NF-κB upregulation through epigenetic silencing of LDOC1 drives tumor biology and specific immunophenotype in Group A ependymoma

Abstract

Background: Inflammation has been identified as a hallmark of high-risk Group A (GpA) ependymoma (EPN). Chronic interleukin (IL)-6 secretion from GpA tumors drives an immune suppressive phenotype by polarizing infiltrating monocytes. This study determines the mechanism by which IL-6 is dysregulated in GpA EPN.

Methods: Twenty pediatric GpA and 21 pediatric Group B (GpB) EPN had gene set enrichment analysis for MSigDB Hallmark gene sets performed. Protein and RNA from patients and cell lines were used to validate transcriptomic findings. GpA cell lines 811 and 928 were used for in vitro experiments performed in this study.

Results: The nuclear factor-kappaB (NF-κB) pathway is a master regulator of IL-6 and a signaling pathway enriched in GpA compared with GpB EPN. Knockdown of NF-κB led to significant downregulation of IL-6 in 811 and 928. NF-κB activation was independent of tumor necrosis factor alpha (TNF-α) stimulation in both cell lines, suggesting that NF-κB hyperactivation is mediated through an alternative mechanism. Leucine zipper downregulated in cancer 1 (LDOC1) is a known transcriptional repressor of NF-κB. In many cancers, LDOC1 promoter is methylated, which inhibits gene transcription. We found decreased LDOC1 gene expression in GpA compared with GpB EPN, and in other pediatric brain tumors. EPN cells treated with 5AZA-DC, demethylated LDOC1 regulatory regions, upregulated LDOC1 expression, and concomitantly decreased IL-6 secretion. Stable knockdown of LDOC1 in EPN cell lines resulted in a significant increase in gene transcription of v-rel avian reticuloendotheliosis viral oncogene homolog A, which correlated to an increase in NF-κB target genes.

Conclusion: These results suggest that epigenetic silencing of LDOC1 in GpA EPN regulates tumor biology and drives inflammatory immune phenotype.

Keywords: LDOC1; NF-κB; ependymoma; inflammation; methylation.

Fig. 1

NF-κB is hyperactivated in GpA EPN. (A) MSigDB HALLMARK_TNF_SIGNALING_NFKB gene set enriched in GpA (n = 19) to GpB (n = 20) EPN. (B) RELA gene expression significantly higher in GpA versus GpB. * P < 0.05. (C) Nuclear and cytoplasmic lysates collected from snap frozen EPN patient samples. Ratio of nuclear RELA to cytoplasmic RELA densitometry (n = 9 each GpA and GpB). A representative blot is shown. (D) Immunofluorescence staining of RELA baseline (left) and after 1 h TNF-α stimulation (right) in 2 GpA EPN cell lines, 928 and 811. Images are representative of 3 independent experiments. RELA is stained red and the nucleus is stained with DAPI in blue.

Fig. 2

NF-κB regulates IL-6 secretion and tumor cell survival in GpA EPN cell lines. (A) Western blot validation of RELA knockdown using lentiviral delivery of shRNA targeting RELA in 811 and 928. (B) IL-6 secretion after RELA knockdown. IL-6 secretion was normalized to nontarget shRNA (NT). (C) Neurosphere area after 10 days of growth following RELA knockdown. Experiments were repeated in triplicate. * denotes P < 0.05. (D) Cell proliferation after RELA knockdown measured using the xCelligence system.

Fig. 3

LDOC1 is identified as the only gene underexpressed in GpA EPN. (A) Limma analysis of 228 patient samples comparing GpA with all other pediatric brain tumors and normal brain identified LDOC1 underexpressed in GpA. (B) PFS for LDOC1 mRNA expression in PF EPN. Median survival for high expression patient was 51 months, for mid-expression 54.6 months, and for low expression patients 23.7 months. P-value determined using Mantel‒Cox test. (C) LDOC1 protein expression in GpA (n = 8) and GpB (n = 7) EPN. Densitometry ratio of LDOC1 to actin. *P < 0.05. (D) LDOC1 gene transcript comparing GpA cell lines with common pediatric brain tumor cell lines. (E) LDOC1 protein expression in GpA EPN cell lines compared with DAOY and DIPG iv. Densitometry ratio of LDOC1 to actin.

Fig. 4

LDOC1 gene expression is silenced through DNA methylation. (A) LDOC1 gene expression measured by qRT-PCR after 7 days of 5AZA-DC treatment. Cells were treated with 10 μM 5AZA-DC refreshed every 3 days. Experiments were completed in triplicate and * denotes P < 0.05. (B) Immunofluorescence staining of LDOC1 (green) after 7 days of 10 μM 5AZA-DC refreshed every 3 days. Experiments were completed in triplicate and images are representative. (C) Quantification of LDOC1 expression after 5AZA-DC treatment ratio of nuclear LDOC1 to DAPI signal.

Fig. 5

5AZA-DC treatment led to decreased RELA transcription and suppression of NF-κB activity. (A) Treatment with 10 μM 5AZA-DC of EPN cell lines for 7 days with refreshing every 3 days. RELA gene transcription by qRT-PCR normalized to untreated (UT) control. (B) IL-6 secretion measured by ELISA (right) in pg/mL. Experiments were repeated in triplicate and * denoted P < 0.05. (C) Quantification of RELA expression after 5AZA-DC treatment ratio of nuclear RELA to DAPI signal. (D) Immunofluorescence staining of RELA after 10 μM 5AZA-DC treatment; 100 ng/mL TNF-α stimulation for 1 h. RELA staining in green and nucleus stained with DAPI (blue).

Fig. 6

LDOC1 regulates RELA transcription and activation of NF-κB signaling in GpA EPN. (A) IL-6 secretion measured from ex vivo patient-derived organotype slice cultures. Red indicates patients with low LDOC1 gene expression and blue denotes patients with high LDOC1. Replicate points denote cultures when material allowed for more than 1 well to be plated. (B) LDOC1 knockdown by lentiviral delivery of shRNA targeting LDOC1. Knockdown was validated by qRT-PCR. (C) IL-6 secretion by ELISA from cells after LDOC1 knockdown. (D) RELA gene expression after LDOC1 knockdown by qRT-PCR, which was normalized to shNT. Experiments were completed in triplicate and * denotes P < 0.05. (E) NF-κB target genes upregulated following LDOC1 knockdown. Experiments were performed in triplicate. Venn diagram displays number of genes upregulated with each construct compared with shNT.