The Role,Mechanism And Clinical Significance Of CHAF1A In Proliferation Of Gastric Cancer

Gastric cancer(GC)has the second morbidity and mortality in all malignancies in China,with great threats to health and life of Chinese people.GC is a complex disease with significant biological and genetic heterogeneity among patients,which limits the use of personalized therapy and contributes to difficulty in treatment,various outcomes and a lack of target agents.Therefore,it is crucial to identify novel prognostic markers and therapeutic targets based on a detailed molecular understanding of GC in order to improve therapy efficacy.Results from The Cancer Genome Atlas(TCGA),as a great breakthrough in cancer research,have expanded our understanding of the characteristics of GC at the molecular level.And the four molecular subtypes defined by TCGA may facilitate the development of targeted and individual treatments of GC.However,these results are based on multiple high-throughput sequencing platforms which need complex technologies and are costly,and therefore is difficulty for application widely.On the other hand,the functions and clinical significance of most genes in humans remain elusive,and new findings are also constantly emerging from studies of those well-known genes.Therefore,in the post molecular classification era,efforts are still needed to find novel molecular markers for determining the natural course of GC and increasing the survival rate of patients.CHAF1A gene codes p150,a primary component of chromatin assembly factor1.CHAF1A participates the rapid nucleosome assembly on newly replicated DNA and also has wide and important roles in epigenetic modification,cell cycle regulation,DNA replication,DNA damage response and repair,indicating a role of CHAF1A in cancer.Recently,CHAF1A has been found to be associated with the malignant phenotype and prognosis of some tumors such as neuroblastoma,colon cancer,glioblastoma,ovarian cancer,hepatocellular carcinoma,and lung cancer.However,the mechanisms of CHAF1A in tumor biology are still unclear.The role of CHAF1A in GC is unknown.A corresponding study was conducted by us which included the following aspects:1)assays for the expression and function of CHAF1A in GC;2)mechanism studies based on gene and antibody chips;3)analyses for clinical significance of CHAF1A and its underlying molecular basis based on high-throughput m RNA sequencing of tumor tissues.This is the first study to show the roles,mechanisms and clinical significance of CHAF1A in GC.We verify that CHAF1A is a novel prognostic marker and a potential therapeutic target of GC,which may promote the development of individual and precision treatments of GC and has significant scientific and clinical values.Part I.The role of CHAF1A in proliferation of gastric cancerCharpter I.The expression of CHAF1A in gastric cancerPurpose:To preliminary investigate the potential role of CHAF1A in GC by studying the expression of CHAF1A in cell lines and tissues of GC and the associations between expressions of CHAF1A and wild-type p53 and the proliferation marker MKI67.Methods:The expression of CHAF1A in cell lines of MGC-803,SGC-7901,MKN-45 and BGC-823 was evaluated by q RT-PCR.CHAF1A m RNA expression was analyzed in tumor and matched adjacent normal tissues using TCGA data and our data that was based on high-throughput m RNA sequencing of fresh tumor tissues from 34 patients.CHAF1A protein expression was examined by immunohistochemistry(IHC)staining in paraffin-embedded,archived tumor and matched adjacent normal tissues in a clinical cohort of 665 GC patients.The protein expressions of wild-type p53 and MKI67 were tested by IHC staining and their correlations with CHAF1A were analyzed.The correlation of m RNA expression between CHAF1A and MKI67 was also analyzed by m RNA sequencing data.Results:1.CHAF1A is upregulated in GCCHAF1A m RNA was expressed in all 4 of the tested GC cell lines.In TCGA and our sequencing data,CHAF1A m RNA levels in GC tissues were up-regulated in 30 of32 and 22 of 34 cases respectively,compared to matched adjacent normal tissues.The frequency of high CHAF1A protein expression in tumors(44.7%,297 of 665 samples)was significantly higher(P<0.001)than that observed in adjacent normal tissues(13.1%,87 of 665 samples).2.CHAF1A is correlated with the expression of MKI67 and wild-type p53The high expression rate of CHAF1A protein in tumor tissues with high MKI67protein expression was 51.5%which was significantly higher than the 32.2%in those with low MKI67 expression(P<0.001).The high expression rate of CHAF1A protein in tumor tissues with high expression of wild-type p53 was 41.3%which was significantly lower than the 50.2%in those with low expression of wild-type p53(P=0.026).CHAF1A m RNA expression was positively correlated with MKI67 m RNA expression in both TCGA and our m RNA sequencing datasets(r~2=0.344 and 0.298respectively,both P<0.001).Conclusions:CHAF1A expression was upregulated in GC compared to normal tissues,which was negatively correlated with the wild-type p53 