Abstract 181: Therapeutic approaches to metastasis induced by mesenchymal stem cells in the tumor microenvironment
Authors: Chattopadhyay Shrikanta , Huang Cherrie , Mukherjee Siddhartha , Yusuf Rushdia Z. , Viswanathan Vasanthi , Wittner Ben S. , Gentry Jeff , Shamji Alykhan , Ramaswamy Sridhar , Scadden David T. , Schreiber Stuart L. 
Published: May 2015

Abstract
Metastasis is the primary cause of death in non-hematological cancers yet there are no specific therapeutics against it because of a lack of validated targets. The Weinberg lab demonstrated that bone-marrow derived mesenchymal stem cells (MSCs) home to the stroma of breast tumors and induce metastasis. We verified these findings in MDA-MB-231 (MDA) xenografts in which co-injection of human MSCs increased MDA thoracic metastasis by 5-fold. In vitro, MSC co-culture induced GFP-labeled MDA cells to migrate 3-fold faster in a modified ‘wound-healing’ assay, mirroring metastasis in vivo. To identify therapeutic targets within MSC-induced metastasis, we performed gene-expression analysis of MSC-MDA co-cultures separated by flow cytometry compared with cells grown alone. The interferon pathway was found to be the most activated pathway upon co-culture, increasing mainly in MSCs. We studied the relevance of these genes in human cancers by first analyzing 3 different gene-expression datasets comparing human breast cancer stroma with normal stroma. Genes upregulated in breast cancer stroma were then studied in a meta-analysis of 19 whole tumor gene-expression datasets correlating gene-expression changes with survival. We identified 103 genes that are upregulated in MSCs by MSC-MDA interactions, are increased in human breast cancer stroma and are significantly associated with poor survival. To determine if they are necessary for MSC-induced metastatic behavior, we performed shRNA knockdown in MSC-MDA co-cultures measuring effects on in vitro migration. Knockdown of a number of interferon-associated genes significantly reduced migration supporting an unexpected functional role of interferons in metastasis. The top interferon gene, ISG15, is an attractive candidate for therapeutic targeting because it is a secreted ubiquitin-like factor that conjugates a number of cytoskeletal proteins involved in motility.
In parallel, we conducted a small-molecule screen with 1600 compounds on the migration of MSC-MDA co-cultures to identify small-molecule inhibitors of metastasis. Counter screens on highly motile endothelial cells excluded compounds that non-specifically inhibit normal cell migration. Only 1 compound, RSL3, specifically blocked MSC-induced MDA migration with cytotoxicity at &gt;10-fold higher concentrations. RSL3 inhibits the glutathione peroxidase 4 (GPX4) enzyme that metabolizes lipid peroxides including arachidonic acid metabolites participating in inflammatory cascades like interferon gamma signaling. RSL3 activity was completely abrogated by co-treatment with the 5-lipoxygenase inhibitor zileuton consistent with the role of arachidonic acid metabolites in MSC-MDA migration.
In summary, targeting components of interferon and arachidonic acid pathways have been discovered as novel therapeutic approaches against microenvironment-induced breast cancer metastasis.
Citation Format: Shrikanta Chattopadhyay, Cherrie Huang, Siddhartha Mukherjee, Rushdia Z. Yusuf, Vasanthi Viswanathan, Ben S. Wittner, Jeff Gentry, Alykhan Shamji, Sridhar Ramaswamy, David T. Scadden, Stuart L. Schreiber. Therapeutic approaches to metastasis induced by mesenchymal stem cells in the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 181. doi:10.1158/1538-7445.AM2014-181