Leukemia-associated RhoGEF (LARG) Restricts Starvation-induced Autophagy Through Rac1-dependent Pathways In Human Lung Adenocarcinoma Cells

Background: LARG is a member of Rho superfamily guanine nucleotide exchange factor,which regulates the conversion of Rho GTPases GDP bound form to GTP bound form.As a member of 70 Rho GEFs,LARG regulates cellular motility,contractility and mitosis through the activity of Rho GTPases.However,little is known about the role of LARG in the regulation of autophagy.The roles of LARG in leukemia and solid tumors depend on different signaling pathways.LARG functions as oncogene in acuteleukemia whereas it plays as tumor suppressor in breast and colorectal cancers.Thus,more researches need to deeply investigate the role of LARG in solid tumors.In this paper,we will focus the expression of LARG in lung adenocarcinoma and the role of LARG in the regulation of starvation-induced autophagy.Methods: First,we investigated the expression of LARG in lung adenocarcinoma in vivo and in vitro.By using six lung carcinoma cell lines and HBEC cell line,we detected the protein and transcription level of LARG in vitro.Immunostaining of LARG in 30 clinical lung adenocarcinoma samples was used to present the data in vivo.Second,the regulation of starvation-induced autophagy by LARG was investigated.By using HBSS treatment as a model to simulate the stressful environment which cancer cells may encounter during the progression of tumor,we tested the alteration of autophagy markers in the cells engineered by sh RNA interference and overexpression technique.Using GST pull down assay,we hoped to find the specific Rho GTPase which is regulated by LARG and to verify the regulation of autophagy by LARG through this Rho GTPase.Last,we investigated the proliferation and apoptosis in stressful cancer cells to evaluate whether LARG altered the ability of survival in stressful cells.Results: First,the protein and transcription level of LARG was decreased in A549,H1299,H446 and H1993 cell lines compared with HBECs,especially in the stage III lung adenocarcinoma separated H1993 cells.In clinical samples,LARG expressed higher in stage I,II samples than stage III,IV samples.Second,cells expressed LARG existed more properties of autophagy than cells without LARG after the treatment of HBSS,including that the decreased accumulation of p62/SSTM-1 and the increased autolysosomes.Results of GST pull down assay illustrated that LARG modulated the activity of Rac1 rather than Rho A in A549 and H1993 cell lines.LARG regulated the starvation-induced autophagy via active Rac1.Last,LARG modulated the protein level of p53 and p21CDKN1 A through autophagy,thereby changing the proliferation and apoptosis in cancer cells during stressful condition.Conclusions: LARG is downregulated during the progression of lung adenocarcinoma.Loss of LARG facilitates starvation-induced autophagy via the decreased of active Rac1 in lung adenocarcinoma cells.The accumulation of p53 upon stressful condition is eliminated by enhanced autophagy,whereas p21CDKN1 is upregulated.Suppression of apoptosis and proliferation leads to the survival of lung adenocarcinoma cells in stressful environment.Above results show us that LARG may function as tumor suppressor in lung adenocarcinoma through the restriction of autophagy.Moreover,LARG is not the molecular target for the regulation of Rho A in lung adenocarcinoma.