expression but positively correlated with the MKI67 expression.These results indicated that CHAF1A may promote gastric carcinogenesis.Charpter II Impact of CHAF1A on proliferation of gastric cancerPurpose:To investigate the impact of CHAF1A on proliferation of GC by functional assays in GC cell lines.Methods:MGC-803 and SGC-7901 cell models with CHAF1A knockdown or overexpression were established.The Cellomics real time cell growth assay was used to measure cell proliferation,MTT assay was used to determine cell viability,and related results were confirmed by colony formation assay.Flow cytometry was used in cell cycle and apoptosis assays.Using m RNA sequencing data,the expression correlations of CHAF1A with key regulators in cell cycle and apoptosis were examined.Xenografts in nude mice were used in vivo assays for tumor growth.Results:1.CHAF1A promotes the proliferation of GC cellsCHAF1A knockdown significantly decreased the cell growth number and proliferation rate in MGC-803 and SGC-7901 cells compared with that in control cells.Colony formation assays further confirmed these results.By contrast,cell proliferation in CHAF1A-overexpressing MGC-803 and SGC-7901 cells significantly increased.2.CHAF1A regulates the cell cycle transition and inhibits apoptosis in GC cellsCHAF1A knockdown significantly increased the percentage of S-phase cells but did not have a significant effect on the percentage of G2/M-phase cells,suggesting an increase of S-phase arrest(P<0.05).Furthermore,the apoptosis rate was dramatically elevated in CHAF1A knockdown cells compared with that in mock cells(P<0.05).Conversely,overexpression of CHAF1A significantly decreased the percentage of S-phase cells and the apoptosis rate in GC cells(P<0.05).3.CHAF1A is correlated with the expressions of key regulators in cell cycle and apoptosisIn TCGA,CHAF1A was significantly positively correlated with the expressions of cyclin A2(CCNA2),Bcl-xl(BCL2L1),and Survivin(BIRC5)but negatively correlated with p21(CDKN1A)expression(P<0.05)in GC.Some of these results were also confirmed by our m RNA sequencing data but the others may be limited by sample size.4.CHAF1A promotes the tumorigenicity of GC cells in vivoThe tumors formed by CHAF1A knockdown cells grew much more slowly than the tumors formed from control cells.Tumors formed by CHAF1A knockdown cells were also smaller,in both size(at day 24,254.36±109.20 mm3 vs.930.47±262.01mm3 for control)and weight(at day 24,0.17±0.06 g vs.0.58±0.15 g for control)compared to control.Conclusions:CHAF1A promotes the proliferation and tumorigenicity of GC cells by regulating cell cycle transition and inhibiting apoptosis.Part II.Molecular mechanism for the impact of CHAF1A on proliferation of gastric cancerPurpose:To investigate the mechanism for oncogenic role of CHAF1A in GC by gene expression profiling analysis and validation of downstream signaling pathway.Methods:The Affymetrix gene chip was used to identify differentially expressed genes between CHAF1A knockdown and the control GC cells.Pathway enrichment analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes(KEGG)and Bio Carta databases.Gene Ontology(GO)analysis was carried out as described by Gene Set Enrichment Analysis(GSEA)software v3.0.Selected differentially expressed genes were further confirmed by Western blot and m RNA sequencing data.The Path Scan~?Stress and Apoptosis Signaling Antibody Array Kit was used to detect the changes of intracellular signaling after CHAF1A overexpression.A network diagram associated with CHAF1A signaling was finally plotted.Results:1.CHAF1A knockdown regulates oncogenic and anti-oncogenic pathways and metabolism in GC cellsA total of 399 genes were differentially expressed between the CHAF1A knockdown and the control cells.Of these,293 genes were up-regulated,and 106genes were down-regulated.Bioinformatic analyses showed that the significant signaling pathways associated with cancer biology included transcriptional misregulation in cancer,proteoglycans in cancer,the Fox O signaling pathway,the p53 signaling pathway,cytokine-cytokine receptor interactions,pathways in cancer,and so on.Selected genes in the p53 signaling pathway were further tested by Western blot,and the up-regulated protein levels of THBS1,CDKN1A,and IGFBP3 by CHAF1A knockdown were validated.In tumor tissues,CHAF1A was also significantly negatively correlated with the m RNA expression of these genes(P<0.05).GO analyses showed that CHAF1A inhibited cell metabolism associated with oxidative phosphorylation,indicating an increase of glycolytic metabolism,which was consistent with the metabolic phenotype in inhibition of the p53 pathway.2.CHAF1A overexpression regulates stress and apoptosis signaling in GC cellsCHAF1A overexpression significantly activated HSP27,p38 MAPK,NF-κB,Chk1 and Survivin and inhibited Bad in both MGC-803 and SGC-7901 cells,with an additional activation of SAPK/JNK in SGC-7901 cells(P<0.05).Positive correlations between the m RNA expressions of CHAF1A and NF-κB(NFKB1),Chk1(CHEK1)and Survivin,were also found by