Clinical Cancer Research CR Helping Patients AACR Membership
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 13, 27, January 1, 2007. doi: 10.1158/1078-0432.CCR-06-0824
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seliger, B.
Right arrow Articles by Lichtenfels, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seliger, B.
Right arrow Articles by Lichtenfels, R.

Human Cancer Biology

Ubiquitin COOH-Terminal Hydrolase 1: A Biomarker of Renal Cell Carcinoma Associated with Enhanced Tumor Cell Proliferation and Migration[?Q1: Running head: UCHL1, a Biomarker of RCC. Short title OK?Q1]

Barbara Seliger1,2, Alla Fedorushchenko1, Walburgis Brenner3, Angelika Ackermann2, Derek Atkins2,4, Samir Hanash5 and Rudolf Lichtenfels1,2

Authors' Affiliations: 1 Institute of Medical Immunology, Martin-Luther-University Halle-Wittenberg, Halle, Germany; 2 IIIrd Department of Internal Medicine, Johannes-Gutenberg University; 3 Clinic for Urology, University Clinic Mainz, Mainz, Germany; 4 Bayer HealthCare AG, Wuppertal, Germany; and 5 Fred Hutchinson Cancer Research Center, Seattle, Washington

Requests for reprints: Barbara Seliger, Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Magdeburger Strasse 2, 06112 Halle, Germany. Phone: 49-345-557-4054; Fax: 49-345-557-4055; E-mail: Barbara.Seliger{at}medizin.uni-halle.de.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Renal cell carcinoma (RCC) accounts for 2% to 3% of all malignancies. It represents one of the most radiation- and chemotherapy-resistant tumors and surgical resections are only effective in organ-defined disease. However, RCC is an immunogenic tumor with response rates to immunotherapies between 10% and 20% of the treated patients. Due to the currently inefficient therapies and the low 5-year survival rates of RCC patients, novel diagnostic, prognostic, and therapeutic markers are urgently needed for this disease.

Experimental Design: Proteome-based approaches were used to identify (a) differentially expressed proteins in RCC compared with normal kidney epithelium and (b) proteins that are able to induce an antibody response in RCC patients. Based on these experiments, a promising candidate was subsequently validated by reverse transcription-PCR, Western blot analyses, and immunohistochemistry. In addition, functional assays were done in generated transfectants.

Results: The ubiquitin COOH-terminal hydrolase L1 (UCHL1) was found to be differentially expressed in both RCC lesions and RCC cell lines and immunoreactive using patients' sera. UCHL1 expression was often down-regulated in primary RCC when compared with normal kidney epithelium but dependent on the RCC subtype, the von Hippel-Lindau phenotype, and the tumor grading. Moreover, the frequency and the level of UCHL1 expression were higher in metastases when compared with primary RCC lesions. Gain-of-function transfectants exhibited a significant higher proliferation and migration rate than UCHL1-negative RCC cells.

Conclusions: UCHL1 expression seems to be associated with the metastatic phenotype of RCC and therefore might serve as potential biomarker for the diagnosis and prognosis of RCC patients.

The degradation of ubiquitin-conjugated cellular proteins by the proteasome system plays a key role in many cellular processes, including cell cycle control, modulation of the cellular response to stress and extracellular factors, DNA repair, immune and inflammatory responses, regulation of transcription, receptor function, oncoprotein degradation, and antigen presentation. There exists significant information about enzymes that ligate ubiquitin to target proteins. Deubiquitination, the reversal of this modification, also participates in regulatory cellular processes and is carried out by several deubiquitinating molecules (1, 2). Thus far, 60 to 70 members of the ubiquitin-specific protease family have been predicted in the human genome, which can be divided into four major classes. The best characterized are the ubiquitin processing proteins (3) and the ubiquitin COOH-terminal hydrolases (UCH; refs. 4). Because UCH catalyzes the hydrolysis of COOH-terminal ubiquityl esters and amides (5), it removes ubiquitin from ubiquitinated cellular proteins, thereby preventing them from targeted degradation via the proteasome pathway. One member of the UCH family is represented by UCHL1, which has a dual function: a hydrolase activity that removes small COOH-terminal ubiquitin to generate the ubiquitin monomer and a dimerization-dependent ubiquitin ligase activity. UCHL1 expression exhibits a marked tissue specificity (6). UCHL1 is expressed in testis and in neuronal tissues at all stages of neuronal differentiation and plays an important role in the development of specific forms of the Parkinson disease (7–9). Using serial analysis of gene expression and proteome-based approaches, UCHL1 has also been shown to be overexpressed in primary non–small lung carcinoma lesions, lung cancer cell lines, in acute lymphoblastic leukemia, esophageal squamous cell carcinoma, colorectal cancer, thyroid carcinoma, pancreatic cancer, and myeloma (10–22). This aberrant expression is strongly associated with advanced diseases stages and diminished patient survival in colorectal, pancreatic, and non–small lung carcinoma. In addition, increased UCHL1 activity was observed in c-myc-overexpressing cells, which correlated with apoptotic resistance induced by inhibition of the proteasome (20).

