FGD1 Exhibits Oncogenic Properties In Hepatocellular Carcinoma Through Regulating Cell Morphology And Autophagy

Background It is well known that loss of function mutations in Faciogenital Dysplasia 1(Fgd1)leads to Aarskog-Scott syndrome(AAS),a rare X-linked disorder that manifests in distinguishing set of face shape,urogenital,and disproportionately short stature,Gene mapping shows that Fgd1 located in Xp.11.12,encodes a member of guanine nucleotide exchange factors(GEFs),FGD1 protein,which is an activator specific for the cell division cycle 42(Cdc42)GTPase,a master regulator of extracellular matrix(ECM).GEFs activate GTPase by exchanging of GDP for GTP on the GTPases.It is well established that,by activating Cdc42,Fgd1 has been involved in a variety of biological processes,including cytoskeleton restructuring,cell morphology,gene transcription,cell cycle progression,and cell polarity.However,increasing evidence revealed that GEF family proteins are also associated with the development of a variety of cancers.For example,leukemia-associated Rho-GEF and GEF-H1,members of the GEF family that have strong homology to Fgd1,were considered as potential oncogenes,due to their contribution to the tumorigenesis of leukemia and osteosarcoma respectively.Similarly,abnormal expression of Fgd1 was also identified in several types of cancers.In breast and prostatic cancers,Fgd1 are up-regulated,and the expression levels are positively correlated with tumor aggressiveness.In addition,missense mutations of Fgd1 gene were observed in numerous types of cancer,such as ovarian cancer,melanoma,uterine cancer,head and neck cancer.These studies above indicate that Fgd1 is critical for normal cell functions,but aberrant expression of Fgd1 may play a role in the development of cancer.However,little is known about the role of Fgd1 in hepatocellular carcinoma(HCC).Methods The potential clinical relevance of Fgd1 is evaluated using TCGA database.By over-expressing or knocking down of Fgd1,the effects of Fgd1 on the malignant behavior of HCC cells were evaluated both in vitro and in vivo.The cell cycle and apoptosis assay were analysis by flow cytometry.For further study,we used the i TRAQ technology to analysis the downstream signal pathway of Fgd1 and confirmed these data by western blot.We also used the transmission electron microscope(TEM)to assessing the autophagy activity and the immunofluorescence were employed for detected the mitochondrial function.Results RNA sequencing data from TCGA database shows that Fgd1 was significantly up-regulated in HCC tissues when compared to normal liver tissues,which is associated with age,BMI and tumor grade.Patients with high level of Fgd1 expression always mean the poor prognosis.Further investigate from the knockdown Fgd1 cell line Bel-7404 and overexpression Fgd1 cell line Hep3 B show that knockdown of Fgd1 significantly inhibited the migration,invasion,and motility capacity of Bel-7404 cells in-vitro,and overexpression Fgd1 in Hep3 B via lenticirus have oppose effects.We also detect the cell morphology by TEM capability of Fgd1 decline,and observe disorder in morphology of pseudopodia,characterized by clutter and breakage,indicating that Fgd1 exhibits oncogenic properties in HCC cells.Further explore the role of Fgd1 in vivo show that,compared with the control groups,knockdown of Fgd1 remarkably inhibited tumor growth in syngeneic mice.Further study the Fgd1 downstream signal pathways in oncogenic properties by i TRAQ analysis.There are 64 proteins that had changed significantly after Fgd1 down-regulated.Further analysis shows that Fgd1 involved in autophagy through Cdc42 signal pathways and its decline may cause ATP content decrease.Conclusions Fgd1 is elevated in HCC tissues and correlated with poor prognosis.Suppression of Fgd1 cause inhibition in proliferation and migration of HCC cell lines Knockdown of Fgd1 significantly inhibits the malignant behaviors of HCC cells which show the oncogenic properties in HCC.Further studies found that Fgd1 regulated autophagy,and led to ATP decline.