Metabolic reprograming and cancer resistance : an investigation into the metabolic control of cancer-associated fibroblasts on breast cancer cell survival and metastasis

[摘要] ENGLISH ABSTRACT: Introduction: Cancer-associated fibroblasts (CAFs) constitute the most abundantmesenchymal cell type present within the tumour microenvironment. Recent evidencesuggests that nutrient deprived cancer cells survive as a result of their ability to undergoextensive metabolic reprogramming exploiting the metabolic capacities of surroundingCAFs. Additionally, it has been proposed that CAFs also play a role in enhancingtumourigenicity and the metastatic capability of cancer cells. However, the mechanismsunderlying the interactions between epithelial cancer cells and surrounding stromalfibroblasts remain to be elucidated. We therefore hypothesize, that nutrient deprivedbreast cancer epithelial cells could influence cancer-associated fibroblasts (CAF's), toproduce metabolites which may be utilized by cancer cells for survival, chemo-resistanceand enhanced migration.Methods: E0771 cancer cells were subjected to glucose starvation after which cellviability, oxidative stress analysis and cell death was assessed. E0771 conditioned mediawas then generated and proteomic analysis on conditioned media was performed. Thismedia was also used to treat mouse embryonic fibroblast (MEF) cells. The activation of aCAF phenotype was assessed by means of Western blotting and confocal microscopy.Furthermore, cell viability assays, oxidative stress, glucose uptake and GLUT4translocation were assessed. MEF conditioned media was then generated and againproteomic analyses were performed. MEF conditioned media was then used to treatglucose deprived E0771 cells. Where after cell viability, cell death and migration wereassessed. The effects of CAFs on chemotherapy resistance and metastasis wasassessed by treating E0771 cells with doxorubicin and MEF conditioned media, followingwhich, cell viability, apoptosis and migration assays were performed. An in vivo tumourbearing mouse model was established using female C57/BL6 mice treated with doxorubicin. Primary epithelial organoids were isolated from tumours and a 3D branchingmorphogenesis assay was performed.Results: 12 hours of glucose deprivation resulted in no significant changes inmitochondrial reductive capacity or markers of apoptosis, however, a significant increasein mitochondrial oxidative stress was observed. Proteomic analysis of glucose deprivedE0771 conditioned media revealed an increase in proteins associated with exosome-likevesicles and an increased clustering of proteins involved in epithelial-to-mesenchymaltransition and glucose metabolism. 2-NBDG glucose uptake was significantly increasedin conjunction with an increase in the fluorescent intensity of the HA-GLUT4-GFPconstruct following exposure to E0771 conditioned media, indicating the increase inglucose uptake is in part mediated by GLUT4 translocation. Furthermore the treatment ofE0771s with MEF conditioned media lead to a significant increase the speed of migrationand EMT. Furthermore, increased invasiveness of epithelial organoids was observedfollowing exposure to MEF-CM in Dox treated animals, with an increase in a moreepithelial-like phenotype.Conclusion: Our data suggest that glucose deprivation induces a state of oxidative stressin the E0771 cells which is transferred to MEFs leading to the 'activation of a CAF-likephenotype, and that this 'activated phenotype contributes significantly to the pro-survivaland pro-metastatic abilities of breast cancer cells. Furthermore, our results contributesignificantly to the understanding of the molecular mechanisms underlying the interactionbetween epithelial cancer cells and fibroblasts within the tumour microenvironment.

[发布机构] Stellenbosch University