Renal cell carcinoma (RCC) accounts for 2% to 3% of malignancies and represents a heterogeneous disease. The most common histologic subtype accounting for 70% to 80% of cases is the clear cell RCC. This sporadic RCC subtype is often associated with loss of function of the von Hippel-Lindau (VHL) gene expression due to loss of heterozygosity, mutations, and promoter methylation (23, 24). VHL is the substrate of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF)-1{alpha} for ubiquitination- and oxygen-dependent proteasomal degradation (25). HIF-1{alpha} is overexpressed in VHL-deficient renal epithelial cells and clear cell RCC leading to increased transcription of HIF-1-regulated genes involved in adaptive cellular responses to hypoxia and in tumor vascularization like the vascular endothelial growth factor (26–28).

Surgical resection is the most effective treatment for organ-defined disease, whereas, unfortunately, RCC is one of the most radiation- and chemotherapy-resistant tumors (29). However, the development of signal transduction modulators, such as sorafenib, a tyrosine kinase inhibitor, has been approved recently for the treatment of advanced RCC. Despite this progress, novel diagnostic, prognostic, and therapeutic markers are urgently needed for this disease. Thus far, by implementation of classic proteomics and PROTEOMEX, a combination of proteome analysis and serology, a large number of differentially expressed proteins have been described recently in RCC lesions and cell lines when compared with normal kidney epithelium (30–32). These proteins are involved in signal transduction, cytoskeletal formation, antigen processing, cell proliferation, and differentiation or represent metabolic enzymes (25, 33–37). Furthermore, we here show heterogeneous UCHL1 expression in both RCC cell lines and surgically removed RCC lesions of different subtypes, staging, and grading and the VHL tumor phenotype. Its aberrant expression is associated with disease progression, enhanced proliferation, and migration of RCC cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials. Two-dimensional gel electrophoresis (2DE) equipment and supply material were purchased from GE Healthcare Bio-Sciences Europe GmbH (Freiburg, Germany), whereas chemicals were mainly obtained from Applichem (Darmstadt, Germany), Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany), or ICN Biochemicals GmbH (Eschwege, Germany). All chemicals used were of analytic grade.

Cell lines and tissue culture. The different human RCC cell lines used in the studies were established from patients with primary RCC of clear cell type. Corresponding autologous normal kidney epithelium cell lines immortalized by SV40LT transformation have been described in detail elsewhere (33). All RCC cell lines were maintained in high-glucose DMEM supplemented with 10% FCS (Life Technologies, Karlsruhe, Germany), 2 mmol/L glutamine, 100 units/mL streptomycin, 1 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate. The SV40LT transformants were cultured in complete medium containing 600 µg/mL hygromycin B (Roche Diagnostics GmbH, Mannheim, Germany).

Tissue samples and serum. The biopsy specimens used in the studies were either taken from a constantly growing local RCC tumor bank [Table 1 , 2DE panel, reverse transcription-PCR (RT-PCR) panel, and RCC cell line panel], which is composed of a large series of primary RCC lesions, corresponding normal kidney epithelium, and metastases obtained from patients who had undergone radical nephrectomy along with clinical data. None of these patients had received preoperative therapy. In addition, biopsy specimens collected at the Institute of Pathology, University Witten-Herdecke, Witten, Germany (Table 1, primary RCC versus metastasis panel and VHL-IHC panel) were analyzed. Histopathologic classification of each tumor was done according to the criteria proposed by Thoenes et al. (38).


