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Profiling phospho-signaling networks in breast cancer using reverse-phase protein arrays

Oncogene volume 32, pages 3470–3476 (2013)Cite this article

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Abstract

Measuring the states of cell signaling pathways in tumor samples promises to advance the understanding of oncogenesis and identify response biomarkers. Here, we describe the use of Reverse Phase Protein Arrays (RPPAs or RPLAs) to profile signaling proteins in 56 breast cancers and matched normal tissue. In RPPAs, hundreds to thousands of lysates are arrayed in dense regular grids and each grid is probed with a different antibody (100 in the current work, of which 71 yielded strong signals with breast tissue). Although RPPA technology is quite widely used, measuring changes in phosphorylation reflective of protein activation remains challenging. Using repeat deposition and well-validated antibodies, we show that diverse patterns of phosphorylation can be monitored in tumor samples and changes mapped onto signaling networks in a coherent fashion. The patterns are consistent with biomarker-based classification of breast cancers and known mechanisms of oncogenesis. We explore in detail one tumor-associated pattern that involves changes in the abundance of the Axl receptor tyrosine kinase (RTK) and phosphorylation of the cMet RTK. Both cMet and Axl have been implicated in breast cancer, or in resistance to anticancer drugs, but the two RTKs are not known to be linked functionally. Protein depletion and overexpression studies in a ‘triple-negative’ breast cell line reveal cross talk between Axl and cMet involving Axl-mediated modification of cMet, a requirement for cMet in efficient and timely signal transduction by the Axl ligand Gas6 and the potential for the two receptors to interact physically. These findings have potential therapeutic implications, as they imply that bi-specific receptor inhibitors (for example, ATP-competitive small-kinase inhibitors such as GSK1363089, BMS-777607 or MP470) may be more efficacious than the mono-specific therapeutic antibodies currently in development (for example, Onartuzumab).

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Acknowledgements

This study was supported by grants from the National Institutes of Health (R33 CA128726, R21 CA126720, and 5RC1-HG005354) and from the Stand Up to Cancer Project (AACR-SU2C-DT0409). TSG is a Human Frontier Science Program Fellow. RLK is partially supported by NSF 0856285. Supplementary Information accompanies the paper on the Oncogene website.

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Authors and Affiliations

  1. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    T S Gujral, R L Karp, A Finski, M Chan, G MacBeath & P Sorger

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    T S Gujral & G MacBeath

  3. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA

    A Finski

  4. Protein Biotechnologies Inc., Ramona, CA, USA

    P E Schwartz

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  1. T S Gujral
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Correspondence to G MacBeath or P Sorger.

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GM is a Vice President and co-founder, and PKS a co-founder of Merrimack Pharmaceuticals, a biotechnology company that develops anti-cancer drugs. PES is a President and CEO of Protein Biotechnologies, Inc. The remaining authors declare no conflict of interest.

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Supplementary Information accompanies the paper on the Oncogene website

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Gujral, T., Karp, R., Finski, A. et al. Profiling phospho-signaling networks in breast cancer using reverse-phase protein arrays. Oncogene 32, 3470–3476 (2013). https://doi.org/10.1038/onc.2012.378

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