Date of Award

2013

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr Ann M. Hopkins

Funder/Sponsor

Health Research Board of Ireland and Breast Cancer Ireland

Keywords

Breast Neoplasms, Neoplasm Metastasis, Cell Movement

Abstract

The majority of breast cancer-related deaths result from metastasis, a process in which tumour cells must dynamically regulate their adhesive and migratory properties. CD44 is a cell-matrix adhesion protein which regulates cell migration. Alterations in its expression have been linked to tumour progression, yet its contribution to breast cancer metastasis remains incompletely understood. A pool of CD44 localises in cholesterol-enriched regions of the cell membrane known as lipid rafts. The aim of this thesis was to interrogate the relationship between lipid rafts and CD44 localisation / function during breast cancer cell migration. We first compared the raft affiliation status of CD44 and its binding partners in a panel of breast cancer cell lines. Raft affiliation of CD44 increased during migration of non-invasive breast cancer cells, but was significantly reduced during migration of highly-invasive cells. CD44 binding partners were detected exclusively in non-raft fractions. To investigate whether CD44 re-localisation outside lipid rafts was sufficient to drive cancer cell migration, we introduced point-mutations into two CD44 palmitoylation sites which target it to lipid rafts. CD44 raft affiliation was significantly reduced in cells transiently transfected with palmitoylationimpaired mutants, which conferred a motile and invasive phenotype compared to control cells. This phenotype was reversible upon termination of selection. Additionally a panel of CD44 non-synonymous human SNPs was identified that could recapitulate our mutagenesis model in vivo. Global manipulation of palmitoylation with novel enzyme inhibitors revealed a pattern whereby increased CD44 palmitoylation translated into increased lipid raft affiliation and decreased breast cancer cell migration. Accordingly, mining of published gene array datasets revealed a novel correlation between pro-palmitoylated states and improved breast cancer patient survival. Furthermore, increased levels of palmitoylated CD44 in primary breast cancer patient cultures showed a direct correlation with non-aggressive cancer phenotypes, and an inverse correlation with a putative progenitor/stem cell phenotype in a cell line model. Collectively, our results support a novel mechanism whereby sub-membranous localisation of CD44 outside lipid rafts is sufficient to promote migration in invasive breast cancer cells, and could act as a biomarker o f breast cancer aggressiveness. We suggest that pharmacological sequestration of CD44 within lipid rafts represents a novel strategy to reduce breast cancer cell migration and potentially metastasis.

Creative Commons License

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

File Size

19,155 KB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2013.

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