Screening,Function And Mechanism Of Driver Genes In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is one of the deadliest digestive tract cancer,which is characterized by its aggressive clinical course and constitutes a poorprognosis.Due to the limited treatment methods of ESCC,the prognosis and the five-year survival rate of most patients is poor.Therefore,it is significant to explore specific pathogenesis of esophageal squamous cell carcinomas,and select potential therapeutic targets and drugs for improving the prognosis of ESCC patients.Ribosomes are conserved organelles necessary for protein synthesis.However,the increased number of ribosomes is not only necessary for cell growth,but is also sufficient to drive the transformation of malignant tumors.Increased rates of ribosome biogenesis led to an abnormal increase in nucleolar size and number,which have been considered to be a marker of many cancers and are related to poor prognosis.Therefore,we planned to deeply investigate the ribosomal proteins(RPs)that play a malignant role in promoting the progression of esophageal squamous cell carcinoma.In this study,we constructed a CRISPR Synergistic Activation Mediator(SAM)system library targeting 89 RPs,including 54 RPLs and 35 RPSs,in two ESCC-derived cell lines to screen for the most oncogenic functional ribosomal proteins.Based on the screening results,we found that the high expression of ribosomal small subunit protein RPS 15 was associated with the metastatic phenotype of esophageal squamous cell carcinoma.Immunohistochemical(IHC)staining analysis of RPS 15 in 504 ESCC specimens revealed higher expression of RPS 15 in tumors compared with that in normal tissue.Moreover,the increased RPS 15 expression was significantly correlated with lymph node metastasis.Finally,we analyzed the impact of RPS 15 expression on survival time of patients with ESCC in our sample set.The Kaplan-Meier curves showed that high RPS 15 expression in ESCC patients significantly correlated with poor prognosis.Furthermore,multivariate Cox regression analysis showed that high RPS 15 expression was an independent risk factor for metastasis and poor prognosis for patients with ESCC.When studying the function of RPS 15,cell proliferation assay and subcutaneous tumor Mechanistic Investigation of Ribosomal Proteins Promoting the Progression of Esophageal Squamous Cell Carcinoma experiment in nude mice showed that RPS15 promoted the proliferation of ESCC cells in vivo and in vitro.Besides,Boyden Chamber migration and invasion assays and lung and lymph node metastasis experiment also proved that the high expression of RPS15 promoted the metastasis of esophageal squamous cell carcinoma in vivo and in vitro.To analyze the impact of RPS15 on cancer cell growth and metastasis,we used a ptychographic QPI label-free imaging technique to obtain the visualization results.Morphological measurements,including cell-doubling time,total mitosis events,track speed,and displacement,were used to compare differences between the RPS15 overexpression group and control group of ESCC cells.The results showed that RPS15 overexpression promoted cell mitosis events and reduced cell-doubling time.As for motility,overexpression of RPS15 increased the track speed and movement ability of ESCC cells.Taken together,these in vivo results indicated that RPS15 played as an oncogene role in the occurrence and development of ESCC.As components of the ribosome,RPs are reported to have highly coordinated expression to ensure the fidelity of ribosomal biogenesis and assembly.Thus,we combined ribosomal profiling and RNA-seq to find downstream molecules and pathways regulated by RPS 15.The results indicated that overexpression of RPS 15 could give rise to coordinated upregulation of translation efficiency for core RPs.Besides,the results also demonstrated that the bulk of genes in the E2F pathway and p38 MAPK pathway related to cell proliferation,metastasis and cell cycle,especially MKK6 and p38,exhibited higher transcription efficiency(TE)in RPS 15 overexpression cells compared with that in the control cells.To test the role of p38 MAPK pathway in the ESCC malignant phenotype regulated by RPS15,we selected SB203580,a small molecule inhibitor of p38 MAPK,to inhibit the activation of the p38 MAPK pathway.Boyden Chamber migration and invasion assays and growth curves indicated that SB203580 could inhibited both metastasis and proliferation ability of ESCC cells with RPS 15 overexpression.As SB203580 treatment alone inhibited ESCC cell proliferation to a moderate degree,the combination of 8 μM SB203580 and 0.5 μg/mL DDP resulted in inhibition of the proliferation of RPS 15 overexpressing ESCC cells by as much as 69%for KYSE30 cells and 84.6%for KYSE450 cells compared with that of the controls,which represented a further inhibition of 42%for KYSE30 cells and 35.33%for KYSE450 cells compared with that of SB203580 alone.In addition,mass spectrometry(MS)analysis was used to