Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 May;46(5):467-73.
doi: 10.1038/ng.2935. Epub 2014 Mar 30.

Genomic and molecular characterization of esophageal squamous cell carcinoma

De-Chen Lin  1 , Jia-Jie Hao  2 , Yasunobu Nagata  3 , Liang Xu  4 , Li Shang  5 , Xuan Meng  6 , Yusuke Sato  7 , Yusuke Okuno  7 , Ana Maria Varela  6 , Ling-Wen Ding  6 , Manoj Garg  6 , Li-Zhen Liu  6 , Henry Yang  6 , Dong Yin  8 , Zhi-Zhou Shi  5 , Yan-Yi Jiang  5 , Wen-Yue Gu  5 , Ting Gong  5 , Yu Zhang  5 , Xin Xu  5 , Ori Kalid  9 , Sharon Shacham  9 , Seishi Ogawa  7 , Ming-Rong Wang  5 , H Phillip Koeffler  10
Affiliations

Genomic and molecular characterization of esophageal squamous cell carcinoma

De-Chen Lin et al. Nat Genet. 2014 May.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Mutation frequencies and signatures, and significantly mutated genes in 139 ESCCs
(a) The number of somatic mutations of each examined case (top), key clinical parameters (middle, see Supplementary Tables 2a and 2b), and the significantly mutated genes (SMG) colored by the type of mutations and their mutational frequency (bottom). Columns, examined cases; Rows, genes. (b) Trinucleotide contexts of mutations occurring at cytosine nucleotides in ESCC. Font size of the bases at the 5′ and 3′ positions are proportional to their frequencies (see Supplementary Fig. 3b). (c) APOBEC3B mRNA levels calculated from two datasets GSE20347 and GSE23400, both of which examined cDNA microarray from matched normal/tumor ESCC cases.
Figure 2
Figure 2. Dysregulated pathways in ESCC
(a) Representative FISH photos of an ESCC case with amplified FGFR1. Green signals label the centromere 8 probe (CNE8) and red signals label FGFR1 gene probe. Scale bars, 5 μm. (b) Representative IHC photos of FGFR1 protein over-expression in ESCCs (Additional Cohort, 60 cases had matched adjacent normal epithelial tissues, see Supplementary Table 8a). Scale bars, 200 μm. (c) Top panel, schematics of protein alterations in FBXW7 caused by somatic mutations. Black, missense; red, stopgain (X) or frameshift indel (fs); *, discovered by Agrawal et al.. Conserved domains were mapped from UniProt (See URL). Bottom panel, representative FBXW7 IHC results of an ESCC case carrying FBXW7 mutations (Frequency and Additional Cohort, all cases had matched adjacent normal epithelial tissue, see Supplementary Table 8b). Scale bars, 100 μm. (d-f) Significantly dysregulated pathways colored by the type of alterations. Red font denotes a predicted activating alteration; black font denotes a predicted inactivating alteration. (d) RTK-MAPK-PI3K signaling; (e) G1-S cell cycle regulation; (f) Epigenetic modification.
Figure 3
Figure 3. Indentification of ZNF750 as a novel recessive cancer gene in ESCC
(a) Schematics of protein alterations in ZNF750 caused by somatic mutations. Black, missense or inframe (inf); red, stopgain (X); *, discovered by Agrawal et al.. Conserved domains were mapped from UniProt. (b) Top, IGV (Integrative Genomics Viewer) heatmap showing loss of ZNF750 copy number identified from 149 ESCC SNP-array data; Bottom, segmentation map of two tumors with ZNF750 deletions from 59 ESCCs examined with array-CGH. (c) ZNF750 mRNA levels calculated from GSE20347 and GSE23400. (d) Representative IHC photos of ZNF750 protein expression in ESCCs (Additional Cohort, all cases had matched adjacent normal epithelial tissues, see Supplementary Table 8c). Scale bars, 400 μm. (e) Short-term cell proliferations assays of EC109 and KYSE30 cells transfected with either siRNAs against ZNF750 (si-ZNF750) or control siRNA (Scramble). Data represent mean ± SD; N = 3. (f) ESCC cells were treated with TPA (100nM) for 24 hours and lysates were subjected to WB analysis. (g) Under TPA (100nM) treatment, short-term cell proliferations assay of KYSE30 cells ectopically expressing either GFP or ZNF750 proteins. Data represent mean ± SD; N = 3. Blots of (e) and (g) showed the WB results of ZNF750 protein expression in indicated samples. β-Actin was examined as a loading control. *, P < 0.05.