View this table:
[in this window]
[in a new window]

  		Table 1. Histopathologic features of RCC specimen cell lines with follow-up data of patients and UCHL1 expression data

 

Figure 1
View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Dimensional spot pattern of RCC cell lines. Left, representative spot pattern of the RCC cell line MZ1257RC. Two hundred micrograms of the lysate were separated in the first dimension on a nonlinear pH 3 to 10 pH Immobiline DryStrip. Separation in the second dimension was carried out on a 16%T/2.5% acrylamide/PDA gel. The gel series was silver stained and subsequently analyzed with the Proteomweaver software package (Bio-Rad, Munich, Germany). Right, inserts, representative zoomed in sections of the boxed segment in the overview gel for the RCC lines MZ1257RC, MZ1940RC, and MZ2733RC as well as for the corresponding normal kidney cell line MZ2733NN. Arrows and {circ}, the dominant UCHL1 spot P3 as well as the P1 and P2 isoforms of UCHL1 defined by immunostaining with the UCHL1-specific antibody PG9500.

 
All serum samples were isolated from venous blood of either RCC patients or normal volunteers after informed consent was obtained from each individual.

2DE, immunoblotting, and mass spectrometry. 2DE was done as described recently (33) using 200 to 800 µg total protein (cell lines or tissues), which were loaded on immobilized pH gradient strips [pH 3-10 NL (cell lines) or pH 4-7 and pH 6-11 (tissues); Immobiline DryStrips, GE Healthcare Bio-Sciences]. Isoelectric focusing; second-dimensional SDS-PAGE separation; gel stainings with SYPRO Ruby, colloidal Coomassie Blue, and silver stainings; and gel documentation and mass spectrometry were done as described previously (33, 37).

Reverse transcription-PCR analysis. For determination of UCHL1 transcription, RT-PCR was done using the UCHL1-specific primer pairs 5'-TGTGGCACAATCGGACTTAT-3' (sense) and 5'-AGGCATTCGTCCATCAAGTT-3' (antisense) using 35 cycles. RT-PCR with ß-actin-specific primers 5'-GAAGCATTTGCGGTGGACGAT-3' (sense) and 5'-TCCTGTGGCATCCACGAAACT-3' (antisense) served as control. Amplification products were size fractionated on a 1% ethidium bromide containing agarose gel. The resulting expression profiles were documented on Black and White instant Polaroid films (Polaroid 667, Polaroid Europe BV, Enschede, the Netherlands), subsequently digitalized using a flat bed image scanner (GE Healthcare Bio-Sciences), and finally subjected to one-dimensional image evaluation by applying the advanced image data analyzer software package (Image analyzer version 4.11, Raytest GmbH, Berlin, Germany). Relative UCHL1 expression levels are given as arbitrary units by setting the peak values of corresponding ß-actin amplicons to 1.

Western blot analysis. Twenty micrograms of total protein/lane were subjected to Western blot analysis as described previously (35) using the anti-UCHL1-specific polyclonal rabbit antibody (PG9500, Affinity Research Products Ltd., Exeter, United Kingdom). Staining with the anti-ß-actin-specific monoclonal antibody AC15 (ab6276; Abcam Ltd., Cambridge, United Kingdom) served as a loading control. On staining with suited horseradish peroxidase–conjugated secondary antibodies (P0217 or P0260, DAKO, Hamburg, Germany), the immunostainings were visualized using a chemiluminescent detection kit (LumiLight Western blot substrate, Roche Diagnostics GmbH).

Immunohistochemistry. Immunohistochemical stainings were done with an anti-UCHL1-specific polyclonal rabbit antibody (generous gift from S. Hanash, Fred Hutchinson Cancer Research Center, Seattle, WA; ref. 17) as described previously (35) and with the anti-UCHL1-specific monoclonal antibody (PG9500). For antigen retrieval, consecutive sections were incubated for 8 and 6 min in citrate buffer in a microwave oven, respectively, followed by a washing procedure with TBS and an additional incubation with normal swine serum (dilution 1:5; DAKO Diagnostika GmbH, Hamburg, Germany) for 10 min. Slides were incubated with the primary antibody for 1 hour at room temperature. Detection was done using the labeled streptavidin-biotin peroxidase kit and amino-9-ethylcarbazol as described recently (DAKO). Negative controls were done by omitting the primary antibody. The design of the microtissue arrays used for the immunohistochemical evaluations has been described previously (36).

Quantitative analysis of immunohistochemistry for each tumor was done according to the following score: 0% to <25% of stained cells are classified as negative (score: –), >25% to >50% of stained cells exhibit a heterogeneous expression (score: +), whereas >50% of stained cells are classified as positive (score: ++).

Transfection. RCC cells were stably transfected with an UCHL1 expression vector using LipofectAMINE (Invitrogen, Karlsruhe, Germany). UCHL1 transfectants were generated by selection in 1 mg/mL G418. The UCHL1 expression vector is based on plasmid p46 carrying the cytomegalovirus promoter and the neomycin resistance (neoR) gene (39).

Proliferation assays. For proliferation assays, 1 x 104 cells per well were seeded in six-well plates in complete DMEM. At different time points, across days 0 and 21, wild-type (WT) cells and mock and UCHL1 transfectants of two independent wells were harvested after trypsinization and counted using a hemocytometer chamber. The growth rate constants (kp) were calculated by linear regression following semilogarithmic plotting of the cell counts versus the time range. Doubling times were calculated by dividing ln2 with the respective growth rate constants.

Migration assays. Cell migration was determined by seeding 1 x 104 cells in 100 µL DMEM on top of noncoated polyethylene terephthalate of Transwell cell culture inserts (24-well inserts, 8.0-µm pore size; Costar, Corning Inc., Corning, NY), whereas the lower chamber was filled with 0.6 mL DMEM. The 24-well plates were incubated for 24 h at 37°C in a CO2 incubator before the nonmigrating cells in the inserts were scraped off; membranes were fixed in ethanol and stained for 40 min with trypan blue. Cells that had migrated through the membranes were visualized with a microscope and quantified by determination of the cell number in three randomly chosen visual fields at x200 magnification.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Identification of UCHL1 overexpression in RCC lesions using proteome-based technologies. To identify proteins that were differentially expressed in RCC, both RCC lesions and normal kidney epithelium as well as RCC and normal kidney epithelium representing cell lines were subjected to classic proteome analysis using 2DE PAGE. The identities of differentially expressed proteins were determined by mass spectrometry following in-gel trypsin digestion of respective protein spots and subsequent extraction of the resulting peptide fragments (33–35, 37). In particular, the expression pattern and features of one of the differentially expressed proteins, UCHL1, which was initially detected by comparing the expression profiles of cell lines derived from primary RCC of clear cell type with a cell line representing normal kidney epithelium (Fig. 1, spot P3 ) as well as via PROTEOMEX analyses of RCC cell lines (35) revealing that autoantibodies in sera obtained from both RCC patients and healthy volunteers are able to recognize this target structure, led us to extend the validation of this candidate protein. First, the identity of this protein spot was further confirmed by 2DE Western blot analysis of RCC cell lines using an anti-UCHL1-specific antibody, which in line with previous findings established in lung cancer (17) led to the detection of up to two additional UCHL1 variants (data not shown), indicating that UCHL1 is prone to undergo posttranslational modifications. The various UCHL1 variants detectable in renal cell lines/tissues are indicated in the zoomed sections of the respective gel segments shown in Fig. 1 (spots P1 and P2). However, the nature of the given modifications of these additional UCHL1 variants is thus far unknown because of the rather low expression levels. In addition, the 2DE-based expression profiling of a panel of biopsy specimen representing primary RCC lesions of various subtypes along with corresponding normal renal tissue sections was done confirming that UCHL1 is heterogeneously expressed in these samples.

The analysis of 30 primary RCC lesions and adjacent normal kidney epithelia is in line with the previous findings in RCC cell lines. UCHL1 exhibits a variable protein expression pattern, which implies that UCHL1 is often down-regulated in primary tumors when compared with normal kidney epithelium. Twenty-three of 24 normal kidney epithelium tissues, which yielded telling spot pattern at the respective gel areas, exert high levels of UCHL1 expression and only 1 of 24 samples (biopsy 2884) totally lacks UCHL1 expression (Table 1, 2DE panel).

Validation of UCHL1 mRNA and/or protein expression in RCC lesions and RCC cell lines. The heterogeneous UCHL1 protein expression in primary RCC lesions defined by proteomics was further confirmed by RT-PCR and Western blot analyses (Fig. 2 ; Table 1, RT-PCR panel and RCC cell line panel). The mRNA expression profiling of a series of RCC biopsy samples and of corresponding normal renal epithelium (Fig. 2A; Table 1, RT-PCR panel) as well as of cell lines obtained from primary or metastatic RCC (Fig. 2B; Table 1, RCC cell line panel) confirmed heterogeneous UCHL1 expression levels. However, only few samples displayed a complete loss of UCHL1 mRNA expression.


Figure 2
View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Evaluation of UCHL1 mRNA and protein expression. A, representative mRNA profiles of RCC lesions and corresponding normal kidney epithelium. The roman numbers refer to the RT-PCR panel listed in Table 1. B, representative mRNA profiles of established RCC cell lines and a metastasis-derived cell line, as listed in Table 1 in the RCC cell line panel, determined by RT-PCR as described in Materials and Methods. C, representative protein expression profiles of RCC cell lines determined by Western blotting as described in Materials and Methods. Analyses using ß-actin-specific primers or an anti-ß-actin-specific antibody served as loading controls for RT-PCR and Western blots, respectively.

 
In contrast, the frequency of aberrant UCHL1 protein expression was significantly higher both in lesions as defined via proteomic profiling (Table 1, 2DE panel) as well as in cell lines derived from primary or metastatic RCC as determined by Western blot analysis (Fig. 2C; Table 1, RCC cell line panel). Seven of 24 primary RCC lesions revealed a complete loss of UCHL1 protein expression, whereas 12 RCC samples exhibit a reduced UCHL1 expression when compared with the corresponding normal kidney epithelium. However, no association between the UCHL1 expression levels and the survival rate of patients was found (Table 1, 2DE panel and RT-PCR panel). Furthermore, the frequency of deficient UCHL1 protein expression in RCC cell lines (Fig. 2C; Table 1, RCC cell line panel) seems to be higher when compared with the corresponding RT-PCR results (Fig. 2B). For example, UCHL1 specific mRNA, but not protein expression, was found in the RCC cell line MZ1790RC and the metastasis-derived cell line MZ1851LN (Fig. 2B and C). Moreover, MZ1774RC cells express less UCHL1 protein than expected when compared with the corresponding RT-PCR signal. These data suggest that in addition to the occurrence of various of UCHL1 isoforms (Fig. 1) also posttranscriptional regulation of UCHL1 might occur.

Distinct UCHL1 expression pattern in RCC subtypes and normal kidney epithelium. Using RCC tissue microarrays, the cellular distribution of the UCHL1 protein was monitored by immunohistochemistry in 87 primary lesions of different RCC subtypes, 9 oncocytomas, and autologous normal kidney epithelia as described previously (36). Abundant cytoplasmic staining of UCHL1 was observed in normal kidney epithelium, such as the distal and proximal tubule system, the mesangium of the glomerula, and the epithelium of the Bowman's capsule. In particular, high levels of UCHL1 expression were detected in the distal tubule system and to a lesser extent in the proximal tubule, mesangium, and Bowman's capsule (Fig. 3A ). Furthermore, oncocytoma express high levels of UCHL1, whereas primary RCC lesions exhibit heterogeneous UCHL1 protein levels, which seems to be dependent on the tumor grading. To determine the clinical significance of UCHL1, the UCHL1 expression pattern determined by immunohistochemistry was correlated to the histopathologic features of the various RCC lesions analyzed. UCHL1 staining of primary lesions significantly varied between the particular RCC subtypes. RCC of papillary subtype (51.6%), clear cell RCC (25%), chromophobic RCC (12.5%), and benign oncocytomas (22.2%), respectively, exhibited a strong staining intensity with the anti-UCHL1-specific polyclonal antibody (17), suggesting a considerable association between the frequency of UCHL1 expression and the RCC subtype (Fig. 3B). In addition, the frequency of the UCHL1 expression was dependent on the tumor grading in clear cell RCC lesions (Fig. 3A) and papillary RCC lesions, where the number of tissue sections with positive UCHL1 stainings increased from 21%/33% (G1) to 47%/69% (G2) and 45.5%/89% (G3), respectively, but not in chromophobic RCC specimens, where the UCHL1 staining frequencies remained almost stable at 33% (G1) and 29% (G2), respectively. Because ~60% of cases of sporadic clear all type RCCs often are associated with alterations at the VHL gene locus (23, 40), immunohistochemical stainings of 16 RCC specimens with WT VHL and 11 RCC specimens with defects in the VHL gene (Table 1, VHL-IHC panel) revealed that UCHL1 is strongly expressed in 25% of WT VHL RCC lesions (Fig. 4A and B ).


Figure 3
View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. UCHL1-specific immunohistochemical stainings. A, comparison of UCHL1-specific staining pattern of primary RCC lesions of distinct grading, oncocytoma, and normal kidney epithelium. Immunohistochemical staining of RCC lesions and normal renal tissues was done as described in Materials and Methods using the anti-UCHL1-specific polyclonal antibody (17). The results show a heterogeneous UCHL1 expression pattern in RCC of different grading (A) and in the various RCC subtypes (B).

 

Figure 4
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Correlation of UCHL1 expression with the VHL status and the metastatic phenotype. Sixteen primary RCC lesions with WT VHL status and 11 RCC lesions with altered VHL were immunohistochemically analyzed for UCHL1 protein expression. A and B, primary RCC of clear cell type G2 (A, x400 magnification) with WT VHL status shows strong positive reactivity in 25% of the samples, whereas matched RCC of clear cell type G2 (A, x400 magnification) with altered VHL lacks UCHL1 protein expression. C and D, twenty primary RCC lesions and corresponding metastases, as listed in Table 1 in the primary RCC panel versus metastasis panel, were immunohistochemically analyzed for UCHL1 protein expression using the anti-UCHL1-specific polyclonal antibody. Data are expressed in percentage of strong positive (black columns), intermediate (striped columns), and UCHL1-negative (white columns) RCC lesions. The primary RCC of clear cell type G2 (C, x200 magnification) shows strong positive UCHL1-specific reactivity of only a few tumor cells (<25%), whereas the matched lymph node metastasis (C, x200 magnification) exhibited a strong positive cytoplasmic UCHL1 staining of >75%.

 
UCHL1 expression is pronounced in metastases. Because UCHL1 is associated with the metastatic phenotype of certain tumors, immunohistochemistry of 20 primary RCC lesions of clear cell type and corresponding metastases of different localization (Table 1, primary RCC versus metastasis panel) was done using an anti-UCHL1-specific antibody (PG9500). As representatively shown in Fig. 4C, immunohistochemical stainings revealed a strong reactivity with the UCHL1-specific antibody in the metastases when compared with the autologous primary tumor lesion. In general, UCHL1 protein expression was more pronounced in metastatic RCC lesions when compared with the primary tumors: 40% of the metastatic RCC lesions exhibited a strong positive UCHL1 staining, whereas only 5% of the primary RCC lesions showed strong anti-UCHL1 reactivity (Fig. 4D).

Functional role of UCHL1 overexpression in RCC. To determine its functional role, UCHL1 was stably transfected into the UCHL1-deficient MZ1940RC cell line. UCHL1+ and WT UCHL1– MZ1940RC cells as well as UCHL1 mock transfectants were analyzed for their proliferation and migration capacity (Fig. 5 ). Growth rate comparison between these cell lines shows that ectopic UCHL1 expression is associated with a reduced doubling time when compared with vector-transfected and nontransfected WT MZ1940RC cells (Fig. 5A). The doubling time of UCHL1– cells was ~40 h, whereas, in gain-of-function UCHL1+ MZ1940RC cell clones, the doubling time did not exceed 32 h. The increased proliferation rate of UCHL1-expressing cells was paralleled by an enhanced migration capacity when compared with UCHL1– controls (Fig. 5B). These data, which were consistent on analyzing several independent clones, indicate that UCHL1 overexpression alters both the proliferation and migration rate of WT MZ 1940 RC cells. In addition, similar effects were seen when melanoma gain-of-function cell lines were analyzed, respectively (data not shown).