identify the proteins potential interacting with RPS 15.Co-IP assays showed that RPS 15 selectively bound to the KH3-4 domain of insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1).Additionally,a molecular docking model(ClusPro)was used to predict the binding of RPS15 residues to IGF2BP1.This demonstrated that D82 may be the residue of RPS15 that directly bound to IGF2BP1.Co-IP assays showed that the mutating the D82 residue to A82 destroyed the binding of RPS15 to IGF2BP1.IGF2BP1 is a new member of m6A readers that protect m6A-modified mRNAs from decay.To determine the effect of IGF2BP1 on the p38 MAPK pathway,IGF2BP1 RNA immnoprecipitation sequencing(RIP-seq)data from previous reports were re-analyzed to identify binding peaks across MAPK14 and MKK6 mRNA transcripts.The results showed that IGF2BP1 binding peaks accumulated across MAPK14(encoded p38 protein)and MKK6 transcripts,especially in the 3’-UTR,indicating that IGF2BP1 bound directly to MAPK14 and MKK6 mRNA transcripts.Furthermore,we detected the expression of MKK6 and p38 in IGF2BP1-ablated ESCC cells and Western blot analysis showed that knockdown of IGF2BP1 in RPS15-overexpressing ESCC cells diminished the upregulating effect of RPS15 on MKK6 and p38 expression.Taken together,our data demonstrated that RPS15 could affect the translation of MAPK14 and MKK6 mRNA,which were directly bound and recognized by the KH3-4 domain of IGF2BP1,which then enhanced p3 8 and MKK6 TE and promoted the proliferation and metastasis of ESCC cells.Next,through the screening of small molecule inhibitors targeting RPS 15,we found that folic acid promoted RPS 15 degradation in ESCC cells,which further inhibited the proliferation ability in RPS 15 overexpressed cells.These data demonstrated that folic acid had a therapeutic effect on ESCC by promoting RPS 15 degradation,and the effect was augmented by the combination treatment of folic acid with DDP.In conclusion,our study firstly revealed the important role of RPS 15 in promoting progress of esophageal squamous cell carcinoma,and proposed that the combination of folic acid and DDP could be an effective strategy to improve prognosis of patients with ESCC.Esophageal carcinoma(EC)is a kind of common digestive tract tumor with high malignancy,and esophageal squamous cell carcinoma(ESCC)is a prevailing histologic subtype of esophageal cancer in China,which is characterized by high mortality rate,aggressive clinical course and constitutes a poor-prognosis subgroup.Although multimodality therapy has been developed to treat ESCC,the survival rate for ESCC has still not improved due to drug resistance or lymphatic metastasis.Because the molecular classification of ESCC has not been fully elucidated,more novel cancer driver genes and prognostic biomarkers need to be explored urgently.Our previous systematic genome-wide study of 508 patients with ESCC identified three genetic alterations in the SLC35E2 promoter.SLC35E2 is a member of SLC35 family,which belongs to the solute carrier family(SLC family).Survival analysis revealed that the hotspot mutation at the promoter of SLC35E2 was correlated with a worse prognosis.Besides,the overall survival analysis from GEPIA database demonstrated that SLC35E2 overexpression in three digestive tract cancers,including colon adenocarcinoma(COAD),esophageal carcinoma(ESCA)and stomach adenocarcinoma(STAD),was associated with a reduced overall survival time.When studying the function of SLC3 5E2 in ESCC,cell proliferation assay and subcutaneous tumor experiment in nude mice showed that SLC35E2 high expression promoted the proliferation ability of ESCC cells in vivo and in vitro.Taken together,these results indicate that SLC35E2 played as an oncogene role in development of ESCC.Mechanistically,to study whether the promoter mutations of SLC35E2 affect the binding of transcription factors and their self-expression,JASPA and PROMO online database were used to predict transcription factors which can bind to three promoter mutation sites of SLC35E2.Both two databases predicted the binding sites at SLC35E2 promoter region which could bind to KLF4.qRT-PCR and Western blot results showed that KLF4 can inhibit both mRNA and protein expression of SLC35E2.Besides,the luciferase report assay and chromatin immunoprecipitation(ChIP)assay showed that KLF4 repressed SLC35E2 transcriptional activity via-118 site.When the mutation from C to G occurred at the-118 site of SLC35E2,the binding between KLF4 and SLC35E2 promoter region was destroyed,and KLF4 lost the transcriptional inhibition function of SLC35E2,which finally affected the survival time of ESCC patients with SLC35E2 promoter mutation.In conclusion,our study firstly revealed the important role of SLC35E2 in promoting progress of esophageal squamous cell carcinoma,provided new evidence for the regulation of KLF4 on the proliferation of esophageal cancer,and proposed the possibility of SLC35E2 as a new target for esophageal squamous cell carcinoma.