Identification of genes correlated with early-stage bladder cancer progression

Abstract

Transitional cell carcinoma (TCC) of the bladder ranks fourth in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) with DNA microarray technology to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis, and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild-type littermates at 3, 6, 20, and 30 weeks of age. These are ages that correspond to premalignant, carcinoma in situ, and early-stage and later stage invasive TCC, respectively. Our preliminary analysis of the microarray data sets has revealed approximately 1,900 unique genes differentially expressed (> or =3-fold difference at one or more time points) between wild-type and UPII-SV40Tag urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and a proliferation signaling genes that are more strongly expressed in the UPII-SV40Tag bladder urothelium. We show that several of the genes upregulated in UPII-SV40Tag urothelium, including RacGAP1, PCNA, and Hmmr, are expressed at high levels in superficial bladder TCC patient samples. These findings provide insight into the earliest events in the development of bladder TCC as well as identify several promising early-stage biomarkers.

Figure 1

H&E stain (panels a,d, and g) and MR images (b,c,e,f,h, and i) of the bladders of wild type littermates (WT, a–c) and transgenic mice at 6 weeks (d–f) and 38 weeks (g–i) of age. Arrows indicate a thickened mucosa in early stage TCC (f) and invasion into surrounding tissue for advanced TCC (h,i). Panels a, d and g were photographed at 100X magnfication.

Figure 2

Preliminary hierarchical clustering of all four time points 3, 6, 20, and 30 weeks. The colors red, green, and black represent genes that are upregulated, downregulated, or no change, respectively, in the UPII-SV40Tag mice when compared to the WT littermates. The last cluster, cluster 8, has been enlarged and rescaled and represents genes that are highly upregulated at all four time points.

Figure 3

A. Selected genes that were expressed at higher levels in UPII-SV40Tag urothelium relative to WT littermates are shown. Black bars indicate raw expression values for UPII-SV40Tag mice (n=2). Open bars indicate expression values for WT mice. B. Selected genes that were expressed at lower levels in UPII-SV40Tag urothelium relative to WT littermates.

Figure 4

Confirmation of microarray data. RT-PCR was performed using the cDNA made from RNA extracted from the urothelium of 6 week old WT and 3 week old hemizygous mice (+/−) expressing SV40T antigen in the bladder under control of the Uroplakin II promoter.

Figure 5

Gene interaction networks #1–3. Gene expression profiles of 3 week old WT and UPII-SV40Tag bladder urothelial tissue (n=2 for each group) were compared using Ingenuity microarray analysis software. Genes overexpressed and underexpressed in UPII-SV40T bladders compared to WT mice, at the 3 week time point, are shown in red and green, respectively.

Figure 6

A. Differentially expressed genes in human bladder derived cells. Semi-quantitative RT-PCR of 25, 30, and 35 cycles was performed on four human bladder cell lines using primers for four of the genes identified from the UPII-SV40Tag mouse microarray experiment. The relative expression was determined by a ratio of each sample to the most intense sample for that gene, which was assigned a value of one. B. Immunohistochemical detection of differentially expressed genes in paraffin sections of superficial bladder TCC. Sections were probed with antibodies for the indicated proteins and detected colorimetrically using horseradish peroxidase-coupled secondary antibodies, except for the RacGAP1 20X and Hmmr 40X panels which were detected by fluorescently tagged secondary antibodies.