Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma

Abstract

Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23-q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior.

Fig. 1.

TCF21 promoter methylation and transcriptional gene silencing in metastasis melanoma. (A) Composition of the human TCF21 gene on the chromosome 6q23.3. (B) Heat maps display quantitative DNA methylation data. Positions of corresponding amplicons a1–a3 are shown relative to the TSS(+1) of TCF21 and a CpG island (CGI, green bar) located in exon 1. Each column represents a CpG unit (consisting of a single CpG or several CpG dinucleotides), each row represents a sample. Relative methylation levels (mCpG) range from 0.0 (light yellow) to 1.0 (dark blue). Gray indicates unavailable data. Upper part, data of 79 metastasis melanoma skin cancers biopsies sorted by the highest to lowest mean value of amplicon a1 and separated into high methylated (≥50%) and low methylated (<50%). In the bottom part, data of 61 metastatic melanoma cell lines derived from the same metastasized biopsies sorted by the highest to lowest mean value of amplicon a1. (C) Kaplan–Meier curves indicating survival after diagnosis of first metastasis (SFM). Patients were grouped according to amplicon a1 methylation levels. Patients with lower methylation level (<50%) of amplicon a1 had a significantly increased SFM (P = 0.05, log-rank test) as compared with patients with higher methylation level (≥50%). (D) Positive correlation (Spearman coefficient ρ = 0.59, P = 0.0001, n = 61) of mean methylation levels in amplicons a1–a3 of metastatic melanoma tissue (MM tissue) and corresponding cancer cell lines (MM cell). (E) Positive correlation (Spearman coefficient ρ = 0.79, P = 0.0003, n = 16) between TCF21 mRNA re-expression and the degree of loss of DNA methylation on TCF21 promoter assessed by amplicon a1 (mean value). The 16 metastatic cell lines were treated by 5-aza-dC (1.5 μM) for 1 week.

Fig. 2.

TCF21 metastasis suppressor activity is mediated by transactivation of KISS1. (A) Dual color FISH on a metaphase spread of cell line C8161 using chromosome 6-specific probe CEP 6 (green signal) and bacterial artificial chromosome RP11-465P13 which contains the genomic sequence of TCF21 (red signal). The two colors indicate the presence of both chromosomes 6. (B) Complete methylation across part of the TCF21 promoter in cell line C8161 as compared with much lower methylation in normal primary skin melanocytes (PSM) and normal primary skin fibroblasts (PSF). (C) Reactivation of TCF21 mRNA expression in C8161 cells upon treatment with 5-aza-dC but not with Trichostatin A (TSA). Treatment with the indicated concentrations for 48 h, values are means ± standard deviation from three independent experiments and shown as a fold re-expression (normalized to the solvent as a control). (D) Quantitative reverse transcription–PCR shows silencing of TCF21 and KISS1 in C8161 but not in PSM and PSF. (E) Restoration of KISS1 transcription upon retroviral introduction of TCF21 cDNA into cell line C8161 (mycTCF21). 1.neo6q, 2.neo6q and 3.neo6q are C8161 derivatives harboring additional 40 cM of chromosome 6q including TCF21. pBABE is empty vector. (F) Binding of TCF21 5′ to the KISS1 transcription start site. Sketch shows the location of several potential TCF21-binding sites (E-box sequence CANNTG). ChIP with cell lines C8161 pBABE (negative vector control) and C8161 pBABEmycTCF21 (TCF21 positive) and antibodies raised against the myc-tag expressed by vector myctcf21 revealed binding of TCF21 in amplicon regions 1–5 (bands in rightmost lanes of composite gel picture below the sketch). No bands were visible in lanes corresponding to the negative control pBABE and amplicon region 6 harboring no E-box sequence. (G) ChIP–quantitative PCR with cell line C8161 myctcf21 and antibodies against TCF21 shows different binding strengths at putative binding sites in the KISS1 promoter, the primer number indicating the position order as in Figure F. Values were normalized to input and are shown as fold enrichment.

Fig. 3.

TCF21 binds to the core promoter of KISS1 (A) Promoter reporter assay in HEK293 cells with overlapping segments located 5′ to the KISS1 transcription start site revealed sequence Δ2 (−2804 to +48) as having the highest promoter strength relative to cells with vector pGL4 only (RLU, relative luminescence units). (B) Promoter reporter assay with deletion derivatives of sequence Δ2 in which various E-box protein sequences had been eliminated. Deletion of 234 bp including the two most 3′ located E-box protein sequences completely abolished promoter activity, whereas elimination of other E-box protein sequences lead to only slight decrease or even strong increase in promoter strength. (C) TCF21 physically interacts with class B basic helix-loop-helix proteins. Coimmunoprecipitation of flag-tagged class B basic helix-loop-helix candidates (E12/E47, TCF4, TCF12) with HA-tagged TCF21 48 h after transfection in Hek293. Equal amounts of proteins were loaded onto an sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to immunoblotting with anti-HA or anti-flag antibody. Negative controls were HA-TCF21 and Flag-E12 cotransfected cells and IgG immunoprecipitation as a negative antibody. (*) indicating that the negative control as input: HA-TCF21 and flag-E12 as protein lysates; IP: IgG. Molecular weights in kilodaltons are indicated on the right. (D) Increased KISS1 promoter strength (construct pGL4-KISS1, −2804 to +48) by synergistic interaction between TCF21 and TCF12. MM-66a cells were transfected with pGL4-KISS1 and basic helix-loop-helix Class B cDNA alone or together with TCF21. Values are means ± standard deviations of four independent experiments (*P < 0.05; Student’s t-test, compared with pGL4-KISS1).

Fig. 4.

Reduced motility of TCF21 expressing C8161 cells (A) Upper panels show initially seeded cells (i) of control pBABE and of pBABEmycTCF21 cells, cells migrated through Boyden Chamber pores (ii). (B) Quantitation of migrated cells transfected with control vector pBABE or pBABEmycTCF21. Given values are means ± standard deviations from triplicates.