Chemoresistance in a breast cancer animal model: the role of obesity and inflammation

Mentoor, Ilze (2019-12)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Background: Globally an increase in obesity incidence represent a major health concern amongst a rising incidence of impaired treatment outcomes of breast cancer patients. Adipose tissue and/or adipocytes, in the tumour microenvironment serve as an exogenous energy source for the survival of breast cancer cells, especially since adipose tissue is abundant in breast tissue. Breast cancer cells modulate lipid metabolism (de novo fatty acid synthesis and lipolysis), by altering the secretion of adipokines through adipocytes, resulting in the release of free fatty acids to provide energy substrates for breast cancer cells to sustain its high proliferation demand for survival. Evidence on the role of obesity and lipid metabolism especially fatty acids in breast cancer treatment resistance is lacking. This motivates investigation to identify and understand the underlying physiological and molecular mechanisms by which chemotherapeutic treatment resistance is achieved. We therefore hypothesise that obesity-induced inflammation alters lipid metabolism in adipose tissue/adipocytes and contribute to the development of doxorubicin chemotherapeutic treatment resistance in breast cancer cells. Methods: A diet induced obesity animal model was established by feeding female C57BL6 mice a high fat diet for 12 weeks. After developing the diet induced obesity phenotype, breast tumour xenographs were induced by subcutaneous inoculation in the fourth mammary gland with E0771 triple negative breast cancer cells.Once tumours became palpable, mice received either vehicle treatment (Hanks balance salt solution) or doxorubicin treatment (cumulative dose of 12 mg/kg). Plasma inflammatory markers, fatty acid profiles and protein expression of lipid metabolism markers (de novo fatty acid synthesis and lipolysis) was determined in mammary adipose and tumour tissue. To validate the in vivo model findings, we developed an in vitro model using a conditioned media approach. A human adipose tissue derived stem cell line was used for the differentiation of mature adipocytes after which conditioned media was collected to assess the paracrine effect between adipocytes and doxorubicin treated MDA-MB-231 triple negative breast cancer cells. Cell viability was assessed with WST-1 assays. Western blots were used to determined alterations in protein expression of apoptotic and lipid metabolism markers (de novo fatty acid synthesis and lipolysis). An Inflammatory marker as well as free fatty acid profile was also analysed in treatment conditioned media. Results: Diet induced obesity significantly increased tumour growth and decreased doxorubicin treatment efficacy in E0771 triple negative breast tumours (p<0.0001), resulting in treatment resistance. Our findings also showed that diet induced obesity supressed de novo fatty acid synthesis (decreased SCD-1) and lipolysis (decreased HSL) in mammary adipose tissue of doxorubicin treated mice. Conversely an increase in de novo fatty acid synthesis (increased SCD-1) and lipolysis (increased ATGL) was found in tumour tissue, leading to significant changes in FAs composition of both tissues. Diet induced obesity also significantly increased plasma leptin (p=0.025) and resistin levels (p=0.046) and increased NFĸB protein expression in mammary fat of doxorubicin treated mice, thereby inducing systemic and local inflammation. Furthermore, we also report that adipocytes promoted acquired breast cancer treatment resistance by significantly increasing the cell viability of doxorubicin treated MDA-MB-231 triple negative breast cancer cells (Dox+CM vs Dox, p=<0.0001). This was achieved by attenuating doxorubicin’s efficacy to induce apoptosis (decreased cleaved-caspase-3, p<0.05), in a paracrine manner. Adipocytes also induced inflammation (increased leptin and MCP-1) as well as lipolysis (increased HSL) in doxorubicin treated breast cancer cells (Dox vs Dox+CM p=0.03), thereby altering the free fatty acid profile of breast cancer cells. Conclusion: Our data suggest that adipose tissue/adipocytes significantly contribute to treatment resistance in triple negative breast cancer cells. We have demonstrated in both in vivo and in vitro models that adipose tissue/adipocytes secretory factors induce inflammation in the breast tumour microenvironment, which leads to the induction of lipolysis in triple negative breast cancer cells. This resulted in altered metabolic behaviour i.e. increased free fatty acid utilization, which can be utilized as energy substrates or induce lipid saturation in order confer to acquired treatment resistance by evading apoptosis We propose that this could be a novel mechanism by which adipose tissue/adipocytes within the tumour microenvironment can contribute to the development of breast cancer treatment resistance under obesogenic conditions. This study also significantly contributed to the identification and understanding of molecular mechanisms underlying breast cancer treatment resistance in obese patients.

