[Thesis]. Manchester, UK: The University of Manchester; 2012.
C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and
prostate cancer stem cells. In breast cancer, CXCR4 signalling via stromal cell-derived
factor-1 (SDF-1) has been reported to be a mediator of metastasis, and is linked to
poor prognosis. However its role in normal and malignant breast stem cell function
has not been investigated.Anoikis-resistant (AR) cells were collected from mammosphere
culture from 2 immortalised (MCF10A, 226L) and 3 malignant (MCF7, T47D, SKBR3) breast
cell lines. For all cell lines, AR cells had a significantly higher mammosphere forming
efficiency (MFE) than unsorted cells. The AR cells of the normal cell lines also demonstrated
increased formation of 3D structures using the Matrigel assay. In vivo, MCF7 and T47D
AR cells demonstrated increased capacity to form tumours compared with unsorted cells.
This suggests that AR cells are enriched for normal and malignant breast stem cells.We
performed an Agilent custom gene microarray and demonstrated up-regulation of CXCR4
mRNA expression in AR cells. CXCR4 protein expression was also higher in AR cells,
shown by flow cytometry. The effects of AMD3100 (CXCR4 antagonist) and SDF-1 (CXCR4
ligand) on stem cell activity were investigated in the mammosphere assay. In the normal
cell lines, SDF-1 significantly reduced MFE and this decrease was rescued by AMD3100.
Incubation with AMD3100 increased MFE in the estrogen receptor positive breast cancer
cell lines (MCF7 and T47D) and patient-derived metastatic tumour samples. AMD3100
reduced the self-renewal of T47D cells, as assessed by second generation mammospheres.
MCF7 cells were retro-virally transfected to over-express CXCR4 or sorted for CXCR4
cell surface expression. Mammosphere formation was significantly increased in CXCR4+
and CXCR4 over-expressing cells compared with CXCR4- and parental cells. There was
a greater reduction in self-renewal following AMD3100 treatment in the CXCR4 over-expressing
cells compared with parental cells. AMD3100 has been shown to have an agonistic effect
on the novel chemokine receptor CXCR7, a scavenging receptor for SDF-1. All cell lines
demonstrated cell surface expression of CXCR7, measured by flow cytometry and mRNA
expression. Potential interactions between CXCR4, CXCR7 and SDF-1 must be considered
in future investigation of the role of CXCR4 signalling.Our data establish that CXCR4
signalling has contrasting effects on normal and malignant breast stem cell activity.
CXCR4 influences self-renewal of malignant stem cells which may account for its role
in tumorigenesis. CXCR4 signalling may be important in tumour formation at the sites
of metastases as well as in cell migration. Its role in stem cell migration merits
further investigation. In conclusion, CXCR4-targeted therapy, alongside current standards
of care, may improve breast cancer outcomes.