AWARD NUMBER: W81XWH 15 1 0039 TITLE: Targeting Tryptophan Catabolism: A Novel Method to Block Triple Negative Breast Cancer Metastasis
The ability of a cancer cell to develop resistance to anoikis, a programmed cell death process triggered by substratum detachment, is a critical step in the metastatic cascade. Triple negative breast cancers (TNBC) exhibit higher rates of metastasis after diagnosis, relative to estrogen positive breast cancers, but while TNBC cells are relatively more resistant to anoikis, the mechanisms involved are unclear. Through gene expression and metabolomic profiling of TNBC cells in forced suspension culture, we identified a molecular pathway critical for anchorage independent cell survival. TNBC cells in suspension upregulated multiple genes in the kynurenine pathway of tryptophan catabolism, including the enzyme tryptophan 2,3 dioxygenase (TDO2) in an NF kB dependent manner. Kynurenine production mediated by TDO2 in TNBC cells was sufficient to activate aryl hydrocarbon receptor (AhR) an endogenous kynurenine receptor. Notably, pharmacologic inhibition or genetic attenuation of TDO2 or AhR increased cellular sensitivity to anoikis, and also reduced proliferation, migration, and invasion of TNBC cells. In vivo, TDO2 inhibitor treated TNBC cells inhibited colonization of the lung, suggesting that TDO2 enhanced metastatic capacity. In clinical specimens of TNBC, elevated expression of TDO2 was associated with increased disease grade, estrogen receptor negative status, and shorter overall survival. Our results define an NFkB regulated signaling axis that promotes anoikis resistance, suggest functional connections with inflammatory modulation by the kynurenine pathway, and highlight TDO2 as an attractive target for treatment of this aggressive breast cancer subtype. Cancer Res; 75(21) 4651 64. 2015 AACR. Introduction The vast majority of breast cancer deaths are caused by complications from metastases (1 3) A high rate of metastasis is characteristic of triple negative breast cancers (TNBC) which lack expression of estrogen receptor (ER) progesterone receptor (PR) and HER2 amplification, and thus do not respond to current endocrine therapies orHER2 targeted therapies (4).Due in part to the absence of effective targeted therapies, but likely also to inherent properties of this subtype, patientswithmetastatic TNBC have a poor prognosis with a median survival of 13 months (5) Thus, identification of new targeted therapies that inhibit or slow metastasis is of critical importance to improve the prognosis of women with TNBC. Metastasis is a complex process with multiple steps, including detachment of cancer cells from the primary tumor, invasion through local tissue, intravasation into the vasculature and lymphatics, survival while in transit, extravasation, and colonization of secondary sites (6) Normal epithelial cells are programmed to undergo apoptosis if they become detached from the basement membrane, a process termed anoikis (7) Anoikis resistance is thought to facilitate survival of tumor cells that detach from the primary tumor and thereby facilitate metastasis (8, 9) Multiple mechanisms of anoikis resistance have been identified, including deregulation of integrin expression and aberrant activation of prosurvival pathways (as reviewed in ref. 9) as well as altered metabolism (10, 11) However, the present study is the first to globally profile gene expression alterations in forced suspension culturewith the goal of identifying targetable pathways important for survival in suspension. This objective screening approach identified multiple components of the kynurenine pathway of tryptophan catabolism and the aryl hydrocarbon receptor (AhR) which is activated by kynurenine (12) as being upregulated by TNBC in suspension culture. Altered tryptophan metabolism and increased secretion of tryptophan metabolites by solid tumors, including in breast cancer, have long been recognized (13, 14) The essential amino acid tryptophan is required for protein synthesis and is a precursor for the formation of multiple signaling molecules including serotonin (15) The majority of tryptophan catabolism occurs via the kynurenine pathway, leading to synthesis of NADth along with intermediate products, including quinolinic acid and Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Colorado. Note: Supplementary data for this article are available at Cancer Research Online (http:/cancerres.aacrjournals.org/ N.C. D'Amato and T.J. Rogers contributed equally to this article. CorrespondingAuthor: Jennifer K. Richer, University of Colorado at Denver, RC1 North P18 5127 Mail Stop 8104, 12800 E. 19th Ave, Aurora, CO 80045. Phone: 303 724 3735; Fax: 303 724 3712; E mail: Jennifer.Richer@ucdenver.edu doi: 10.1158/0008 5472.CAN 15 2011 2015 American Association for Cancer Research. Cancer Research www.aacrjournals.org 4651 on November 3, 2015. (c) 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst September 11, 2015; DOI: 10.1158/0008 5472.CAN 15 2011