effects in solid tumors, including lung cancer [5]. Recently, histone methyltransferase and demethylase inhibitors targeting histone modification have been reported. More than 50 lysine methyltransferases and demethylases have been identified to date [6], and preliminary in vitro data targeting histone modifying enzymes have shown that they have promising antitumor activity in lung cancer cells [7, 8].

Metformin, a first-line oral hypoglycemic agent for the treatment of type 2 diabetes mellitus, has been reported to exert antitumor properties in numerous types of cancer, including NSCLC. Mounting evidences from in vitro and in vivo studies have suggested the molecular mechanisms that underlie the antitumor effects of metformin [9]. Molecular properties such as the inhibition of reactive oxygen species and the AKT/mTOR pathway and the activation of AMPK have suggested its potential as an antitumor agent. Recent studies also support the notion that metformin is likely to exert its anticancer activity by targeting epigenetic changes such as histone modifications. Metformin activates AMPK and inhibits histone H2B monoubiquitination in T47D breast cancer cells [10]. It also targets histone demethylase KDM6A/UTX and elevates global levels of H3K27me3 in the tumor tissues of mice bearing highly aggressive breast cancer xenografts [11]. The elevation of H3K27me3 via EZH2 overexpression abrogated the effect that metformin had on cell proliferation and apoptosis in SKOV3 and ES2 cells [12].

Recently, we reported that metformin inhibits the proliferation of lung cancer cells by blocking cell cycle progression in the G1 phase through the downregulation of cyclins, CDKs, and E2Fs [13]. However, the mechanisms by which metformin downregulates cell cycle regulators are not fully understood. To understand possible mechanisms underlying the downregulation of cell cycle regulatory genes by metformin, we analyzed histone H3 modifications, DNA methylation, and chromatin accessibility at the promoter regions of cell cycle regulatory genes in lung cancer cells.

Cell culture

The H1299 and A549 lung cancer cell lines were obtained from the American Type Culture Collection. Those cell lines have been authenticated using short tandem repeat profiling in Samsung Medical Center for three years. The cells were cultured in RPMI 1640 medium (Lonza, Allendale, NJ) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were treated with 5 mM metformin (D150959, Sigma-Aldrich, St. Louis, MO) for 72 hours. All experiments were performed with mycoplasma-free cells.

DNA methylation analysis

Genome-wide DNA methylation was analyzed using the SureSelect Methyl-Seq Target Enrichment System (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. Genomic DNA was isolated from cultured H1299 cells and randomly sheared. Library DNA was hybridized to biotinylated RNA probes (SureSelect Methyl-Seq Capture Library, Agilent Technologies) and recovered with streptavidin beads. Eluted DNA was treated with sodium bisulfite, amplified by PCR, and sequenced on an Illumina Genome Analyzer. Mapping of the sequenced reads was performed with the Bowtie2 program. After duplicate removal, the number of mapped reads from cells treated with metformin and untreated controls was 47,803,761 and 54,906,440, respectively. Differentially methylated regions were defined as regions with more than 5 consecutive methylated CpGs and a ratio of observed to expected CpG of greater than or equal to 0.6. Regions with a p-value < 0.001 (Wilcoxon rank sum test) and a fold change |FC| ≥ 1.5 were considered significant.

Global histone H3 modification assay

Global histone H3 modifications (phosphorylation, methylation, and acetylation) were detected using an EpiQuik Histone H3 Modification Multiplex Assay Kit (Catalog No. P-3100-96, Epigentek, Farmingdale, NY). Briefly, total core histones were extracted with an EpiQuik Total Histone Extraction Kit (Epigentek, Farmingdale, NY). The isolated histones were added to wells coated with antibodies specific for 21 different histone H3 modifications.

The captured histones were detected with a detection antibody, followed by a color development reagent. Absorbance intensity was measured at 450 nm with a microplate reader. The amount of a particular histone modification was calculated as a percentage of the total H3 signal.

