Profiling the Evolution of Human Metastatic Bladder Cancer

Abstract

Pulmonary metastases frequently develop in patients with aggressive bladder cancer, yet investigation of this process at the molecular level suffers from the poor availability of human metastatic tumor tissue and the absence of suitable animal models. To address this, we developed progressively more metastatic human bladder cancer cell lines and an in vivo bladder-cancer lung-metastasis model, and we successfully used these to identify genes of which the expression levels change according to the degree of pulmonary metastatic potential. By initially intravenously injecting the poorly metastatic T24T human urothelial cancer cells into nude mice, and then serially reintroducing and reisolating the human tumor cells from the resultant mouse lung tumors, three derivative human lines with increasingly metastatic phenotypes, designated FL1, FL2, and FL3, were sequentially isolated. To identify the genes associated with the most lung-metastatic phenotype, the RNA complement from the parental and derivative cells was evaluated with oligonucleotide microarrays. In doing so, we found 121 genes to be progressively up-regulated during the transition from T24T to FL3, whereas 43 genes were progressively downregulated. As expected, many of the genes identified in these groups could, according to the ascribed functions of their protein product, theoretically participate in tissue invasion and metastasis. In addition, the magnitude of gene expression changes observed during the metastatic transition correlated with the in vivo propensity for earlier lung colonization and decreased host survival. To additionally define which genes found in the experimental system were of relevance to human bladder cancer lung metastasis, we evaluated gene expression profiles of 23 primary human bladder tumors of various stages and grades, and then we compared these gene expression profiles to the altered profiles in our model cell lines. Here we found that the expression of epiregulin, urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)14, and tissue inhibitor of metalloproteinase (TIMP-2) were consistently and progressively upregulated when viewed as a function of tumor stage in tissues of patients versus the metastatic potential seen in the mouse lung model. The strong correlation of these four markers between the experimental and clinical situations helps validate this system as a useful tool for the study of lung metastasis and defines targets of therapy that may reduce the incidence of this process in patients.

Src phosphorylation of RhoGDI2 regulates its metastasis suppressor function

“…To reduce the time required and improve the quantitative evaluation of lung metastasis by human xenografts in murine hosts, we developed a DNA PCR assay (14). Using this assay, the amount of human DNA in the lung following tail-vein inoculation of cancer cells was found to correlate with surrogates of murine survival (14).…”

“…Using this assay, the amount of human DNA in the lung following tail-vein inoculation of cancer cells was found to correlate with surrogates of murine survival (14). Here, we sought to adapt this assay to determine the effect of RhoGDI2 on lung colonization, defined as the growth of human tumors in the lung to a sufficient size for visible detection by using imaging.…”

Identification of a metastasis signature and the DLX4 homeobox protein as a regulator of metastasis by combined transcriptome approach

“…Twenty-seven genes were overexpressed, whereas 18 were significantly reduced in association with the acquisition of a metastatic capability during in vivo selection of LNM35 (Table 1). The 27 examples overexpressed in LNM35 included various genes previously suggested to play a functional role in metastasis such as chemokine (C-X-C motif) ligand (Heaney et al, 2000;Bernal et al, 2002), immediate early response 3 (IER3) (Goswami et al, 2004;Nicholson et al, 2004), defender against cell death 1 (DAD1) (Ayala et al, 2004;Goswami et al, 2004;Kim et al, 2006) and methionyl aminopeptidase 2 (METAP2) (Dasgupta et al, 2005;Morowitz et al, 2005). Similarly, genes with higher expression in N15 also included some previously implicated in metastasis such as cadherin 1 (E-cadherin) (Cavallaro and Christofori, 2004) and lectin galactoside-binding soluble 3 (galectin-3) (Liu and Rabinovich, 2005).…”

Expression profiling of Ral-depleted bladder cancer cells identifies RREB-1 as a novel transcriptional Ral effector

“…Significance of Ral-regulated genes in human bladder cancer By combining previously published gene expression profiling data sets of human bladder cancer (Dyrskjot et al, 2004;Nicholson et al, 2004) and our Ral siRNA data, we evaluated the number of probe sets that were altered as a function of Ral modulation and differentially expressed in human bladder cancer using standard statistics. More detail is provided in the Supplementary Materials.…”

“…Having elucidated the sets of genes regulated in response to RalA and/or RalB depletion in a human bladder cancer cell line, we sought to ground these in vivo by determining the extent to which the RalA/ RalB-driven transcriptional regulation contributes to human bladder cancer. To do this, we took advantage of 2 previously published data sets of gene expression profiling of human bladder tumors (Dyrskjot et al, 2004;Nicholson et al, 2004), which, when combined, result in a cohort of 65 bladder tumors and 20 normal bladder epithelial samples profiled on HG-U133A GeneChips. From this cohort, we identified a total of 607 probe sets that were differentially expressed in human bladder cancer vs normal tissue.…”