Suppression of the grainyhead transcription factor 2 gene (GRHL2) inhibits the proliferation, migration, invasion and mediates cell cycle arrest of ovarian cancer cells

Abstract

Previously, we have identified the Grainyhead transcription factor 2 gene (GRHL2) as notably hypomethylated in high-grade (HG) serous epithelial ovarian tumors, compared with normal ovarian tissues. GRHL2 is known for its functions in normal tissue development and wound healing. In the context of cancer, the role of GRHL2 is still ambiguous as both tumorigenic and tumor suppressive functions have been reported for this gene, although a role of GRHL2 in maintaining the epithelial status of cancer cells has been suggested. In this study, we report that GRHL2 is strongly overexpressed in both low malignant potential (LMP) and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Suppression of the GRHL2 expression led to a sharp decrease in cell proliferation, migration and invasion and induced G1 cell cycle arrest in epithelial ovarian cancer (EOC) cells displaying either epithelial (A2780s) or mesenchymal (SKOV3) phenotypes. However, no phenotypic alterations were observed in these EOC cell lines following GRHL2 silencing. Gene expression profiling and consecutive canonical pathway and network analyses confirmed these data, as in both these EOC cell lines, GRHL2 ablation was associated with the downregulation of various genes and pathways implicated in cell growth and proliferation, cell cycle control and cellular metabolism. Taken together, our data are indicative for a strong oncogenic potential of the GRHL2 gene in EOC progression and support recent findings on the role of GRHL2 as one of the major phenotypic stability factors (PSFs) that stabilize the highly aggressive/metastatic hybrid epithelial/mesenchymal (E/M) phenotype of cancer cells.

Keywords: CRISPR/Cas9; GRHL2; cancer cell migration/invasion; epithelial ovarian cancer; epithelial-to-mesenchymal transition; microarrays; shRNA.

Figure 1.

Analysis of GRHL2 expression in serous EOC tumors by IHC. A. Representative IHC images of GRHL2 protein expression in normal ovarian tissues, low-malignant potential (LMP) tumors and high-grade (HG) tumors. B. Box-plot presentation of GRHL2 protein expression levels in normal ovarian tissues, LMP tumors and HG tumors. C. Western-blot analysis of endogenous GRHL2 protein expression in different EOC cell lines.

Figure 2.

Analyses of alterations in functional phenotypes upon CRISPR/Cas9-mediated GRHL2 knockout (KO) in A2780s cells. (A) effect of GRHL2 KO on cell proliferation. (B, C) Representative images from one of the 3 independent experiments showing migration (B, left) and invasion (C, left) in the control clone and GRHL2-KO clones A1 and A2 (at magnification ×400). The bar graphs in panels B (right) and C (right) are quantitative determinations of data obtained by selecting 10 random fields per filter (at magnification× 40) under phase contrast microscopy. Differences between control cells and KO cells were determined by a Student's t-test. Error bars denote ± SEM and *indicates statistical significance (p ≤ 0.05).

Figure 3.

Effect of GRHL2 suppression on cell cycle control in EOC cells. (A) Effect of GRHL2 suppression on cell cycle control in A2780s cells. Cell-cycle profile was examined by flow cytometry and percentages of cells in G0/G1, S, and G2/M phase in the GRHL2 KO clone A1 were compared with the mock-transfected control (Ctrl) clone. Propidium iodide staining shows an increased fraction of cells in the G1-phase and a decrease of cells in the S-phase at 0 h, and especially at 6 and 12 h post hydroxyurea removal in the A1 clone, when compared with the control clone (Ctrl). (B) Effect of GRHL2 suppression on cell cycle control in SKOV3 cells. Cell-cycle profiles were examined by flow cytometry and percentages of cells in G0/G1, S, and G2/M phase in the shRNA-GRHL2 clone 2 (sh-S2) were compared with the mock-transfected control (Ctrl) clone. Propidium iodide staining shows a significantly increased fraction of cells in the G1-phase and a strong decrease of cells in the S-phase at 0, 6 and 12 h post hydroxyurea removal in the sh-S2 clone, when compared with the control clone (Ctrl).

Figure 4.

Analyses of alterations in functional phenotypes upon shRNA-mediated GRHL2 knockdown (KD) in SKOV3 cells. (A) Effect of GRHL2 KD clones on cell proliferation compared with the control clone (B, C) Representative images from one of the 3 independent experiments showing migration (B, left) and invasion (C, left) in the control clone and GRHL2-knockdown clones sh-S2 and sh-S5 (at magnification ×400). The bar graphs in panels B (right) and C (right) are quantitative determinations of data obtained by selecting 10 random fields per filter (at magnification× 40) under phase contrast microscopy. Differences between control cells and KD cells were determined by a Student's t-test. Error bars denote ± SEM and *indicates statistical significance (p ≤ 0.05).

Figure 5.

Top canonical pathways that were significantly dysregulated upon GRHL2 knockout (KO) in A2780s cells. A. Canonical pathways associated with upregulated genes upon GRHL2 KO; B. Canonical pathways associated with downregulated genes upon GRHL2 KO. Top functions are displayed that meet the Benjamini-Hochberg (B-H) multiple testing correction p-value of 0.05.

Figure 6.

Top canonical pathways that were significantly dysregulated upon GRHL2 knockdown (KD) in SKOV3 cells. A. Canonical pathways associated with upregulated genes upon GRHL2 KD; B. Canonical pathways associated with downregulated genes upon GRHL2 KD. Top functions are displayed that meet the B-H multiple testing correction p-value of 0.05.

Figure 7.

Top canonical pathways that were significantly dysregulated upon GRHL2 overexpression in SKOV3 cells. A. Canonical pathways associated with upregulated genes upon GRHL2 overexpression; B. Canonical pathways associated with downregulated genes upon GRHL2 overexpression. Top functions are displayed that meet the B-H multiple testing correction p-value of 0.05.