ChIP-seq assay

Chromatin immunoprecipitation (ChIP) was performed with a SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (#9003, Cell Signaling Technology, Danvers, MA) according to the manufacturer's instructions. Briefly, cells were cross-linked with 1% formaldehyde (Sigma‐Aldrich) and lysed. The cross-linked chromatin was digested with micrococcal nuclease to a length of 150–900 bp of DNA/protein fragments. The fragments were immunoprecipitated with antibodies against H3K4me3 (C42D8, Cell Signaling Technology), H3K27me3 (C36B11, Cell Signaling Technology), or normal rabbit IgG with protein G magnetic beads. The protein–DNA cross-links were reversed, and purified DNA was then subjected to next-generation sequencing (NovaSeq 6000 System). Raw data were checked using the FastqQC program and trimmed with Trimmomatic 0.38. The sequence library was mapped to reference genome UCSC hg19 using the Bowtie1 program. Peaks were identified using the MACS2 algorithm. Comparisons between paired samples were performed in the MAnorm program, and the M, A, and P values were calculated [14]. The M-value indicates the fold change in normalized read densities, the A-value denotes the average signal strength of the normalized read densities, and the p-value shows the significance of read intensity differences between two samples. Regions with p-values < 0.001 (-log10 p-value greater than 3) were considered statistically significant.

RNA-seq assay

Total RNA was isolated from cultured H1299 cells using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA). The RNA was broken into short fragments after removing genomic DNA contamination with DNase I. The RNA fragments were reverse-transcribed into cDNA, ligated to adaptors, amplified by PCR, and sequenced using a next-generation sequencing device (NovaSeq 6000 System). The quality of the raw sequence data was checked using the FastQC program; low-quality reads and PCR duplicates were removed using Trimmomatic 0.38. Trimmed sequences were aligned to the reference transcripts using the HISAT2 program. The number of mapped reads was 83,118,285 and 73,808,022 for cells treated with metformin and untreated controls, respectively. Finally, the expression level of each transcript was estimated by counting the reads mapped to the transcripts. The read count was normalized into fragments per kilobase of transcript per million mapped reads value, and reads with a value of zero were excluded. Of the 27,685 genes, 10,654 were removed, and the remaining 17,031 genes were further analyzed. Significantly up- or downregulated genes were identified using a cutoff value of |FC| ≥ 1.5.

Gene-set enrichment analysis

A Gene Ontology search and Gene Set Enrichment Analysis were performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis [15, 16].

ATAC-seq assay

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed by Active Motif (Carlsbad, CA) as previously described [17]. Peaks were identified using the MACS2 algorithm at a cutoff of p-value < 1E-7 with the non-model option. Significantly different regions were generated by applying the cutoffs MaxTags ≥ 100 and log2 ratio > 1 or < -1.

Quantitative real-time PCR (qRT- PCR)

Total RNA was isolated using a PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA) and reverse-transcribed using a SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). qRT-PCR was performed with SYBR green dye (4385614, Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation for 5 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The PCR primer sequences are listed in Supplementary Table S1.

Western blot analysis

Total protein was extracted from cultured cells using lysis buffer containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). The lysates were heated for 5 min at 95 °C, loaded on 10% sodium dodecyl sulfate-polyacrylamide gels, and transferred to a PVDF membrane (Immobilon-P, Millipore, Medford, MA). After blocking with a 3% solution of fetal bovine serum, the membranes were probed with the antibodies listed in Supplementary Table S2. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and visualized with an Immun-Star Western Kit (Bio-Rad, Hercules, CA).

Small interfering RNA (siRNA)-mediated gene silencing

To knock down target genes, cells were transiently transfected with 40 nM gene-specific siRNA (BioNeer, DaeJeon, Korea) or non-targeting siRNA (BioNeer) as a negative control.

The siRNA sequences or their commercial IDs are listed in Supplementary Table S3. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used to transfect the siRNA into cells according to the manufacturer’s protocol. At 48 hours post-transfection, gene expression was measured using quantitative real-time PCR or western blot analyses.

Cell viability assay

Cells were cultured in 6-well plates (10,000 cells/well) and transfected with specific siRNA at a concentration of 40 nM. Cell viability was measured using a WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] assay kit (Biomax, Seoul, Korea) according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA).

