Multifocal urothelial cancers with the mutator phenotype are of monoclonal origin and require panurothelial treatment for tumor clearance

Purpose: UC is a disease of the entire urothelium, characterized by multiplicity and multifocality. The clonal relationship among multiple UCs has implications regarding adjuvant chemotherapy. It has been investigated in studies of chromosomal alteration and single gene mutation. However, these genetic changes can occur in unrelated tumors under similar carcinogenic selection pressures. Tumors with high MSI have numerous DNA mutations, of which many provide no selection benefit. While these tumors represent an ideal model for studying UC clonality, their low frequency has prevented their previous investigation. Materials and Methods: We investigated 32 upper and lower urinary tract UCs with high MSI and 4 nonUC primary cancers in 9 patients. We used the high frequency and specificity of individual DNA mutations in these tumors (MSI at 17 loci) and the early timing of epigenetic events (methylation of 7 gene promoters) to investigate tumor clonality. Results: Molecular alterations varied among tumors from different primary organs but they appeared related in the UCs of all 9 patients. While 7 patients had a high degree of concordance among UCs, in 2 the UCs shared only a few similar alterations. Genetic and epigenetic abnormalities were frequently found in normal urothelial samples. Conclusions: Multiple UCs in each patient appeared to arise from a single clone. The molecular order of tumor development varied from the timing of clinical presentation and suggested that residual malignant cells persist in the urinary tract despite apparent curative surgery. These cells lead to subsequent tumor relapse and new methods are required to detect and eradicate them.

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

Purpose: Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. Experimental design: We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Results: Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. Conclusions: In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma. (C) 2009 Elsevier Ltd. All rights reserved.

Bystander-induced differentiation: a major response to targeted irradiation of a urothelial explant model.

A ureter primary explant technique, using porcine tissue sections was developed to study bystander effects under in vivo like conditions where dividing and differentiated cells are present. Targeted irradiations of ureter tissue fragments were performed with the Gray Cancer Institute charged particle microbeam at a single location (2 microm precision) with 10 3He2+ particles (5 MeV; LET 70 keV/microm). After irradiation the ureter tissue section was incubated for 7 days allowing explant outgrowth to be formed. Differentiation was estimated using antibodies to Uroplakin III, a specific marker of terminal urothelial differentiation. Even although only a single region of the tissue section was targeted, thousands of additional cells were found to undergo bystander-induced differentiation in the explant outgrowth. This resulted in an overall increase in the fraction of differentiated cells from 63.5+/-5.4% to 76.6+/-5.6%. These changes are much greater than that observed for the induction of damage in this model. One interpretation of these results is that in the tissue environment, differentiation is a much more significant response to targeted irradiation and potentially a protective mechanism.

Recurrence of Urothelial Carcinoma of the Bladder: A Role for Insulin-Like Growth Factor-II Loss of Imprinting and Cytoplasmic E-Cadherin Immunolocalization

Purpose:This study documents the frequency of insulin-like growth factor-II (IGF-II) loss of imprinting (LOI) in a series of 87 bladder tissues. E-cadherin (CDH1) immunolocalization was also investigated due to the known redistribution of this adherence protein to the cytoplasm following exogenous exposure to IGF-II.
Experimental Design: Informative IGF-II cases were identified following DNA-PCR amplification and subsequent sequencing of the transcribable ApaI RFLP in exon 9 of IGF-II. Similar approaches using primer-specific cDNA templates identified the imprinting status of IGF-II in these informative cases. CDH1cellular localization was assessed on a tissue microarray platform of 114 urothelial carcinoma of the bladder (UCB) cases (70 pTanoninvasive and 44 pT1laminapropria invasive) using the commercially available Novocastra antibody.
Results: IGF-IILOI was evident in 7 of17 (41%) UCB tumors and 4 of11 (36%) tumor-associated normal urothelial samples.Two of four pT1grade 3 tumors, the subject of much debate concerning their suitability for radical cystectomy, showed LOI at the IGF-II locus. In those tumors showing IGF-II LOI, 4 of 7 (57%) displayed concomitant CDH1cytoplasmic staining. In contrast, only 3 of 10 (30%) IGF-IImaintenance ofimprinting tumorshad concomitant CDH1cytoplasmiclocalization. UCB cell lines displaying cytoplasmic CDH1immunolocalization expressed significantly higher levels of IGF-II (CAL29, HT1376, and RT112) compared with RT4, a cell line displaying crisp membranous CDH1staining. Finally, cytoplasmic CDH1staining was an independent predictor of a shorter time to recurrence independent of tumor grade and stage.
Conclusions: We suggest that CDH1 cytoplasmic immunolocalization as a result of increased IGF-II levels identifies those nonmuscle invasive presentations most likely to recur and therefore might benefit from more radical nonconserving bladder surgery

Urothelial cancer gene regulatory networks inferred from large-scale RNAseq, Bead and Oligo gene expression data

BACKGROUND: Urothelial pathogenesis is a complex process driven by an underlying network of interconnected genes. The identification of novel genomic target regions and gene targets that drive urothelial carcinogenesis is crucial in order to improve our current limited understanding of urothelial cancer (UC) on the molecular level. The inference of genome-wide gene regulatory networks (GRN) from large-scale gene expression data provides a promising approach for a detailed investigation of the underlying network structure associated to urothelial carcinogenesis.

METHODS: In our study we inferred and compared three GRNs by the application of the BC3Net inference algorithm to large-scale transitional cell carcinoma gene expression data sets from Illumina RNAseq (179 samples), Illumina Bead arrays (165 samples) and Affymetrix Oligo microarrays (188 samples). We investigated the structural and functional properties of GRNs for the identification of molecular targets associated to urothelial cancer.

RESULTS: We found that the urothelial cancer (UC) GRNs show a significant enrichment of subnetworks that are associated with known cancer hallmarks including cell cycle, immune response, signaling, differentiation and translation. Interestingly, the most prominent subnetworks of co-located genes were found on chromosome regions 5q31.3 (RNAseq), 8q24.3 (Oligo) and 1q23.3 (Bead), which all represent known genomic regions frequently deregulated or aberated in urothelial cancer and other cancer types. Furthermore, the identified hub genes of the individual GRNs, e.g., HID1/DMC1 (tumor development), RNF17/TDRD4 (cancer antigen) and CYP4A11 (angiogenesis/ metastasis) are known cancer associated markers. The GRNs were highly dataset specific on the interaction level between individual genes, but showed large similarities on the biological function level represented by subnetworks. Remarkably, the RNAseq UC GRN showed twice the proportion of significant functional subnetworks. Based on our analysis of inferential and experimental networks the Bead UC GRN showed the lowest performance compared to the RNAseq and Oligo UC GRNs.

CONCLUSION: To our knowledge, this is the first study investigating genome-scale UC GRNs. RNAseq based gene expression data is the data platform of choice for a GRN inference. Our study offers new avenues for the identification of novel putative diagnostic targets for subsequent studies in bladder tumors.

AURKA overexpression accompanies dysregulation of DNA-damage response genes in invasive urothelial cell carcinoma

Invasive urothelial cell carcinoma (UCC) is characterized by increased chromosomal instability and follows an aggressive clinical course in contrast to non-invasive disease. To identify molecular processes that confer and maintain an aggressive malignant phenotype, we used a high-throughput genome-wide approach to interrogate a cohort of high and low clinical risk UCC tumors. Differential expression analyses highlighted cohesive dysregulation of critical genes involved in the G(2)/M checkpoint in aggressive UCC. Hierarchical clustering based on DNA Damage Response (DDR) genes separated tumors according to a pre-defined clinical risk phenotype. Using array-comparative genomic hybridization, we confirmed that the DDR was disrupted in tumors displaying high genomic instability. We identified DNA copy number gains at 20q13.2-q13.3 (AURKA locus) and determined that overexpression of AURKA accompanied dysregulation of DDR genes in high risk tumors. We postulated that DDR-deficient UCC tumors are advantaged by a selective pressure for AURKA associated override of M phase barriers and confirmed this in an independent tissue microarray series. This mechanism that enables cancer cells to maintain an aggressive phenotype forms a rationale for targeting AURKA as a therapeutic strategy in advanced stage UCC.

MT1-MMP regulates urothelial cell invasion via transcriptional regulation of Dickkopf-3

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a zinc-binding endopeptidase, which plays a crucial role in tumour growth, invasion and metastasis. We have shown previously that MT1-MMP has higher expression levels in the human urothelial cell carcinoma (UCC) tissue. We show here that siRNA against MT1-MMP blocks invasion in UCC cell lines. Invasion is also blocked by broad-spectrum protease and MMP inhibitors including tissue inhibitor of metalloproteinase-1 and -2. Membrane type-1-MMP can also regulate transcription. We have used expression arrays to identify genes that are differentially transcribed when siRNA is used to suppress MT1-MMP expression. Upon MT1-MMP knockdown, Dickkopf-3 (DKK3) expression was highly upregulated. The stability of DKK3 mRNA was unaffected under these conditions, suggesting transcriptional regulation of DKK3 by MT1-MMP. Dickkopf-3 has been previously shown to inhibit invasion. We confirm that the overexpression of DKK3 leads to decreased invasive potential as well as delayed wound healing. We show for the first time that the effects of MT1-MMP on cell invasion are mediated in part through changes in DKK3 gene transcription.

Comprehensive profiling and localisation of the matrix metalloproteinases in urothelial carcinoma

The matrix metalloproteinases (MMPs) are endopeptidases which break down the extracellular matrix and regulate cytokine and growth factor activity. Several MMPs have been implicated in the promotion of invasion and metastasis in a broad range of tumours including urothelial carcinoma. In this study, RNA from 132 normal bladder and urothelial carcinoma specimens was profiled for each of the 24 human MMPs, the four endogenous tissue inhibitors of MMPs (TIMPs) and several key growth factors and their receptors using quantitative real time RT-PCR. Laser capture microdissection (LCM) of RNA from 22 tumour and 11 normal frozen sections was performed allowing accurate RNA extraction from either stromal or epithelial compartments. This study confirms the over expression in bladder tumour tissue of well-documented MMPs and highlights a range of MMPs which have not previously been implicated in the development of urothelial cancer. In summary, MMP-2, MT1-MMP and the previously unreported MMP-28 were very highly expressed in tumour samples while MMPs 1, 7, 9, 11, 15, 19 and 23 were highly expressed. There was a significant positive correlation between transcript expression and tumour grade for MMPs 1, 2, 8, 10, 11, 12, 13, 14, 15 and 28 (P < 0.001). At the same confidence interval, TIMP-1 and TIMP-3 also correlated with increasing tumour grade. LCM revealed that most highly expressed MMPs are located primarily within the stromal compartment except MMP-13 which localised to the epithelial compartment. This work forms the basis for further functional studies, which will help to confirm the MMPs as potential diagnostic and therapeutic targets in early bladder cancer.