| Background:Esophageal carcinoma is a malignant tumor originating from normal esophageal epithelial cells.The latest study show that in all kinds of cancer,the incidence and mortality of esophageal cancer are in the forefront.In addition,in terms of regions,Asia is a region with a high incidence of esophageal cancer,and China in East Asia is one of the countries with a high incidence of esophageal cancer.In terms of the pathological type of esophageal cancer in china,the vast majority of patients are esophageal squamous cell carcinoma(ESCC),so the study of ESCC is of great significance.According to molecular classification,esophageal squamous cell carcinoma can be divided into immunomodulatory type,immunosuppressive type,cell cycle pathway activation type and NRF2 pathway activation type.At present,traditional surgical resection,radiotherapy and chemotherapy are still the main treatment methods for esophageal cancer.In addition,immune checkpoint blocking therapy has also been applied in clinical treatment.However,since the onset of esophageal squamous cell carcinoma is insidious and the early symptoms are not obvious,most patients are already in the stage of disease progression when they see a doctor,often with metastasis to nearby lymph nodes and distant organs,resulting in poor prognosis of patients with esophageal squamous cell carcinoma.Glutamine(Gln)is one of the most abundant amino acids in human body.It is an important nitrogen source and metabolic fuel,and participates in the synthesis of many biological macromolecules and the regulation of life activities in human body.Studies have shown that cancer cells prefer to use glutamine as an energy source,and glutamine plays critical roles in regulating cancer cell proliferation and metastasis.For example,in KRAS-mutated lung cancer cells,glutamine can promote the progression of the cell cycle phase S to G2,leading to the malignant phenotype of lung cancer.Recently,increased Glutamine metabolism was reported in two independent studies when comparing ESCC with the normal esophageal tissues,implying that targeting Glutamine-addiction might offer a promising avenue to treat human ESCC.Serine/threonine kinase STK11(LKB1)was commonly known as a crucial tumor suppressor with a high frequency of genetic alterations in multiple subtypes of nonsmall cell lung carcinomas(NSCLC),cervical carcinomas and breast cancer.Accumulating evidence demonstrated that LKB1-loss leads to the progression of distinct cancers,such as NSCLC,cervical cancer,pancreatic cancer,melanoma,the myeloproliferative neoplasms and ESCC.The tumor suppressive activity of LKB1 is mainly controlled by its phosphorylated substrate AMPK,which is a master regulator in cell survival and metabolic processes,including Glutamine-addiction.AMPK was reported to phosphorylate PPARδ,inhibiting the transcription of glutamine transporter SLC1A5.Moreover,LKB1 deletion could render KRAS-mutant NSCLC or polycystic kidney disease,relying on glutamine metabolism in a context-dependent manner.Meanwhile,the activation of LKB1-AMPK pathway was tightly associated with autophagy,apoptosis and cell senescence in response to genetic or environmental stimulus.Cellular senescence is usually triggered by diverse forms of cellular stresses,resulting in an irreversible proliferation arrest.A variety of factors,including telomere damage,oncogene overexpression,DNA damage caused by ROS,mitochondrial dysfunction and inflammation,can lead to cellular senescence.Senescent cells usually present numerous specifically phenotypes,such as flattened and enlarged changes in cell morphology,elevated senescence-associated β-galactosidase(SA-β-gal)activity,increased levels of ROS,and changes in gene expression of multiple cell-cycle inhibitors.Importantly,senescence is capable of inhibiting cancer cell proliferation,thus to provide an early barrier for tumorigenesis and potentially suppress cancer progression.Meanwhile,studies have shown increased glutamine metabolism in ESCC compared with normal esophageal epithelial cells.RNA-binding proteins(RBPs)are a family of proteins that manage the life of m RNAs.Importantly,RBPs play vital roles in the regulation of gene expression,and malfunction of RBPs results in various diseases,including human cancer.However,the function and molecular mechanistic details of whether RBPs affect initiation and progression of ESCC remains largely unknown.The RNA-binding protein,RNAbinding motif 4(RBM4),is a nuclear and plasma shuttle protein that plays an important role in pre-m RNA splicing,gene silencing and m RNA translation.The function of RBM4 depends on its ability to recognize and bind to m RNA.For example,in NSCLC,RBM4 can regulate the alternative splicing of Bcl-x,promote the generation of short form Bcl-x S,lead to increased apoptosis of tumor cells,and inhibit the progression and development of lung cancer.RBM4 can regulate the progression of multiple cancers.However,the function and roles of RBM4 in ESCC were not been reported.Methods:1.The bioinformatics methods were used to analysis the ESCC dataset of TCGA database.The expression of 124 known RBPs in ESCC were analyzed,and the expression of RBPs was verified in GEO database.2.The expression level of RBM4 by immunohistochemistry staining of 75 paired ESCC and normal clinical samples.The expression of RBM4 in ESCC cell lines was detected by western blotting and the expression of RBM4 in tissue microarray was detected by immunohistochemical.3.Cell lines with knockdown or overexpression of RBM4 were constructed,and conduct a series of phenotypic experiments,such as cell proliferation,migration and clone.In addition,the effect of RBM4 on tumor growth was verified by xenograft tumor models in nude mice.4.The obtained stable cell lines with low RBM4 expression were used toextractm RNA.The transcriptional alterations of RBM4-depleted KYSE150 cells by the m RNA-seq we re analyzed to find the molecular biological mechanism.5.To verify the sensitivity of ESCC cells with high expression of RBM4 to CB-839.Ex ploring the possibility of RBM4 as a potential therapeutic target.Results:1.Through the analysis of TCGA database,we found that a variety of RBPs expressed abnormally in ESCC,including RBM4,it was overexpression in ESCC.This conclusion was verified in two independent data sets of GEO database.In addition,RBM4 expression was increased in ESCC tissues and precancerous lesions.2.Overexpression of RBM4 promoted the progression of ESCC,while depletion of RBM4 inhibited the progression of ESCC.3.Depletion of RBM4 leads to senescence and inhibits ESCC progression by LKB1-AMPK-P27 axis.4.The glutamine-addiction of ESCC cells were affected by RBM4,dependening on LKB1-AMPK axis.5.In ESCC tissues,the expression of RBM4 was increased,while the expression of LKB1 was inhibited,and they were negatively correlated.Moreover,cells with high expression of RBM4 were more sensitive to the glutaminase inhibitor CB-839.6.In vivo experiments on nude mice showed that CB-839 could significantly inhibit tumor growth caused by overexpression of RBM4.Conclusion:RBM4 has high expression level and RBM4 might function as a tumor-promoting RBP in ESCC.Depletion of RBM4 can lead to cell senescence and inhibit the progression of ESCC.In addition,RBM4 can inhibit the expression of LKB1 and promote the metabolic reprogramming of ESCC cells.Depletion of RBM4 can increase the expression of LKB1 and inhibit the uptake of glutamine and the production of glutamate.Cells with high expression of RBM4 are more sensitive to CB-839,and RBM4 is expected to be a potential target for ESCC treatment. |
PDF Full Text Request