Statistical analysis

Univariate analyses were performed using the Student’s t-test or Wilcoxon rank sum test and Pearson's chi-square test or Fisher’s exact test for continuous and categorical variables, respectively. Linear relationships between two continuous variables were analyzed using the Pearson correlation coefficient. The effect of MLL2 overexpression on survival was estimated using Kaplan-Meier survival curves, and the difference between the survival times of two independent groups was evaluated by the log-rank test. A Cox proportional hazards analysis was conducted to estimate the hazard ratios for MLL2 overexpression on survival after controlling for potential confounding factors. Statistical analyses were conducted using R software (version 3.6.1).

Metformin reduces histone H3K4me3 at the promoter regions of positive cell cycle regulatory genes in lung cancer cells

Global alterations of histone H3 were analyzed using an enzyme-linked immunosorbent assay. Metformin resulted in histone H3 modifications in lung cancer cells, including methylation at H3K4, but the effect was minimal (Fig. 1a). To more clearly evaluate the effect of metformin on histone modifications, we analyzed the global levels of H3K4me3, H3K9me2, and H3K27me3, which are all important in lung cancer pathogenesis, using western blotting (Fig. 1b). Metformin reduced H3K4me3 and H3K9me2 in both A549 and H1299 cells and H3K27me3 in H1299 cells. ChIP-seq was performed to find the DNA regions in which metformin induced the observed alterations in H3K4me3 and H3K27me3. The ChIP-seq data are available at the Gene Expression Omnibus (GEO) with the ID: GSE141053. More than 80% of H3K4me3 was detected at gene promoters, whereas less than 10% of H3K27me3 was observed at promoters. Most (70%) of H3K4me3 was enriched at a transcription start site (TSS) ± 1 kb (Fig. 1c), and H3K27me3 was largely located outside of gene promoters (Fig. 1d). Metformin significantly reduced the H3K4me3 levels at the promoter regions of 1113 genes and increased the levels at the promoter regions of 1492 genes. The Gene Set Enrichment Analysis revealed that H3K4me3 levels decreased at the promoters of 88 cell cycle regulatory genes in response to metformin in lung cancer cells (Supplementary Table S4). At the promoter regions of CDKN1A (p21) and CDKN1B (p27), H3K4me3 was upregulated in response to metformin, but the difference was statistically significant only in CDKN1A (Supplementary Table S5). Representative ChIP-seq images of H3K4me3 reduction are shown in Fig. 1E.

H3K4me3 reduction at the promoter regions of positive cell cycle regulatory genes is associated with mRNA downregulation

To identify genes whose transcription was significantly altered by metformin, we performed RNA-seq in H1299 cells. The RNA-seq data are available at the GEO with the ID: GSE141052. The expression levels of 1,114 genes were altered by 1.5 times or more in cells treated with metformin: the mRNA levels of 499 genes were downregulated, and those of 615 genes were upregulated (Fig. 2a). A KEGG enrichment analysis showed that the genes significantly up- and downregulated in response to metformin were involved in the cell cycle, apoptosis, cellular senescence, and p53 signaling pathways (Fig. 2b). Eighty-five genes involved in the cell cycle are listed in Supplementary Table S6. Positive cell cycle regulators such as cyclin A2, cyclin E2, CDK1, E2F2, E2F6, and E2F8 were downregulated, whereas negative cell cycle regulators such as CDKN2B (p15), CDKN1A (p21), DDIT4, and GADD45 were upregulated. Of the 85 genes, 24 genes (28%), including MKI67, E2F8, CDK1, CDC7, and UHRF1, showed reduction of H3K4me3 at their promoter regions (Fig. 2c and Supplementary Table S7), and those H3K4me3 levels were positively related to mRNA levels (Fig. 2d).

The effect of metformin on histone H3 methyltransferase is independent of AMPK

We investigated whether metformin regulates the expression of histone H3 methyltransferase and demethylase in lung cancer cells. Metformin downregulated the protein (Figs. 3a and 3b) and mRNA (Fig. 3c) expression of EHMT1, EHMT2, EZH1, EZH2, MLL1, MLL2, and WDR82 in lung cancer cells. Metformin also reduced the expression of H3K4 demethylase KDM5A (JARID1A) and H3K9 demethylase KDM4B (JMJD2B) at the levels of protein (Fig. 3d) and mRNA (Fig. 3e) in A549 and H1299 cells. Because metformin is known to exert its antitumor action partly through AMPK activation, we analyzed whether its effect on the expression of histone H3 methyltransferases depended on AMPK. AMPK activation using 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) did not suppress mRNA expression of MLL2, WDR82 (Fig. 3f), DOT1L, EHMT2, or EZH2 (data not shown), but AMPK inhibition using dorsomorphin suppressed the expression of those genes (Fig. 3g). Thus, the downregulation of histone H3 methyltransferases by metformin might not depend on AMPK.

