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Gene expression analysis in renal cell carcinoma

ABSTRACT: Renal cell carcinoma (RCC) tumors express varying gene profiles, dependent on VHL and HIF status, as well as other events. Here we analyzed a series of RCC tumors for HIF-alpha expression and VHL genotype, in relation to overal gene expression profiles. Keywords: Patient sample study

ORGANISM(S): Homo sapiens

PROVIDER: GSE11985 | GEO | 2008/10/18

SECONDARY ACCESSION(S): PRJNA105869

REPOSITORIES: GEO

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Project description:Renal cell carcinoma (RCC) accounts for around 2-3% of all adult neoplasms and its incidence is increasing. RCC is largely resistant to standard cancer treatment regimens including chemotherapy and radiation therapy, whilst immunotherapy modestly provides benefit to only a small subset of patients. As a result, prognosis for RCC patients with advanced disease is very poor, with a 5-year survival rate of less than 10%. An early event in the development of familial and up to 90% of sporadic RCC is the loss and/or mutation/methylation of the von Hippel-Lindau (VHL) tumour suppressor gene. The tumor suppressor role of VHL relies primarily on its ability to target members of the hypoxia-inducible factors (HIF) for proteosomal degradation, however it has also been implicated in other biological processes linked to oncogenesis, such as the extracellular matrix assembly and the regulation of actin cytoskeleton, that are less clearly defined. Targeting the VHL/HIF pathway may dramatically improve RCC treatment, as demonstrated by an increasing number of novel agents that have shown promising activity in recent clinical trials6. Identification of other possible proteins regulated by VHL that may be involved in tumour suppression will improve our understanding of RCC pathobiology and also provide novel disease biomarkers or potential targets. A number of our studies have identified changes in glycosylation in RCC, at least some of which appear to alter as a result of changes in the VHL gene. Lectin binding immunohistochemistry shows altered reactivity with a number of lectins in the malignant tissue compared with the normal tissue. Comparison of plasma membrane protein enriched fractions from human RCC cells stably transfected with control vector or with wild type VHL led to the identification of VHL-associated changes in the forms of several plasma membrane proteins which we beelive to be due to differential glycosylation. For one of these we have now shown that it is differentially O-glycosylated and this has been confimed in cell lysates and patient tissue by Western blotting. A parallel approach using 2D-PAGE analysis of RCC cell line conditioned medium (CM) has also identified a tumour-specific glycoform of a secretory protein that appears to undergo differential N-glycosylation in RCC and this has been confirmed in analysis of some matched primary tumour/normal pairs. These findings are potentially very interesting, as these proteins could serve as prognostic or diagnostic markers, or even as targets for improving RCC therapy. We are therefore keen to explore the underlying mechanism of differential glycosylation that may be associated to VHL dependent gene expression. As there are an immense number of enzymes involved in the conjugation of N- and particularly O-glycans to proteins, the glyco-microarray is an ideal tool to elucidate this further. For chip analyses we have three cell lines generated from human RCC cells (RCC4, 786-0 and UMRC2) that have been stably transfected with control vector or with wild type VHL i.e. 6 cell lines in total. Whole RNA was extracted from each cell line in 3 replicates per line.

Project description:Renal cancer accounts for 2% to 3% of all adult malignancies in the US. In 2011 more than 64,770 new cases and 13,000 deaths were reported and the incidence is steadily increasing by 2.5% per year. Clear cell renal carcinoma (ccRCC) represents the majority (85% to 90%) of adult kidney cancers and is the most malignant. This cancer is characterized by an early loss of the von Hippel-Lindau tumor suppressor (VHL) on short arm of chromosome 3 in the majority of tumors (80%). It is a classic knowledge that loss of VHL results in an increased accumulation and activity of the hypoxia-inducible transcription factor (HIF), which in turns activates expression of genes promoting tumor growth. In that respect, induction of angiogenic factors induces blood flow through the tumors and delivery of nutrients and oxygen to cancer cells, while induction of genes inducing anaerobic glycolysis supports adaptation to reduced nutrients availability and shifts towards metabolic pathways promoting tumor growth. Recently we have discovered another tumor suppressing pathway regulated by VHL. We found that VHL is a major regulator of the process of macroautophagy (autophagy) (ref). We identified that VHL induces expression of a microRNA, miR-204, which in turn inhibits activity of a major regulator required for the formation of autophagosome, LC3B. LC3B-mediated autophagy is necessary for the ccRCC tumor growth, and inhibition of this process by VHL and miR-204 significantly contributes to the tumor suppression. Most importantly, we discovered that VHL by inhibiting activity of HIF, induced expression and activity of an LC3B paralog, LC3C. In contrast to LC3B, LC3C acts as a tumor suppressor, indicating the specificity and complexity of different autophagic programs. Integrating these novel data with the established role of VHL in regulating angiogenesis produces a more global picture of VHL as a master tumor suppressor that regulates nutrient access for renal cancer cells from both extracellular and intracellular sources. In the process of searching for other genes induced by VHL that could participate in the tumor suppressing activity of VHL, we discovered that reconstitution of VHL in RCC cells induces enrichment of genes expressed from the Smith-Magenis locus (SM), located on the short arm of chromosome 17p11.2. Smith-Magenis syndrome is a complex neurobehevioural disorder, characterized by intellectual impairment, craniofacial and skeletal anomalies and sleep disturbance. Recently, deletions or mutations of one specific gene in the SM locus, RAI1, (retinoic acid induced 1), were shown to be responsible for the syndrome. Interestingly, this locus contains another kidney tumor suppressor gene, folliculin (FLCN), of which activity is lost in the genetic dominant disorder, Birt-Hogg-Dube syndrome (BHD). BHD is characterized by skin fibrofolliculomas, pulmonary cysts and spontaneous pneumothorax, as well as renal cancer of mixed histological types, including chromophobe, oncocytic, clear cell and papillary type. Here we show that VHL regulates expression of FLCN, and in turn FLCN contributes to the VHL-mediated suppression of tumor growth. Two-condition experiment, VHL(-) vs. VHL(+). 4 biological replicates in each group. Two dual-channel arrays with VHL(+) on Cy5 and VHL(-) on Cy3, and two dual-channel arrays with flipped samples.

Project description:Altered metabolism is an important part of malignant transformation of tumor cells. Oncogenic transformation may reprogram tumor metabolism and render tumor cells addicted to extracellular nutrients. Such nutrient addictions associated with oncogenic mutations may offer therapeutic opportunities; however, it remains difficult to predict these nutrient addictions. Here, we performed a nutrigenetic screen to determine the phenotypes of isogenic pairs of clear-cell renal cancer cells (ccRCC) with or without VHL upon the deprivation of individual amino acids. We identified that cystine deprivation triggered rapid programmed necrosis in VHL-deficient RCC, but not in their VHL-restored counterparts. Similar cystine addiction was also observed in VHL-deficient primary RCC tumors cells. Blockage of cystine uptake significantly delayed xenograft growth of ccRCC. Importantly, cystine deprivation triggered similar metabolic changes regardless of VHL status. Therefore, metabolic differences due to cystine deprivation are not different enough to readily explain the distinct fate of life vs. death in VHL-deficient and restored cell.. Instead, we found that increased levels of TNFÎ± associated with VHL loss in the VHL-deficient RCC force them to rely on intact RIPK1 to inhibit apoptosis. However, this pre-existing elevated TNFÎ± in the VHL-deficient ccRCC renders these cells susceptible to the necrosis signaling triggered by cystine deprivation. In addition, we identified that cystine-deprived necrosis in VHL-deficient RCC depends on reciprocal amplification of the Src-p38-Noxa signaling and TNFÎ±-RIP1/3-MLKL necrosis pathways that culminate in MLKL oligomerization and programmed necrosis. Together, our data reveal that the contextual cystine-addictions in VHL-deficient ccRCC is dependent on activating pre-existing oncogenic pathways to trigger programmed necrosis. RNA was extracted by RNAeasy kits (Qiagen) from the RCC4 Vec and VHL-reconstituted cells which were exposed to the control full DMEM or cystine deprived DMEM media for 6 hours (three replicates in each condition).

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Gene expression analysis in renal cell carcinoma

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