Deregulation of Rab and Rab Effector Genes in Bladder Cancer

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

Rab and Rab-interacting proteins.

<p>Example of the Rab27 cluster. The Rab27 cluster is comprised of the two <i>RAB27</i> isoforms (<i>RAB27A</i> and <i>RAB27B</i>), the GEF <i>MADD</i>, the GAP <i>TBC1D10A</i> and 12 effector proteins. The other Rab and Rab-interacting proteins are shown in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone.0039469.s001" target="_blank">Figure S1</a>.</p

Deregulated genes correlated with differentiation markers.

<p>The Pearson correlation coefficient (r) of the expression of the deregulated genes with the expression of urothelial differentiation markers in <i>FGFR3</i>-mutated superficial tumors (TaG1G2) (n = 28 samples) and <i>FGFR3</i>-non-mutated muscle-invasive tumors (T2–4) (n = 63) is presented. Correlation with p<1% (|r| above 0.479 for Ta-T1 tumors, and |r| above 0.323 for <i>FGFR3</i>-non-mutated T2–4 tumors) are written in bold.</p

Flow chart of the different analysis steps.

<p>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 <i>FGFR3</i> 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 <i>FGFR3</i>-mutated tumors and <i>FGFR3</i>-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.</p

Genes specifically deregulated in one of the two pathways of bladder cancer pathogenesis.

<p>SAM analysis of the deregulated genes was performed comparing their expression, for a given stage, <i>FGFR3</i>-mutated tumors and <i>FGFR3</i>-non-mutated tumors. Only the genes found differentially expressed are presented. <i>SYTL1</i>, which is down-regulated in <i>FGFR3</i>-non-mutated T2–4 tumors compared to normal urothelium (first 2 columns and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone-0039469-t002" target="_blank">Table 2</a>) is also specifically down-regulated in <i>FGFR3</i>-non-mutated T2–4 tumors compared to <i>FGFR3</i>-mutated T2–4 tumors (last 2 columns). <i>LEPRE1</i>, <i>MICAL2</i>, <i>RAB23</i> and <i>STXBP1</i>, which are up-regulated in <i>FGFR3</i>-non-mutated T2–4 tumors compared to normal urothelium (first 2 columns and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone-0039469-t002" target="_blank">Table 2</a>) are also specifically up-regulated in <i>FGFR3</i>-non-mutated T2–4 tumors compared to <i>FGFR3</i>-mutated T2–4 tumors (last 2 columns). FC: Fold Change.</p

Several deregulated genes have their expression correlated with the expression of <i>MKI67</i>, a proliferation marker gene.

<p>The Pearson correlation coefficient (r) between the expression of the deregulated genes and the expression of proliferation marker gene, <i>MKI67</i>, was calculated for <i>FGFR3</i>-mutated tumors in the TaG1G2 group (n = 28) and for <i>FGFR3</i>-non-mutated tumors in the T2–4 group (n = 63). The expression of the deregulated genes as a function of <i>MKI67</i> expression is shown in TaG1G2 <i>FGFR3</i>-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 <i>FGFR3</i>-mutated tumor group and above 0.323 for the <i>FGFR3</i>-non-mutated tumor group).</p

Deregulated genes during bladder cancer pathogenesis in the <i>FGFR3</i>-non-mutated tumor pathway.

<p>From line 1 to line 19, genes are down-regulated. From line 20 to line 31, genes are up-regulated. The results in bold passed the thresholds: FC<0.667 (for down-regulation) or >1.5 (for up-regulation) and q-value <5%.</p><p>FC: Fold Change.</p><p>Number of FGFR3-non-mutated tumor samples: 3 TaG3, 25 T1, 63 T2–4.</p><p>Number of normal urothelial samples: 4.</p

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

<p>The expression of the 13 up-regulated genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone-0039469-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone-0039469-t003" target="_blank">3</a>) (<i>CAV1</i>, <i>ITGA5</i>, <i>KIF20A</i>, <i>LEPRE1</i>, <i>MICAL1</i>, <i>MICAL2</i>, <i>RAB23</i>, <i>RAB31</i>, <i>RABAC1</i>, <i>SDC1</i>, <i>STXBP1</i>, <i>TMEM22</i> and <i>ZWINT</i>) 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.</p

Results obtained after the different analysis steps.

<p>The results of each analysis step are shown in the flow chart presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone-0039469-g001" target="_blank">Figure 1</a>.</p

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

<p>Expression of <i>SYTL1</i>, <i>LEPRE1</i>, <i>MICAL2</i>, <i>RAB23</i> and <i>STXBP1</i> measured by Affymetrix array in normal samples (n = 4) and <i>FGFR3</i>-mutated tumor samples (TaG1G2, n = 28; T1, n = 13; T2-4, n = 9), and <i>FGFR3-</i>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. <i>SYTL1</i> is down-regulated in <i>FGFR3</i>-non-mutated tumors, whereas <i>LEPRE1</i>, <i>MICAL2</i>, <i>RAB23</i> and <i>STXBP1</i> are up-regulated.</p