MLL2 knockdown significantly reduces the expression of H3K4me3 and positive cell cycle regulators

To find the H3K4 methyltransferases that were most significantly involved in the reduction of H3K4me3 in cells treated with metformin, we knockdowned the H3K4 methyltransferases MLL1, MLL2, WDR5, and WDR82 using siRNA in NSCLC cells. The knockdown of histone H3K4 methyltransferases by siRNA was confirmed by real-time PCR analysis (Fig. 4a). MLL2 knockdown most significantly reduced global levels of H3K4me3 (Figs. 4b & 4c) and cell proliferation (Fig. 4d) in both A549 and H1299 cells. The number of cells on the third day after siMLL2 transfection decreased significantly by 66% and 51% compared with the siControl condition in A549 cells and H1299 cells, respectively (Fig. 4d). MLL2 knockdown also significantly downregulated the expression of both positive cell cycle regulators that showed significant H3K4me3 reduction in lung cancer cells (Fig. 4f) and those that did not (Fig. 4f).

Metformin does not significantly change DNA methylation or chromatin accessibility at the promoter regions of positive cell cycle regulatory genes

To understand whether the observed changes in the mRNA levels of cell cycle regulatory genes in response to metformin are associated with DNA methylation, we analyzed the methylation levels of CpGs at the promoter regions of the genes using the SureSelect Methyl-Seq Target Enrichment System. Some regions at the genome level were found to be hyper- or hypomethylated, but most of the cell cycle regulatory genes did not show significantly altered DNA methylation like that seen in CDK1 and E2F8 (Fig. 5a). ATAC-seq was performed to investigate whether the downregulation of positive cell cycle regulators in cells treated with metformin was associated with changes in chromatin accessibility. The ATAC-seq data are available at the GEO with ID: GSE141059. Chromatin accessibility in cells treated with metformin decreased in 159 regions and increased in 124 regions. Most regions with changes in chromatin accessibility were outside of promoter regions, defined as TSS ± 3 kb. Tag distribution (Fig. 5b) in the promoter regions and the peak tag number in the total merged peak (Fig. 5c) were found to be similar. The regions with the most severe increases and decreases in chromatin accessibility were located 4,667 bp downstream of CHAC1 (Fig. 5d) and 15,111 bp downstream of ERRFI1 (Fig. 5e), respectively. We identified 54 genes whose chromatin accessibility was significantly changed at the promoter regions, but most of those genes were not related to cell cycle regulation (Supplementary Table S8).

Clinicopathological characteristics of MLL2 overexpression in primary NSCLC

We used previously reported data to analyze the clinicopathological significance of MLL2 overexpression in 42 primary NSCLCs [18]. MLL2 was defined to be overexpressed when its mRNA level was ≥ 1.5 fold higher in tumor tissues than in matched normal tissue. MLL2 overexpression was found in 14 (32%) of the 42 NSCLCs tested and was not associated with patient age, pathologic stage, or differentiation (data not shown). However, MLL2 overexpression was found to be highly prevalent in females (P = 0.04, Fig. 6a). Although MLL2 overexpression was not statistically associated with smoking status (P = 0.34; Fig. 6b), it tended to occur more frequently in never-smokers: the number of pack-years for patients with and without MLL2 overexpression was 17 ± 21 and 34 ± 28, respectively (P = 0.04; Fig. 6c). MLL2 overexpression was also found in adenocarcinoma at a higher prevalence than in squamous cell carcinoma, though that difference was not statistically significant (P = 0.27; Fig. 6d). The effect of MLL2 overexpression on patient survival was analyzed in the 42 NSCLCs. The median follow-up period for the patients was 5.3 years. MLL2 overexpression was significantly associated with poor recurrence-free survival in adenocarcinoma (p = 0.02; Fig. 6e) but not in squamous cell carcinoma (p = 0.87). The five-year recurrence-free survival rate of 27 adenocarcinoma patients with and without MLL2 overexpression was 34% and 78%, respectively. A Cox proportional hazards analysis showed that the recurrence-free survival of adenocarcinoma patients with MLL2 overexpression was approximately 1.32 (95% CI = 1.12–4.57; p = 0.01) times poorer than in patients without MLL2 overexpression after controlling for sex and pathologic stage (Supplementary Table S9).

