Dysregulation at multiple points of the kynurenine pathway is a ubiquitous feature of renal cancer: implications for tumour immune evasion.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), the first step in the kynurenine pathway (KP), is upregulated in some cancers and represents an attractive therapeutic target given its role in tumour immune evasion. However, the recent failure of an IDO inhibitor in a late phase trial raises questions about this strategy. METHODS: Matched renal cell carcinoma (RCC) and normal kidney tissues were subject to proteomic profiling. Tissue immunohistochemistry and gene expression data were used to validate findings. Phenotypic effects of loss/gain of expression were examined in vitro. RESULTS: Quinolate phosphoribosyltransferase (QPRT), the final and rate-limiting enzyme in the KP, was identified as being downregulated in RCC. Loss of QPRT expression led to increased potential for anchorage-independent growth. Gene expression, mass spectrometry (clear cell and chromophobe RCC) and tissue immunohistochemistry (clear cell, papillary and chromophobe), confirmed loss or decreased expression of QPRT and showed downregulation of other KP enzymes, including kynurenine 3-monoxygenase (KMO) and 3-hydroxyanthranilate-3,4-dioxygenase (HAAO), with a concomitant maintenance or upregulation of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in the NAD+ salvage pathway. CONCLUSIONS: Widespread dysregulation of the KP is common in RCC and is likely to contribute to tumour immune evasion, carrying implications for effective therapeutic targeting of this critical pathway.

An analysis of the impact of pre-analytical factors on the urine proteome: Sample processing time, temperature, and proteolysis.

PURPOSE: We have examined the impact of sample processing time delay, temperature, and the addition of protease inhibitors (PIs) on the urinary proteome and peptidome, an important aspect of biomarker studies. EXPERIMENTAL DESIGN: Ten urine samples from patients with varying pathologies were each divided and PIs added to one-half, with aliquots of each then processed and frozen immediately, or after a delay of 6 h at 4°C or room temperature (20-22°C), effectively yielding 60 samples in total. Samples were then analyzed by 2D-PAGE, SELDI-TOF-MS, and immunoassay. RESULTS: Interindividual variability in profiles was the dominant feature in all analyses. Minimal changes were observed by 2D-PAGE as a result of delay in processing, temperature, or PIs and no changes were seen in IgG, albumin, ß2 -microglobulin, or α1 -microglobulin measured by immunoassay. Analysis of peptides showed clustering of some samples by presence/absence of PIs but the extent was very patient-dependent with most samples showing minimal effects. CONCLUSIONS AND CLINICAL RELEVANCE: The extent of processing-induced changes and the benefit of PI addition are patient- and sample-dependent. A consistent processing methodology is essential within a study to avoid any confounding of the results.

Predicting response to bevacizumab in ovarian cancer: a panel of potential biomarkers informing treatment selection.

PURPOSE: The aim of this study was to identify and validate novel predictive and/or prognostic serum proteomic biomarkers in patients with epithelial ovarian cancer (EOC) treated as part of the phase III international ICON7 clinical trial. EXPERIMENTAL DESIGN: ICON7 was a phase III international trial in EOC which showed a modest but statistically significant benefit in progression-free survival (PFS) with the addition of bevacizumab to standard chemotherapy. Serum samples from 10 patients who received bevacizumab (five responders and five nonresponders) were analyzed by mass spectrometry to identify candidate biomarkers. Initial validation and exploration by immunoassay was undertaken in an independent cohort of 92 patients, followed by a second independent cohort of 115 patients (taken from across both arms of the trial). RESULTS: Three candidate biomarkers were identified: mesothelin, fms-like tyrosine kinase-4 (FLT4), and α1-acid glycoprotein (AGP). Each showed evidence of independent prognostic potential when adjusting for high-risk status in initial (P < 0.02) and combined (P < 0.01) validation cohorts. In cohort I, individual biomarkers were not predictive of bevacizumab benefit; however, when combined with CA-125, a signature was developed that was predictive of bevacizumab response and discriminated benefit attributable to bevacizumab better than clinical characteristics. The signature showed weaker evidence of predictive ability in validation cohort II, but was still strongly predictive considering all samples (P = 0.001), with an improvement in median PFS of 5.5 months in signature-positive patients in the experimental arm compared with standard arm. CONCLUSIONS: This study shows a discriminatory signature comprising mesothelin, FLT4, AGP, and CA-125 as potentially identifying those patients with EOC more likely to benefit from bevacizumab. These results require validation in further patient cohorts.

