Abstract 4033: Identification and characterization of a truncated RUNX3 gene variant with putative dominant-negative function

Abstract

Abstract Stem cells and cancer stem cells show a distinct proliferative potential for forming clonal floating colonies in vitro, termed tumor spheres. We have generated tumor spheres from a clinical gastric cancer sample (GCIC38) using serum-free culture, and expression of EpCAM and cytokeratin proteins confirmed the epithelial origin of these cells. GCIC38 sphere-derived cells were able to initiate tumors in NOD/SCID mice that recapitulate the tumor in the gastric cancer patient. Real-time PCR analysis showed that in contrast to human gastric cancer cell lines AGS, SNU16 and NCI-N87, GCIC38 sphere-derived cells express relatively high levels of stem cell markers such as SOX2, Musashi-1, and Bmi-1. Surprisingly, mRNA expression of the well-known tumor suppressor for gastric cancer, RUNX3, was found to be 4.6 and 16-fold higher than that of SNU16 and NCI-N87 cells respectively. Consistently, western blot analyses showed that GCIC38 sphere-derived cells express high levels of RUNX3 compared to a panel of gastric cancer cell lines. Immunofluorescence staining confirmed that RUNX3 was expressed in the nuclei of these cells, ruling out the possibility of RUNX3 protein mislocalization. To further characterize RUNX3 in GCIC38 sphere-derived gastric cancer cells, we cloned and identified a novel truncated 0.4kb RUNX3 variant (RUNX3-LG) that is expressed concurrently with the 1.2kb full length RUNX3 transcript (wt-RUNX3) in these cells. Sequence analysis revealed that RUNX3-LG is a truncated form of RUNX3 variant 2 encoded by a transcript utilizing the P2 promoter. There are no mutations in the wt-RUNX3 transcript whilst exons 3, 4 and 5 are spliced out in the RUNX3-LG transcript resulting in the deletion of RUNT domain in the RUNX3-LG protein. Immunoprecipitation studies using RUNX3 antibodies showed endogenous expression of a 15kDa protein in GCIC38 sphere-derived cells, concordant with the predicted size of RUNX3-LG protein. Preliminary data show that ectopic over-expression of RUNX3-LG in AGS gastric cancer cells results in reduced expression of RUNX3 target genes Claudin-1, S100A2 and p21. We propose that RUNX3-LG may be a dominant negative variant in gastric cancer cells with high wt-RUNX3 expression, which may abrogate wt-RUNX3 gene function including its tumor-suppressive effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2011-4033