Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas

Abstract

Rationale: Oncogenic STAT3 signaling activation and 3p22-21.3 locus alteration are common in multiple tumors, especially carcinomas of the nasopharynx, esophagus and lung. Whether these two events are linked remains unclear. Our CpG methylome analysis identified a 3p22.2 gene, DLEC1, as a methylated target in esophageal squamous cell (ESCC), nasopharyngeal (NPC) and lung carcinomas. Thus, we further characterized its epigenetic abnormalities and functions. Methods: CpG methylomes were established by methylated DNA immunoprecipitation. Promoter methylation was analyzed by methylation-specific PCR and bisulfite genomic sequencing. DLEC1 expression and clinical significance were analyzed using TCGA database. DLEC1 functions were analyzed by transfections followed by various cell biology assays. Protein-protein interaction was assessed by docking, Western blot and immunoprecipitation analyses. Results: We defined the DLEC1 promoter within a CpG island and p53-regulated. DLEC1 was frequently downregulated in ESCC, lung and NPC cell lines and primary tumors, but was readily expressed in normal tissues and immortalized normal epithelial cells, with mutations rarely detected. DLEC1 methylation was frequently detected in ESCC tumors and correlated with lymph node metastasis, tumor recurrence and progression, with DLEC1 as the most frequently methylated among the established 3p22.2 tumor suppressors (RASSF1A, PLCD1 and ZMYND10/BLU). DLEC1 inhibits carcinoma cell growth through inducing cell cycle arrest and apoptosis, and also suppresses cell metastasis by reversing epithelial-mesenchymal transition (EMT) and cell stemness. Moreover, DLEC1 represses oncogenic signaling including JAK/STAT3, MAPK/ERK, Wnt/β-catenin and AKT pathways in multiple carcinoma types. Particularly, DLEC1 inhibits IL-6-induced STAT3 phosphorylation in a dose-dependent manner. DLEC1 contains three YXXQ motifs and forms a protein complex with STAT3 via protein docking, which blocks STAT3-JAK2 interaction and STAT3 phosphorylation. IL-6 stimulation enhances the binding of DLEC1 with STAT3, which diminishes their interaction with JAK2 and further leads to decreased STAT3 phosphorylation. The YXXQ motifs of DLEC1 are crucial for its inhibition of STAT3 phosphorylation, and disruption of these motifs restores STAT3 phosphorylation through abolishing DLEC1 binding to STAT3. Conclusions: Our study demonstrates, for the first time, predominant epigenetic silencing of DLEC1 in ESCC, and a novel mechanistic link of epigenetic DLEC1 disruption with oncogenic STAT3 signaling in multiple carcinomas.

Keywords: 3p22; DLEC1; STAT3; carcinoma; methylation.

Figure 1

CpG methylome study identified DLEC1 as a methylated target downregulated in multiple carcinomas. (A) Methylome analysis showed signal enrichment in the DLEC1 promoter CGI in ESCC, NPC and lung cancer by MeDIP and Illumina 27k methylation array (bottom panels). Positive MeDIP signal peaks (blue) are marked. (B) Broad expression of DLEC1 in human normal adult tissues by semi-quantitative RT-PCR, with GAPDH as a control. (C) Reduction and silencing of DLEC1 in ESCC and lung carcinoma cell lines. M: methylated; U: unmethylated. (D) Pharmacological demethylation with 5-Aza and TSA restored DLEC1 expression in methylated and silenced carcinoma cell lines. (E) Representative BGS results of carcinoma cell lines. Each single allele of the DLEC1 CGI is shown as an individual row, with filled circle representing methylated while open circles representing unmethylated CpG sites. ESCC: esophageal squamous carcinoma; NPC: nasopharyngeal carcinoma; Ca: carcinoma.