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

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 ß-dystroglycan, the transmembrane subunit of the DAG1 gene product. VHL-dependent changes in the form of ß-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.

Proteomic analysis of formalin-fixed paraffin-embedded renal tissue samples by label-free MS: assessment of overall technical variability and the impact of block age.

PURPOSE: Protein profiling of formalin-fixed paraffin-embedded (FFPE) tissues has enormous potential for the discovery and validation of disease biomarkers. The aim of this study was to systematically characterize the effect of length of time of storage of such tissue blocks in pathology archives on the quality of data produced using label-free MS. EXPERIMENTAL DESIGN: Normal kidney and clear cell renal cell carcinoma tissues routinely collected up to 10 years prior to analysis were profiled using LC-MS/MS and the data analyzed using MaxQuant. Protein identities and quantification data were analyzed to examine differences between tissue blocks of different ages and assess the impact of technical and biological variability. RESULTS: An average of over 2000 proteins was seen in each sample with good reproducibility in terms of proteins identified and quantification for normal kidney tissue, with no significant effect of block age. Greater biological variability was apparent in the renal cell carcinoma tissue, possibly reflecting disease heterogeneity, but again there was good correlation between technical replicates and no significant effect of block age. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that archival storage time does not have a detrimental effect on protein profiling of FFPE tissues, supporting the use of such tissues in biomarker discovery studies.

Impact of pre-analytical factors on the proteomic analysis of formalin-fixed paraffin-embedded tissue.

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a tremendous potential resource for biomarker discovery, with large numbers of samples in hospital pathology departments and links to clinical information. However, the cross-linking of proteins and nucleic acids by formalin fixation has hampered analysis and proteomic studies have been restricted to using frozen tissue, which is more limited in availability as it needs to be collected specifically for research. This means that rare disease subtypes cannot be studied easily. Recently, improved extraction techniques have enabled analysis of FFPE tissue by a number of proteomic techniques. As with all clinical samples, pre-analytical factors are likely to impact on the results obtained, although overlooked in many studies. The aim of this review is to discuss the various pre-analytical factors, which include warm and cold ischaemic time, size of sample, fixation duration and temperature, tissue processing conditions, length of storage of archival tissue and storage conditions, and to review the studies that have considered these factors in more detail. In those areas where investigations are few or non-existent, illustrative examples of the possible importance of specific factors have been drawn from studies using frozen tissue or from immunohistochemical studies of FFPE tissue.

Proteomics and the search for biomarkers for renal cancer.

Worldwide, over 273,000 people are diagnosed with renal cancer each year. Approximately one third of patients present with locally advanced or metastatic disease and although surgery is largely curative in those with localised disease, about one third of these patients will subsequently relapse. Renal cancer is resistant to conventional chemotherapy and radiotherapy but increased understanding of the underlying tumour biology is leading to the use and development of targeted therapies, such as tyrosine kinase inhibitors targeting pathways downstream of the von Hippel Lindau tumour suppressor gene. There are no biomarkers in routine clinical use in renal cancer but they are urgently needed for enabling earlier diagnosis, differential diagnosis of histological subtypes and their variants, risk stratification to allow personalised follow-up and prediction and monitoring of response and toxicity to targeted therapies. Several groups are now applying proteomic strategies in biomarker discovery in renal cancer. We review the progress being made in these studies and discuss the strategies needed to ensure effective translation of findings to the clinic.

Determination of the consequences of VHL mutations on VHL transcripts in renal cell carcinoma.

Genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumour suppressor gene are common in sporadic conventional (clear cell) renal cell carcinoma (ccRCC). The effects on VHL expression are unknown but increased understanding may be relevant clinically, either in terms of prognosis or in therapy selection. We have examined the expression of VHL mutant RNA in 84 ccRCC tumours previously screened for mutations in genomic DNA, 56 of which contained 52 unique mutations or polymorphisms. Based on the predicted change to the primary amino acid sequence, 24 of the mutations were missense, 11 resulted in frameshifts with premature truncation, 9 resulted in immediate truncation at the site of the mutation and 2 were frameshifts which extended the reading frame beyond the normal stop codon. Nine tumours had intronic variants, including substitution of invariant residues at splice sites, deletion of nucleotides spanning the exon-intron junction, an intronic variant of unknown function and the polymorphism c.463+43A>G. Four variants were identified which were present in genomic DNA but not in mRNA. Three of these, all encoding apparent missense changes to the primary amino acid sequence, were located close to the ends of exons, reduced the strength of the splice site and function as null rather than missense variants. One nonsense variant was not detectable in mRNA but all other mutations resulting in premature truncation codons (PTCs) were, suggesting truncating VHL mutations may potentially generate truncated VHL protein. An intronic variant, c.341­11T>A, previously regarded as of unknown function, is associated with an increased level of skipping of exon 2 and may, therefore, reduce production of pVHL. Our data show that the biological consequences of VHL mutations are not necessarily predictable from the sequence change of the mutation and that for the majority of VHL mutations, the potential for the generation of mutant protein exists.

Shotgun proteomics of human bile in hilar cholangiocarcinoma.

The need to find biomarkers for hepatobiliary diseases including cholangiocarcinoma (CCA) has led to an interest in using bile as a proximal fluid in biomarker discovery experiments, although there are inherent challenges both in its acquisition and analysis. The study described here greatly extends previous studies that have started to characterise the bile proteome. Bile from four patients with hilar CCA was depleted of albumin and immunoglobulin G and analysed by GeLC-MS/MS. The number of proteins identified per bile sample was between 378 and 741. Overall, the products of 813 unique genes were identified, considerably extending current knowledge of the malignant bile proteome. Of these, 268 were present in at least 3 out of 4 patients. This data set represents the largest catalogue of bile proteins to date and together with other studies in the literature constitutes an important prelude to the potential promise of expression proteomics and subsequent validation studies in CCA biomarker discovery.

Upregulation of Hic-5 in glomerulosclerosis and its regulation of mesangial cell apoptosis.

Glomerulosclerosis is characterized by the loss of glomerular cells by apoptosis and deposition of collagen type I into the normal collagen IV-containing mesangial matrix. We sought to determine the alterations that might contribute to these changes by performing proteomic analysis of rat mesangial cell lysates comparing cells cultured on normal collagen type IV to those grown on abnormal collagen type I surfaces. Subculture on collagen type I was associated with changed expression of several proteins, including a significant upregulation of the paxillin-like LIM protein, hydrogen-peroxide-induced clone 5 (Hic-5), and increased the susceptibility of the cells to apoptosis in response to physiological triggers. When we knocked down Hic-5 (using siRNA), we found mesangial cells grown on collagen type I were protected from apoptosis to the same degree as untreated cells grown on collagen type IV. Further we found that the level of Hic-5 in vivo was almost undetectable in control rats but increased dramatically in the glomerular mesangium of remnant kidneys 90 and 120 days after subtotal nephrectomy. This induction of Hic-5 paralleled the upregulation of mesangial collagen type I expression and glomerular cell apoptosis. Our results suggest that Hic-5 is pivotal in mediating the response of mesangial cells to attachment on abnormal extracellular matrix during glomerular scarring.

Analysis of VHL Gene Alterations and their Relationship to Clinical Parameters in Sporadic Conventional Renal Cell Carcinoma.

PURPOSE: This study aimed to carry out a comprehensive analysis of genetic and epigenetic changes of the von Hippel Lindau (VHL) gene in patients with conventional (clear cell) renal cell carcinoma and to determine their significance relative to clinicopathologic characteristics and outcome. EXPERIMENTAL DESIGN: The VHL status in 86 conventional renal cell carcinomas was determined by mutation detection, loss of heterozygosity (LOH), and promoter methylation analysis, extending our original cohort to a total of 177 patients. Data were analyzed to investigate potential relationships between VHL changes, clinical parameters, and outcome. RESULTS: LOH was found in 89.2%, mutation in 74.6%, and methylation in 31.3% of evaluable tumors; evidence of biallelic inactivation (LOH and mutation or methylation alone) was found in 86.0% whereas no involvement of VHL was found in only 3.4% of samples. Several associations were suggested, including those between LOH and grade, nodal status and necrosis, mutation and sex, and methylation and grade. Biallelic inactivation may be associated with better overall survival compared with patients with no VHL involvement, although small sample numbers in the latter group severely limit this analysis, which requires independent confirmation. CONCLUSIONS: This study reports one of the highest proportions of conventional renal cell carcinoma with VHL changes, and suggests possible relationships between VHL status and clinical variables. The data suggest that VHL defects may define conventional renal cell carcinomas but the clinical significance of specific VHL alterations will only be clarified by the determination of their biological effect at the protein level rather than through genetic or epigenetic analysis alone. (Clin Cancer Res 2009;15(24):7582-92).

Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel-Lindau transfectant cell line model.

The von Hippel-Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL-defective RCC cell line UMRC2 by transfection with vector control or VHL (-/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin-affinity chromatography and analysis by GeLC-MS/MS, VHL-associated changes in plasma membrane proteins were analysed. Comparative analysis of -/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the alpha 3 and beta1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL-defective cells. Western blotting confirmed these changes and also revealed VHL-dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient-matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL-dependent proteins that may be important in tumorigenesis.

Understanding and managing renal cell carcinoma: can proteomic studies contribute to clinical practice?

Renal cell carcinoma (RCC) is associated with a poor prognosis and there is a need for biomarkers to assist at all stages of disease management including diagnosis, prognosis, monitoring for relapse and predicting response to therapy. Additionally, identification of new therapeutic targets is a priority. Increased understanding of disease pathogenesis and the molecular changes underlying tumour formation is essential to assist in the rational design of such molecules. As the technologies underlying proteomics-based research have developed, they have been applied extensively to the analysis of cancers including RCC, with tissues, cell lines and biological fluids being used for analysis. A number of approaches have been adopted including two-dimensional polyacrylamide gel electrophoresis and mass spectrometry profiling of intact proteins, shotgun mass spectrometry-based profiling at the peptide level, antibody arrays and strategies analysing the immune response to tumours with a view to identifying tumour-associated antigens. Although these studies are still at a relatively early stage, promising results have been reported with some being taken forward to preliminary validation. The challenge now is to build on these initial efforts, focusing particularly on interrogating the less readily accessible, lower abundance proteome and implementing large-scale validation studies to develop potential markers, antigens and targets and facilitate translation of suitable findings into the clinic.

Circulating markers of biliary malignancy: opportunities in proteomics?

Cholangiocarcinoma, a primary liver tumour that arises from biliary epithelial cells, is increasing in incidence and has poor prognosis. Diagnosis is difficult, particularly in patients with primary sclerosing cholangitis, who are at risk of developing the disease. Timely diagnosis is essential because surgical resection in early disease remains the only cure. The lack of a sensitive and specific early diagnostic marker and of alternative treatments are the main reasons why patients have limited survival. The use of proteomic-based approaches, which analyse the physiological or pathological complement of proteins (ie, the proteome) in cells, tissues, or biological fluids, has received substantial interest in biomarker discovery. Proteomics complements genomic studies and examines functional end-units quantitatively and qualitatively, including post-translational modifications which might vary with disease and might have key roles in protein function or localisation. Major advances in technology and bioinformatics have enhanced proteomic studies, resulting in increased understanding of the pathogenesis of many diseases and in biomarker discovery with effective use of tissues, cell lines, and biological fluids. We review the current status and promise of proteomic-based approaches in biomarker discovery for cholangiocarcinoma.

Proteomic profiling identifies afamin as a potential biomarker for ovarian cancer.

