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

Previously, we have identified cytosolic form of the grainyhead transcription factor 2 gene (GRHL2) as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that GRHL2 is strongly overexpressed in both LMP and HG serous EOC tumors, thus suggesting that epigenetic mechanisms might be implicated in GRHL2 overexpression in serous epithelial ovarian cancer (EOC). Knockdown of the GRHL2 expression in EOC cells led to sharp decrease of cell proliferation and induced G1-phase cell cycle arrest. Additionally, GRHL2 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon BCAT1 suppression, while some tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the GRHL2 gene in advanced EOC and identify this transaminase as a novel EOC biomarker and putative EOC therapeutic target. To better understand the molecular mechanisms of GRHL2 gene action in ovarian cancer cells, we employed the Agilent Whole Human Genome microarrays, containing ~ 44,000 genes to identify global gene expression changes upon GRHL2 suppression in A2780s cells and GRHL2 suppression and overexpression in SKOV3 cells. We compared the gene expression of the previously selected CRISPR/Cas9-mediated GRHL2 knockout A2780s clones KOA1 and KOA1 against the corresponding control (CA) A2780s clone, and the shRNA-mediated GRHL2-knockdown SKOV3 clones sh-S2 and sh-S5 against the corresponding control (CS) SKOV3 clone. Additionally, we compared gene expression of the previously selected GRHL2 overexpressing SKOV3 clones OV1 and OV2 against the corresponding control (CSov) SKOV3 clone. The microarray experiments were performed in duplicates, as four hybridizations were carried out for the GRHL2-suppressing/overexpressing cell clones against the corresponding control, using a fluorescent dye reversal (dye-swap) technique.