Date of Award
PhD (Doctor of Philosophy)
Dr Ann M. Hopkins
Health Research Board of Ireland and Breast Cancer Ireland
Breast Neoplasms, Neoplasm Metastasis, Cell Movement
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.
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Babina, IS. A novel role of lipid rafts in regulating CD44-dependent breast cancer cell migration. [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2013.