Abstract
Individuals with gallbladder carcinoma (GBC), the most aggressive malignancy of the biliary tract, have a poor prognosis. Here we report the identification of somatic mutations for GBC in 57 tumor-normal pairs through a combination of exome sequencing and ultra-deep sequencing of cancer-related genes. The mutation pattern is defined by a dominant prevalence of C>T mutations at TCN sites. Genes with a significant frequency (false discovery rate (FDR) < 0.05) of non-silent mutations include TP53 (47.1%), KRAS (7.8%) and ERBB3 (11.8%). Moreover, ErbB signaling (including EGFR, ERBB2, ERBB3, ERBB4 and their downstream genes) is the most extensively mutated pathway, affecting 36.8% (21/57) of the GBC samples. Multivariate analyses further show that cases with ErbB pathway mutations have a worse outcome (P = 0.001). These findings provide insight into the somatic mutational landscape in GBC and highlight the key role of the ErbB signaling pathway in GBC pathogenesis.
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Acknowledgements
This study was supported by the National Natural Science Foundation of China (81172026, 81272402, 81301816, 81172029, 81370728, 81125020, 81328022 and 81302507), the National High-Technology Research and Development Program (863 Program, 2012AA022606; 2012BAK01B00), the Foundation for Interdisciplinary Research of Shanghai Jiao Tong University (YG2011ZD07), the Shanghai Science and Technology Commission Intergovernmental International Cooperation Project (12410705900), the Shanghai Science and Technology Commission Medical-Guiding Project (12401905800), the China Postdoctoral Science Foundation (2013M541513), the Program for Changjiang Scholars and the Leading Talent program of Shanghai.
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H. Wang, Yun Liu and Yingbin Liu conceived the study. C.L., B.S., B.L., Y.E.C., L.H., H. Wang, Yun Liu and Yingbin Liu directed the study. M.L., Z.Z., X.L., H. Wang, Yun Liu and Yingbin Liu contributed to the project design. M.L., X.L., D. Zhou, T.W., X. Wu, X.-A.W. and Qichen Ding performed experiments. Z.Z., J.Y., D. Zhang, X. Weng and H.Z. performed bioinformatics data analysis. W.W., K.Q., H. Weng, Qian Ding, P.C., T.L., Y.H. and W.L. contributed samples, data and comments on the manuscript. M.L., Z.Z., X.L., Z. Tan, J.M., W.G., W.T. and Y. Zheng analyzed and interpreted data. Y.S., R.B., Y.C., L.J., P.D., J.G., W.S., J.L., Z. Tang, Y. Zhang and X. Wang contributed reagents, materials and/or analysis tools. M.L., Z.Z. and X.L. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Research strategy of this study.
Whole-exome sequencing was performed for 32 GBC pairs, and ultra-deep targeted gene sequencing was performed for 51 GBC pairs. The recurrently mutated genes, as determined from the exome data, were included in the targeted gene panels. Recurrently mutated genes and pathways were evaluated for the two data sets. By combining the 2 data sets, 21 of 57 GBC patients carrying non-silent somatic mutation(s) of ErbB pathways were identified. Oncology studies of the ErbB family on GBC cell lines were conducted, and associations between ErbB pathway mutations and prognosis were assessed.
Supplementary Figure 2 Comparison between exome and targeted sequencing.
(a) The samples included in the exome and targeted sequencing are shown in a Venn diagram. In total, 57 samples were utilized in this study, 26 of which were processed with both exome and targeted sequencing. (b) In the 26 overlapped samples, somatic non-silent mutations found in the shared coding region of exome and targeted sequencing are compared and shown in a Venn diagram. Seventy-one somatic mutations were identified by both methods; 22 and 96 were discovered only by exome or targeted sequencing, respectively.
Supplementary Figure 3 Schematic diagram of TP53 and KRAS somatic mutations.
(a,b) The relative positions of somatic mutations in TP53 and KRAS are shown with the gene structure. An orange box indicates a non-silent mutation, and a blue box indicates a mutation in an intron or UTR.
Supplementary Figure 4 The oncogenic effect of ERBB3 mutants in OCUG-1 cells.
OCUG-1 cells were transiently transfected with vector expressing ERBB3-WT or mutants, and cell viability was determined by MTT assay. Data represent the means ± s.e.m. of three independent experiments (*P < 0.05, **P < 0.01 compared to cells transfected with the control vector; §P < 0.05, §§P < 0.01 compared to cells expressing ERBB3 WT).
Supplementary Figure 5 The efficacies of RNA interference against ERBB1, ERBB2 and ERBB3 in GBC cells as determined by RT-PCR.
(a–d) GBC cells were respectively transfected with RNAi against two independent regions of ERBB1, ERBB2 and ERBB3, and the mRNA expressions of these genes was determined with real-time quantitative PCR 48 h later. β-actin was used as an internal control.
Supplementary Figure 6 Analysis of cell viability of GBC cells after knocking down ERBB1, ERBB2 and ERBB3.
(a–d) GBC cells were transfected with two siRNAs against ERBB1, ERBB2 and ERBB3, and cell viability was determined at days 0, 2, 4 and 6. The data shown are representative of values from three independent experiments (mean ± s.e.m.; *P < 0.05, **P < 0.01 versus the control siRNA group).
Supplementary Figure 7 Knocking down of ERBB3 and ERBB2 impaired GBC cell migration.
The effect of ERBB3 and ERBB2 on cell migration was determined for NOZ and OCUG-1 cells using RNAi. Representative images indicate three independent experiments with similar results.
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Li, M., Zhang, Z., Li, X. et al. Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway. Nat Genet 46, 872–876 (2014). https://doi.org/10.1038/ng.3030
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DOI: https://doi.org/10.1038/ng.3030