C. Subramanian3, S.W. Croslow1, D. Hess3, A. Bindra3, B.B. Blagg2, J.V. Sweedler1, M.S. Cohen3 1University of Illinois Urbana Champaign, Chemistry, Urbana, IL, USA 2University of NotreDame, Chemistry And Biochemistry, NotreDame, INDIANA, USA 3Carle Illinois College of medicine, Urbana, IL, USA
Introduction: While survival rates for estrogen receptor-positive (ER+) breast cancer has improved with hormonal therapies and CDK4/6 inhibitors, patients often develop resistance or recurrence, that can be due to changes in cancer cell energy metabolism. Cruentaren A (CA) is a natural compound that blocks ATP synthase and has been shown to inhibit energy metabolism more selectively in cancer cells. We hypothesize that CA will effectively target ER+ breast cancers by influencing mitochondrial metabolism and cell cycle progression. To test this hypothesis, we evaluated the efficacy and metabolic mechanism of CA in targeting ER+ breast cancer MCF7 cells and validated with patient-derived xenograft organoids (PDXOs, to more accurately replicate tumor heterogeneity).
Methods: Validated MCF7 breast cancer cells were grown in appropriate culture medium. ER+ PDXOs HCI011 and HCI017 (which maintain functional ER and retain endocrine sensitivity) were developed in Matrigel domes that were then disrupted for RNA isolation and subjected to RNAseq analysis and RT-PCR. Dissociated organoids were cryo-sectioned and analyzed using MALDI-2 on a Bruker timsTOF FleX system. Mass Spectrometry (MS) imaging was performed and executed with TIMS for separation of isobaric species. Mitochondrial and glycolytic stress tests were conducted on an XFe96 Extracellular Flux Analyzer.
Results:RNA sequencing analysis on MCF7 cells and PDXOs subjected to IC50 CA concentrations demonstrated over 9000 significantly(>2 fold from baseline) differentially regulated genes including 750 genes involved in metabolic pathways (Bonferroni padj<0.05). CA treatment significantly targeted genes in cell cycle, MAPK signaling, oxidative phosphorylation (OXPHOS), and necroptosis pathways. To validate the changes in metabolism, bioenergetic measurements using Seahorse indicated a significant (p<0.01) decrease from baseline in both glycolysis and OXPHOS after CA treatment. RT-PCR further confirmed a 70% down-regulation (p<0.05) of glycolytic genes such as PKM1 and PFK, as well as the cell cycle protein MCM2. Several genes associated with the mitochondrial OXPHOS pathway, including UQCRC1, COX6C, and others, were significantly downregulated after CA treatment(p<0.01). MS imaging to identify changes in lipid metabolism revealed significant upregulation of phosphatidyl choline PC(34:1, 38:4, and 36:1) and serine PS(O-38.0), and downregulation of diacylglycerol DG(32:2) and phosphatase PA(32:2 and 34:3); p<0.05 each.
Conclusion:CA treatment inhibits growth ER+ breast cancer cells and organoids by altering energy metabolism and cell cycle pathways. Further translational validation will be required to define its future therapeutic potential.