Abstract

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

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Acknowledgements

This work was supported by National Cancer Institute (NCI) CPTAC awards U24CA160034 (Broad Institute; Fred Hutchinson Cancer Research Center), U24CA160036 (Johns Hopkins University), U24CA160019 (Pacific Northwest National Laboratory), U24CA159988 (Vanderbilt University), U24CA160035 (Washington University, St. Louis; University of North Carolina, Chapel Hill). P.W. and F.P. were also supported by SUB-R01GM108711 and MJE by CPRIT grant RR140033. M.J.E. is also a McNair Foundation Scholar. D.F. was supported by Leidos contract 13XS068. Primary genomics data for this study were generated by The Cancer Genome Atlas pilot project established by the NCI and the National Human Genome Research Institute. Resequencing of select samples conducted in this study was supported by National Cancer Institute (NCI) CPTAC award U24CA160035. Information about TCGA and the investigators and institutions that constitute the TCGA research network can be found at http://cancergenome.nih.gov/. We also acknowledge the expert assistance of J. Snider, P. Erdmann-Gilmore and R. Connors for the preparation of the tumour tissues for solubilization. We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, for the use of the Tissue Procurement Core, which provided accessioning, histologic processing and review for the TCGA samples included in this study. The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant #P30 CA91842 (see more at http://www.siteman.wustl.edu/ContentPage.aspx?id=243#sthash.mEU0QuXx.dpuf). We also thank the HAMLET Core at The Washington University in St. Louis for providing breast cancer xenograft tumors. The HAMLET Core was supported in part by grants from NIH/NCRR Washington University-ICTS (UL1 RR024992) and Susan G. Komen for the Cure (KG 090422). F.M. was also supported by The Swedish Research Council (Dnr 2014-323). We also thank A. Subramanian, C. Flynn and J. Asiedu at the Broad Institute for their guidance and assistance in accessing LINCS to run a large number of enrichment queries.

Author information

Author notes

    • Philipp Mertins
    • , D. R. Mani
    • , Kelly V. Ruggles
    •  & Michael A. Gillette

    These authors contributed equally to this work.

Affiliations

  1. The Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Philipp Mertins
    • , D. R. Mani
    • , Michael A. Gillette
    • , Karl R. Clauser
    • , Jana W. Qiao
    • , Filip Mundt
    • , Karsten Krug
    •  & Steven A. Carr
  2. Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, New York 10016, USA

    • Kelly V. Ruggles
    • , Emily Kawaler
    •  & David Fenyö
  3. Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Michael A. Gillette
  4. Department of Genetics and Genomic Sciences, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai New York, New York 10029, USA

    • Pei Wang
    • , Francesca Petralia
    •  & Zhidong Tu
  5. Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA

    • Xianlong Wang
    • , Chenwei Lin
    • , Ping Yan
    •  & Amanda G. Paulovich
  6. Department of Medicine, McDonnell Genome Institute, Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri 63108, USA

    • Song Cao
    • , Venkata Yellapantula
    • , Kuan-lin Huang
    • , Michael D. McLellan
    •  & Li Ding
  7. Department of Oncology-Pathology, Karolinska Institute, 171 76 Stockholm, Sweden

    • Filip Mundt
  8. Lester and Sue Smith Breast Center, Dan L. Duncan Comprehensive Cancer Center and Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA

    • Jonathan T. Lei
    •  & Matthew J. Ellis
  9. Department of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

    • Michael L. Gatza
    • , Matthew Wilkerson
    •  & Charles M. Perou
  10. Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA

    • Sherri R. Davies
    •  & R. Reid Townsend
  11. Biostatistics Center, Massachusetts General Hospital Cancer Center, Boston, Massachusetts 02114, USA

    • Steven J. Skates
  12. Department of Biomedical Informatics and Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA

    • Jing Wang
    •  & Bing Zhang
  13. National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Christopher R. Kinsinger
    • , Mehdi Mesri
    •  & Henry Rodriguez

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    A list of participants and their affiliations appears in the Supplementary Information..

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Contributions

P.M., D.R.M., M.A.G., K.R.C., and S.A.C. designed the proteomic analysis experiments, data analysis workflow, and proteomic-genomic data comparisons. P.M., M.A.G., J.W.Q., and S.A.C. directed and performed proteomic analysis of breast tumour and quality control samples. P.M., D.R.M., K.V.R., K.R.C., P.W., X.W., S.C., E.K., F.P., Z.T., J.T.L., M.L.G., M.W., V.Y., K.H., C.L., M.D.M., P.Y., J.W., B.Z., and D.F. performed proteomic-genomic data analyses. D.R.M., P.W., and S.J.S. provided statistical support. D.R.M., K.V.R., K.R.C., K.K. and D.F. performed analyses of mass spectrometry data and adapted algorithms and software for data analysis. S.R.D., R.R.T and M.J.E. developed and prepared breast xenografts used as quality control samples. P.M. and F.M. prepared and analyzed cell lines for correlative functional annotation of frequently mutated genes. P.M., D.R.M., M.A.G., and S.A.C designed strategy for quality control analyses. M.A.G., S.R.D., C.R.K., M.M., and H.R. coordinated acquisition, distribution and quality control evaluation of TCGA tumour samples. P.M., M.A.G., C.M.P., L.D., A.G.P., and M.J.E. interpreted data in the context of breast cancer biology. P.M., D.R.M, M.A.G., K.R.C., P.W., A.G.P, M.J.E. and S.A.C. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Philipp Mertins or Matthew J. Ellis or Steven A. Carr.

All primary mass spectrometry data are deposited at the CPTAC Data Portal as raw and mzML files and complete protein assembly data sets for public access (https://cptac-data-portal.georgetown.edu/cptac/s/S029). In addition, a set of ancillary files such as dataset G1/P1, G3/P3, G4/P4, G5/P5, G7/P7, CNA correlation tables for CNA–mRNA, CNA–proteome and CNA–phosphoproteome, CNA data, and RNA-seq expression data have also been deposited at the CPTAC Data Coordinating Center (DCC). Two browsers for the results: one provides track hubs for viewing the identified peptides in the UCSD genome browser (http://fenyolab.org/cptac_breast_ucsc); the other is an online tool for proteogenomic data exploration, accessed at http://prot-shiny-vm.broadinstitute.org:3838/BC2016/ (see Supplementary Methods for descriptions).

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    Supplementary Information

    This file contains a Supplementary Discussion, Supplementary Methods, the legends for Supplementary Tables 1-19 (see separate zipped file), Supplementary References and Supplementary Notes (see Contents for more details).

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    Supplementary Tables

    This zipped file contains Supplementary Tables 1-19.

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https://doi.org/10.1038/nature18003

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