Abstract P1-07-12: Defining the role of the kynurenine pathway in mediating anoikis resistance in triple negative breast cancer
Authors: Rogers Thomas , D'Amato Nicholas , Nemkov Travis , Gordon Michael , Hansen Kirk , Richer Jennifer 
Published: May 2015

Background: Anoikis resistance is thought to be a critical trait of metastatic cancer cells, enabling them to leave the primary tumor and travel through extracellular matrix, intravasate, and survive in the vasculature or lymphatics in transit to a metastatic site. This is particularly important for the triple-negative breast cancer (TNBC) subtype, which has a peak risk of recurrence within the first three years post-diagnosis and an increased mortality rate in the first five years as compared to other subtypes. We performed global profiling of TNBC cells in attached versus forced suspension culture conditions (using poly-HEMA coated plates) for 24 hours. These data revealed that TNBC cells surviving in suspension upregulate multiple genes involved in tryptophan catabolism, also known as the kynurenine pathway (KP), including the rate limiting enzyme tryptophan 2,3,-dioxygenase (TDO) and kynureninase (KYNU). A key metabolite of this pathway has been found to activate the aryl hydrocarbon receptor (AhR), which was also up-regulated in suspended cells.

Hypothesis: We hypothesize that the ability to upregulate the kynurenine pathway (KP) facilitates TNBC cell survival in suspension and mediates the migratory/invasive potential of TNBC.

Methods: We assessed mRNA and protein levels of TDO2, KYNU and AhR by RT-qPCR and western blot. AhR luciferase reporter activity, as well as known AhR regulated genes, were measured in suspension compared to adherent conditions. TNBC cells were treated with small molecule inhibitors of AhR and TDO2. Additionally, secretion of endogenous kynurenine was measured by high performance liquid chromatography (HPLC). Purified kynurenine was added to rescue AhR activity following TDO2 inhibition. Finally, anoikis sensitivity and migratory potential were measured following pharmacological inhibition of TDO2 and AhR.

Results: Relative mRNA levels of TDO2, KYNU, and AhR increase 9, 7, and 2 fold respectively in suspended TNBC cells compared to adherent conditions (P&lt;0.0001). Estrogen receptor positive breast cancer cells lines do not significantly upregulate these genes. AhR reporter activity (P&lt;0.0001) and nuclear localization increase in suspended TNBC cells. Additionally, AhR reporter activity (P&lt;0.0001) and AhR target gene expression (CYP1A1, CYP1B1: P&lt;0.0001) decreased in TNBC cells treated either AhR antagonist (CH223191) or selective TDO2 inhibitor (680C91). Conversely, when purified kynurenine was added to TNBC cells in vitro, AhR activity increased (P&lt;0.05). Furthermore, kynurenine secretion, as measured by HPLC, increased 2.5 fold in suspended TNBC cells and this increase in kynurenine was reduced by addition of 680C91 (P&lt;0.0001). Finally, targeting TDO2 or AhR increased anoikis sensitivity and decreased migration of attached and suspended TNBC cells.

Conclusions: Collectively, these results suggest that the kynurenine pathway may play a critical role in metastatic TNBC. Further mechanistic studies will focus on how the kynurenine pathway is mediating these tumorigenic properties either through the de novo synthesis of NAD+ and/or activation of AhR by kynurenine. Targeting the kynurenine pathway in the clinic may provide a therapeutic strategy to reduce TNBC mortality rates.

Citation Format: Thomas Rogers, Nicholas D'Amato, Travis Nemkov, Michael Gordon, Kirk Hansen, Jennifer Richer. Defining the role of the kynurenine pathway in mediating anoikis resistance in triple negative breast cancer [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 P1-07-12.