Silencing of GRHL2 induces epithelial‑to‑mesenchymal transition in lung cancer cell lines with different effects on proliferation and clonogenic growth

Abstract

Grainyhead-like 2 (GRHL2) is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT). It has been previously shown that GRHL2 can confer both oncogenic and tumor-suppressive roles in human cancers, including breast, pancreatic and colorectal cancers. However, its role in lung cancer remains elusive. In the present study, a meta-analysis of multiple gene expression datasets with clinical data revealed that GRHL2 expression was increased in lung cancer compared with that in the normal tissues. Copy number analysis of GRHL2, performed using datasets of whole exome sequencing involving 151 lung cancer cell lines, revealed frequent amplifications, suggesting that the increased GRHL2 expression may have resulted from gene amplification. A survival meta-analysis of GRHL2 using The Cancer Genome Atlas (TCGA) dataset showed no association of GRHL2 expression with overall survival. GRHL2 expression was found to be associated with EMT status in lung cancer in TCGA dataset and lung cancer cell lines. GRHL2 knockdown induced partial EMT in the hTERT/Cdk4-immortalized normal lung epithelial cell line HBEC4KT without affecting proliferation measured by CCK-8 assays. In addition, GRHL2 silencing caused three lung cancer cell lines, H1975, H2009 and H441, to undergo partial EMT. However, the proliferative effects differed significantly. GRHL2 silencing promoted proliferation but not colony formation in H1975 cells whilst suppressing colony formation without affecting proliferation in H2009 cells, but it did not affect proliferation in H441 cells. These results suggest cell type-dependent effects of GRHL2 knockdown. Downstream, GRHL2 silencing enhanced the phosphorylation of AKT and ERK, assessed by western blotting with phospho-specific antibodies, in HBEC4KT, H1975 and H2009 cell lines but not in the H441 cell line. By contrast, transient GRHL2 overexpression did not affect A549 cell proliferation, which lack detectable endogenous expression of the GRHL2 protein. However, GRHL2 overexpression did suppress E-cadherin expression in A549 cells. These results suggested that GRHL2 does not only function as a tumor suppressor of EMT but can also behave as an oncogene depending on the lung cancer cell-type context.

Keywords: The Cancer Genome Atlas; epithelial-to-mesenchymal transition; human bronchial epithelial cell line.

Figure 1.

GRHL2 is expressed at high levels in lung cancer. (A) Expression analysis of GRHL2 in lung adenocarcinoma and squamous cell carcinoma in TCGA dataset. (B) Meta-analysis of GRHL2 expression in lung adenocarcinoma and squamous cell carcinoma from six studies. T, tumor, N, normal, RE, random effects. (C) DNA copy number analysis of GRHL2 showing frequent increases in the copy number of GRHL2 in NSCLC cell lines. (D) Kaplan-Meier survival curves for patients with high or low GRHL2 expression (the cut-off value is the median expression level). (E) Survival meta-analysis for GRHL2 in adenocarcinoma (left) and squamous cell carcinoma (right). In each forest plot, the name of each study is followed by the number of total tumor samples. GRHL2, Grainyhead-like 2; SMD, standardized mean difference; TE, estimated treatment effect; seTE, standard error of treatment effect; HR, hazard ratio; CI, confidence interval, TCGA, The Cancer Genome Atlas; NSCLC, non-small cell lung cancer.

Figure 2.

GRHL2 is associated with an epithelial phenotype in lung cancer. (A) Western blotting of GRHL2, E-cadherin and Vimentin in a panel of lung cancer and immortalized bronchial epithelial cell lines. Values below bands indicate the semi-quantitation of band intensities normalized to β-actin. (B) Correlations between bands intensities of GRHL2 and E-cadherin (left graph) or Vimentin (right graph) in (A). Correlation analysis between the expression of GRHL2, E-cadherin, VIMENTIN and six master epithelial-to-mesenchymal transition regulator genes in (C) adenocarcinomas and (D) squamous cell carcinomas in The Cancer Genome Atlas dataset. The degrees of positive or negative correlation, revealed by Pearson's correlation coefficients, are colored by the darkness of purple or blue, respectively. GRHL2, Grainyhead-like 2; ZEB, Zinc finger E-box-binding homeobox; SNAI, snail homolog.