Figure 2

DLEC1 downregulation and methylation in esophageal and other carcinomas. (A) DLEC1 expression levels in esophageal, lung and head/neck tissues through GENT online analysis (upper panel). C: cancer; N: normal. (B) DLEC1 protein expression was detected by Western blot in human normal tissues of esophageal, lung and testis, immortalized esophageal epithelial cell lines and ectopically expressed tumor cell lines. (C) Analyses of TCGA datasets revealed an inverse correlation between DLEC1 mRNA expression and promoter methylation in esophageal and lung cancers. (D) Promoter methylation and expression of DLEC1 were detected in esophageal, nasopharyngeal and lung primary carcinomas, but rarely in matched adjacent esophageal and nasopharynx tissues. DLEC1 expression in representative primary ESCC, NPC tumors samples and normal tissues, as measured by semi-quantitative RT-PCR. (E) Detailed BGS analysis of representative tumor samples. (F) Association of DLEC1 methylation and expression with overall survival was analyzed in ESCC and lung cancer samples from TCGA datasets. ESCC: esophageal squamous carcinoma; NPC: nasopharyngeal carcinoma; Ca: carcinoma.

Figure 3

DLEC1 inhibits the growth of esophageal and other carcinoma cells. (A) Representative colony formation assay by monolayer culture in DLEC1-expressing ESCC, NPC and lung carcinoma cells. Quantitative analyses of colony numbers are shown as values of mean ± SD. ***p < 0.001. (B) DLEC1 expression was measured by Western blot. (C) Cell proliferation assay comparing the proliferation rates of multiple carcinoma cells stably-expressing DLEC1. At each indicated time point, cell viability was determined and represented as the degree of absorbance at 570 nm using MTT assay. The mean ± SD absorbance (triplicate wells) for each time point is plotted against days after seeding. C1: clone 1, C2: clone 2. (D) Representative cell cycle and flow cytometry data are shown (left panel). Cell apoptosis was examined by flow cytometry analysis with Annexin V-FITC and propidium iodide (PI) doubles staining (right panel). Values are the mean ± SD of three independent experiments. *p < 0.05, **p < 0.05. (E) Upregulation of apoptotic markers (cleaved-PARP and caspase 3) detected by Western blot.

Figure 4

DLEC1 suppresses cell migration and invasion of esophageal and other carcinoma cells, with transwell (A) migration and (B) invasion assay of DLEC1-expressing tumor cells. Migrated and invaded cells at the lower surface of the transwell filter were stained and counted. **p < 0.001, ***p < 0.0001. Original magnification, ×252. Scale bar 100 μm. (C) Morphology changes of H1299 cells after ectopic DLEC1 expression. Original magnification, ×252. Scale bar 100 μm. (D) Impaired actin stress fiber organization in DLEC1-expressing H1299 cells. (E) Indirect immunofluorescence detecting the expression of E-cadherin and vimentin. Original magnification, ×400. Scale bar 20 μm. (F) Western blot showing the expression of EMT markers (E-cadherin, vimentin) and metastasis markers (MMP7, Twist) in DLEC1- or vector-transfected cells. For the internal controls refer to Figure 3B. (G) Downregulation of representative stem cell markers in DLEC1-expressing carcinoma cells by qPCR. *p < 0.05, **p < 0.01.

Figure 5

DLEC1 as a cytoplasmic protein regulates multiple cell signaling pathways. (A) Subcellular localization of DLEC1 (green) in cell cytoplasm of carcinoma cells. DAPI counterstaining (blue) was used to visualize nuclear DNA. Images of osteosarcoma cell line U-2 OS were retrieved from Human Protein Atlas database. Original magnification, ×400. Scale bar 10 μm. (B) Promoter luciferase activities of NF-κB, AP-1, STATs-bs, TopFlash, SRE and PAI-1 reporters were normalized to the values of Renilla luciferase activity in tumor and immortalized normal cell lines. Results are expressed as fold reduction of activity and shown as means ± SD of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01. (C) DLEC1 inhibited the phosphorylation levels of ERK, AKT and STAT3. Western blot was used to examine protein levels of total ERK, p-ERK (Thr202/Tyr204), total AKT, p-AKT(ser473), total STAT3, p-STAT3 (Tyr705, Ser727), total β-catenin and active β-catenin in DLEC1-expressing multiple carcinoma cells. (D) Results are expressed as ratio between phosphorylated/activated (p-ERK, p-AKT, p-STAT3 (Tyr705), Active-β-catenin) and non-phosphorylated/total (ERK, AKT, STAT3, β-catenin) forms. Three different experiments were performed and data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

