Date of Award

2013

Document type

Thesis

Degree Name

MD (Medical Doctor)

First Supervisor

Dr Anne Hopkins

Second Supervisor

Professor A.D.K. Hill

Keywords

Breast Neoplasms, Sodium, Potassium, Exchange ATPase, Therapeutic Use

Abstract

Breast cancer pathogenesis is modulated by complex signalling pathways centred on cholesterol-enriched membrane domains termed lipid rafts. Caveolae, a subtype of rafts, express the protein caveolin-1, which has been suggested as both as a tumour suppressor and promoter. One protein that interacts with caveolin-1 is the sodium potassium ATPase (Na+ K+ ATPase) ion channel. Na+ K+ ATPase has recently been implicated in cell signalling via interaction with its natural inhibitors, cardiac glycosides (CGs). Although predominantly used for heart failure indications, CGs reportedly have anti-proliferative properties in tumour cells. Given the importance of caveolin-1 in malignant behaviours, we hypothesized that CGs exert anti-breast cancer effects by modulating functional associations between Na+ K+ ATPase and caveolae. We first demonstrated concentration- and time-dependent anti-proliferative responses to the CGs digoxin, ouabain and oleandrin in breast cell lines and primary cultures. These effects were most pronounced in primary cell cultures, and least in ER/PR/HER2 triple-negative cells. We next showed concentration-dependent reductions in cell migration in response to CGs, particularly in ER-positive cells. CGs arrested the cell cycle at both G1 and S phase in ER-positive cells, with partial arrest at G1 in ER-negative cells. This was paralleled by increased p21 expression at both gene and protein level in ER-positive cells, and decreased p53 expression in ERnegative cells. CGs slightly decreased Na+ K+ ATPase association with caveolae in ER-positive cells, but increased that in ER-negative cells. Knockdown of caveolin-1 (but not the planar lipid raft marker flotillin-1) potentiated the effects of CGs in ER-negative but not ER-positive cells. Pharmacological lipid raft disruption abrogated the effects of CGs in ER-positive cells, while raft augmentation did the opposite. In contrast, raft disruption did not alter CG effects in ER-negative cells, but augmentation potentiated their effects. Finally, we showed that ouabain transiently activated both Src and ERK in ER-negative cells, but induced delayed Src and ERK activation in ER-positive cells. Taken together, our results provide novel evidence that the powerful anti-proliferative effects of CGs in breast cancer cells involve modulations of the functional association between Na+ K+ ATPase and caveolin-1. We suggest that the Na+ K+ ATPase, caveolin-1 and their interaction in caveolae represent exciting targets for consideration in future breast cancer drug development strategies.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

12.7 MB

Comments

A thesis submitted to the Royal College of Surgeons in Ireland for the degree of Doctor of Medicine from the National University of Ireland in 2013.

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