CLIC4 Knockdown Promotes Cancer Cell Stemness And Epithelial Mesenchymal Transition In Gastric Cancet

Objective:Although CLIC4(Chloride intracellular channel 4)has been implicated in several types of cancers,its role in gastric cancer remains unknown.Our present study focuses on the role of CLIC4 as a suppressor of gastric cancer tumor growth and progression.Methods:We applied tandem mass spectrometry by using isobaric tags for relative and absolute quantitation(ITRAQ)technology on a panel of 4 pairs of gastric adenocarcinoma samples(stage ?-?,Her-2-negative),and identified CLIC4 as a marker that is differentially regulated in gastric cancer(GC).We then expanded the study by analyzing the expression of CLIC4 in additional 102 gastric cancer samples and their adjacent normal gastric tissues by Western blotTo assess the role of CLIC4 in tumor development,we examined the expression level of CLIC4 in 3 different GC cell lines MKN45,N87,and AGS.GC cells were transfected with lentivirus that carries shRNA targeting CLIC4,or overexpressing CLIC4 and stable lines were selected by puromycin.Next,we explored the role of CLIC4 in migration and invasion using lentivirus-mediated stable overexpression or knockdown of CLIC4 in GC cells by transwell and wound healing assayTumor resistance to chemotherapy is mostly due to the presence of cancer stem cells.MKN45,N87,and AGS cells contain different amounts of cancer stem cells that express CD44 and OCT4.To investigate the effect of CLIC4 on cancer stem cells,we validated the effect of overexpression or knockdown of CLIC4 on the expression of CD44 and OCT4 in gastric cancer cells by RT-PCR.Then we explored the effect of CLIC4 knockdown or overexpression on chemoresistance of 5-Fu and Etoposide.The ability to grow and form colonies in soft agar is one of the hallmarks of cancer stem cells,so we studied the role of CLIC4 on the anchorage-independent growth of gastric cancer cells by soft agar colony formation assay.EMT(Epithelial-Mesenchymal Transition)is one of the mechanisms that generate cancer stem cells.We sought to determine whether CLIC4 modulation could affect EMT.So we explored the role of knockdown or overexpression of CLIC4 in EMT by Western blot and immunofluorescence.We also validated the effects of overexpression or knockdown of CLIC4 on EMT markers,such as Snail,Snai2,Snai3,Zeb1,Zeb2,etc.To assess the effect of CLIC4 modulation on tumor growth in vivo,we injected MKN45 cells that were transfected with a lentivirus overexpressing CLIC4 or N87 cells transfected with lentivirus that carry shRNA subcutaneously into nude mice to observe the effect of CLIC4 on tumor growth.Results:1?We identified CLIC4 as a new marker in gastric cancer(GC)by 1TRAQ technology.The expression of CLIC4 is reduced in GC tumor tissues compared to adjacent normal tissues,and the expression levels of CLIC4 correlate inversely with the clinical stage of GC 2?RT-PCR and Western blot analysis showed an inverse relationship between the expression levels of CLIC4 and the tumorigenic potentials of MKN45,N87,and AGS cells,suggesting that CLIC4 may be a tumor suppressor.CLIC4 knockdown in N87 cells promoted migration and invasion,while overexpression of CLIC4 in MKN45 has the opposite effect.The results from wound healing assay using N87 and AGS cells further suggested the inhibitory role of CLIC4 on tumor cell migration.3?Our results show that the more aggressive MKN45 cells express much higher amount of CD44 and OCT4 compared to N87 and AGS cells.The expression of these markers was inhibited by CLIC4 overexpression but enhanced by CLIC4 knockdown,suggesting an inhibitory role of CLIC4 on cancer cell stemness.MKN45 cells formed more and larger colonies than N87 and AGS cells.The colony number and size of MKN45 cells were significantly reduced when CLIC4 was overexpressed.In contrast,colony formation ability was enhanced in N87 cells when CLIC4 was knocked down,suggesting that CLIC4 knockdown promotes anchorage-independent growth.Consistent with the expression levels of CD44 and OCT4,MKN45 cells were more resistant to 5-Fluorouracil(5-Fu)and Etoposide treatment.Furthermore,MKN45 cells transfected with a lentivirus that overexpresses CLIC4 are more sensitive to treatment with 5-Fu,whereas N87 cells transfected with lentivirus that expresses CLIC4 shRNA are more resistant to 5-Fu.4?Western blot and immunofluorescence studies showed upregulation of E-cadherin and downregulation of vimentin when CLIC4 was overexpressed,implying that CLIC4 overexpression inhibited EMT.In contrast,CLIC4 knockdown in N87 cells promoted EMT.CLIC4 knockdown in AGS cells resulted in upregulation of Snai3.5?Tumor growth was decreased by CLIC4 overexpression and increased by CLIC4 knockdown.Conclusions:We identified CLIC4 as a marker that is differentially regulated in GC by ITRAQ technology.Our data revealed that the expression levels of CLIC4 correlate inversely with the clinical stage of GC and the expression levels of cancer stem cell markers CD44 and OCT4.Knockdown of CLIC4 promoted migration,invasion,and EMT through upregulation of the transcription repressor Snai3.Anchorage-independent growth of GC cells was decreased and the cells became more sensitive to 5-Fu and Etoposide treatment when CLIC4 was overexpressed.The ability of N87 cells to form tumors in nude mice was enhanced when CLIC4 was silenced.In conclusion,for the first time,we were able to demonstrate that CLIC4 suppresses gastric cancer tumor growth by inhibiting cancer cell stemness and EMT.These findings shed new insights into the pathogenesis of gastric cancer and could lead to the development of new targeted therapies.