AFRIKAANSE OPSOMMING: Agtergrond: Wêreldwyd verteenwoordig die toenemende insidensie van vetsugtigheid ‘n groot gesondheids risikoen beperkook behandelings uitkomste van borskankerpasiënte. Vetweefsel, en/of adiposiete in die tumormikro-omgewing dien as eksogene energiebron vir die oorlewing van borskankerselle, veral omrede daar ‘n oorvloed van vetweefsel in borsweefsel voorkom. Borskankerselle moduleer lipiedmetabolisme (de novo vetsuur sintese en lipolise), deur die wysiging van adipokiensekresie via adiposiete wat vryvetsure lewer om as energiesubstraat vir borskankerselle dien om sodoende die hoë proliferasie tempo van hierdie selle vir oorlewing te handhaaf. Daar is min inligting oor die rol van lipiedmetabolisme, spesifieke verwysing na vryvetsure in borskanker-behandelingsweerstandigheid. Hierdie feit dien as motivering om die onderliggende fisiologiese en molekulêre meganismes te identifiseer en om die rol van vetsure in chemoterapeutiese behandelingsweerstandigheid te ondersoek. Ons hipotese is dus dat vetsug-geïnduseerde inflammasie, lipiedmetabolisme in vetweefsel/adiposiete wysig en ‘n bydrae lewer tot die ontwikkeling van doksorubisien chemoterapeutieseweerstand in borskankerselle. Metodes: ‘n Dieetgeïnduseerde vetsug-diermodel is gevestig deur vroulike C57BL6 muise met ‘n hoë vet dieet vir 12 weke te voer. Nadat ‘n dieetgeïnduseerde vetsug fenotipe ontwikkel is, is hererologe borskanker gewasse geïnduseer deur middel van ‘n subkutane inokulering in die vierde borsklier met E0771 trippel negatiewe borskankerselle.Nadat die gewassesigbaar begin word het, het die muise òf ‘n draerbehandeling (Hanks gebalanseerde soutoplossing), òf doksorubisienbehandeling (kumulatiewe dosis van 12 mg/kg) ontvang. Plasma inflammatoriese merkers, vetsuurprofiele en proteïenuitdrukking van lipiedmetabolisme merkers (de novo vetsuur sintese en lipoliese) is in borsvetweefsel en tumorweefsel ondersoek. Om die in vivo model se bevindinge te bevestig, is ‘n in vitro model ontwikkel waarby kondisionerings media ingesluit is. ‘n Stamsellyn vanuit menslike vetweefsel is gebruik om volwasse adiposiete te differensieer waarna kondisionerings media versamel is om die parakrien effek tussen adiposiete en doksorubisien behandelde MDA-MB-231 trippel negatiewe kankerselle te ondersoek. Die aantal lewende selle is met behulp van die WST-1 toets bepaal. Westerse kladtegniek is gebruik om wysigings in proteïenuitdrukking van apoptotiese en lipiedmetabolisme merkers te bepaal (de novo vetsuur sintese en lipolise). ‘n Inflammatoriese merker en vryvetsuurprofiel is vir die behandelings-kondisionerings media ondersoek. Resultate: Die vetsuggeïnduseerde dieet het gewas groei betekenisvol versnel en die doksorubisien behandelings effektiwiteit in E0771 trippel negatiewe borskanker gewasse verlaag (p<0.0001), wat tot behandelingsweerstand gelei het. Ons resultate toon verder dat dieetgeïnduseerde vetsug beide de novo vetsuur sintese (verlaagde SCD-1) asook lipolise (verlaagde HSL) in borsweefsel van doksorubisien behandelde muise onderdruk. Die teenoorgestelde is by tumorweefsel waargeneem waar de novo vetsuur sintese (verhoogde SCD-1) en lipolise (verhoogde ATGL) tot betekenisvolle veranderinge in vetsuursamestelling van beide weefsels aanleiding gegee het. Dieetgeïnduseerde vetsug het plasma leptien (p=0.025) en resistienvlakke (p=0.046) betekenisvol verhoog, en NFĸB proteïenuitdrukking in borsvetweefsel van die doksorubisien behandelde muise verhoogwat tot sistemiese en locale inflammasie gelei het. Adiposiete bevorder ook borskanker behandelingsweerstand deur die aantal lewende selle van doksorubisien behandelde MDA-MB-231 trippel negatiewe borskankerselle (Dox+CM vs Dox, p=<0.0001) te verhoog. Hierdie effek is verkry deur die effektiwiteit van doksorubisien te verlaag om sodoende sel dood (apoptose) te induseer (verlaagde gesplyte-kaspase-3, p<0.05), in ‘n parakriene wyse. Adiposiete het inflammasie induseer (verhoogde leptien en MCP-1) asook lipolise (verhoogde HSL) in die doksorubisien behandelde borskankerselle (Dox vs Dox+CM p=0.03), en hierdeur die vryvetsuurprofiel van borskankerselle gewysig. Gevolgtrekking: Ons data wys dat vetweefsel/adiposiete ‘n betekenisvolle bydrae lewer tot behandelingsweerstandigheid in trippel negatiewe borskanker selle. Ons het beide in vivo en in vitro aangetoon dat vetweefsel/adiposiete sekretoriese faktore induseer om inflammasie in die borskanker mikro-omgewing te veroorsaak, en sodoende lipolise in trippel negatiewe borskankerselle induseer. Dit het die metaboliese gedrag gewysig, i.e. verhoogde vetsuur verbruik wat of kan dien as energiesubstraat of lipiedversadiging induseer om apoptose te ontwyk. Ons is van mening dat hierdie ‘n nuwe meganisme is waarby vetweefsel/adiposiete binne die tumormikro-omgewing ‘n bydrae lewer tot die ontwikkeling van borskankerbehandelingsweerstand. Hierdie studie het verder ‘n betekenisvolle bydrae gelewer om die molekulêre meganismes te identifiseer en te verklaar wat onderliggend tot borskankerbehandelingsweerstand in vetsugtige pasiënte is.

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