Abstract 3954: A role for the cholecystokinin 2 receptor (CCK-2R) in promoting cancer progression

Abstract

Abstract Background and aims: 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 a potential role for CCK-2R in promoting survival of cancer stem cells using RNAi combined with a sensitive method to detect CCK-2R mRNA. Materials and methods: A panel of cancer cell-lines was used, including GI, glioblastoma and small cell lung cancer (SCLC), with CCK-2R-transfected cells as a positive control. Linear-after-the-Exponential (LATE)-PCR was used to quantify CCK-2R gene expression and its sensitivity compared with a Taqman-based assay. Flow cytometry was used to investigate receptor protein expression. The activity of CCK-2R promoter reporters constructed in pGL4, using between 250 and 2000bp of DNA upstream of the CCK-2R start codon, was quantified using luciferase assays. Results: LATE-PCR for CCK-2R gene expression is 1000-fold more sensitive than the Taqman-based assay. Cell-lines from the panel, including HCT116 (colorectal) and H209 (SCLC), in which CCK-2R mRNA was not detectable by the Taqman assay, were positive using the LATE-PCR. CCK-2R siRNAs resulted in up to 86% knockdown of the receptor in CCK-2R-transfected AGS cells, confirming the specificity of the LATE-PCR. FACS analysis for the CCK-2R protein suggests the presence of a small population of cells within HCT116 (colorectal) and AGS (gastric) cell-lines that express CCK-2R very highly. CCK-2R expression was enriched when cells were grown as colospheres and RNAi demonstrated a role for the CCK2R in promoting cell survival. The CCK-2R promoter constructs were active in cancer cell-lines, indicating that the 250bp proximal to the CCK-2R start codon are most crucial for transcription. However, transcriptional activity did not always correlate with gene expression. Conclusions: LATE-PCR provides a highly sensitive method for detection of genes such as CCK-2R which have important biological functions but low expression. An element just upstream of the CCK-2R transcriptional start site appears to be controlling transcription in cell-lines showing low levels of endogenous expression of the CCK-2R genes, but lack of correlation between the RNA levels and transcriptional activity suggests possible post-transcriptional regulation of this gene. CCK-2R protein expression is elevated in a subset of cells, and may play a role in promoting survival of cancer stem cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3954.