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111 COMBINED, MULTI-DATASET MICROARRAY META-ANALYSIS IDENTIFIES GENE EXPRESSION SIGNATURES THAT DISCRIMINATE RENAL ONCOCYTOMAS FROM CHROMOPHOBE RENAL CELL CARCINOMAS
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
clear cell type tumor and adjacent normal tissue) by real time RT-PCR. For gain-of-function studies, miR-1- and miR-133a-transfected RCC cells were subjected to cell proliferation assay, flow cytometry, and invasion assay. To find common target genes of miR-1 and miR-133a, we analyzed the gene expression profile of their transfectants. The luciferase reporter assay was carried out to confirm the actual binding sites between the miRNAs and the candidate target gene. We checked mRNA expression level of the gene in the clinical specimens and performed loss-of-function studies using si-RNA method in RCC cell lines. RESULTS: The expression levels of miR-1 and miR-133a were significantly lower in the RCC cell lines and the RCC specimens than normal tissues (each, P ⬍ 0.001). The gain-of-function studies demonstrated significant inhibition of cell proliferation (P ⬍ 0.001) and invasion (P ⬍ 0.05) in both miR-1 and miR-133a transfectants. A flow cytometry analysis revealed that miR-1 and miR-133a restoration induced apoptosis and cell cycle arrest in RCC cell lines. We focused on the transgelin-2 (TAGLN2) as a common target of miR-1 and miR-133a on the basis of the gene expression profile of their transfectants. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1 and miR-133a transfectants (P ⬍ 0.05). The expression level of TAGLN2 mRNA was significantly higher in the RCC specimens (P ⬍ 0.001), moreover, there was a statistically significant inverse correlation between TAGLN2 and both miR-1 and miR-133a expression (r ⫽ ⫺0.414, P ⫽ 0.0002 and r ⫽ ⫺0.485, P ⬍ 0.0001). The loss-of-function studies demonstrated significant inhibitions of cell proliferation (P ⬍ 0.001) and invasion (P ⬍ 0.05) in the si-TAGLN2-transfected RCC cell lines. CONCLUSIONS: Our data indicates that up-regulation of the oncogenic TAGLN2 was due to down-regulation of the tumor suppressive miR-1 and miR-133a in human RCC. This novel molecular network may be a critical role for RCC development and serve as a novel therapeutic target in RCC. Source of Funding: None
111 COMBINED, MULTI-DATASET MICROARRAY META-ANALYSIS IDENTIFIES GENE EXPRESSION SIGNATURES THAT DISCRIMINATE RENAL ONCOCYTOMAS FROM CHROMOPHOBE RENAL CELL CARCINOMAS Vladimir Valera*, Beatriz Walter, Maria Merino, Bethesda, MD INTRODUCTION AND OBJECTIVES: Renal oncocytomas and chromophobe renal cell carcinomas are two closely related entities that differ mostly in the malignant potential of the latter. Several attempts have been made to identify unique markers for each of these entities. From the molecular standpoint, either protein expression by immunohistochemistry or gene expression profiling based on microarray analysis are the most common approaches. The results so far are limited by the reduced number of cases included in each study. In this work, we aimed to evaluate the gene expression profile of renal oncocytomas and chromophobe RCC by combining publicly available microarray data to overcome such limitations. METHODS: Datasets containing the MeSH identifiers “Chromophobe” and “Oncocytoma” were queried in the NCBI Gene Espression Ommibus (GEO) database. Original, raw intensity files from singleplatform studies were retrieved. Only non-redundant samples were used for analysis. Quality control probes were used to ensure comparability. After data preprocessing, gene expression signatures were investigated by unpaired T-test analysis with stringent conditions for false discovery rate and gene fold-changes. A classifier based on gene expression pattern was built and externally validated. Analyses were conducted with Genespring and R/Bioconductor software. RESULTS: Seventeen datasets were initially identified. From these, 10 were based on Affymetrix gene microarray technology, and 4 non-redundant databases were used for analysis, comprising 34 chromophobe tumors, 40 oncocytomas, and 38 normal samples. After uniform array data preprocessing, 5291 genes were found differentially
THE JOURNAL OF UROLOGY姞
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expressed. After data reduction by PCA, 121 genes responsible for the highest variability among samples were used for building a classifier. Phosphoenolpyruvate carboxykinase 1 (PCK1, 15.1-fold), Apolipoprotein E (APOE, 3.9-fold) and Aquaporin 6 (9.4-fold) showed higher expression in oncocytomas, while Osteoactivin (GPNMB) and Claudin 8 (CLDN8) showed increased expression in chromophobe tumors of 9.2 fold and 14.2-fold respectively. The combination of genes helped to accurately classify tumors with an overall accuracy of 94%. CONCLUSIONS: The results demonstrate that unique gene expression profiles can be used to differentiate with high accuracy benign renal oncocytomas from malignant chromophobe RCCs by analyzing systematically public microarray databases. Source of Funding: Intramural program, National Cancer Institute.
