EGFR-ERK Induced Activation Of GRHL1 Promotes Cell Cycle Progression By Up-regulating Cell Cycle Related Genes In Lung Cancer

Objective: Grainyhead-like 1(GRHL1)is a transcription factor involved in embryonic development.However,little is known about the biological function of GRHL1 in cancer.Here we investigated the function and molecular mechanism of GRHL1 in non-small cell lung cancer(NSCLC).Methods: The expression of GRHL1 in non-small cell lung cancer(NSCLC)specimens and cells and its relationship with the prognosis of patients was determined by tissue microarray and protein blot experiment.Cell growth and colony formation assays were used to investigate the function of GRHL1 on promoting the proliferation of NSCLC cells.RNA-seq assay and flow cell cycle experiment were used to explore the target genes regulated by GRHL1 and the influence of GRHL1 in cell cycle pathway.Q-PCR,western blot,Ch IP,and luciferase assays were conducted to determine the direct regulation of GRHL1 target genes.EGF stimulation,ERK inhibition,nuclearplasma separation experiment,immunoprecipitation and,point mutation were used to explore the regulation of GRHL1 by EGFR-ERK.GRHL1 knockdown NSCLC cell lines were established to text the tumor growth in the NSCLC xenograft model.Results: GRHL1 was upregulated in non-small cell lung cancer(NSCLC)and correlated with poor survival of patients.GRHL1 overexpression promoted the proliferation of NSCLC cells.RNA sequencing showed that a series of cell cyclerelated genes were altered when knocking down GRHL1.This study further demonstrated that GRHL1 could regulate the expression of cell cycle-related genes by binding to the promoter regions and increasing the transcription of the target genes.Besides,EGF stimulation could activate GRHL1 and promote its nuclear translocation.We identified the key phosphorylation site at Ser76 on GRHL1 that is regulated by the EGFR-ERK axis.Serine 76 of GRHL1 was the key phosphorylation site regulated by the EGFR-ERK signaling axis.Conclusions: These findings elucidate a new function of GRHL1 on regulating the cell cycle progression and point out the potential role of GRHL1 as a drug target in NSCLC.