Dataset Information

VHL-dependent regulation of a ?-dystroglycan glycoform and glycogene expression in renal cancer.

ABSTRACT: Identification of novel biomarkers and targets in renal cell carcinoma (RCC) remains a priority and one cellular compartment that is a rich potential source of such molecules is the plasma membrane. A shotgun proteomic analysis of cell surface proteins enriched by cell surface biotinylation and avidin affinity chromatography was explored using the UMRC2- renal cancer cell line, which lacks von Hippel-Lindau (VHL) tumour suppressor gene function, to determine whether proteins of interest could be detected. Of the 814 proteins identified ~22% were plasma membrane or membrane-associated, including several with known associations with cancer. This included ?-dystroglycan, the transmembrane subunit of the DAG1 gene product. VHL-dependent changes in the form of ?-dystroglycan were detected in UMRC2-/+VHL transfectants. Deglycosylation experiments showed that this was due to differential sialylation. Analysis of normal kidney cortex and conventional RCC tissues showed that a similar change also occurred in vivo. Investigation of the expression of genes involved in glycosylation in UMRC2-/+VHL cells using a focussed microarray highlighted a number of enzymes involved in sialylation; upregulation of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) was validated in UMRC2- cells compared with their +VHL counterparts and also found in conventional RCC tissue. These results implicate VHL in the regulation of glycosylation and raise interesting questions regarding the extent and importance of such changes in RCC.

SUBMITTER: Aggelis V

PROVIDER: S-EPMC3823392 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

VHL-dependent regulation of a β-dystroglycan glycoform and glycogene expression in renal cancer.

Aggelis Vassilis V Craven Rachel A RA Peng Jianhe J Harnden Patricia P Schaffer Lana L Hernandez Gilberto E GE Head Steven R SR Maher Eamonn R ER Tonge Robert R Selby Peter J PJ Banks Rosamonde E RE

International journal of oncology 20130821 5

Identification of novel biomarkers and targets in renal cell carcinoma (RCC) remains a priority and one cellular compartment that is a rich potential source of such molecules is the plasma membrane. A shotgun proteomic analysis of cell surface proteins enriched by cell surface biotinylation and avidin affinity chromatography was explored using the UMRC2- renal cancer cell line, which lacks von Hippel-Lindau (VHL) tumour suppressor gene function, to determine whether proteins of interest could be ...[more]

PMID: 23970118

Similar Datasets

Project description:The commonest histological type of renal cancer, clear cell renal cell carcinoma (cc RCC), is associated with genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumour suppressor. VHL inactivation leads to induction of hypoxia-inducible factors (HIFs) and a hypoxic pattern of gene expression. Differential levels of specific microRNAs (miRNAs) are observed in several tumours when compared to normal tissue. Given the central role of VHL in renal cancer formation, we examined the VHL-dependent regulation of miRNAs in renal cancer.VHL-dependent miRNA expression in cc RCC was determined by microarray analysis of renal cell line RCC4 with mutated VHL (RCC4-VHL) and reintroduced wild-type VHL (RCC4 + VHL). Five miRNAs highly upregulated in RCC4 + VHL and five miRNAs highly downregulated in RCC4 + VHL were studied further, in addition to miR-210, which is regulated by the HIF-VHL system. miRNA expression was also measured in 31 cc RCC tumours compared to adjacent normal tissue.A significant increase in miR-210, miR-155 and miR-21 expression was observed in the tumour tissue. miR-210 levels also showed a correlation with a HIF-regulated mRNA, carbonic anhydrase IX (CAIX), and with VHL mutation or promoter methylation. An inverse correlation was observed between miR-210 expression and patient survival, and a putative target of miR-210, iron-sulfur cluster assembly protein (ISCU1/2), shows reciprocal levels of mRNA expression in the tumours.We have identified VHL-regulated miRNAs and found that for some the regulation is HIF-dependent and for others it is HIF-independent. This pattern of regulation was also seen in renal cancer tissue for several of these miRNAs (miR-210, miR-155, let-7i and members of the miR-17-92 cluster) when compared with normal tissue. miR-210 showed marked increases in expression in renal cancer and levels correlated with patient survival. The inverse correlation between miR-210 levels and ISCU1/2 provides support for the hypothesis that ISCU1/2 is a target of miR-210 and that it may contribute to the anaerobic respiration seen in renal (and other) tumours.See Commentary: http://www.biomedcentral.com/1741-7015/8/65.

Project description:The loss of von Hippel-Lindau (VHL) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2alpha (HIF2alpha) instead of hypoxia-inducible factor-1alpha (HIF1alpha). MS/MS data generated by data dependent acquisition via the LTQ-FT were extracted by BioWorks version 3.3 and searched against a composite IPI human protein sequence database (IPI _human_v 3.73.par) containing both forward and randomized sequences using Mascot version 2.2 (Matrix Science, Boston, MA) search algorithm. Mascot was searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 15.0 ppm assuming trypsin as the digestion enzyme with the possibility of one missed cleavage. Carbamidomethylation of cysteine was specified as a fixed modification, while oxidation of methionine, N-terminal protein acetylation, N-terminal peptide and lysine carbamoylation, and phosphorylation of serine and threonine were specified as variable modifications. The analysis of VHL-wt (Caki-2) human RCC and VHL-mut (786-O) cells was carried out from three independent, biological samples from each cell line (tryptic digests of Caki-2 and 786-O, respectively) and duplicate injections were made for each sample. Data processing was accomplished using two software tools: Scaffold (version Scaffold 3.0, Proteome Software Inc., Portland, OR) and Progenesis LC-MS (version 3.0, Nonlinear Dynamics, Durham, NC). Scaffold was employed to validate MS/MS-based peptide and protein identifications from Mascot database searching. Initial peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm [20]. Protein probabilities were assigned by the Protein Prophet algorithm [21]. Protein identifications were accepted, if they reached greater than 99% probability and contained at least 2 identified unique peptides. These identification criteria typically established < 0.01% false discovery rate based on a decoy database search strategy at the protein level. Extracted peptide intensities were evaluated with Progenesis LC-MS to obtain label-free relative quantification from the raw data files. The software created a single aggregate run based on LC retention time of each analysis to contain all MS data with distinguished peptide ions and, then, further feature outline maps were generated for detection and quantification of peptide ions from individual analyses using default program parameters. Peptide quantifications were performed by summing peak intensities followed by normalization, data filtering to retain only ions with positive charges (z) of two and three, and exporting peak lists to query against the IPI human protein sequence database using Mascot (see Database Search subsection above). Proteins were accepted as differentially expressed following analysis of variance (ANOVA, P<0.05), requiring at least a twofold change in expression and also passing validation by Peptide Prophet and Protein Prophet using protein identifications from Scaffold.

Dataset Information

VHL-dependent regulation of a ?-dystroglycan glycoform and glycogene expression in renal cancer.

Publications

VHL-dependent regulation of a β-dystroglycan glycoform and glycogene expression in renal cancer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure