BACKGROUND The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers,which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways.Sever...BACKGROUND The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers,which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways.Several therapeutic anti-GFRA1 antibody-drug conjugates are under development.Demethylation(or hypomethylation)of GFRA1 CpG islands(dmGFRA1)is associated with increased gene expression and metastasis risk of gastric cancer.However,it is unknown whether dmGFRA1 affects the metastasis of other cancers,including colon cancer(CC).AIM To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target.METHODS CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study.The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing.Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients.Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo.The phosphorylation of AKT and ERK proteins was examined by Western blot analysis.RESULTS The proportion of dmGFRA1 in CC,surgical margin,and normal colon tissues by MethyLight was 68.4%,73.4%,and 35.9%(median;nonparametric test,P=0.001 and<0.001),respectively.Using the median value of dmGFRA1 peak area proportion as the cutoff,the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or welldifferentiated samples(92.3%%vs 55.8%,Chi-square test,P=0.002)and significantly higher in CC samples with distant metastasis than in samples without(77.8%vs 46.0%,P=0.021).The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC(adjusted hazard ratio=0.49,95%confidence interval:0.24-0.98),especially for 89 CC patients with metastatic CC(adjusted hazard ratio=0.展开更多
为检测正常胃粘膜及胃癌组织和正常结肠及结肠癌组织中GFRa1 m RNA表达量,以及两种剪接体GFRa1a和GFRa1b的m RNA含量。初步明确胃及结肠组织中GFRa1剪接体存在形式以及与胃癌、结肠癌发生的相关性。采用收集23例正常胃粘膜/胃炎组织、2...为检测正常胃粘膜及胃癌组织和正常结肠及结肠癌组织中GFRa1 m RNA表达量,以及两种剪接体GFRa1a和GFRa1b的m RNA含量。初步明确胃及结肠组织中GFRa1剪接体存在形式以及与胃癌、结肠癌发生的相关性。采用收集23例正常胃粘膜/胃炎组织、20对胃癌及其切缘组织和6例非癌患者正常结肠活检组织、20对结肠癌及其切缘组织的方法。选择并提取结肠癌细胞系RKO,正常人胃粘膜上皮细胞系GES-1和人胃癌细胞SGC-7901这3种细胞系的RNA。以Alu m RNA为内参照,用定量RT-PCR的方法对胃组织、结肠组织以及所选的细胞系中GFRa1总m RNA含量进行分析。运用DHPLC技术区分GFRa1不同的剪接体,定量测定两种转录本剪接体的比值。结果显示,胃及结肠组织中,与正常及癌组织相比,切缘组织GFRa1总m RNA、剪接体a及剪接体b的m RNA相对拷贝数均显著增加(P<0.05)。所有组织中GFRa1剪接体b的m RNA相对拷贝数均高于剪接体a。细胞系RKO、GES-1和SGC-7901均检测到GFRa1剪接体b的表达,而未检测到剪接体a。由此可知,胃和结肠组织以及相应的细胞系中GFRa1主要以剪接体b的形式存在;GFRa1剪接体b的高表达可能在胃及结肠组织癌变过程发挥作用。展开更多
基金Supported by the National Natural Science Foundation of China A3Foresight Program,No.31261140372Beijing Science and Technology Commission,No.Z151100001615022the Science Foundation of Peking University Cancer Hospital,No.2017-25
文摘BACKGROUND The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers,which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways.Several therapeutic anti-GFRA1 antibody-drug conjugates are under development.Demethylation(or hypomethylation)of GFRA1 CpG islands(dmGFRA1)is associated with increased gene expression and metastasis risk of gastric cancer.However,it is unknown whether dmGFRA1 affects the metastasis of other cancers,including colon cancer(CC).AIM To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target.METHODS CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study.The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing.Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients.Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo.The phosphorylation of AKT and ERK proteins was examined by Western blot analysis.RESULTS The proportion of dmGFRA1 in CC,surgical margin,and normal colon tissues by MethyLight was 68.4%,73.4%,and 35.9%(median;nonparametric test,P=0.001 and<0.001),respectively.Using the median value of dmGFRA1 peak area proportion as the cutoff,the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or welldifferentiated samples(92.3%%vs 55.8%,Chi-square test,P=0.002)and significantly higher in CC samples with distant metastasis than in samples without(77.8%vs 46.0%,P=0.021).The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC(adjusted hazard ratio=0.49,95%confidence interval:0.24-0.98),especially for 89 CC patients with metastatic CC(adjusted hazard ratio=0.
文摘为检测正常胃粘膜及胃癌组织和正常结肠及结肠癌组织中GFRa1 m RNA表达量,以及两种剪接体GFRa1a和GFRa1b的m RNA含量。初步明确胃及结肠组织中GFRa1剪接体存在形式以及与胃癌、结肠癌发生的相关性。采用收集23例正常胃粘膜/胃炎组织、20对胃癌及其切缘组织和6例非癌患者正常结肠活检组织、20对结肠癌及其切缘组织的方法。选择并提取结肠癌细胞系RKO,正常人胃粘膜上皮细胞系GES-1和人胃癌细胞SGC-7901这3种细胞系的RNA。以Alu m RNA为内参照,用定量RT-PCR的方法对胃组织、结肠组织以及所选的细胞系中GFRa1总m RNA含量进行分析。运用DHPLC技术区分GFRa1不同的剪接体,定量测定两种转录本剪接体的比值。结果显示,胃及结肠组织中,与正常及癌组织相比,切缘组织GFRa1总m RNA、剪接体a及剪接体b的m RNA相对拷贝数均显著增加(P<0.05)。所有组织中GFRa1剪接体b的m RNA相对拷贝数均高于剪接体a。细胞系RKO、GES-1和SGC-7901均检测到GFRa1剪接体b的表达,而未检测到剪接体a。由此可知,胃和结肠组织以及相应的细胞系中GFRa1主要以剪接体b的形式存在;GFRa1剪接体b的高表达可能在胃及结肠组织癌变过程发挥作用。
