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

Mitchell, Megan Irvette (2018-03)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Introduction: Cancer-associated fibroblasts (CAFs) constitute the most abundant mesenchymal cell type present within the tumour microenvironment. Recent evidence suggests that nutrient deprived cancer cells survive as a result of their ability to undergo extensive metabolic reprogramming exploiting the metabolic capacities of surrounding CAFs. Additionally, it has been proposed that CAFs also play a role in enhancing tumourigenicity and the metastatic capability of cancer cells. However, the mechanisms underlying the interactions between epithelial cancer cells and surrounding stromal fibroblasts remain to be elucidated. We therefore hypothesize, that nutrient deprived breast cancer epithelial cells could influence cancer-associated fibroblasts (CAF’s), to produce metabolites which may be utilized by cancer cells for survival, chemo-resistance and enhanced migration. Methods: E0771 cancer cells were subjected to glucose starvation after which cell viability, oxidative stress analysis and cell death was assessed. E0771 conditioned media was then generated and proteomic analysis on conditioned media was performed. This media was also used to treat mouse embryonic fibroblast (MEF) cells. The activation of a CAF phenotype was assessed by means of Western blotting and confocal microscopy. Furthermore, cell viability assays, oxidative stress, glucose uptake and GLUT4 translocation were assessed. MEF conditioned media was then generated and again proteomic analyses were performed. MEF conditioned media was then used to treat glucose deprived E0771 cells. Where after cell viability, cell death and migration were assessed. The effects of CAFs on chemotherapy resistance and metastasis was assessed by treating E0771 cells with doxorubicin and MEF conditioned media, following which, cell viability, apoptosis and migration assays were performed. An in vivo tumour bearing mouse model was established using female C57/BL6 mice treated with doxorubicin. Primary epithelial organoids were isolated from tumours and a 3D branching morphogenesis assay was performed. Results: 12 hours of glucose deprivation resulted in no significant changes in mitochondrial reductive capacity or markers of apoptosis, however, a significant increase in mitochondrial oxidative stress was observed. Proteomic analysis of glucose deprived E0771 conditioned media revealed an increase in proteins associated with exosome-like vesicles and an increased clustering of proteins involved in epithelial-to-mesenchymal transition and glucose metabolism. 2-NBDG glucose uptake was significantly increased in conjunction with an increase in the fluorescent intensity of the HA-GLUT4-GFP construct following exposure to E0771 conditioned media, indicating the increase in glucose uptake is in part mediated by GLUT4 translocation. Furthermore the treatment of E0771s with MEF conditioned media lead to a significant increase the speed of migration and EMT. Furthermore, increased invasiveness of epithelial organoids was observed following exposure to MEF-CM in Dox treated animals, with an increase in a more epithelial-like phenotype. Conclusion: Our data suggest that glucose deprivation induces a state of oxidative stress in the E0771 cells which is transferred to MEFs leading to the “activation” of a CAF-like phenotype, and that this “activated” phenotype contributes significantly to the pro-survival and pro-metastatic abilities of breast cancer cells. Furthermore, our results contribute significantly to the understanding of the molecular mechanisms underlying the interaction between epithelial cancer cells and fibroblasts within the tumour microenvironment.

AFRIKAANSE OPSOMMING: Inleiding: Kanker-geassosieërde fibroblaste (KGFe) maak die grootste deel uit van die mesenkiemale selle wat in die tumor mikro-omgewing voorkom. Onlangse bewyse stel voor dat nutriënt-ontneemde kankerselle oorleef as gevolg van hul vermoë om omvattende metaboliese herprogrammering te ondergaan en die metabolise kapasiteit van omliggende KGFe te benut. Daar word ook verder voorgestel dat KGFe ‘n rol speel om die tumorigenisiteit en die metastatiese kapasitiet van kankerselle te verhoog. Die meganismes onderliggend aan hierdie interaksie tussen die epiteliale kankerselle en die omliggende fibroblaste moet egter nog ontrafel word. Ons hipotese is dus dat nutriëntontneemde borskanker epiteelselle KGFe beïnvloed om metaboliete te produseer wat deur die kankerselle vir oorlewing, chemo-weerstand en verhoogde migrasie benut kan word. Metodes: E0771 borskankerselle is blootgestel aan media wat van glukose ontneem is, waarna sellewensvatbaarheid, oksidatiewe stres en merkers van seldood is geanaliseer. Gekondisioneeerde media uit E0771 selle gegenereer waarna proteomiese analises daarop uitgevoer is. Hierdie media is ook gebruik om muis embrioniese fibroblaste (MEFe) te behandel. Die aktivering van die KGF fenotipe is bepaal deur middel van westelike kladtegniek en konfokale mikroskopie. Verder is sel-lewensvatbaarheid, oksidatiewe stres, glukose opname en GLUT4 translokering, geasseseer. MEF-gekondisioneerde media is daarna gegenereer waarop daar ook proteomiese analises uitgevoer is. MEFgekondisioneerde media is ook gebruik om glukose-ontneemde E0771 kankerselle te behandel waarna sellewensvatbaarheid, L-laktaat konsentrasies, ATP produksie en seldood gemeet is. Die effek van KGFe op chemoterapie weerstand en metastase is geasseseer deur E0771 kankerselle met doxorubicin in kombinasie met MEF gekondisioneerde media te behandel, waarna sellewensvatbaarheidsanalise, apoptose asook ‘n migrasie bepalings uitgevoer is. Verder is daar ook ‘n in vivo tumor-draende muismodel gebruik waar C57/BL6 muise met doxorubicin behandel is. Primêre epiteel organoïede is vanuit tumore geïsoleer waarop daar ‘n 3D vertakkingsmorfogenese gedoen is. Resultate: 12 ure van glukose-ontneming het geen veranderinge in mitokondriale reduktiewe kapasitiet of in merkers van apoptose teweeggebring nie, alhoewel ‘n insiggewende verhoging in mitokondriale oksidatiewe stress op daardie tydspunt waargeneem is. Proteomiese analise van glukose-ontneemde E0771-gekondisioneerde media toon ‘n toename in proteïene aan wat geassosieer word met eksosoom vesikels; proteïene wat betrokke is by epiteel-tot-mesenkiemale oorgang asook proteïene betrokke by glukose metabolisme. 2-NBDG glukose opname is ook insiggewend verhoog tesame met ‘n toename in fluoriserende intensiteit van die HA-GLUT4-GFP fragment na blootstelling aan E0771-gekondisioneerde media, wat aandui dat glukose opname gedeeltelik bemiddel is deur GLUT4 translokering. Die behandeling van E0771 selle met MEF-gekondisioneerde media het tot ‘n insiggewende verhoging in die spoed van migrasie gelei, asook ‘n toename in EMO. Daar is ook ‘n toename in ‘n epiteelfenotipe en indringing in die epiteel-organoiede waargeneem na blootstelling aan MEFgekondisioneerde media in Dox-behandelde muise. Gevolgtrekking: Ons data bewys dat glukose-ontneming oksidatiewe stress in E0771 kankerselle teweegbring wat dan veroorsaak dat die omliggende MEFe geaktiveer word om na ‘n KGF fenotipe te verander. Hierdie geaktiveerde fenotipe dra dan op hulle beurt insiggewend by tot die oorlewing en pro-metastatiese vermoë van nutriënt-ontneemde borskankerselle. Ons resultate maak ‘n insiggewende bydrae tot die verstaan van die molekulêre meganismes betrokke by die interaksie tussen kankerselle en fibroblaste in die tumor mikro-omgewing.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103267
This item appears in the following collections: