Down-Regulation of Homeobox Gene GBX2 Expression Inhibits Human Prostate Cancer Clonogenic Ability and Tumorigenicity1

Previously, we have demonstrated that GBX genes, a homeobox-containing human family of DNA-binding transcription factors consisting of GBXl and GBX2, are overexpressed in a panel of human prostatic cancer cell lines (i.e., TSU-prl, PC3, DU145, and LNCaP) compared to normal prostate. In the present studies, specific primer sets were designed for reverse transcription-PCR detection of the expression of GBXl versus GBX2 in human prostate cancer. These studies demonstrated that the GBX2 gene, but not the GBXl gene, is consistently overexpressed in this panel of human prostate cancer cell lines compared to normal human prostate. Using a quantitative-competitive PCR analysis, GBX2 mRNA was expressed as 3 x HI1 copies//*g RNA in normal prostate tissue and 4 x IO4 copies/jig RNA in the immortalized normal neonatal prostate epithelial cell line 267B-1, as compared to 6 x IO5, 5 x IO5, 3 x IO5, and 1 x IO5 copies/fig RNA in TSU-prl, DU145, LNCaP, and PC3 prostate cancer cell lines, respectively. To examine the importance of GBX2 ex pression for prostate cancer malignancy, GBX2-overexpressing TSU-prl and PC3 human prostatic cancer cells were transacted with a eukaryotic expression vector containing an antisense GBX2 homeobox domain cDNA. Stable translatant clones with 5-10-fold decreased levels of GBX2 mRNA expression were obtained. When tested in vitro, the clonogenic ability of the GBX2 antisense transfectants was reduced by approximately 50% in both cell lines. When implanted s.c. into nude mice, the tumorigenicity of the antisense GBX2 transfectants from both human prostatic cancer cell lines was inhibited by more than 70% compared to the parental cells. These results suggest that expression of (¡HX2gene is required for ma lignant growth of human prostate cells.