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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.

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