pubmed.ncbi.nlm.nih.gov

Epigenetic regulation of L1CAM in endometrial carcinoma: comparison to cancer-testis (CT-X) antigens - PubMed

  • ️Tue Jan 01 2013

Epigenetic regulation of L1CAM in endometrial carcinoma: comparison to cancer-testis (CT-X) antigens

Uwe Schirmer et al. BMC Cancer. 2013.

Erratum in

Abstract

Background: L1CAM was originally identified as an adhesion molecule involved in neural development. In many human carcinomas L1CAM is over-expressed and is associated with a bad prognosis. We previously reported that L1CAM was absent in the vast majority of endometrioid endometrial carcinomas (ECs) (type 1) but was strongly expressed in the more aggressive serous and clear-cell ECs (termed type 2). The differential regulation of L1CAM in ECs is not well understood. Recent evidence suggests that it can be regulated by epigenetic mechanisms. Here we investigated the role of DNA-methylation of the L1CAM promoter for expression. We also studied the relationship to cancer testis (CT-X) antigens that co-localize with L1CAM on chromosome Xq28, a region that is often activated in human tumors.

Methods: We used EC cell lines and primary tumor tissues for our analysis. For expression analysis we employed RT-PCR and Western blotting. DNA-Methylation of the L1CAM promoter was determined after bisulfite conversation and DNA sequencing. Tumor tissues were examined by immunohistochemical (IHC) staining.

Results: We demonstrate that the treatment of L1CAM low/negative expressing EC cell lines with 5'-Azacytidine (5-AzaC) or knock-down of DNMT1 (DNA methyltransferase 1) as well as the HDAC (histone deacetylase) inhibitor Trichostatin A (TSA) up-regulated L1CAM at the mRNA and protein level. The L1CAM gene has two promoter regions with two distinct CpG islands. We observed that the expression of L1CAM correlated with hypermethylation in promoter 1 and 5-AzaC treatment affected the DNA-methylation pattern in this region. The CT-X antigens NY-ESO-1, MAGE-A3 and MAGE-A4 were also strongly up-regulated by 5-AzaC or knock-down of DNMT1 but did not respond to treatment with TSA. Primary EC tumor tissues showed a variable methylation pattern of the L1CAM promoter. No striking differences in promoter methylation were observed between tumor areas with L1CAM expression and those without expression.

Conclusions: L1CAM expression correlated with methylation of the L1CAM promoter in EC cell lines. In negative cell lines L1CAM expression is up-regulated by epigenetic mechanism. Although genes localized on Xq28 are often re-expressed by human tumors, L1CAM and CT-X antigens show distinct regulation in response to HADC inhibitors and 5-AzaC.

PubMed Disclaimer

Figures

Figure 1
Figure 1

L1CAM expression in endometrial carcinoma cell lines. (A) mRNA was isolated from the indicated cell lines, transcribed to cDNA and subjected to quantitative RT-PCR analysis. β-actin served as internal standard. (B) FACS analysis of selected cell lines. Cells were stained with mAb L1-9.3 (solid line) to the ectodomain of L1CAM followed by PE-conjugated goat anti mouse IgG. For background control (shaded curves) the primary antibody was omitted.

Figure 2
Figure 2

Regulation of L1CAM expression by epigenetic mechanisms. (A) RT-PCR analysis of cells treated for 5 days with the indicated concentration of 5-AzaC, TSA or both compounds. DMSO was used as a mock control. Cells were lysed and mRNA was isolated and transcribed into cDNA. β-actin served as internal standard. (B) Cells were treated as described above and cell lysates were prepared for Western blot analysis. MAb L1-11A was used as a primary antibody followed by peroxidase conjugated Goat anti mouse IgG and ECL detection. (C) TSA and VA up-regulate L1CAM expression. Cells were treated and analyzed as described in (B).

Figure 3
Figure 3

MethyLight analysis of promoter 1 of the L1CAM promoter region. (A) The indicated cell lines were treated with 5-AzaC or DMSO and the L1CAM promoter was subjected to MethyLight PCR. (B) MethyLight analysis of the L1CAM promoter region in EC cell lines. The analyzed region comprised fragment PP1 (see Figure 4A).

