Yanjie Zhao1, Lixuan Wei1, Mingming Shao1, Xudong Huang1, Jiang Chang2, Jian Zheng3, Jiahui Chu1, Qionghua Cui1, Linna Peng1, Yingying Luo1, Wenle Tan1, Wen Tan1, Dongxin Lin4, Chen Wu5. 1. Department of Etiology and Carcinogenesis, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. 2. Key Laboratory for Environment and Health, Ministry of Education, School of Public Health, Huazhong University of Science and Technology, Wuhan, China. 3. Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, China. 4. Department of Etiology and Carcinogenesis, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, China; Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China. Electronic address: lindx@cicams.ac.cn. 5. Department of Etiology and Carcinogenesis, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: chenwu@cicams.ac.cn.
Abstract
BACKGROUND & AIMS: We performed a screen for genes whose expression correlates with invasiveness of esophageal squamous cell carcinoma (ESCC) cells. We studied the effects of overexpression and knockdown of these genes in cell lines and expression levels in patient samples. METHODS: We selected genes for analysis from 11 loci associated with risk of ESCC. We analyzed the effects of knocking down expression of 47 of these genes using RNA interference on-chip analysis in ESCC cells and HeLa cells. Cells with gene overexpression and knockdown were analyzed in migration and invasion assays or injected into nude mice and metastasis of xenograft tumors was quantified. We collected ESCC and non-tumor esophageal tissues from 94 individuals who underwent surgery in China from 2010 and 2014; clinical information was collected and survival time was measured from the date of diagnosis to the date of last follow-up or death. Levels of messenger RNAs (mRNAs) were quantified by RNA sequencing, and levels of proteins were determined from immunoblot analyses. Patient survival was compared with mRNA levels using Kaplan-Meier methods and hazard ratios were calculated by Cox models. RESULTS: We identified 8 genes whose disruption increased migration and 10 genes whose disruption reduced migration. Knockdown of BRCA1-associated protein gene (BRAP) significantly reduced migration of KYSE30, KYSE150, and HeLa cells. In patient tumors, 90% of ESCCs examined had higher levels of BRAP protein than paired non-tumor tissues, and 63.8% had gains in BRAP DNA copy number. Levels of BRAP mRNA in ESCC tissues correlated with patient survival time, and high expression increased risk of death 2.4-fold compared with low expression. ESCCs that had metastasized to lymph node had significantly higher levels of BRAP mRNA than tumors without metastases. Knockdown of BRAP in ESCC and HeLa cell lines significantly reduced migration and invasiveness; these cell lines formed less metastases in mice than control cells. Nuclear translocation of the nuclear factor-κB (NF-κB) P65 subunit and phosphorylation of inhibitor of NF-κB kinase subunit β (IKBKB or IKKβ) increased in cells that overexpressed BRAP and decreased in cells with BRAP knockdown. In immunoprecipitation assays, BRAP interacted directly with IKKβ. Expression of matrix metalloproteinase 9 and vascular epithelial growth factor C, which are regulated by NF-κB, was significantly reduced in cells with knockdown of BRAP and significantly increased in cells that overexpressed BRAP. CONCLUSIONS: Expression of BRAP is increased in ESCC samples compared with non-tumor esophageal tissues; increased expression correlates with reduced patient survival time and promotes metastasis of xenograft tumors in mice. BRAP overexpression leads to increased activity of NF-κB and expression of matrix metalloproteinase 9 and vascular epithelial growth factor C.
BACKGROUND & AIMS: We performed a screen for genes whose expression correlates with invasiveness of esophageal squamous cell carcinoma (ESCC) cells. We studied the effects of overexpression and knockdown of these genes in cell lines and expression levels in patient samples. METHODS: We selected genes for analysis from 11 loci associated with risk of ESCC. We analyzed the effects of knocking down expression of 47 of these genes using RNA interference on-chip analysis in ESCC cells and HeLa cells. Cells with gene overexpression and knockdown were analyzed in migration and invasion assays or injected into nude mice and metastasis of xenograft tumors was quantified. We collected ESCC and non-tumor esophageal tissues from 94 individuals who underwent surgery in China from 2010 and 2014; clinical information was collected and survival time was measured from the date of diagnosis to the date of last follow-up or death. Levels of messenger RNAs (mRNAs) were quantified by RNA sequencing, and levels of proteins were determined from immunoblot analyses. Patient survival was compared with mRNA levels using Kaplan-Meier methods and hazard ratios were calculated by Cox models. RESULTS: We identified 8 genes whose disruption increased migration and 10 genes whose disruption reduced migration. Knockdown of BRCA1-associated protein gene (BRAP) significantly reduced migration of KYSE30, KYSE150, and HeLa cells. In patienttumors, 90% of ESCCs examined had higher levels of BRAP protein than paired non-tumor tissues, and 63.8% had gains in BRAP DNA copy number. Levels of BRAP mRNA in ESCC tissues correlated with patient survival time, and high expression increased risk of death 2.4-fold compared with low expression. ESCCs that had metastasized to lymph node had significantly higher levels of BRAP mRNA than tumors without metastases. Knockdown of BRAP in ESCC and HeLa cell lines significantly reduced migration and invasiveness; these cell lines formed less metastases in mice than control cells. Nuclear translocation of the nuclear factor-κB (NF-κB) P65 subunit and phosphorylation of inhibitor of NF-κB kinase subunit β (IKBKB or IKKβ) increased in cells that overexpressed BRAP and decreased in cells with BRAP knockdown. In immunoprecipitation assays, BRAP interacted directly with IKKβ. Expression of matrix metalloproteinase 9 and vascular epithelial growth factor C, which are regulated by NF-κB, was significantly reduced in cells with knockdown of BRAP and significantly increased in cells that overexpressed BRAP. CONCLUSIONS: Expression of BRAP is increased in ESCC samples compared with non-tumor esophageal tissues; increased expression correlates with reduced patient survival time and promotes metastasis of xenograft tumors in mice. BRAP overexpression leads to increased activity of NF-κB and expression of matrix metalloproteinase 9 and vascular epithelial growth factor C.