ELF3 is an antagonist of oncogenic-signalling-induced expression of EMT-TF ZEB1

Abstract

Background: Epithelial-to-mesenchymal transition (EMT) is a key step in the transformation of epithelial cells into migratory and invasive tumour cells. Intricate positive and negative regulatory processes regulate EMT. Many oncogenic signalling pathways can induce EMT, but the specific mechanisms of how this occurs, and how this process is controlled are not fully understood. Methods: RNA-Seq analysis, computational analysis of protein networks and large-scale cancer genomics datasets were used to identify ELF3 as a negative regulator of the expression of EMT markers. Western blotting coupled to siRNA as well as analysis of tumour/normal colorectal cancer panels was used to investigate the expression and function of ELF3. Results: RNA-Seq analysis of colorectal cancer cells expressing mutant and wild-type β-catenin and analysis of colorectal cancer cells expressing inducible mutant RAS showed that ELF3 expression is reduced in response to oncogenic signalling and antagonizes Wnt and RAS oncogenic signalling pathways. Analysis of gene-expression patterns across The Cancer Genome Atlas (TCGA) and protein localization in colorectal cancer tumour panels showed that ELF3 expression is anti-correlated with β-catenin and markers of EMT and correlates with better clinical prognosis. Conclusions: ELF3 is a negative regulator of the EMT transcription factor (EMT-TF) ZEB1 through its function as an antagonist of oncogenic signalling.

Keywords: Oncogenic signalling; RNA-seq; Wnt signalling; epithelial-mesenchymal transition; protein networks.

Figure 1

Comparative transcriptome analysis identifies ELF3 as a candidate regulator of oncogenic signalling-induced EMT (A) Selected enriched Gene Ontology terms in mutant or wild-type β-catenin cells. Significant Gene Ontology terms (p < 0.05) in either mutant or wild-type cells pertaining to Wnt signalling or EMT are shown (B, C) Gene-expression ratios (log2 ratio of mutant/wild-type β-catenin cells from RNA-Seq) for (B) known direct β-catenin/TCF transcriptional targets and for (C) Epithelial-Mesenchymal-Transition components. Significance (indicated by asterices) is fdr-adjusted Student’s t-test (D) Highest scoring sub-network showing connected gene products with significantly differential (p < 0.05) abundance between mutant and wild-type β-catenin cells. Network nodes are coloured according to the mutant/wild-type expression ratio (red = mutant> wild-type), (green = wild-type> mutant). Transcription factors enriched in this network are shown in the Table, and are ranked according to the degree of enrichment. The highest scoring network is significantly enriched for KLF family and ELF3 transcription factors. Asterices indicate those genes predicted to be regulated by ELF3 (from the transcription factor enrichment analysis).

Figure 1 (Continued).

Figure 2

ELF3 expression and correlation across large-scale tumour datasets (A) ELF3 and EMT marker ZEB1 show anti-correlation of gene-expression across colorectal, pancreatic and stomach adenocarcinoma TCGA datasets. Colorectal values are microarray z-scores and stomach and pancreatic values are RNA-Seq RSEM values (B) Heatmap showing gene-expression values from colorectal cancer TCGA (270 samples) study (Muzny et al, 2012). The 25 most-correlated (Pearson’s r) gene-expression profiles for ELF3 and CTNNB1 are shown as indicated and the histogram indicates the distribution of correlation values across this dataset for ELF3.

Figure 3

Reduced ELF3 expression in colorectal adenocarcinoma (CRC) and correlation with poor patient survival (A) Representative ELF3 staining pattern (High or Low ELF3) in 86 human CRC tissue microarray cores. Scale bar: 200 μm. (B) Graph showing IHC scores of ELF3 staining in 86 paired human CRC and adjacent normal tissues (P < 0.0001) (C) A Kaplan–Meier survival curve shows significant association between low levels of ELF3 and poor survival in CRC patients (P = 0.022). The y axis indicates percentage of patients surviving at the indicated time points.

Figure 4

ELF3 expression is repressed by oncogenic signaling (A) Relative ELF3 mRNA expression in HCT116-CTNNB1^Δ45/- and HCT116-CTNNB1^WT/- cells from RNA-Seq analysis (n = 3 replicates for each cell-line). (B) Western blot analysis of ELF3, β-catenin and EMT markers ZEB1 and E-cadherin in HCT116-CTNNB1^Δ45/- and HCT116-CTNNB1^WT/- in cell-lines. μ-tubulin was used as a loading control. (C) ELF3 mRNA levels in HCT-116 cells compared with HKe-3 and HKh-2 cells, in which mutant KRAS is removed. (n = 3 replicates for each cell-line). (D) Western blot analysis of ELF3, phosphor-ERK (p-ERK) and an EMT marker ZEB1 in HKe3 ER:HRAS V12 cells with the indicated treatment. β-tubulin was used as a loading control. Graph showing relative expression of ZEB1 or ELF3, normalized to β-tubulin in HKe3 ER:HRAS V12 cells with the indicated treatment (n = 3).

Figure 5

ELF3 is an antagonist of oncogenic EMT and Wnt signaling (A) Western blot analysis of ZEB1, ELF3, phospho-ERK (p-ERK) and phospho-AKT (p-AKT) in HKe3 ER:HRAS V12 cells with the indicated treatment. β-tubulin was used as a loading control. (B) Expression of human ELF3 inhibits Super8XTOPFlash luciferase reporter activity in HEK293T and in HCT116 cells (C) Western blot analysis of ZEB1, ELF3 and β-catenin in HKe3 ER:HRAS V12 cells with the indicated treatment. β-actin was used as a loading control.