Enrichment of genes associated with squamous differentiation in cancer initiating cells isolated from urothelial cells transformed by the environmental toxicant arsenite.

Arsenic is an environmental toxicant with long-term exposure associated with the development of urothelial carcinomas. Our lab has developed an in-vitro model of urothelial carcinoma by exposing the immortal, but non-tumorigenic bladder cell line, the UROtsa, to arsenite (As3+). These transformed cells form tumors in immune-compromised mice, which resemble urothelial carcinomas with components of the tumor exhibiting squamous differentiation. The goal of the present study was to determine the differences in global gene expression patterns between the As3+-transformed UROtsa cells and the urospheres (spheroids containing putative cancer initiating cells) isolated from these cell lines and to determine if the genes involved in the development of squamous differentiation were enriched in the urospheres. The results obtained in this study show an enrichment of genes such as KRT1, KRT5, KRT6A, KRT6B, KRT6C, KRT14 and KRT16 associated with squamous differentiation, a characteristic feature seen in aggressive basal subtypes of urothelial cell carcinoma (UCC) in the urospheres isolated from As3+-transformed UROtsa cells. In addition, there is increased expression of several of the small proline-rich proteins (SPRR) in the urospheres and overexpression of these genes occur in UCC's displaying squamous differentiation. In conclusion, the cancer initiating cells present in the urospheres are enriched with genes associated with squamous differentiation.

The expression of keratin 6 is regulated by the activation of the ERK1/2 pathway in arsenite transformed human urothelial cells.

Urothelial cancers have an environmental etiological component, and previous studies from our laboratory have shown that arsenite (As+3) can cause the malignant transformation of the immortalized urothelial cells (UROtsa), leading to the expression of keratin 6 (KRT6). The expression of KRT6 in the parent UROtsa cells can be induced by the addition of epidermal growth factor (EGF). Tumors formed by these transformed cells have focal areas of squamous differentiation that express KRT6. The goal of this study was to investigate the mechanism involved in the upregulation of KRT6 in urothelial cancers and to validate that the As+3-transformed UROtsa cells are a model of urothelial cancer. The results obtained showed that the parent and the As+3-transformed UROtsa cells express EGFR which is phosphorylated with the addition of epidermal growth factor (EGF) resulting in an increased expression of KRT6. Inhibition of the extracellular-signal regulated kinases (ERK1/2) pathway by the addition of the mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 kinase inhibitor U0126 resulted in a decrease in the phosphorylation of ERK1/2 and a reduced expression of KRT6. Immuno-histochemical analysis of the tumors generated by the As+3-transformed isolates expressed EGFR and tumors formed by two of the transformed isolates expressed the phosphorylated form of EGFR. These results show that the expression of KRT6 is regulated at least in part by the ERK1/2 pathway and that the As+3-transformed human urothelial cells have the potential to serve as a valid model to study urothelial carcinomas.

Human renal tubular cells contain CD24/CD133 progenitor cell populations: Implications for tubular regeneration after toxicant induced damage using cadmium as a model.

The proximal tubules of the kidney are target sites of injury by various toxicants. Cadmium (Cd+2), an environmental nephrotoxicant can cause adverse effects and overt renal damage. To decipher the mechanisms involved in nephrotoxicity, an in vitro model system is required. Mortal cultures of human proximal tubule (HPT) cells have served, as models but are difficult to acquire and do not lend themselves to stable transfection. The immortalized human proximal tubule cell line HK-2, has served as a model but it lacks vectorial active transport and shows signs of lost epithelial features. Recently a new proximal tubule cell line was developed, the RPTEC/TERT1, and the goal of this study was to determine if this cell line could serve as a model to study nephrotoxicity. Global gene expression analysis of this cell line in comparison to the HK-2 and HPT cells showed that the RPTEC/TERT1 cells had gene expression patterns similar to HPT cells when compared to the HK-2 cells. The HPT and the RPTEC/TERT1 cell line had an increased population of stem/progenitor cells co-expressing CD24 and CD133 when compared to the HK-2 cells. The level of expression of cadherins, claudins and occludin molecules was also similar between the RPTEC/TERT1 and the HPT cells. Acute exposure to Cd+2 resulted in necrosis of the RPTEC/TERT1 cells when compared to the HK-2 cells which died by apoptosis. Thus, the RPTEC/TERT1 cells are similar to HPT cells and can serve as a good model system to study mechanisms involved in toxicant induced renal damage.

Protein interactions with metallothionein-3 promote vectorial active transport in human proximal tubular cells.

Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, ß-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.

Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines.

BACKGROUND: Epithelial 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. METHODS: Real-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. RESULTS: This 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. CONCLUSIONS: The 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.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC.

BACKGROUND: This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3)) and cadmium (Cd(+2))-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2)-and As(+3)-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. METHODS: Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3)-and Cd(+2)-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. RESULTS: It was shown that the As(+3)-and Cd(+2)-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. CONCLUSIONS: Tumor initiating cells isolated from SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.