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Abstract
Metastatic progression of tumors is the major cause of death in patients with triple neg-
ative breast cancer (TNBC). However, since metastasis is a multi-step process, unraveling
its complexity is a major challenge. One eective way of tackling this question is to study
natural blockers of the metastatic process, metastasis suppressors, and identify the mecha-
nisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP, also known
as PEBP1), a protein that regulates kinase activity, is a physiological suppressor of TNBC
metastasis. Although RKIP inhibits the activity of Raf-1, other kinase targets of RKIP in
tumors are not known. To address this question, we used a mass spectrometry approach
involving inhibitor-conjugated beads to identify kinases that are down-regulated by RKIP in
human TNBC xenograft tumors. Our results identied a network of stress kinases targeted
by RKIP, including kinases that have not been previously reported as RKIP targets. By
using a high-throughput invasion assay, we developed a low-dose multi-drug cocktail of ki-
nase inhibitors targeting stress MAP kinases p38, JNK, MLK, and MEK. This combination
treatment mimicked RKIP's anti-metastatic role in cultured cell, as well as xenograft and
syngeneic models of TNBC. In order to unravel the eect of this stress network on metastatic
gene expression, we conducted an RNA-seq analysis comparing metastatic xenograft tumors
to non-metastatic RKIP-overexpressing tumors. Genes downregulated by RKIP in these
tumors were enriched in motility and invasion related gene sets, and their expression was
induced under stress conditions. Several of these genes were directly regulated by BACH1, a
pro-metastatic factor targeted by RKIP. Interestingly, the same genes were negatively corre-
lated with RKIP expression across multiple TCGA cancer types and with other metastasis
suppressor genes within the breast cancer cohort, highlighting the clinical relevance of the
RKIP-network. Elucidating RKIP function at a systems level reveals the interplay between
key metastatic signaling cascades, particularly in relation to cell motility and invasion. Our
ndings suggest that a low-dose, multi-drug combination therapy that targets a network of
stress kinases is a viable anti-metastatic therapy for TNBC patients.