Figure 4
Figure 4

Methylation of the L1CAM promoter in EC cell lines. (A) Schematic illustration of the L1CAM promoter region according to the Ensembl database. Exon 1 contains the ATG and the transcription start sites. The upstream sequence of appr. 4100 bp was identified as promoter 2. The non-coding Exon 0 (82 bp) is located appr. 10 kb upstream and is followed by an appr. 4.5 kb promoter region (promoter 1). CpG islands are also indicated. PP1, 2 and 3 indicates fragments that were analysed by sequencing after bisulfite conversion. CpG sides are indicated. (B) Schematic illustration of methylation sides in promoter 1 and promoter 2 of the cell lines analysed.

Figure 5
Figure 5

L1CAM promoter Methylight analysis of EC tumor tissues. (A) Representative staining examples of EC tissues with L1CAM negative and L1CAM positive staining areas. (B) Results of the Methylight reaction from paired tumor samples and normal endometrium (numbered from 1 to 14) are shown. DNAs were extracted from punched areas (1 mm diameter) that were selected according to IHC staining. Note that normal endometrium is L1CAM negative.

Figure 6
Figure 6

Analysis of MAGEA and NY-ESO-1 expression. RT-PCR analysis of cells treated for 5 days with the indicated concentration of 5-AzaC, TSA or both compounds. DMSO was used as a mock control. Cells were lysed and mRNA was isolated and transcribed into cDNA. β-actin served as internal standard.

Figure 7
Figure 7

Analysis of expression after DNMT1 knock-down. (A) DNMT1 was depleted by siRNA in HEC-1A and ECC1 cells and the efficacy of the knock-down was analyzed by RT-PCR. (B) Effect of DNMT1 depletion on the expression of the indicated gene products as analyzed by RT-PCR. siGFP was used as control. (C) Cells were treated as described above and cell lysates were prepared for Western blot analysis. Primary antibodies to the indicated proteins were used followed by peroxidase conjugated Goat anti mouse IgG and ECL detection. Representative blots from n = 2 experiments are shown.

Figure 8
Figure 8

IHC analysis of testis and EC tissues. Expression of NY-ESO-1 and MAGE-A4 but absence of L1CAM in normal human testis tissue. Conversely, L1CAM is expressed in type II EC but NY-ESO-1 and MAGE-A4 are undetectable. Note that a representative case of n = 5 is shown. Sequential tissue sections were analysed by IHC.

Similar articles

Cited by

References

    1. Gavert N, Ben-Shmuel A, Raveh S, Ben-Ze’ev A. L1-CAM in cancerous tissues. Expert Opin Biol Ther. 2008;8(11):1749–1757. doi: 10.1517/14712598.8.11.1749. - DOI - PubMed
    1. Schafer MK, Altevogt P. L1CAM malfunction in the nervous system and human carcinomas. Cell Mol Life Sci. 2010;67(14):2425–2437. doi: 10.1007/s00018-010-0339-1. - DOI - PMC - PubMed
    1. Kiefel H, Bondong S, Hazin J, Ridinger J, Schirmer U, Riedle S, Altevogt P. L1CAM: a major driver for tumor cell invasion and motility. Cell Adh Migr. 2012;6(4):374–384. doi: 10.4161/cam.20832. - DOI - PMC - PubMed
    1. Fogel M, Gutwein P, Mechtersheimer S, Riedle S, Stoeck A, Smirnov A, Edler L, Ben-Arie A, Huszar M, Altevogt P. L1 expression as a predictor of progression and survival in patients with uterine and ovarian carcinomas. Lancet. 2003;362(9387):869–875. doi: 10.1016/S0140-6736(03)14342-5. - DOI - PubMed
    1. Fogel M, Mechtersheimer S, Huszar M, Smirnov A, Abu-Dahi A, Tilgen W, Reichrath J, Georg T, Altevogt P, Gutwein P. L1 adhesion molecule (CD 171) in development and progression of human malignant melanoma. Cancer Lett. 2003;189(2):237–247. doi: 10.1016/S0304-3835(02)00513-X. - DOI - PubMed

Publication types

MeSH terms

Substances