Abstract 1882 Integrin α6β4 Regulates Tryptophan Metabolism through the Kynurenine Pathway in Triple Negative Breast Cancer

The laminin receptor integrin α6β4 is overexpressed in over 80% of triple negative breast cancer (TNBC) cases and contributes to the aggressive nature of TNBC, in part, through gene regulation. The kynurenine pathway, which degrades tryptophan to NAD+, is often upregulated in TNBC and is associated with poorer outcomes. While the role of the kynurenine pathway has been well described in the context of immunosuppression, the significance of de novo NAD+ generation in TNBC has yet to be elucidated. In this study, we seek to identify how the kynurenine pathway is regulated by integrin α6β4 and its impact on tumor growth in TNBC. To investigate these events, we performed RNA sequencing on BT549 cells that stably express integrin β4 or an empty vector (control). We found that compared to control cells, integrin α6β4-expressing cells have significant upregulation of key kynurenine pathway enzymes including IDO1, KYNU, and QPRT, which were confirmed via qPCR and Western blot. These data suggest that integrin α6β4 drives tryptophan metabolism through the kynurenine pathway toward de novo NAD+ synthesis. To further investigate this relationship, we knocked-down integrin β4 in other TNBC cell lines and found that integrin β4 knock-down decreases expression of IDO1, the rate limiting enzyme of tryptophan degradation, thus suggesting that IDO1 expression is regulated in an integrin α6β4-dependent manner. In support of this observation, we found a positive correlation between integrin β4 and IDO1 expression in TNBC through analysis of the TCGA Breast Invasive Carcinoma PanCancer Atlas dataset. To test if integrin α6β4 upregulation of IDO1 impacts tumor growth in a physiologically relevant condition, we treated cells in 3-dimensional (3D) culture with Epacadostat, an IDO1 inhibitor. Upon treatment with Epacadostat, integrin α6β4-expressing cells exhibit significantly decreased invasive growth in 3D culture compared to controls; however, no basal differences in cell viability were observed in 2D culture. Metabolite assessment shows that integrin β4 knock-in results in a decrease in the NAD+/NADH ratio, suggesting that consumption of NAD+ in these cells is potentially higher. In summary, our data suggests that integrin α6β4 promotes the kynurenine pathway through transcriptional regulation of IDO1 to facilitate TNBC tumor growth. Moving forward, we will explore the potential of this alteration of tryptophan metabolism towards the kynurenine pathway to increase de novo NAD+ synthesis. Notably, the largest consumers of NAD+ are PARPs, which utilize NAD+ to recruit base excision repair machinery to the sites of DNA damage. Given that TNBC patients are treated with DNA-damaging chemotherapeutics and most TNBC tumors express integrin α6β4, the upregulation of the kynurenine pathway by integrin α6β4 has significant implications with regards to tumor progression and treatment efficacy for TNBC patients. This work was supported by the National Institutes of Health under grant number R01 CA223164 (to Kathleen O'Connor) and the University of Kentucky Center for Cancer and Metabolism, funded through the NIH/NIGMS COBRE program under grant number P20 GM121327 (to Kathleen O'Connor and Min Chen).