TWIST-1 is overexpressed in neoplastic choroid plexus epithelial cells and promotes proliferation and invasion

Abstract

The pathogenesis of choroid plexus papillomas, intraventricular papillary neoplasms most often occurring sporadically in children and young adults, remains poorly understood. To identify pathways operative in the development of choroid plexus papillomas, gene expression profiles obtained from laser-microdissected human choroid plexus papilloma cells (n = 7) were compared with that of normal choroid plexus epithelial cells laser microdissected from autopsy tissue (n = 8). On DNA microarray data analysis, 53 probe sets were differentially expressed in choroid plexus papilloma tumor cells (>7-fold). Up-regulation of TWIST1, WIF1, TRPM3, BCLAF1, and AJAP1, as well as down-regulation of IL6ST was confirmed using quantitative reverse transcription-PCR. Knockdown of Twist1 gene expression in the rat choroid plexus epithelial cell line Z310 significantly reduced proliferation as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell invasion in a Matrigel assay, whereas cell migration was not affected. Screening for expressional changes of cancer-related genes upon Twist1 knockdown revealed up-regulation of Cdkn1a, Cflar, and Serpinb2 and down-regulation of Figf. To conclude, using gene expression profiling, several genes differentially expressed in human choroid plexus papillomas could be identified. Among those, TWIST1 is highly expressed in choroid plexus papillomas and promotes proliferation and invasion.

Figure 1.

Identification and validation of differentially expressed genes in choroid plexus papilloma cells. Expression levels (arbitrary units) of 53 genes found to be differentially expressed (>7-fold) in laser-microdissected human choroid plexus papilloma cells compared with normal choroid plexus (A), validation of expression of selected genes using quantitative RT-PCR (B; fold-change compared with nonneoplastic choroid plexus; columns, mean; bars, SE; n = 4–6; *, P < 0.05), and confirmation of differential expression of TWIST-1 on protein level in choroid plexus papillomas compared with nonneoplastic choroid plexus (C). Insets, representative stainings of choroid plexus papilloma and normal choroid plexus.

Figure 2.

Expression of TWIST-1 in immortalized rat choroid plexus epithelial cells and effect on proliferation and migration. Expression of TWIST-1 protein along with choroid plexus markers transthyretin and Kir7.1 in the choroid plexus epithelial cell line Z310 (A). Upon transfection with the two specific siRNAs TWIST1_1 and TWIST1_3, TWIST-1 mRNA expression is inhibited for up to 72 h compared with control scrambled siRNA (B; columns, mean; bars, SE; n = 4; **, P < 0.01;*, P < 0.05) and proliferative activity as measured by MTT assay is significantly reduced (C; columns, mean; bars, SE; n = 3; **, P < 0.01; *, P < 0.05). Silencing of Tp53 (Tp53 XScr) for 24 h results in reduced expression of p53 protein compared with control scrambled siRNA (Scr X Scr) and increased proliferative activity (D; columns, mean results from one representative of two independent experiments, each performed in octuplicate; bars, SE; *, P < 0.05). Upon simultaneous silencing of Tp53 and Twist1 (Tp53 X Twist1_1), the antiproliferative effect of Twist1 knockdown is abolished (##, P < 0.01). Cell migration is not significantly affected upon silencing of Twist1 expression with the two specific siRNAs TWIST1_1 and TWIST1_3, (E; columns, mean; bars, SE; n = 3).

Figure 3.

Effect of Twist1 knockdown on cell invasion and differential expression of cancer-related genes. Gene silencing with siRNA TWIST1_1 affects cell invasion compared with control in a Matrigel assay (A; columns, mean representative experiment performed in quadruplicate; bars, SE; **, P < 0.01) and results in differential expression (>1.5-fold) of 5 of 84 cancer-related genes examined by quantitative RT-PCR (B, mean from two independent experiments).