112 CHARACTERIZATION OF CANCER STEM CELL-LIKE CELLS AND STROMAL CELLS IN RENAL CELL CARCINOMA Daniel Rottke*, Hamburg, Germany; Verena Bo¨rger, Peter Albers, Du¨sseldorf, Germany; Margit Fisch, Hamburg, Germany; Rolf Ackermann, Ru¨diger Sorg, Du¨sseldorf, Germany INTRODUCTION AND OBJECTIVES: Cancer stem cells are crucial to the development and progression of tumors. Non-tumorigenic tissue stem cells such as mesenchymal stem cells (MSC)-like cells constitute components of tumor stroma and may also contribute to these processes. We have characterized the stem cell features of two cell lines with mesenchymal morphology, DH-1 and GF-1, derived from metastases of clear cell renal cell carcinomas (RCC), and one cell line, MG-1, with epithelial appearance derived from a primary papillary type 1 RCC. METHODS: Cell lines were characterized by morphology, flow cytometry, cytogenetics, RT-PCR expression, their differentiation potential and by xenotransplantation into mice. RESULTS: The mesenchymal cell lines lacked cytogenetic aberrations typical for RCC and did not form tumors in mice. GF-1 and DH-1 cells had an immunophenotype in common with bone marrowderived MSC. Like MSC, both cell lines showed osteogenic differentiation. When co-cultured with MCF7 carcinoma cells, GF-1 cells but only to a lesser extent DH-1 cells or MSC induced epithelial-to-mesenchymal transition with change in morphology and down-regulation of Ecadherin in MCF7 cells. Upon co-transplantation, only GF-1 cells promoted tumor formation in the various mouse models. The epithelial cell line MG-1 had a similar immunophenotype, but in contrast to the mesenchymal cells, they were CD90-, CD133⫹, CD326⫹ and cytokeratin 8/18⫹ and revealed chromosomal aberrations typical for papillary RCC. They formed spheres and showed osteogenic but not adipogenic differentiation. When transplanted into mice, they formed tumors from which tumorigenic cells could be regrown. Subcloning of CD133⫹ and CD133- subpopulations of MG-1 cells resulted in immunophenotypically similar cells with osteogenic differentiation potential. However, tumor-formation was observed for CD133- clones only, with lower numbers of cells initiating tumor formation than in the parental line, suggesting that in papillary RCC cancer stem cells reside in the CD133population. CONCLUSIONS: Thus, the newly established epithelial RCC cell line MG-1- subclones share typical features with cancer stem cells, including immunophenotype, tumorigenicity, self-renewal and differentiation potential. In contrast, the mesenchymal cell lines DH-1 and GF-1 show characteristics of MSC and apparently are derived from RCC stroma. However, only GF-1 cells but not DH-1 cells or MSC have potent tumor stroma activity and induce epithelial-to-mesenchymal transition in vitro and promote tumor-formation by a normally nontumorigenic RCC cell line in vivo. Source of Funding: Stiftung urologische Forschung Forschungskomission Universita¨t Du¨sseldorf.
