TRIO amplification and abundant mRNA expression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer

Abstract

Studies by comparative genome hybridization have suggested that 5p amplification is related to tumor progression in urinary bladder cancer. In this study seven genes (TAS2R, ADCY2, DNAH5, CTNND2, TRIO, ANKH, and MYO10) located to 5p15.31-5p15.1 were analyzed by fluorescence in situ hybridization using a tissue microarray containing samples from tumors and cell lines with known 5p amplification by comparative genome hybridization. Amplification frequency was highest for TRIO, which maps to 5p15.2 and encodes a protein with a putative role in cell-cycle regulation. To further investigate the role of TRIO amplification in bladder cancer, a tissue microarray containing samples from 2317 bladder tumors was used for fluorescence in situ hybridization analysis. TRIO amplification was strongly associated with invasive tumor phenotype, high tumor grade, and rapid tumor cell proliferation (Ki67 LI) (P < 0.0001 each). Only 7 of 456 pTaG1/G2 tumors (1.5%) but 62 of 485 pT1-4 carcinomas (12.8%) had TRIO amplification. TRIO amplification was not associated with poor prognosis. Using a frozen bladder tumor tissue microarray RNA in situ hybridization confirmed that TRIO is up-regulated in amplified tumors. It is concluded that TRIO up-regulation through amplification has a potential role in bladder cancer progression.

Figure 1

CGH results in tumors with 5p amplification. CGH profile examples of two primary bladder cancers showing distinct peaks in the 5p15.31-p15.1 region suggesting amplification. The number of observations is shown on the bottom of each profile. The vertical lines indicate the base line ratio (1.0), and the threshold selected to define relative chromosomal gains (1.2, right) and losses (0.8, left).

Figure 2

TRIO analysis on a bladder cancer tissue microarray. A: TRIO amplification: tumor cell showing clusters of green TRIO signals and two red centromere 6 signals. B: Nonamplified tumor cell with two copies each of TRIO (green) and centromere 6 (red).

Figure 3

TRIO expression and amplification in bladder cancer. A: H&E-stained section of a TMA containing 80 bladder tumors from fresh frozen tissues. B: RISH phosphor image of the TMA hybridized with TRIO-specific oligomers after 48 hours of exposure time. C: Autoradiography of a bladder tumor array element showing tumor cells with high TRIO mRNA expression. D: Schematic illustration of the RISH result. E: Schematic illustration of the FISH result obtained from a consecutive TMA section. F: Northern blot analysis of three bladder tumor cell lines. Abundant TRIO mRNA expression (9.4 kb) in 5p amplified 5-HTB and low expression in the not amplified cell lines 3-HTB and 4-HTB. The Northern blot was rehybridized with GAPDH probe as a control.