Mayne, Cerys Mary
(2011)
The role of the cholecystokinin 2 receptor in cancer.
PhD thesis, University of Nottingham.
Abstract
The gastrointestinal (GI) hormone, gastrin, promotes cancer progression and its down-regulation has been linked to reduced cancer stem cell numbers. Gastrin acts through the cholecystokinin-2 receptor (CCK-2R) and its biological effects are blocked by CCK-2R inhibitors. We investigated the regulation of the CCK-2R and its potential role in promoting survival of cancer stem cells (CSC).
A panel of cancer cell-lines, including GI, glioblastoma and lung, with CCK-2R-transfected cells as a positive control, were grown either as monolayers, or, to provide a 3D in vitro tumour model, as spheres. Linear-after-the-Exponential (LATE)-PCR was used to quantify CCK-2R gene expression and this was validated using siRNAs. Flow cytometry was used to investigate receptor protein expression. Activity of CCK-2R promoter reporters was quantified using luciferase assays.
LATE-PCR for CCK-2R gene expression is 10,000-fold more sensitive than the Taqman-based assay, and provides a highly precise method for detection of genes which have important biological functions but low expression. This assay showed that primary non-small-cell lung tumours have significantly more expression than normal lung tissue, indicating a potential therapeutic marker. CCK-2R siRNAs resulted in up to 97% (p<0.05) knockdown of the receptor in cancer cells, confirming the specificity of LATE-PCR and offering a therapeutic possibility.
The CCK-2R promoter constructs were active in lung, glioma and colorectal cancer cell-lines, demonstrating a potential drug target; however, transcriptional activity did not correlate with gene expression suggesting post-transcriptional or translational regulation is a factor affecting CCK-2R expression.
Flow cytometry suggests the presence of a small population of cells within each of these cell-lines which expresses CCK-2R very highly, which was not correlated to CSC markers. However, CCK-2R expression was enriched when cells were grown as spheres, and inhibition caused a delay in sphere-forming, implying that the CCK-2R may play a role in tumour, and CSC, expansion. Thus, CCK2R provides a potential target for therapeutic intervention in cancer.
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