PURPOSE: To discover and validate serum glycoprotein biomarkers in ovarian cancer using proteomic-based approaches. EXPERIMENTAL DESIGN: Serum samples from a "discovery set" of 20 patients with ovarian cancer or benign ovarian cysts or healthy volunteers were compared by fluorescence two-dimensional differential in-gel electrophoresis and parallel lectin-based two-dimensional profiling. Validation of a candidate biomarker was carried out with Western blotting and immunoassay (n = 424). RESULTS: Twenty-six proteins that changed significantly were identified by mass spectrometric sequencing. One of these, confirmed by Western blotting, was afamin, a vitamin E binding protein, with two isoforms decreasing in patients with ovarian cancer. Validation using cross-sectional samples from 303 individuals (healthy controls and patients with benign, borderline, or malignant ovarian conditions and other cancers) assayed by ELISA showed significantly decreased total afamin concentrations in patients with ovarian cancer compared with healthy controls (P = 0.002) and patients with benign disease (P = 0.046). However, the receiver operating characteristic areas for total afamin for the comparison of ovarian cancer with healthy controls or benign controls were only 0.67 and 0.60, respectively, with comparable figures for CA-125 being 0.92 and 0.88 although corresponding figures for a subgroup of samples analyzed by isoelectric focusing for afamin isoform 2 were 0.85 and 0.79. Analysis of a further 121 samples collected prospectively from 9 patients pretreatment through to relapse indicated complementarity of afamin with CA-125, including two cases in whom CA-125 was noninformative. CONCLUSIONS: Afamin shows potential complementarity with CA-125 in longitudinal monitoring of patients with ovarian cancer, justifying prospective larger-scale investigation. Changes in specific isoforms may provide further information.

Proteomic evaluation of pathways associated with dexamethasone-mediated apoptosis and resistance in multiple myeloma.

We have used global protein expression analysis to characterize the pathways of dexamethasone-mediated apoptosis and resistance in myeloma. Analysis of MM.1S cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) identified a series of proteins that were up- and downregulated following dexamethasone treatment. Downregulated proteins included proteins involved in cell survival and proliferation, whereas upregulated proteins were involved in post-translational modification, protein folding and trafficking. A comparison with published gene expression studies identified FK binding protein 5 (FKBP5) (also known as FKBP51), a key regulatory component of the Hsp90-steroid-receptor complex to be increased at the mRNA and protein level postdexamethasone exposure. Quantitative real time polymerase chain reaction and 2D-PAGE analysis of the dexamethasone resistant cell line MM.1R demonstrated no increase in FKBP5, consistent with its association with dexamethasone-mediated apoptosis. Western blot analysis of FKBP5 and other members of the Hsp90-receptor complex showed an increase in FKBP5 whilst FKBP4 (also known as FKBP52) and Hsp90 expression remained constant. No changes were observed in MM.1R. In conclusion, we demonstrated that following steroid receptor signalling, the cell carries out a number of adaptive responses prior to cell death. Interfering with these adaptive responses may enhance the myeloma killing effect of dexamethasone.

Key clinical issues in renal cancer: a challenge for proteomics.

Renal cancer has many clinical challenges which proteomics is ideally placed to address. The issues cover all aspects of the disease including diagnosis, prognosis, treatment selection and monitoring to detect metastatic disease. In all cases novel biomarkers would considerably help in clinical management and with the relative resistance to conventional chemotherapy and radiotherapy, a better understanding of the underlying pathogenesis may contribute to the much needed development of novel therapeutic targets and the better use of promising new anti-angiogenic treatments. This review briefly highlights some of the clinical issues and describes proteomics-based approaches generally, before focussing on reviewing the proteomic studies to date in this area.

S. pombe CLASP needs dynein, not EB1 or CLIP170, to induce microtubule instability and slows polymerization rates at cell tips in a dynein-dependent manner.

The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.Delta and tip1.Delta disrupted linear growth, corrupting peg1 (+) did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 (+) manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170.

Proteomic identification of a role for the von Hippel Lindau tumour suppressor in changes in the expression of mitochondrial proteins and septin 2 in renal cell carcinoma.

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes. One of the findings confirmed by Western blotting was up-regulation of the mitochondrial protein ubiquinol cytochrome c reductase complex core protein 2 following VHL transfection, a change that was also observed in two other cell line backgrounds. A marked decrease in expression of this and several other mitochondrial proteins was demonstrated in RCC tissues and using VHL-transfectants, several were shown to exhibit VHL-dependent regulation. Thus, VHL may contribute to the decreased mitochondrial function seen in RCC. A form of septin 2 down-regulated following VHL transfection was also identified. Septin 2 was up-regulated in 12/16 RCCs, while alteration of the form present was also observed in 1/3 tumours analysed. Thus, increased expression of septin 2 is a common event in RCC and protein modification may also alter septin 2 function in a subset of tumours.