Downregulation and Hypermethylation of GABPB1 Is Associated with Aggressive Thyroid Cancer Features

Abstract

Promoter mutations of the telomerase reverse transcriptase (TERT) gene occur frequently in thyroid carcinoma (TC), including papillary (PTC) and anaplastic subtypes (ATC). Given that the ETS family transcription factors GABPA and GABPB1 activate the mutant TERT promoter and induce TERT expression for telomerase activation, GABPB1 has been proposed as a cancer therapeutic target to inhibit telomerase. Here, we sought to determine the role of GABPB1 in TC pathogenesis. In TC-derived cells carrying the mutated TERT promoter, GABPB1 knockdown led to diminished TERT expression but significantly increased invasive potentials in vitro and metastatic potential in a xenograft zebrafish model and altered expression of markers for epithelial-to-mesenchymal transition. GABPB1 expression was downregulated in aggressive TCs. Low GABPB1 expression correlated with its promoter hypermethylation, which in turn was also associated with shorter disease-free survival. Consistently, DNA methylation inhibitors enhanced GABPB1 expression, as observed upon reduced promoter methylation. Our results suggest that GABPB1 is required for TERT expression and telomerase activation, but itself serves as a tumor suppressor to inhibit TC progression. Furthermore, aberrant DNA methylation leads to GABPB1 silencing, thereby promoting TC aggressiveness. Thus, caution is needed if targeting GABPB1 for cancer therapy is considered.

Keywords: DNA methylation; GABPA; GABPB1; TERT; telomerase; thyroid carcinoma.

Figure 1

GABPB1 depletion leads to diminished TERT and DICER1 expression but promotes invasiveness and distant metastasis of TC cells. U-hth-74 and U-hth-104 cells were transfected with control and GABPB1 RNAis, respectively, and harvested for analyses at 48 h. Three independent experiments were performed. (A) Verification of GABPB1 knockdown efficiency using immunoblotting. RNAi to GABPA was also included as an additional control to support the RNAi specificity. Original Images for Western blots are shown in Figure S7. (B) Downregulation of TERT mRNA expression in GABPB1-depleted cells. qPCR was performed to determine mRNA levels. (C,D) Proliferation analyses of GABPB1-depleted U-hth-74 and U-hth-104 cells. (E,F) Enhanced invasion of GABPB1-depleted U-hth-74 and U-hth-104 cells. Matrigel assays were used to determine invasive potentials of cells. (G) Knocking down of GABPB1 in U-hth-104 cells promotes distant metastasis in a zebrafish xenograft model. Representative fluorescence (Magenta) images of U-hth-104 cells injected into perivitelline space (red arrow) of zebrafish larvae 48 h post fertilization under 10X objective. White arrow marks distant tumor foci quantified after 24 h post injection. Scale bar denotes 200 μm (H) Numbers of tumor foci found in the tails of zebrafish larvae. (I) Downregulation of DICER1 mRNA expression in GABPB1-depleted cells. C: control; GA: GABPA; GB: GABPB1; ns: not significant; no: number.

Figure 2

GABPB1 mRNA expression levels are associated with aggressive TCs, and positively correlated with GABPA and DICER1 expression. Gene expression levels were determined using qPCR. (A,B) Significantly lower expression of GABPB1−All and GABPB1L in ATC_K than in PTC_K, respectively. (C) Correlation matrix for analyses between GABPB1−All, GABPB1L, GABPA, DICER1, and TERT mRNA levels in PTC_K. Spearman correlation r values are illustrated in color from blue (−1) to red (1). GABPB1−All: total amount of GABPB1 mRNAs; including all transcript variants of GABPB1; GABPB1L: long variant of GABPB1 mRNA; * p < 0.05; *** p < 0.001; **** p < 0.0001.

Figure 3

The methylation landscape of the GABPB1 loci across TCGA Pan-cancer. (A) Unclustered heatmap showing the methylation density (β value) of the GABPB1 gene locus at various CpG sites based on methylation 450K across 33 cancer types. (B) Methylation of cg14821257 CpG site. Data are presented in Tukey boxplots with ranked order of cancer types based on median β value.

Figure 4

GABPB1 promoter hypermethylation contributes to the downregulation of GABPB1 expression and predicts shorter disease-free survival in thyroid cancer. Analyses of GABPB1 promoter methylation density, gene expression, and survival in PTC_TCGA were performed. Cellular experiments using a DNA methylation inhibitor were performed to verify the causal relationship between GABPB1 promoter methylation and gene expression. (A–D) Identification of cg14821257 at the GABPB1 promoter as the predominant CpG affecting GABPB1 expression. (B,C) Significantly increased cg14821257 methylation in PTC_TCGA tumors as compared to adjacent non-cancerous thyroid tissues (NC_TCGA), but no significant difference for GABPB1 expression. (D) The inverse correlation between methylation of cg14821257 and GABPB1 mRNA levels. (E,F) Hypermethylation of the GABPB1 promoter was significantly associated with shorter disease-free survival but not with overall survival. (G) Upregulation of GABPB1 expression in U-hth-74 and U-hth-104 cells treated with the DNA methylation inhibitor 5-Aza. (H) Reduced cg14821257 methylation in U-hth-74 and U-hth-104 cells treated with the DNA methylation inhibitor 5-Aza. cg14821257 methylation was determined using pyrosequencing.