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The urothelial cell line UROtsa transformed by arsenite and cadmium display basal characteristics associated with muscle invasive urothelial cancers.

ABSTRACT: Muscle invasive urothelial carcinomas are divided into various molecular subtypes with basal and luminal subtypes being the prominent ones. The basal muscle-invasive urothelial carcinomas are generally more aggressive at presentation and significantly enriched with squamous features. Our laboratory has developed an in-vitro model of urothelial cancer by transforming the immortalized cell line UROtsa with arsenite (As3+) and cadmium (Cd2+). In this study, we characterized the tumors formed by these transformed cell lines as more basal-like based on their gene expression patterns with increased expression of KRT1, KRT5, KRT6, KRT14, KRT16, KRT17 and CD44. In addition, histological examination of these tumor transplants showed squamous features enriched in basal muscle invasive urothelial carcinomas. The expression of these genes increased in the transformed cell lines as well as in the urospheres, which are putative cancer initiating cells/stem cells derived from the cell lines. There was also increased expression of these genes in the urospheres derived from the parent UROtsa cell line. Thus, our data shows that the As3+ and Cd2+-transformed cell lines and their derived tumor transplants have gene expression profiles similar to the basal subtype of muscle invasive bladder carcinomas with tumors having enriched squamous features. The increased expression of basal markers in the urospheres suggests that stem cells may be involved in the development of squamous differentiation seen in some of the muscle invasive bladder carcinomas.

SUBMITTER: Hoggarth ZE

PROVIDER: S-EPMC6294394 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEpithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown.MethodsReal-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice.ResultsThis study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin.ConclusionsThe finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth. Mesenchymal to epithelial transition and E-cadherin are viewed as necessary for a cell to establish a new metastatic site. The lack of N-cadherin expression in tumor transplants is consistent with E-cadherin expressing cells "seeding" a site for tumor growth. The study shows that a minority population of cultured cells can be the initiators of tumor growth.

Project description:There is growing evidence that molecular subtypes (e.g. luminal and basal subtypes) affect the prognosis and treatment response in patients with muscle invasive urinary bladder cancer (invasive urothelial carcinoma, iUC). Modeling these subtypes in pre-clinical animal studies is essential, but it is challenging to produce these subtypes, along with other critical host and tumor features, in experimentally-induced animal models. This study was conducted to determine if luminal and basal molecular subtypes are present in naturally-occurring canine iUC, a cancer that mimics the human condition in other key aspects. RNA sequencing was performed on 29 canine treatment naive iUC tissue samples and on four normal canine bladder mucosal samples. Data were aligned to CanFam 3.1, and differentially expressed genes were identified. Unsupervised hierarchical clustering of these genes revealed two distinct groups (n = 13, n = 16). When genes that distinguish basal and luminal subtypes in human cancer (n = 2015) were used to probe genes differentially expressed between normal canine bladder and iUC, 829 enriched signature genes were identified. Unsupervised hierarchical clustering of these genes revealed two distinct groups comprised of 18 luminal subtype tumors and 11 basal subtype tumors. The enriched genes included MMP9, SERPINE2, CAV1, KRT14, and RASA3 in basal tumors, and PPARG, LY6E, CTSE, CDK3, and TBX2 in luminal tumors. In supervised clustering, additional genes of importance in human iUC were identified in canine iUC associated with claudin-low and infiltrated tumors. A smaller panel of genes (n = 60) was identified that distinguished canine luminal and basal iUC with overall 93.1% accuracy. Immune signature patterns similar to those in human iUC were also identified with the greatest enrichment of immune genes being in the basal subtype tumors. These findings provide additional compelling evidence that naturally-occurring canine iUC is a highly relevant and much needed model of human iUC for translational research.

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The urothelial cell line UROtsa transformed by arsenite and cadmium display basal characteristics associated with muscle invasive urothelial cancers.

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