A nucleosome is composed of genomic DNA wrapped around an octamer that contains two copies of each of the histone proteins H2A, H2B, H3, and H4. Five lysines in histone H3 (K4, K9, K27, K36, K79) and a lysine in histone H4 (K20) are known to be modulated by methylation. H3K4me3 is present at the promoters of active genes and leads to low nucleosome density at those promoters, and H3K36me3 is associated with elongating RNA polymerase II and occurs in the bodies of active genes. H3K9me2/3 is associated with constitutive heterochromatin formation at promoters and strongly associated with gene repression. H3K27me3 and H4K20me3 are also tightly associated with heterochromatin as clear markers of gene repression. In this study, we investigated the effect of metformin on epigenetic alterations (DNA methylation, histone H3 modification, and chromatin accessibility) in lung cancer cells. Although the effects of metformin on DNA methylation and chromatin accessibility were minimal, metformin downregulated H3K4me3, especially at the promoter regions of positive regulatory genes of cell cycle.

H3K4me3 is found in ∼75% of all human active gene promoters in several cell types [19], and the presence of this mark at the promoters of protein coding genes is known to correlate with an active state of gene expression [20]. Many studies have reported the molecular mechanisms that underlie the regulation of gene expression by H3K4me3. H3K4me3 is known to positively regulate gene expression through chromatin remodeling. Recruitment of the chromatin remodeling complex NURF by H3K4me3 allows the transcription factors more access to DNA. Transcriptional pre-initiation complex formation is facilitated by recruiting transcription factor II D (TFIID) via the TATA binding protein associated factor 3 (TAF3) [21]. TAF3, as a component of TFIID, interacts with H3K4me3 and provides the mechanism by which TFIID is recruited to the promoter. TAF3 binding sites are significantly enriched at cell cycle regulatory genes [21]. In contrast, the loss of H3K4me3 inhibits transcription by reducing TFIID binding to promoters [22, 23]. In this study, approximately 80% of H3K4me3 was found to be enriched in gene promoters, and 24 (27%) of 88 cell cycle genes with downregulated H3K4me3 in response to metformin also showed downregulated mRNA levels. Based on these observations, it is likely that metformin affects the expression of cell cycle regulatory genes at least partially through the reduction of H3K4me3 at their promoter regions.

AMPK activation is known to be one of the mechanisms underlying the antineoplastic effect of metformin. Metformin can inhibit cancer cell growth by suppressing mammalian target of rapamycin complex 1 (mTORC1) in AMPK-dependent and AMPK-independent pathways [24]. Several groups have reported that metformin could inhibit mTORC1 activation in the absence of AMPK. Metformin inhibited mTORC1 in a GTPase-dependent manner in HEK293T cells and did not have a pronounced effect on the energy status of AMPK dKO MEFs in the absence of AMPK [25]. AMPK inhibition had a minor impact on the metformin effect, which induced mTOR inhibition and cell cycle arrest through REDD1 in prostate cancer cells [26]. In addition, metformin suppressed the proliferation of glioma cells through PRAS40-mediated mTOR inhibition independent of AMPK [27]. In this study, we also found that the effect of metformin on H3K4 methyltransferase expression did not depend on AMPK activation (Figs. 3f & 3 g). Accordingly, the ability of metformin to downregulate histone H3K4 methyltransferase in lung cancer cells could be independent of AMPK activation despite the contribution of the AMPK/mTOR signaling pathway to metformin-induced anticancer therapy.

The transfer of methyl groups to lysine residues on histone H3K4 is known to be principally catalyzed by methyltransferase. MLL1 and MLL2 are members of the SET1/COMPASS complex and control the expression of actively transcribed genes by regulating H3K4me3 [28]. WDR82, as a core subunit of the SET1A complex, mediates recruitment of the SET1A histone methyltransferase complex to transcription start sites [29]. In this study, metformin significantly inhibited the expression of MLL1, MLL2, and WDR82 in lung cancer cells (Fig. 3A). H3K4me3 was most significantly downregulated in A549 and H1299 cells treated with siMLL2 rather than siMLL1 or siWDR5 (Figs. 4b & 4c), suggesting that MLL2 could function as a core factor regulating H3K4me3 in lung cancer cells. MLL2 is located at 12q12-13 and is also known as KMT2D/ALR/MLL4. MLL2 is known to function as an oncogene or a tumor suppressor, depending on the cancer type. Several groups have reported that MLL2 plays an oncogenic role in human cancer. MLL2 overexpression compared with adjacent benign epithelium was reported in invasive carcinomas of the breast and colon [30]. MLL2 knockdown resulted in a significant decrease in the migration of esophageal squamous cell carcinoma cells. Clinically, a high level of MLL2 is significantly associated with early-stage ESCC lymph node metastasis [31]. shRNA-mediated knockdown of KMT2D attenuated neoplastic cell migration [32]. Knockdown of MLL2 using siRNA or lentiviruses suppressed the proliferation of gastric cancer cells [33]. In this study, MLL2 knockdown also significantly inhibited the proliferation of lung cancer cells (Fig. 4d) and downregulated positive cell cycle regulators (Figs. 4e & 4f). These observations suggest that MLL2 might function as an oncogene in some kinds of human cancer and play a role in reducing H3K4me3 at the promoter regions of positive cell cycle regulatory genes in response to metformin in lung cancer cells.

MLL2 is known to be involved in tumor progression and is associated with poor prognosis in a variety of cancers. MLL2 knockout suppressed cell cycle progression by inducing cell cycle arrest at the G1 stage in esophageal squamous cell carcinoma cells in vitro and inhibited cell migration [34]. In addition, high MLL2 expression predicts poor prognosis and promotes tumor progression by inducing the EMT in esophageal squamous cell carcinoma [34]. MLL2 maintains overall H3K4me2/me3 levels in MLL-AF9 leukemia cells, and deletion of MLL2 reduces the survival of those cells in vitro and in vivo [35]. MLL2 overexpression positively affected cell migration in pheochromocytoma and was negatively associated with patient survival in gastrointestinal diffuse large B-cell lymphoma and breast cancer [36–38]. NSCLC patients with an MLL2 mutation showed reduced overall and progression-free in NSCLC compared with those who had wild-type MLL2 [39]. Consistent with those results, we found that MLL2 overexpression was significantly associated with poor recurrence-free survival in lung adenocarcinoma.

The human lysine demethylase 5 (KDM5) subfamily consists of four family members designated KDM5A–D and specifically catalyzes the demethylation of H3K4. KDM5A, also known as retinoblastoma binding protein (RBP2), was initially identified as a binding partner of retinoblastoma protein, and its overexpression has been observed in cancers such as glioblastoma, gastric cancer [40], hepatocellular carcinoma, and lung cancer [41]. Increasing evidence suggests that KDM5A plays an oncogenic role in tumorigenesis and the progression of human cancer. KDM5A promotes small cell lung cancer tumorigenesis by repressing NOTCH signaling and sustaining ASLC1 expression [42]. Wang et al. [43] reported that KDM5A downregulated the expression of E-cadherin and that KDM5A overexpression induced the epithelial-mesenchymal transition in NSCLC cells. KDM5A promotes cell cycle progression by repressing p27 and activating cyclins D1 and E1 in lung cancer cells [41]. In lung cancer cells, KDM5A binds directly to the promoter regions of cyclin D1 and cyclin-dependent kinase inhibitor p27 (KIP1) and promotes G₁-S progression by activating cyclins D1 and E1 and suppressing p27 (CDKN1B) [41]. KDM5A also promotes the angiogenesis of NSCLC cells by upregulating VEGF and activating HIF-1α via PI3K/Akt signaling [44]. KDM5A depletion enhanced the promoter activities of p16 (INK4A), p21 (CIP1), and p27 (KIP1) and upregulated H3K4me3 levels in gastric cancer cells [40]. In this study, metformin suppressed KDM5A expression and upregulated H3K4me3 at the promoter regions of p21 and p27 (Supplementary Table S5). Thus in lung cancer cells, metformin might increase p21 and p27 expression by upregulating H3K4me3 at the promoter region of each gene through the downregulation of KDM5A.

