No abstract

Breast cancer metastasis occurs via blood and lymphatic vessels. Breast cancer cells ‘educate’ lymphatic endothelial cells (LECs) to support tumor vascularization and growth. However, despite known metabolic alterations in breast cancer, it remains unclear how lymphatic endothelial cell metabolism is altered in the tumor microenvironment and its effect in lymphangiogenic signaling in LECs. We analyzed metabolites inside LECs in co-culture with MCF-7, MDA-MB-231, and SK-BR-3 breast cancer cell lines using $$^1\hbox {H}$$ 1 H nuclear magnetic resonance (NMR) metabolomics, Seahorse, and the spatial distribution of metabolic co-enzymes using optical redox ratio imaging to describe breast cancer-LEC metabolic crosstalk. LECs co-cultured with breast cancer cells exhibited cell-line dependent altered metabolic profiles, including significant changes in lactate concentration in breast cancer co-culture. Cell metabolic phenotype analysis using Seahorse showed LECs in co-culture exhibited reduced mitochondrial respiration, increased reliance on glycolysis and reduced metabolic flexibility. Optical redox ratio measurements revealed reduced NAD(P)H levels in LECs potentially due to increased NAD(P)H utilization to maintain redox homeostasis. $$^{13}\hbox {C}$$ 13 C -labeled glucose experiments did not reveal lactate shuttling into LECs from breast cancer cells, yet showed other $$^{13}\hbox {C}$$ 13 C signals in LECs suggesting internalized metabolites and metabolic exchange between the two cell types. We also determined that breast cancer co-culture stimulated lymphangiogenic signaling in LECs, yet activation was not stimulated by lactate alone. Increased lymphangiogenic signaling suggests paracrine signaling between LECs and breast cancer cells which could have a pro-metastatic role.

Biocatalytic C–H activation has the potential to merge enzymatic and synthetic strategies for bond formation. Fe II /αKG-dependent halogenases are particularly distinguished for their ability both to control selective C–H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for the selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how site-selectivity and chain length selectivity are achieved. We now report the crystal structure of the HalB and HalD, revealing the key role of the substrate-binding lid in positioning the substrate for C 4 vs C 5 chlorination and recognition of lysine vs ornithine. Targeted engineering of the substrate-binding lid further demonstrates that these selectivities can be altered or switched, showcasing the potential to develop halogenases for biocatalytic applications.

Breast cancer is the second most commonly diagnosed cancer among women worldwide. Patient death is typically caused by metastasis development rather than the primary tumor. Metastasis in breast cancer has been shown to occur via blood and lymphatic vessels. Research shows that breast cancer cells ‘educate' lymphatic and blood endothelial cells to support tumor growth by stimulating growth factor secretion and directing tumor dissemination. In addition, cell metabolism is altered during malignant transformation. Cancer cells have increased energy and macromolecule biosynthesis requirements to sustain rapid proliferation. However, it remains unclear how tumor endothelial cell metabolism is altered and how metabolism contributes to tumor metastasis. Therefore, this study aims to use 1H NMR metabolomics to identify breast cancer-endothelial cell metabolic interactions in a high-throughput manner. Firstly, we studied if in vitro co-culture with breast cancer altered endothelial cell metabolism. We identified distinct metabolic profiles for lymphatic endothelial cells (LECs) grown in monoculture or co-culture with three different breast cancer cell lines. Principal component analysis revealed LECs co-cultured with breast cancer cells clustered separately from control LECs. Interestingly, LECs co-cultured with triple negative breast cancer cells clustered together with LECs co-cultured with HER2+ breast cancer cell lines indicating that the metabolic changes occurring in these LECs were similar. One-way ANOVA with Tukey's HSD post-hoc analysis revealed 17 significantly different metabolites between all five conditions analyzed including: lactate, glucose, phosphocholine, aspartate, acetate, glycerophosphocholine, lysine and methionine. Quantitative metabolite set enrichment analysis revealed 13 metabolic pathways that were significantly enriched between control LECs and LECs co-cultured with breast cancer cell lines. Enriched pathways included: glycolysis, gluconeogenesis, pyruvate metabolism and protein biosynthesis. Together, these results indicate that culturing LECs with certain breast cancer lines causes significant changes in endothelial metabolism. Alterations in endothelial cell metabolism in response to breast cancer co-culture can further our understanding of tumor-vascular interactions and may lead to identification of metabolic biomarkers and a better understanding of breast cancer metastasis. Citation Format: Suehelay Acevedo-Acevedo, Douglas C. Millar, Sean P. Palecek. Elucidating the metabolic crosstalk between lymphatic endothelial cells and breast cancer using 1H NMR metabolomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3481.

Biocatalytic C–H activation has the potential to merge enzymatic and synthetic strategies for bond formation. FeII/αKG-dependent halogenases are particularly distinguished for their ability both to control selective C-H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how regioselectivity and chain length selectivity are achieved. We now report the crystal structure of the HalB and HalD, revealing the key role of the substrate-lid in positioning the substrate for C4 vs C5 chlorination and recognition of lysine vs ornithine. Targeted engineering of the substrate-binding lid further demonstrates that these selectivities can be altered or switched, showcasing the potential to develop halogenases for biocatalytic applications.