Figure 4
Figure 4. Inactivation of FAT1 through multiple mechanisms
(a) Schematics of protein alterations in FAT1, FAT2 and FAT3 caused by somatic mutations. Black, missense; red, stopgain (X), Splicing site (sp) or frameshift indel (fs); *, discovered by Agrawal et al.. Conserved domains were mapped from UniProt. (b) Mutually exclusive analysis of FAT1, FAT2 and FAT3 mutations. Columns, examined cases; Rows, genes; Black, missense; red, stopgain, Splicing site or frameshift indel; *, P <0.01. (c) Top, IGV heatmap showing loss of FAT1 copy number identifiedfrom 149 ESCC SNP-array data; Bottom, segmentation map of two tumors with FAT1 deletions from 59 ESCC array-CGH data. (d) Representative IHC photos of FAT1 protein expression in ESCCs (Additional Cohort, all 18 cases had matched adjacent normal epithelial tissues, see Supplementary Table 8e). Scale bars, 400 μm. (e) Short-term cell proliferations were measured in EC109 and KYSE150 cells transfected with either siRNAs against FAT1 (si-FAT1) or control (Scramble). Value represent mean ± SD; N = 4. Blots showed the WB results of FAT1 protein level in indicated samples. β-Actin was detected as a loading control. (f) KYSE150 cells stably expressing either control shRNA (Scramble) or shRNA targeting FAT2 (shFAT2) were injected subcutaneously on the upper flanks of NOD/SCID mice. After 19 days, mice were sacrificed, and the tumors were analyzed. (g) Relative levels of mRNAs of FAT2 quantified with q-PCR in either Scramble or shFAT2 KYSE150 cells. Value represent mean ± SD; N = 3.*, P< 0.05.
Figure 5
Figure 5. Targeting XPO1 in ESCC
(a) Crystal structural modeling of D624G mutation in XPO1 protein (grey ribbon) and its relationship with Snurportin (red ribbon). Dashed yellow lines represent hydrogen bonds. (b) Representative IHC photos of XPO1 protein expression in ESCCs (ESCC-D18 was from Discovery Cohort; the rest of the cases were from Additional Cohort, all cases had matched adjacent normal epithelial tissues, see Supplementary Table 8d). Scale bars, 200 μm. (c) XPO1 mRNA levels examined from GSE20347 and GSE23400. (d) ESCC cells were infected with lentivirus encoding shRNA against either XPO1 (shXPO1) or control shRNA (Scramble), and their proliferation was measured, and (e) cell lysates were subjected to WB analysis with indicated antibodies. (f) ESCC cells were treated with KPT-330 at indicated concentrations for 72 hours, and cell proliferation and apoptosis (g) were measured, and (h) cell lysates were subjected to WB analysis with indicated antibodies. β-Actin was assayed as a loading control. Values of (d, f, g) represent mean ± SD. N = 4. **, P < 0.01; *, P < 0.05.
See this image and copyright information in PMC

Comment in

References

    1. Zhao P, Dai M, Chen W, Li N. Cancer trends in China. Jpn J Clin Oncol. 2010;40:281–5. - PubMed
    1. Pennathur A, Gibson MK, Jobe BA, Luketich JD. Oesophageal carcinoma. Lancet. 2013;381:400–12. - PubMed
    1. Yang YL, et al. Amplification of PRKCI, located in 3q26, is associated with lymph node metastasis in esophageal squamous cell carcinoma. Genes Chromosomes Cancer. 2008;47:127–36. - PubMed
    1. Bass AJ, et al. SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas. Nat Genet. 2009;41:1238–42. - PMC - PubMed
    1. Luo ML, et al. Amplification and overexpression of CTTN (EMS1) contribute to the metastasis of esophageal squamous cell carcinoma by promoting cell migration and anoikis resistance. Cancer Res. 2006;66:11690–9. - PubMed

Publication types

MeSH terms

Substances