tumor m RNA sequencing in patients(P<0.05).Together with the changes of gene expression by CHAF1A knockdown,the above results were used to plot a network diagram associated with CHAF1A signaling.Conclusions:CHAF1A promotes malignant processes of GC by regulation of gene expression involved with cell growth,apoptosis,survival,stress response and metabolism.Part III.Clinical significance of CHAF1A in gastric cancer and corresponding molecular basisPurpose:To explore the clinic significance of CHAF1A by studying the associations of CHAF1A protein level with clinicopathologic characteristics and prognosis of GC patients.To investigate the underlying molecular basis of the clinical significance of CHAF1A by analyses of tumor m RNA sequencing data.Methods:IHC and m RNA sequencing had been described in Part I.Survival follow-up was conducted.Survival analysis was performed using the Kaplan-Meier method plus the Log-rank test.Prognostic factors were analyzed using univariate and multivariate Cox proportional hazard models,and hazard ratio were calculated.Results:1.CHAF1A protein level is associated with specific clinical characteristics of GC patientsHigh CHAF1A protein expression was more common in non-cardia than in cardia GC;the difference was close to statistical significance(48.0 vs 41.1%;P=0.073).A significantly lower incidence of perineural invasion(PNI)in tumors with high versus low CHAF1A protein expression was found(38.1 vs 51.9%;P<0.001).CHAF1A protein expression was also associated with tumor size(P=0.014)and histology grade(P=0.005).2.CHAF1A protein level is an independent prognostic indicator of survival in non-cardia GC patientsIn the overall cohort,CHAF1A protein level was prognostic for OS(P=0.018)and DFS(P=0.048).Univariate analysis showed that CHAF1A protein level significantly impact OS(P=0.035)and had a trend to significantly impact DFS(P=0.050).However,multivariate analysis indicated that CHAF1A protein level was not an independent prognosticator for OS and DFS(P>0.05).In subgroup analyses,the prognostic significance of CHAF1A protein level for OS and DFS occurred in non-cardia GC(both P<0.001)but not in cardia GC(P=0.383 and 0.215,respectively).Univariate analysis showed that CHAF1A protein level significantly impact both OS and DFS in non-cardia GC(both P<0.001).Multivariate analysis found that CHAF1A protein level was an independent predictor for both OS(P=0.015)and DFS(P=0.002)in non-cardia GC.In addition,CHAF1A protein level was also prognostic for OS of patients with small(size≤4.5 cm)and PNI-negative tumors(P=0.015 and 0.005,respectively).3.CHAF1A promotes the Warburg effect and modulates biological activities of GC depending on clinical characteristics of patientsTumor m RNA sequencing data from our 34 patients were used to further explore the changes in gene expression matched with clinical relevance of CHAF1A.Patients were divided into high and low CHAF1A expression groups by the median sequencing value of CHAF1A m RNA.GSEA analysis showed that the pathways for cell cycle,DNA replication,DNA repair,and biological metabolism had the most significant changes in the comparison of high with low CHAF1A expression.In accordance with our results from gene chip,oxidative phosphorylation was inhibited by high CHAF1A expression.In TCGA,all of glycolytic enzymes were significantly up-regulated in tumors with high CHAF1A expression(P<0.05).In addition,expressions of oncogenes such as m TOR,MYC and AKT that are crucial for glycolytic regulation were also significantly up-regulated in tumors with high CHAF1A expression(P<0.05).These results that CHAF1A depressed oxidative phosphorylation and increased glycolysis verified that CHAF1A promoted the Warburg effect in GC.The GO results showed that CHAF1A also modulated gene expression,immune response,protein function and localization,cell senescence and fate,cell differentiation and tissue development(included various epithelial and nerve cells).The mechanisms of CHAF1A within clinical subgroups were further investigated.We found that CHAF1A:had additional,stronger effects on immunomodulation other than cell proliferation and survival in cardia than in non-cardia GC;had increased influences on neural activities and decreased impacts on cell proliferation and survival in PNI-positive compared to PNI-negative GC;and induced different metabolism types between large(size>4.5 cm)and small tumors that oxidative phosphorylation and related pathways were up-regulated in large tumors and down-regulated in small tumors.Collectively,CHAF1A can be a prognostic marker in patients whose CHAF1A expression in tumors promotes glycolysis or has main and strong effects on cell proliferation.At the same time,these results also suggested that the role of CHAF1A is context-dependent.Conclusions:CHAF1A protein level is associated with specific clinical characteristics of GC patients and is an independent predictor of prognosis in non-cardia GC patients.CHAF1A may promote the Warburg effect and impact tumor biology of GC depending on patient characteristics.