The cellular response of triple-negative breast cancer to short-term starvation: implications for chemosensitivity

Prangley, Charne (2021-03)

Thesis (MSc)--Stellenbosch University, 2021.


ENGLISH ABSTRACT: Introduction: Breast cancer is currently the most common cancer among women globally. Triplenegative breast cancer (TNBC) is an aggressive and often drug-resistant sub-type of breast cancer that is correlated with poor patient outcomes. As a result, adjuvant therapies that may improve drug sensitivity are currently being sought. Due to the unique metabolic hallmarks of cancer, metabolic adjuvant therapies have become an area of increasing interest. We therefore set out to investigate the effect of short-term starvation (STS) on the growth, viability, and metabolism of TNBC cells and a benign breast epithelial cell line. We also investigated the effect of STS on chemotherapyinduced cytotoxicity in these cells to determine whether STS may enhance the effect of doxorubicin in TNBC. Methods: Three cell lines were utilised for this study: a benign breast epithelial cell line (MCF- 12A), and two triple-negative breast cancer cell lines (BT-549 and MDA-MB-231). Western blotting was employed to determine the effect of starvation over time on growth and proliferation signalling pathways (PI3K/Akt) and markers of autophagy (Atg5, p62 and LC3-II). Immunocytochemistry was utilised to quantify autophagic puncta. Cell cycle progression and viability were assessed using flow cytometry and a WST1 assay, respectively. The effect of STS on chemosensitivity was then established by incubating cells in standard or starvation-mimicking media for 24 hours, whereafter they received doxorubicin at a concentration of 2.5 μM. Chemosensitivity was then established in terms of live cell number, cell death and viability, and cell cycle progression. Results and Discussion: In response to STS, the MCF-12A cells downregulated pro-growth signalling pathways, while the MDA-MB-231 cells showed significant upregulation. A 24-hour starvation period had no significant effects on these pathways or on autophagic flux in the BT-549 cells. Both the MCF-12A and MDA-MB-231 cell lines significantly upregulated autophagic flux in response to STS, with the latter achieving the most significant effect at 24-hours. This may have offered protection to these cells, as a period of starvation prior to drug administration reduced doxorubicin-induced G2/M arrest. Additionally, STS had no other significant effects on chemosensitivity in these cells. In the BT-549 cells, however, starvation was able to significantly increase the percentage of dead cells in the group that received STS prior to doxorubicin treatment. As autophagy was not significantly increased in this cell line during starvation, this suggests that autophagy may indeed play a role in drug resistance. Conclusion: In summary, the cell lines which displayed an upregulation of autophagy at 24 hours of starvation were not sensitised to doxorubicin in terms of cell death, and also experienced amelioration of doxorubicin-induced G2/M arrest. This supports the notion that autophagic upregulation may protect cancer cells from doxorubicin-induced cytotoxicity and contribute to drug resistance. However, to gain a more thorough understanding of this phenomenon, future studies investigating the mechanisms by which autophagy promotes chemoprotection are recommended.

AFRIKAANSE OPSOMMING: Inleiding: Borskanker is die mees algemene kanker in vroue wêreldwyd. Trippel negatiewe borskanker (TNBK) is ‘n aggressiewe en menigmaal ‘n middel-weerstandige subtipe borskanker wat met ‘n swak prognose geassosieer word. As gevolg hiervan, word adjuvante terapeutiese opsies ondersoek om middel sensitiwiteit te verbeter. Metaboliese adjuvante terapeutitese opsies word toenemend ondersoek as gevolg van die unieke metaboliese kenmerke van kanker. Die doel van hierdie studie was om die effek van kort termyn uithongering (KTU) op selgroei, sel lewensvatbaarheid en metabolisme in TNBK selle en benigne bors epiteelselle te ondersoek. Die effek van KTU op chemosensitiwiteit in hierdie selle is ook ondersoek om te bepaal of dit as ‘n moontlike behandelingsopsie kan dien. Metodes: Drie sellyne is in hierdie studie gebruik: ‘n benigne borsepiteel sellyn (MCF-12A), en twee TNBK sellyne (BT-549 en MDA-MB-231). Westelike kladtegniek is gebruik om die effek van uithongering oor tyd op selgroei seinoordragpaaie (PI3K/Akt) en merkers van autofagie te bepaal (Atg5, p62 en LC3-II). Immunositochemie is gebruik om autofagie punktae te kwantifiseer. Selsiklus progressie en sel lewensvatbaarheid is deur middel van vloeisitometrie en ‘n WST-1 toets onderskeidelik bepaal. Die effek van KTU op chemosensitiwiteit is bepaal deur selle in standaard- en uithongering nabootsende media vir 24 uur te inkubeer waarna dit doxorubicin teen ‘n konsentrasie van 2.5 μM ontvang het. Chemosensitiwiteit is daarna vasgestel deur middel van die bepaling van die aantal lewende selle, seldood en sel lewensvatbaarheid, asook selsiklus progressie. Resultate en Bespreking: Die MCF-12A selle het groei seinoordragpaaie afgereguleer in reaksie op KTU, terwyl die MDA-MB-231 selle weer ‘n insiggewende opregulering van hierdie paaie getoon het. ‘n Vier-en-twintig uur uithongeringsperiode het geen insiggewende effek of hierdie paaie of autofagie vloei in die BT-549 selle gehad nie. Beide die MCF-12A en die MDA-MB-231 sellyne het autofagie vloei insiggewend opgereguleer in reaksie op KTU, met die MDA-MB-231 selle wat die mees insiggewende effek na 24 uur getoon het. Dit kon beskerming aan hierdie selle gebied het, aangesien die uithongeringstydperk voor die toediening van doxorubicin, die doxorubicin- geinduseerde G2/M inperking verminder het. KTU het geen verdere effek ten opsigte van chemosensitiwiteit in hierdie sellyne gehad nie. In die BT-549 selle, het uithongering die persentasie dooie selle insiggewend verhoog in die groep wat KTU ondergaan het voor doxorubicin behandeling. Autofagie vloei was nie insiggewend verhoog tydens uithangering in hierdie groep nie wat moontlik kan beteken dat autofagie inderdaad ‘n rol in middelweerstandigheid in hierdie sellyne speel. Gevolgtrekking: Ter opsomming, die sellyne wat ‘n opregulering in autofagie vloei getoon het na 24 uur uithongering, is nie gesentitiseer vir doxorubicin in terme van seldood nie en het verder ook vermindering in doxorubicin-geinduseerde G2/M inperking getoon. Dit ondersteun die begrip dat autofagie opregulering kankerselle teen doxorubicin- geinduseerde sitotoksisiteit beskerm en middelweerstandigheid kan bewerkstellig. Om ‘n meer breedvoerige verstaan van hierdie fenomeen te verkry, word verdere studies aanbeveel wat die meganismes ondersoek waardeur autofagie beskerming van kankerselle teen chemoterapie verleen.

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