Figure 3.

GRHL2 silencing induces a partial EMT in hTERT/Cdk4-immortalized normal lung epithelial cell line (HBEC4KT) without affecting its growth. (A) Western blotting of GRHL2, E-cadherin and Vimentin in the immortalized normal bronchial epithelial cell line HBEC4KT transfected with either control or GRHL2 siRNA. Values below bands indicate quantitation of band intensities normalized to β-actin. Quantification of band intensities is shown in the right graph. (B) Reverse transcription-quantitative PCR of E-cadherin, VIMENTIN and four master EMT regulators in HBEC4KT cells, transfected with either control (si-NC) or GRHL2 siRNA. (C) Proliferation and (D) colony formation assays for HBEC4KT cells transfected with either control or GRHL2 siRNA. Results are shown as the mean ± SD (n=3), where comparisons were performed with one-way ANOVA followed by the Dunnett test. **P<0.01 and ***P<0.001 vs. si-NC. GRHL2, Grainyhead-like 2, siRNA, small-interfering RNA; NC, negative control; n.s., non-significant; EMT, epithelial-to-mesenchymal transition; ZEB, Zinc finger E-box-binding homeobox; SNAI, snail homolog.

Figure 4.

GRHL2 silencing induces a partial EMT in lung cancer but its effects on growth are cell type-dependent. (A) Western blotting of GRHL2, E-cadherin and vimentin in the lung cancer cell lines H1975 and H2009 transfected with control or GRHL2 siRNA. Values below bands indicate quantitation of band intensities normalized to β-actin. Quantification of band intensities is shown in the bottom graph. (B) Reverse transcription-quantitative PCR of E-cadherin, vimentin and four master EMT regulator genes in H1975 and H2009 cells transfected with control (si-NC) or GRHL2 siRNA. (C) Proliferation and (D) colony formation assays of H1975 and H2009 cells transfected with either control or GRHL2 siRNA. Results are shown as the mean ± SD, assessed by one-way ANOVA followed by the Dunnett test. *P<0.05, **P<0.01 and ***P<0.001 vs. si-NC. GRHL2, Grainyhead-like 2; siRNA, small-interfering RNA; NC, negative control; n.s., non-significant; EMT, epithelial-to-mesenchymal transition; ZEB, Zinc finger E-box-binding homeobox; SNAI, snail homolog.

Figure 5.

GRHL2 knockdown promotes the phosphorylation of AKT and ERK in HBEC4KT and lung cancer cell lines. Western blotting of GRHL2, p- or total AKT and p- or total ERK in HBEC4KT, H1975 and H2009 cells transfected with control or GRHL2 siRNA. Values below bands indicate the semi-quantitation of band intensities normalized to AKT for p-AKT (ser473), ERK for p-ERK or β-actin for GRHL2. GRHL2, Grainyhead-like 2; siRNA, small-interfering RNA; NC, negative control; p-, phosphorylated.

Figure 6.

GRHL2 overexpression does not affect proliferation in A549 cells. (A) Western blotting of GRHL2, E-cadherin and Vimentin in the lung cancer cell line A549 transiently transfected with GRHL2- or LacZ-expressing vectors. Quantification of band intensities is shown in the right graph. (B) Reverse transcription-quantitative PCR of E-cadherin, vimentin and four master epithelial-to-mesenchymal regulator genes in A549 transiently transfected with GRHL2- or LacZ-expressing (control) vectors. **P<0.01 vs. control. (C) Proliferation assay for A549 transiently transfected with GRHL2- or LacZ-expressing vectors. Results are shown as the means ± SD (n=3), where comparisons with controls were performed by the unpaired t-test. n.s., indicates ‘no significant difference’. GRHL2, Grainyhead-like 2; ZEB, Zinc finger E-box-binding homeobox; n.s., not significant; SNAI, snail homolog.