Deregulation of Rab and Rab effector genes in bladder cancer

Abstract

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer.

Figure 1. Flow chart of the different analysis steps.

The first step is the identification through public data bases and expert knowledge of the genes of interest to study, here the Rabs and their effectors. The second step consists of selecting subgroups of tumors and analysing the expression of the different genes selected in the first step in these subgroups compared to the normal urothelium. The subgrouping here has been done taking into account the FGFR3 mutation status, the stage and the grade, separating the tumors into two pathways. A comparison of the expression observed in bladder cancer cell lines and in cultured normal human urothelial cells allowed discarding of genes for which the expression could be possibly due to the presence of stroma (in comparison to normal cells, upregulation in bladder tumors but not in bladder tumor cell lines). Different types of analysis were then performed on the selected deregulated genes: 1) a comparison of the expression in FGFR3-mutated tumors and FGFR3-non-mutated tumors allowed the identification of genes specifically deregulated in one of the two pathways of bladder cancer pathogenesis; 2) by grouping genes into cluster of genes (here the Rab clusters), we identified clusters with deregulated expression; 3) by analysing the possible correlation between the expression of the deregulated genes and the expression of proliferation or differentiation marker genes, we identified the deregulated genes associated with proliferation or differentiation.

Figure 2. Results obtained after the different analysis steps.

The results of each analysis step are shown in the flow chart presented in Figure 1.

Figure 3. The Rab cycle.

Rab GTPases cycle between an active GTP-bound form and an inactive GDP-bound form. Rab activation is mediated by a guanine exchange factor (GEF). The hydrolysis of bound GTP is catalyzed by a GTPase activating protein (GAP) resulting in the inactivation of the Rab protein. In its active form, the Rab is associated with membranes and can interact with a variety of effector proteins. In its inactive form the protein is cytosolic and is in complex with a GDP dissociation inhibitor (GDI) protein.

Figure 4. Rab and Rab-interacting proteins.

Example of the Rab27 cluster. The Rab27 cluster is comprised of the two RAB27 isoforms (RAB27A and RAB27B), the GEF MADD, the GAP TBC1D10A and 12 effector proteins. The other Rab and Rab-interacting proteins are shown in supplementary Figure S1.

Figure 5. Model of bladder cancer.

The “FGFR3 model” of bladder cancer progression distinguishes a FGFR3-non-mutated tumor pathway and a FGFR3-mutated tumor pathway. Cis: Carcinoma in situ. Stages were defined by the 1997 TNM classification and grades by the 1973 World Health Organization classification (see Material and Methods for reference).

Figure 6. Up-regulated gene expression in normal human urothelium (NHU) cells and bladder tumor cell lines.

The expression of the 13 up-regulated genes (Table 2 and 3) (CAV1, ITGA5, KIF20A, LEPRE1, MICAL1, MICAL2, RAB23, RAB31, RABAC1, SDC1, STXBP1, TMEM22 and ZWINT) was measured by Affymetrix array in 7 bladder cancer cell lines (KK47, MGHU3, RT112, RT4, SCaBER, SD48 and T24) and normal human urothelial (NHU) cells grown in culture. The threshold for genes to be considered as up-regulated in tumor cell lines (2 fold the expression measured in NHU cells) is represented by a black line.

Figure 7. Expression in normal samples and in the two groups of tumors (mutated and non-mutated for FGFR3) classified according to stage, of the genes found to be specifically deregulated in one of the two pathways of bladder tumor pathogenesis.

Expression of SYTL1, LEPRE1, MICAL2, RAB23 and STXBP1 measured by Affymetrix array in normal samples (n = 4) and FGFR3-mutated tumor samples (TaG1G2, n = 28; T1, n = 13; T2-4, n = 9), and FGFR3-non-mutated samples (TaG3, n = 3; T1, n = 25; and T2-4, n = 63). Are represented the 10th percentile (below bar), the 25th percentile (box bottom), the median (bar in the box), the 75^th percentile (box top) and the 90^th percentile (upper bar). The points represent the outlier samples. SYTL1 is down-regulated in FGFR3-non-mutated tumors, whereas LEPRE1, MICAL2, RAB23 and STXBP1 are up-regulated.

Figure 8. Several deregulated genes have their expression correlated with the expression of MKI67, a proliferation marker gene.

The Pearson correlation coefficient (r) between the expression of the deregulated genes and the expression of proliferation marker gene, MKI67, was calculated for FGFR3-mutated tumors in the TaG1G2 group (n = 28) and for FGFR3-non-mutated tumors in the T2–4 group (n = 63). The expression of the deregulated genes as a function of MKI67 expression is shown in TaG1G2 FGFR3-mutated tumors (upper figures) and in T2–4 non-mutated tumors (lower figures). Only the plots for the correlated genes are presented (p<1%, which corresponds to a correlation coefficient, |r| above 0.479 for the FGFR3-mutated tumor group and above 0.323 for the FGFR3-non-mutated tumor group).

Figure 9. Gene expression in differentiated or non differentiated normal urothelial cells of the genes found to be associated with proliferation or differentiation in tumors.

The expression of the genes found to be associated with proliferation or differentiation in tumors were measured in normal urothelial cells (NHU) in culture at four different times after passage: 6 hours, 1 day, 3 days and 6 days in differentiating conditions (in the presence of an inhibitor of EGFR and an activator of PPARγααμμ in the NHU medium) or in non-differentiating conditions (NHU medium only).