Speckle-type POZ protein functions as a tumor suppressor in non-small cell lung cancer due to DNA methylation

Abstract

Background: Tumor suppressor epigenetic silencing plays an important role in non-small cell lung cancer (NSCLC) development and progression. Previously, the expression of speckle-type POZ protein (SPOP) has been found to be significantly inhibited in NSCLC. Our research aimed to investigate the molecular mechanisms, clinical significance and epigenetic alteration of SPOP in NSCLC.

Materials and methods: Bisulfite sequencing PCR and methylation-specific PCR were performed to test gene methylation. Chromatin immunoprecipitation (ChIP) was performed to detect transcription factor C/EBPα combinations and the promoter of the SPOP gene. Furthermore, we evaluated the effects of C/EBPα siRNA on SPOP expression, tumor cell migration and proliferation via MTT and Transwell assays in vitro and tumor growth in vivo. The relationship between the methylation status of the SPOP gene and clinicopathologic characteristics was investigated.

Results: Hypermethylation was found in the CpG island of the SPOP gene promoter in NSCLC tissues, and this methylation was found to be correlated with SPOP expression. SPOP promoter methylation was associated with the pathology grade. The transcriptional activities were significantly inhibited by the hypermethylation of specific CpG sites within the SPOP gene promoter, while 5-aza-2'-deoxycytidine significantly increased SPOP gene expression. C/EBPα also played a key role in SPOP regulation. Five C/EBPα binding sites in the CpG island of the SPOP gene promoter were identified by ChIP. Inhibition of C/EBPα significantly reduced SPOP expression. SPOP mediated the C/EBPα-regulated suppression of invasion, migration and proliferation in vitro and tumor growth in vivo.

Conclusions: SPOP function and expression in NSCLS were regulated by DNA methylation and C/EBPα transcriptional regulation combination effects, indicating that the SPOP promoter methylation status could be utilized as an epigenetic biomarker and that the C/EBPα-SPOP signaling pathway could be a potential therapeutic target in NSCLC.

Keywords: C/EBPα; DNA methylation; NSCLC; SPOP; Transcriptional regulation.

Fig. 1

Hypermethylation of the CpG island in the SPOP gene promoter in NSCLC. a Scheme of the location of the CpG islands in the promoter of the SPOP gene. The CpG sites are indicated by vertical red lines. The regions used for MS-PCR and BSP are also indicated. b Representative results of methylation analysis by MS-PCR in NSCLC tissues (T) and adjacent normal lung tissues (N). U unmethylation; M, methylation. c Statistical results showing that the methylation level in the NSCLC tissues was higher than that in the adjacent normal tissues. d Correlation analysis of the MSP results. *P < 0.05. Data represent the results of three independent experiments

Fig. 2

Hypermethylation of specific CpG sites within the SPOP gene promoter inhibits transcriptional activities. a Schematic of the regions used for BSP showing the locations of the 17 CpG sites in the SPOP gene promoter area. b Bisulfite sequencing analysis of the methylation status of the SPOP gene promoter. Each oval indicates clones from different cell lines. Eight clones were subjected to bisulfite sequencing. The methylated clones of individual CpG sites are labeled in black. c The mRNA levels of SPOP in different cell lines. d Treatment with two luciferase constructs with SssI methylase in vitro. *P < 0.05. Data represent the results of three independent experiments

Fig. 3

SPOP expression is induced by AZA treatment in methylated lung cancer cells. a DNA methylation status of the SPOP gene proximal promoter was analyzed by bisulfite sequencing in AZA-treated cells. b SPOP mRNA and protein levels were determined by qPCR and western blotting in the indicated cells treated with varying concentrations of AZA. c Effect of AZA on lung cancer cell growth. MTT assay was used to detect the growth of cell treated with different concentrations of AZA. The data are presented as the mean ± SD of three independent experiments. *P < 0.05 as compared with control (Non-treated with AZA)

Fig. 4

C/EBPα binding the SPOP proximal promoter region is abrogated by DNA methylation and induces SPOP expression in lung cancer cells. a Location of the predicted C/EBPα binding sites in the SPOP gene promoter region. The sequences depicted in boldface and underlined denote the predicted binding sites of C/EBPα, and the TSS is labeled. b ChIP assay demonstrating the direct binding of C/EBPα to the SPOP gene promoter in NSCLC tissues. The ChIP-enriched DNA fragments of the SPOP gene promoter using IgG and an anti-C/EBPα antibody were amplified by PCR. Total input was used as a positive control. c Sequential deletion and substitution mutation analyses identified C/EBPα binding sites in the SPOP gene promoter region. Serially truncated and mutated SPOP gene promoter constructs were cotransfected with siC/EBPα or siControl into A427 cells, and the relative luciferase (luc.) activities were determined. Following the transfection of the C/EBPα expression plasmid (d) or siC/EBPα (e) into the A427 and H1299 cells, the C/EBPα and SPOP mRNA and protein levels were determined by qPCR and western blotting. The data are presented as the mean ± SD of three independent experiments. *P < 0.05

Fig. 5

SPOP mediates C/EBPα-regulated proliferation, migration and invasion of lung cancer cells in vitro and in vivo. a C/EBPα and SPOP expression in A429 and H1299 cells treated as indicated was detected by western blotting. b Cell proliferation ability was assessed by MTT assays in A429 and H1299 cells treated as indicated. c, d Cell Migration and invasion abilities were assessed using Transwell and Matrigel-coated Transwell assays, respectively, in A427 (c) and H1299 (d) cells treated as indicated. The data are presented as the mean ± SD of three independent experiments. *P < 0.05 versus A427 or H1299 cells cotransfected with siRNA and vector control (siControl + Vector); ^#P < 0.05 versus A427 cells cotransfected with siSPOP and vector control (Vector + siSPOP) or H1299 cells cotransfected with siControl and SPOP expression plasmid (siControl + SPOP). e Representative xenograft tumors in nude mice (upper left panel), and representative photographs of tumors removed from bodies 28 days after implantation (bottom left panel). f Tumor growth curves showing that the C/EBPα depletion significantly enhanced xenograft tumor growth, and these effects were reversed by the SPOP overexpression in the nude mice. *P < 0.05 versus siRNA and vector control group; ^#P < 0.05 versus siSPOP and vector control group