This study was limited by several factors. First, the clinical significance of MLL2 expression was retrospectively studied in only a small number of samples. Those results need to be confirmed in a prospective study with a large number of patients. Second, the effects of MLL2 and KDM5A on histone H3K4me3 were analyzed separately. H3K4me3 alteration needs to be analyzed after double knockdown of MLL2 and KDM5A.

The present study suggests that metformin may suppress the expression of positive cell cycle regulatory genes by reducing H3K4me3 at their promoter regions through MLL2 downnregulation in lung cancer cells. And, MLL2 may be a therapeutic target for reducing recurrence after surgery in lung adenocarcinoma.

CHAC1, ChaC glutathione specific gamma-glutamylcyclotransferase 1; CCNA, cyclin A; CCNB, cyclin B; CDK1, cyclin dependent kinase 1; CCNE, cyclin E; DNMTi, DNA methyltransferases inhibitor; DOT1L, DOT1 like histone lysine methyltransferase; E2F8, E2F transcription factor 8; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; ERRFI1, ERBB receptor feedback inhibitor 1; EHMT1, euchromatic histone lysine methyltransferase 1; GADD45, growth arrest and DNA damage-inducible 45; HDACi, histone deacetylases inhibitor; H3K9me2, histone H3 dimethylation on lysine 9; H3K4me3, histone H3 trimethylation on lysine 4; H3K27me3, histone H3 trimethylation on lysine 27; KDM4B, lysine-specific demethylase 4B; KDM5A, lysine-specific demethylase 5A; MLL1, lysine methyltransferase 2A; NSCLC, non-small cell lung cancer; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; AMPK, protein kinase AMP-activated catalytic subunit alpha 1; UHRF1, ubiquitin like with PHD and ring finger domains 1; WDR82, WD repeat domain 82.

Ethics approval and consent to participate: This study was approved by the Institutional Review Board (IRB#: 2010-07-204) at the Samsung Medical Center, Seoul, Korea, and conducted in accordance with the Declaration of Helsinki Ethical Principles. Written informed consent for the research use of pathological specimens was obtained from all patients before surgery. Patients' clinical information are not publicly available due to privacy and ethical restriction.

Consent for publication: Not applicable

Availability of data and materials: The datasets analyzed during the current study are available at the Gene Expression Omnibus (GEO) with the IDs (GSE141053 and GSE141052).

Competing interests: The authors declared that they have no competing interests.

Funding: This work was supported by an NRF grant funded by the Korean government (No. 2018R1D1A1B07050658), Republic of Korea.

Authors’ contributions: DK made the hypothesis for this study, conducted most of the experiments, and wrote the manuscript. YK and BBL analyzed gene expression in fresh-frozen tumor and matched normal tissues from NSCLC patients. EYC and JH made histological diagnosis of patients and provided tissue samples, and YMS performed surgery on 42 lung cancer patients. D-HK directed and supervised all studied. All authors read and approved the final manuscript.

Acknowledgements: We thank Eunkyung Kim and Jin-Hee Lee for data collection and management and Hoon Suh for sample collection.

Authors’ information

¹Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Korea 440-746

²Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 135-710

³Department of Thoracic and Cardiovascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 135-710

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Metformin Reduces Histone H3K4me3 at the Promoter Regions of Positive Cell Cycle Regulatory Genes in Lung Cancer Cells

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Journal Publication

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Abstract

Figures

Background

Materials And Methods

Cell culture

DNA methylation analysis

Global histone H3 modification assay

ChIP-seq assay

RNA-seq assay

Gene-set enrichment analysis

ATAC-seq assay

Quantitative real-time PCR (qRT- PCR)

Western blot analysis

Small interfering RNA (siRNA)-mediated gene silencing

Cell viability assay

Statistical analysis

Results

The effect of metformin on histone H3 methyltransferase is independent of AMPK

Clinicopathological characteristics of MLL2 overexpression in primary NSCLC

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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Journal Publication

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