clear cell type tumor and adjacent normal tissue) by real time RT-PCR. For gain-of-function studies, miR-1- and miR-133a-transfected RCC cells were subjected to cell proliferation assay, flow cytometry, and invasion assay. To find common target genes of miR-1 and miR-133a, we analyzed the gene expression profile of their transfectants. The luciferase reporter assay was carried out to confirm the actual binding sites between the miRNAs and the candidate target gene. We checked mRNA expression level of the gene in the clinical specimens and performed loss-of-function studies using si-RNA method in RCC cell lines. RESULTS: The expression levels of miR-1 and miR-133a were significantly lower in the RCC cell lines and the RCC specimens than normal tissues (each, P ⬍ 0.001). The gain-of-function studies demonstrated significant inhibition of cell proliferation (P ⬍ 0.001) and invasion (P ⬍ 0.05) in both miR-1 and miR-133a transfectants. A flow cytometry analysis revealed that miR-1 and miR-133a restoration induced apoptosis and cell cycle arrest in RCC cell lines. We focused on the transgelin-2 (TAGLN2) as a common target of miR-1 and miR-133a on the basis of the gene expression profile of their transfectants. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1 and miR-133a transfectants (P ⬍ 0.05). The expression level of TAGLN2 mRNA was significantly higher in the RCC specimens (P ⬍ 0.001), moreover, there was a statistically significant inverse correlation between TAGLN2 and both miR-1 and miR-133a expression (r ⫽ ⫺0.414, P ⫽ 0.0002 and r ⫽ ⫺0.485, P ⬍ 0.0001). The loss-of-function studies demonstrated significant inhibitions of cell proliferation (P ⬍ 0.001) and invasion (P ⬍ 0.05) in the si-TAGLN2-transfected RCC cell lines. CONCLUSIONS: Our data indicates that up-regulation of the oncogenic TAGLN2 was due to down-regulation of the tumor suppressive miR-1 and miR-133a in human RCC. This novel molecular network may be a critical role for RCC development and serve as a novel therapeutic target in RCC. Source of Funding: None
111 COMBINED, MULTI-DATASET MICROARRAY META-ANALYSIS IDENTIFIES GENE EXPRESSION SIGNATURES THAT DISCRIMINATE RENAL ONCOCYTOMAS FROM CHROMOPHOBE RENAL CELL CARCINOMAS Vladimir Valera*, Beatriz Walter, Maria Merino, Bethesda, MD INTRODUCTION AND OBJECTIVES: Renal oncocytomas and chromophobe renal cell carcinomas are two closely related entities that differ mostly in the malignant potential of the latter. Several attempts have been made to identify unique markers for each of these entities. From the molecular standpoint, either protein expression by immunohistochemistry or gene expression profiling based on microarray analysis are the most common approaches. The results so far are limited by the reduced number of cases included in each study. In this work, we aimed to evaluate the gene expression profile of renal oncocytomas and chromophobe RCC by combining publicly available microarray data to overcome such limitations. METHODS: Datasets containing the MeSH identifiers “Chromophobe” and “Oncocytoma” were queried in the NCBI Gene Espression Ommibus (GEO) database. Original, raw intensity files from singleplatform studies were retrieved. Only non-redundant samples were used for analysis. Quality control probes were used to ensure comparability. After data preprocessing, gene expression signatures were investigated by unpaired T-test analysis with stringent conditions for false discovery rate and gene fold-changes. A classifier based on gene expression pattern was built and externally validated. Analyses were conducted with Genespring and R/Bioconductor software. RESULTS: Seventeen datasets were initially identified. From these, 10 were based on Affymetrix gene microarray technology, and 4 non-redundant databases were used for analysis, comprising 34 chromophobe tumors, 40 oncocytomas, and 38 normal samples. After uniform array data preprocessing, 5291 genes were found differentially
THE JOURNAL OF UROLOGY姞
e47
expressed. After data reduction by PCA, 121 genes responsible for the highest variability among samples were used for building a classifier. Phosphoenolpyruvate carboxykinase 1 (PCK1, 15.1-fold), Apolipoprotein E (APOE, 3.9-fold) and Aquaporin 6 (9.4-fold) showed higher expression in oncocytomas, while Osteoactivin (GPNMB) and Claudin 8 (CLDN8) showed increased expression in chromophobe tumors of 9.2 fold and 14.2-fold respectively. The combination of genes helped to accurately classify tumors with an overall accuracy of 94%. CONCLUSIONS: The results demonstrate that unique gene expression profiles can be used to differentiate with high accuracy benign renal oncocytomas from malignant chromophobe RCCs by analyzing systematically public microarray databases. Source of Funding: Intramural program, National Cancer Institute.
