Genomic and molecular characterization of esophageal squamous cell carcinoma


Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide and particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified previously uncharacterized mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to those already known (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Our approaches also uncovered many druggable candidates, and XPO1 was further explored as a therapeutic target because it showed both gene mutation and protein overexpression. Our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

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Figure 1: Mutation frequencies and signatures, and significantly mutated genes in 139 ESCCs.
Figure 2: Dysregulated pathways in ESCC.
Figure 3: Identification of ZNF750 as a recessive cancer gene newly implicated in ESCC.
Figure 4: Inactivation of FAT1 through multiple mechanisms.
Figure 5: Targeting XPO1 in ESCC.

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We thank P. Tan and Z. Zang for generously sharing related facilities. We also thank B. Koegler and S. Koegler for their generous support. This work is supported by National High-Tech R&D Program of China 2012AA02A503 and 2012AA02A209 (M.-R.W.), National Natural Science Foundation of China 81330052 (M.-R.W.), US National Institutes of Health grant R01CA026038-35 (H.P.K.), National Research Foundation Singapore and the Singapore Ministry of Education under the Research Centers of Excellence initiative (H.P.K.), and Singapore Ministry of Health's National Medical Research Council under its Singapore Translational Research (STaR) Investigator Award to H.P.K.

Author information




D.-C.L., M.-R.W. and H.P.K. designed the study and wrote the manuscript. D.-C.L., J.-J.H., Y.N., Y.S., Y.O., X.M., L.X., A.M.V., L.-W.D. and M.G. performed experiments. D.Y, J.-J.H., Z.-Z.S., L.S., Y.-Y.J., W.-Y.G., T.G., Y.Z. and X.X. coordinated sample collection and processing. O.K. and S.S. provided KPT-330 and structurally analyzed XPO1 point mutation. D.-C.L., J.-J.H., Y.N., S.O., M.-R.W. and H.P.K. analyzed and discussed the data. Y.N., H.Y., L.-Z.L., Y.S. and Y.O. performed bioinformatical analysis.

Corresponding authors

Correspondence to De-Chen Lin or Ming-Rong Wang.

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Competing interests

O.K. and S.S. are employees of Karyopharm Therapeutics Incorporated. The remaining authors declare no conflict of interest.

Integrated supplementary information

Supplementary Figure 1 Somatic mutation frequencies detected in exomes from ESCC and other human cancers.

Each dot represents an examined case (tumor-germline pair), and tumor types are ordered by their median somatic mutation frequencies (indicated by the black dash line). The number beneath X-axis indicates the number of examined cases of each cancer. Except ESCC, all of the other cancers were sequenced and published elsewhere, and their data were re-analyzed by Lawrence et al. CLL, chronic lymphocytic leukemia; MM, multiple myeloma; GBM, glioblastoma multiforme; DLBCL, diffuse large B-cell lymphoma; HNSCC, head and neck squamous cell carcinoma; EAC, esophageal adenocarcinoma; Lung AC, lung adenocarcinoma; Lung SCC, lung squamous cell carcinoma. Source data

Supplementary Figure 2 Intratumoral clonality analysis.

Intratumoral clonality plots of four representative ESCC cases from Discovery Cohort. ESCC-D13, D15, D17 tumors were developed bi-clonaly whereas ESCC-D14 tumor showed multi-clonal structure. Variant allele frequency was calculated with copy-number neutral variants, which was further used to plot intratumoral clonal architectures. Source data

Supplementary Figure 3 Mutation types and signatures in ESCC.

(a) The occurrence of the six types of base-substitution mutations observed in ESCC. (b) The occurrence of cytosine mutations at each group of trinucleotide, which shows biases toward mutations of TCN motifs. (c) Genes mutated in over 5% (≥ 7 cases) of the entire cohort. Source data

Supplementary Figure 4 Down-regulation of FBXW7 protein expression in ESCC.

Representative FBXW7 IHC results of one ESCC case from Additional Cohort (Upper panel) and one ESCC case from Frequency Cohort with FBXW7 mutation. Scale bars, 200 μm.

Supplementary Figure 5 Identification of ZNF750 as a novel recessive cancer gene in ESCC.

(a) Box plot showing relative ZNF750 mRNA levels in 947 human cancer cell lines across 21 cancer types analyzed from CCLE database (see URL for the description of Box Plot). Number in the parentheses indicates the number of cell lines analyzed. (b) Summary of somatic mutations affecting ZNF750 across different tumor types including mutation data reported by The Cancer Genome Atlas (TCGA, see URL) and Catalogue Of Somatic Mutations In Cancer (COSMIC, see URL). Number in the parentheses indicates the number of cases sequenced. Concerning C-Mel/CSCC, in TCGA pathological reports, several cutaneous melanoma (C-Mel) patients with ZNF750 mutations also had cutaneous squamous cell carcinoma (CSCC). UASCC, upper aerodigestive squamous cell carcinoma; HNSCC, head and neck squamous cell carcinoma; CRC, colon and rectum adenocarcinoma; LG Glioma, lower grade glioma; AML, acute myeloid leukemia; GBM, glioblastoma multiforme; AG, autonomic ganglia carcinoma. Squamous cell carcinomas were highlighted with Orange Square. (c) Upper panel, mRNA expression of indicated terminal differentiation-related genes quantified with q-PCR in EC109 and KYSE180 cells transfected with either siRNAs against ZNF750 (siZNF750) or control siRNA (Scramble), as well as (lower panel) infected with lentivirus encoding either of ZNF750 protein (Flag-ZNF750) or GFP (GFP-control). WB results showing ZNF750 protein level in indicated samples. β-Actin was used as a loading control. Value represent mean ± s.d. N = 5. (d) Short-term cell proliferation assay of ESCC cells treated with control medium (DMSO) or TPA (100nM) for 5 days. N = 3. **, P < 0.01; *, P < 0.05. Source data

Supplementary Figure 6 FAT gene mutations and loss of heterozygosity.

(a) Summary of somatic mutations affecting FAT1, FAT2 and FAT3 across different tumor types from COSMIC. Number in the parentheses indicates the number of cases sequenced. (b) Inactivation of both alleles of FAT1 in two ESCC cases. Upper panel, SNP-array data showing FAT1 loss of heterozygosity in both tumors (ESCC-D10 and ESCC-D20, which were from Discovery Cohort). Blue line, total gene dosage; Green and red line, alleles-specific gene dosage. The number on the left indicates log2 tumor/reference ratio. Lower panel, Sanger sequencing signals confirmed the somatic mutations of FAT1 in both cases. Arrows indicate the beginning of frameshift mutation bases in tumor DNA and the corresponding bases in germline DNA. Source data

Supplementary Figure 7 The growth-suppressive properties of FAT1 in ESCC cells.

KYSE150 cells stably expressing either FAT1 protein (Flag-FAT1) or GFP (GFP-control) were subjected to (a) short-term cell proliferation assay or (b) soft-agar colony formation assay. (c) WB results showing FAT1 protein level. β-Actin was used as a loading control. Value represent mean ± SD. N = 3. **, P < 0.01. *, P < 0.05. Source data

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Supplementary Figures 1–7 and Supplementary Tables 1–13 (PDF 15766 kb)

Supplementary Table 14

Chromosome regions for targeted capture with SureSelect cRNA baits (Frequency Cohort). (XLSX 899 kb)

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Lin, DC., Hao, JJ., Nagata, Y. et al. Genomic and molecular characterization of esophageal squamous cell carcinoma. Nat Genet 46, 467–473 (2014).

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