Abstract P1-07-05: Triple negative breast cancer metastases: Protein signal transduction networks within the tumor-stromal microenvironment complement genomic analysis and stratify local versus distant metastasis
Authors: Espina Virginia , Levin Maren K , Carpten John D , Craig David W , Von Hoff Daniel , Liotta Lance A , O'Shaughnessy Joyce 
Published: May 2015

Background: Genomic profiling of primary TNBC may not reflect the lethal metastatic (mTNBC) lesions that constitute a selected subclone of the primary tumor. Moreover, while the stroma microenvironment of primary tumors has been the subject of study, almost nothing is known about the stromal microenvironment of metastasis. We conducted proteomic signal pathway network analysis of microdissected tumor cells and stroma from mTNBC samples for which whole genome sequencing was known. We addressed the following questions: 1) Can potentially actionable driver mutations in the metastasis be inferred from the activated/ suppressed protein signaling pathways in the tumor cells? 2) Are the tumor cell signaling pathways different between local and distant metastasis? 3) Does the proteomic signaling profile of the stroma provide strategies for stromal therapy of mTNBC?

Methods: Enriched populations of mTNBC cells and adjacent stromal cells were collected by Laser Capture Microdissection from 13 fresh frozen mTNBC samples. Signal pathway profiling of 130 protein and/or phosphoprotein endpoints were quantified by reverse phase protein arrays (RPPA). The samples represented local (regional lymph node and chest wall) (n=7) and distant (n=6) metastasis. Spearman rho correlation analysis identified pair-wise protein linakges (rho=0.8, p&lt;0.01). Whole genome sequencing was also performed on the mTNBC samples (Craig DW. Mol Cancer Ther, 2012).

Results: We compared the protein signaling network of the tumor and matched stroma to investigate tumor-stroma crosstalk, and also to the genomic alterations (GAs) in the same tumor. GAs in mTNBC fell into two major categories: DNA repair or cell cycle/growth factor signaling. Proteomic analysis confirmed the down-regulation of endocrine (ER, AR, PR) and HER2 pathways. PhosphoJAK1/2 and phosphoHer3 were markedly down-regulated in mTNBCs, while EGFR and Her4 signaling were not. Spearman rho pairwise correlations revealed differences between local and distant metastases as well as between local and distant stroma. Cyclin D1 in locally metastatic tumor cells was linked with Autotaxin, PLCgamma and RUNX1, whereas in distant metastatic tumor cells, Cyclin D1 was linked with HIF-1alpha, phosphoJAK1, and HSP70. Growth factor signaling via phosphoEFGR was more prevalent in distant mTNBC tumor samples (322 linkages) compared to local mTNBC tumor samples (146 linkages). Distant stromal cell linkages were dominated by growth factor (phospho mTOR) and immune cell crosstalk, e.g. 21 linkages between CD63 in distant stromal samples compared to 1 CD63 linkage in local stromal cells.

Discussion: Proteomic signal pathway data can stratify metastatic lesions into functionally important groups based on a) clinical phenotype (distant versus local metastasis) and b) GA subtype (DNA repair vs growth factor signaling), thereby providing new actionable strategies for novel therapies, e.g. anti-JAK1 and anti-HSP70, and providing a means to prioritize potential driver mutations. Profiling the signal pathway network of mTNBC stroma, for the first time, provides novel strategies for targeting immune cell and mTOR pathways, for new classes of stromal therapy.

Citation Format: Virginia Espina, Maren K Levin, John D Carpten, David W Craig, Daniel Von Hoff, Lance A Liotta, Joyce O'Shaughnessy. Triple negative breast cancer metastases: Protein signal transduction networks within the tumor-stromal microenvironment complement genomic analysis and stratify local versus distant metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-05.