112 CHARACTERIZATION OF CANCER STEM CELL-LIKE CELLS AND STROMAL CELLS IN RENAL CELL CARCINOMA Daniel Rottke*, Hamburg, Germany; Verena Bo¨rger, Peter Albers, Du¨sseldorf, Germany; Margit Fisch, Hamburg, Germany; Rolf Ackermann, Ru¨diger Sorg, Du¨sseldorf, Germany INTRODUCTION AND OBJECTIVES: Cancer stem cells are crucial to the development and progression of tumors. Non-tumorigenic tissue stem cells such as mesenchymal stem cells (MSC)-like cells constitute components of tumor stroma and may also contribute to these processes. We have characterized the stem cell features of two cell lines with mesenchymal morphology, DH-1 and GF-1, derived from metastases of clear cell renal cell carcinomas (RCC), and one cell line, MG-1, with epithelial appearance derived from a primary papillary type 1 RCC. METHODS: Cell lines were characterized by morphology, flow cytometry, cytogenetics, RT-PCR expression, their differentiation potential and by xenotransplantation into mice. RESULTS: The mesenchymal cell lines lacked cytogenetic aberrations typical for RCC and did not form tumors in mice. GF-1 and DH-1 cells had an immunophenotype in common with bone marrowderived MSC. Like MSC, both cell lines showed osteogenic differentiation. When co-cultured with MCF7 carcinoma cells, GF-1 cells but only to a lesser extent DH-1 cells or MSC induced epithelial-to-mesenchymal transition with change in morphology and down-regulation of Ecadherin in MCF7 cells. Upon co-transplantation, only GF-1 cells promoted tumor formation in the various mouse models. The epithelial cell line MG-1 had a similar immunophenotype, but in contrast to the mesenchymal cells, they were CD90-, CD133⫹, CD326⫹ and cytokeratin 8/18⫹ and revealed chromosomal aberrations typical for papillary RCC. They formed spheres and showed osteogenic but not adipogenic differentiation. When transplanted into mice, they formed tumors from which tumorigenic cells could be regrown. Subcloning of CD133⫹ and CD133- subpopulations of MG-1 cells resulted in immunophenotypically similar cells with osteogenic differentiation potential. However, tumor-formation was observed for CD133- clones only, with lower numbers of cells initiating tumor formation than in the parental line, suggesting that in papillary RCC cancer stem cells reside in the CD133population. CONCLUSIONS: Thus, the newly established epithelial RCC cell line MG-1- subclones share typical features with cancer stem cells, including immunophenotype, tumorigenicity, self-renewal and differentiation potential. In contrast, the mesenchymal cell lines DH-1 and GF-1 show characteristics of MSC and apparently are derived from RCC stroma. However, only GF-1 cells but not DH-1 cells or MSC have potent tumor stroma activity and induce epithelial-to-mesenchymal transition in vitro and promote tumor-formation by a normally nontumorigenic RCC cell line in vivo. Source of Funding: Stiftung urologische Forschung Forschungskomission Universita¨t Du¨sseldorf.