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).