DLEC1 inhibits STAT3 signaling activation through suppressing its phosphorylation in tumor cells. Expression levels of p-STAT3 (Tyr705, Tyr727) and STAT3 were assessed by Western blot in cells transfected with (A) both DLEC1 and STAT-3C; and (B) different dose of DLEC1 expression plasmid. (C) Overexpression of DLEC1 decreases IL-6-induced p-STAT3 level. KYSE150 and H1299 cells were treated with or without IL-6 (10 ng/mL) for 30 min. Phosphorylated and total STAT3 levels were examined. (D) Heat-map showing the expression pattern of DLEC1 and IL-6/STAT3 signaling molecules in ESCC and lung carcinoma patients (Oncomine database).

Figure 7

DLEC1 binds to STAT3 and blocks its interaction with JAK2. (A) Predicted models of DLEC1-STAT3 protein docking. 3D structure of DLEC1 and STAT3 PDB are shown. The N-terminal of DLEC1 (residues 108-165) resembles the solution structure of the C-terminal PapD-like domain of human HYDIN protein (protein phosphatase 1) (PDB id: 2E6J), while its C-terminal (residues 806-857) yields 7 matches to respiratory complex I or bovine mitochondrial super-complex. Fourteen structural similarities were observed for STAT3 PDBs, with only three covering the tyrosine residue Y705 (PDB id: 1BG1, 1BF5, 4E68). (B, C) Three dimensional protein models of interaction between DLEC1 and STAT3 proteins. DLEC1 (PDB id: 2E6J) and STAT3 (PDB id: 1BG1, 1BF5 and 4E68) were predicted by ZDOCK server. Grey represents DLEC1 PDB; Color represents STAT3 PDB. (D) Association of endogenous DLEC1 and STAT3 proteins in HEK293T cells. Lysates of HEK293T cells were subjected to IP with DLEC1, STAT3 or JAK2 antibodies. DLEC1 interacts and interferes with STAT3-JAK2 complex. Flag-DLEC1 was transfected in (E-G) multiple carcinoma cells and (H) HEK293T cells for IP with anti-STAT3, anti-Flag or anti-JAK2 antibody. 5% of DLEC1-transfected cell lysis for each IP was used as input.

Figure 8

Roles of the YXXQ motif in DLEC1-mediated inhibition of STAT3 phosphorylation/activation. (A) Localization of the consensus STAT3-binding motifs (YXXQ) in the DLEC1 protein (upper panel). Schematic of the DLEC1-YXXQ mutant was shown (lower panel). (B) DLEC1-YXXQ mutant retained the phosphorylation level of STAT3, which was decreased in DLEC1-expressing cells. KYSE150 and H1299 cells were treated with or without IL-6 (10 ng/mL) for 30 min. Phosphorylated and total STAT3 levels were examined by Western blot. (C-E) The interaction of DLEC1 and its YXXQ mutant with JAK2-STAT3 under IL-6 stimulation by co-IP assay. DLEC1 or DLEC1-YXXQ mutant (DLEC1-3Y) was transfected in HEK293T, KYSE150 and H1299 cells for the IP experiment. 5% of DLEC1-transfected cell lysis for each IP was used as input. (F) A schematic model showing the regulation of oncogenic STAT3 signaling activation by DLEC1. DLEC1 suppresses STAT3 phosphorylation through binding to STAT3 via YXXQ motif and further interring with JAK2-STAT3 interaction.