Abstract
Nasopharyngeal carcinoma (NPC) has extremely skewed ethnic and geographic distributions, is poorly understood at the genetic level and is in need of effective therapeutic approaches. Here we determined the mutational landscape of 128 cases with NPC using whole-exome and targeted deep sequencing, as well as SNP array analysis. These approaches revealed a distinct mutational signature and nine significantly mutated genes, many of which have not been implicated previously in NPC. Notably, integrated analysis showed enrichment of genetic lesions affecting several important cellular processes and pathways, including chromatin modification, ERBB-PI3K signaling and autophagy machinery. Further functional studies suggested the biological relevance of these lesions to the NPC malignant phenotype. In addition, we uncovered a number of new druggable candidates because of their genomic alterations. Together our study provides a molecular basis for a comprehensive understanding of, and exploring new therapies for, NPC.
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Acknowledgements
We thank M.-S. Zeng (Sun Yat-sen University Cancer Center), T. Chan (Memorial Sloan-Kettering Cancer Center), Z.J. Zang (National Cancer Centre Singapore) and P. Tan for generously sharing materials as well as relevant facilities. This work is supported by US National Institutes of Health grant R01CA026038-35 (H.P.K.), the National Research Foundation Singapore and the Singapore Ministry of Education under the Research Centres of Excellence initiative (H.P.K.) and the Singapore Ministry of Health's National Medical Research Council under its Singapore Translational Research (STaR) Investigator Award to H.P.K. This work is also supported by The Terry Fox Foundation International Run Program through funds raised by the Terry Fox Singapore Run to the National University Cancer Institute, Singapore (NCIS).
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D.-C.L. and H.P.K. designed the study and wrote the manuscript. D.-C.L., X.M., M.H., Y.N., Y.S., A.M.V., L.X., L.-W.D. and A.S. performed experiments. C.S.H., B.F.P., B.C.G., S.C.L., F.G.Y. and K.S.L. coordinated sample collection and processing. D.-C.L., Y.N., Y.S., L.-Z.L., H.Y. and S.O. performed bioinformatical analysis. D.-C.L., X.M., Y.N., P.M., M.Z.O., B.C.G., S.C.L., F.G.Y., H.Y., S.O., K.S.L. and H.P.K. analyzed and discussed the data.
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Integrated supplementary information
Supplementary Figure 1 Somatic coding mutation rates of NPC and other types of human cancers
Scatter plot showing somatic coding mutation rates with each dot representing a tumor-germline pair and the horizontal bar representing median value. The number under the X-axis indicates the number of tumor-germline pairs sequenced. All of the cancers except for NPC have been published elsewhere, and were analyzed by Lawrence et al. 21. GBM, glioblastoma multiforme; CLL, chronic lymphocytic leukemia; MM, multiple myeloma; DLBCL, diffuse large B-cell lymphoma; HNSCC, head and neck squamous cell carcinoma; Lung SCC, lung squamous cell carcinoma. EAC, esophageal adenocarcinoma; Lung AC, lung adenocarcinoma.
Supplementary Figure 2 Analysis of intratumoral clonality
Intratumoral clonality plots of three representative NPC cases subjected to whole-exome sequencing. Both NPC2F and NPC15F tumors showed bi-clonal architecture and NPC7D tumor was multi-clonal. VAF was calculated with copy-number neutral variants as described previously1,2,4,22,23.
Supplementary Figure 3 Mutational spectrum and signature of NPC
(a) Frequency of the six types of base substitutions caused by somatic mutations in NPC. (b) Detailed breakdown of mutations at each group of trinucleotides. (c) Relative expression of APOBEC3B mRNA analyzed from two datasets (GDS334124 and GSE3457325) which performed cDNA microarrays on NPC cases. Horizontal bar, mean value; N.S., no significance.
Supplementary Figure 4 Tumor suppressive function of ARID1A in NPC cells
NPC cells were transiently transfected with either negative control siRNA (Scramble) or pooled siRNAs against ARID1A (siARID1A). 48 hours later, cells were analyzed by (a) WB with indicated antibodies, and (b) migration assay. (c) NPC cells stably expressing either negative control shRNA (Scramble) or pooled shRNAs against ARID1A (shARID1A) were subjected to WB analysis. (d) HONE1 cells stably expressing either negative control shRNA (Scramble) or pooled shRNAs against ARID1A (shARID1A) were subjected to migration assay, (e) anchorage-independent colony formation assay and (f) xenograft growth assay. Horizontal bar, mean value. (g) Quantification of c-Myc protein levels of Fig. 4d. (h) SNP-array data of chromosome 1p of HONE1 cells. Blue line, total gene dosage; green and red lines, alleles-specific gene dosage. The value on the left indicates copy number. (i) MSP analysis of ARID1A promoter region in HONE1 cells. Upper panel, potential CpG sites were predicted by MethPrimer17. Vertical orange bars, CpG sites. Lower panel, PCR products were amplified by either methylated-specific (“M”) or unmethylated-specific (“U”) primers. (j) Relative ARID1A mRNA level of NPC cells was measured by real-time RT-PCR and normalized with HONE1 cells. (k) HONE1 cells were treated with either DMSO (Control), Vorinostat (5 μM, histone deacetylase inhibitor), or MG-132 (10 μM, proteasome inhibitor) for 12 hours and subject to WB analysis with indicated antibodies. Both β-Actin and α-Tubulin were used as loading controls for WB. Data from (b, d, e, g, j) represent mean ± SD; N = 3. *, P < 0.05; **, P < 0.01. N.S., no significance.
Supplementary Figure 5 Exploration of the function of autophagy pathway and FAT1 in NPC cells
(a) SUNE1 cell were treated with either DMSO (control), chloroquine (15 μM), cisplatin (20 μM), or chloroquine (15 μM) together with cisplatin (20 μM) for 48 hours, and apoptotic cells were detected with Annexin-V staining. (b) NPC cells were transfected with control siRNAs (Scramble) or siRNAs against FAT1 and subjected to either short-term proliferation assay or WB analysis with indicated antibodies. β-Actin was examined as a loading control. (c) A panel of isogenic NPC cells stably over-expressing either GFP or FAT1 (FAT1-OE) were examined by xenograft growth assay. Data of (a, b) represent mean ± SD; N = 3. Horizontal bar, mean value; *, P < 0.05; N.S., no significance.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Tables 4,5 and 7–10. (PDF 1905 kb)
Supplementary Table 1
(a) Coverage analysis of whole exome sequencing (Discovery Cohort and 5 NPC cell lines). (b) Coverage analysis of targeted sequencing (Prevalence Cohort). (XLSX 24 kb)
Supplementary Table 2
(a) Clinical parameters of NPCs analyzed with whole exome sequencing and SNP-array (Discovery Cohort). (b) Clinical parameters of NPCs analyzed with targeted sequencing (Prevalence Cohort). (XLSX 23 kb)
Supplementary Table 3
(a) Summary of somatic mutations of 56 NPCs analyzed with whole exome sequencing. (b) Summary of probable somatic mutations in NPC cell lines. (c) Summary of somatic mutations of 66 NPCs analyzed with targeted sequencing (XLSX 170 kb)
Supplementary Table 6
Genes for targeted capture in Prevalence Cohort (XLSX 27 kb)
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Lin, DC., Meng, X., Hazawa, M. et al. The genomic landscape of nasopharyngeal carcinoma. Nat Genet 46, 866–871 (2014). https://doi.org/10.1038/ng.3006
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DOI: https://doi.org/10.1038/ng.3006
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