Figure 5
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Altered functional characteristics in the UCHL1-transfected RCC cell line MZ1940RC. A, growth curves of WT (solid line), mock-transfected (broken line), and UCHL1-transfected (dotted line) MZ1940RC cells. Time points taken (days; X axis) and the corresponding cell numbers (Y axis). B, comparison of migration rates of WT (black columns), mock-transfected (white columns), and UCHL1-transfected (striped columns) MZ1940RC cells. The number of cells passing Transwell filters (Y axis). SDs for each of the cell lines analyzed (bars).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
There is increasing evidence that ubiquitin-specific proteases play an important role in pathologic processes (41). Changes in the ubiquitin-specific protease profile has been detected in a panel of human (tumor) cells (42). In the present study, we used 2DE to identify markers, which might be associated with the development of RCC. Using this approach, UCHL1 was identified to be heterogeneously expressed in RCC lesions and adjacent normal kidney epithelium. Using RT-PCR, Western blot analysis, and/or immunohistochemistry, UCHL1 mRNA and protein expression was detected in >90% of normal kidney tissues, which is in accordance with D'Andrea et al. (43). In addition, the majority of primary RCC lesions and RCC cell lines expressed heterogeneous UCHL1 mRNA and protein expression levels (Figs. 2-4; Table 1, 2DE panel, RT-PCR panel, and RCC cell line panel). Compared with normal kidney epithelium, UCHL1 expression is often down-regulated in primary RCC lesions. These results are comparable with Hoek et al. (44), describing a predominant down-regulation of UCHL1 expression in melanoma when compared with normal melanocytes.

Furthermore, there exists an association between the VHL status and the expression of UCHL1. WT VHL RCC lesions exhibit UCHL1 expression at a higher frequency (Fig. 4A and B; Table 1, VHL-IHC panel) than lesions with altered or lacking VHL. This is in accordance with a recent report of Craven et al. (32) also showing the induction of UCHL1 expression in VHL+ RCC cell lines following WT VHL gene transfer. The VHL tumor suppressor gene is the substrate binding subunit of the E3 ubiquitin ligase complex, which targets HIFs, such as HIF-1{alpha}, for proteasomal degradation under normoxid conditions. Thus, a loss of VHL function is linked to the stabilization of the HIF-1{alpha}/HIF-1ß transcription factor complex triggering the expression of several genes associated with the development of RCC (e.g., promotion of angiogenesis via secretion of vascular endothelial growth factor and establishment of an autocrine loop via secretion of transforming growth factor-{alpha}). In addition, a VHL-dependent regulation of mitochondrial proteins, in particular metabolic enzymes, have been reported (32). Although VHL seems to contribute to the changes in the UCHL1 expression pattern in RCC, it requires further investigation to which extent it contributes to its deregulated expression. One possible mode of action for UCHL1 in VHL-expressing tumor cells might be the counteracting of a VHL-driven HIF-1{alpha} ubiquitination leading to a stabilization of the HIF transcription factor and supporting the tumorigenicity of the renal cancer cells, but this hypothesis still needs to be addressed in more detail.

However, both the frequency and the level of UCHL1 expression in RCC were dependent on the RCC subtype and tumor grading (Fig. 3), at least for RCC of clear cell type and papillary RCC. This is at variance to lung cancer, where no significant correlation between the presence of UCHL1 and histologic subtype exists (11). Furthermore, UCHL1 expression is significantly increased in RCC metastases when compared with autologous primary RCC lesions (Fig. 4C and D; Table 1, primary RCC versus metastasis panel; refs. 35, 43), which is in line with the concept that the establishment of metastases should benefit from both functional effects associated with the expression of UCHL1, an increased proliferation rate, and an improved migration capacity (Fig. 5). Based on these findings, UCHL1 expression might play an important role in disease progression of RCC and might serve as a diagnostic and prognostic marker for this disease. Indeed, in lung, pancreatic, esophageal, and colon carcinoma, UCHL1 overexpression is associated with poor clinical outcome and reduced patients' survival (11–14, 22). In contrast, reduced UCHL1 expression in melanoma was significantly associated with poor survival of patients (44).

UCHL1 has been shown recently to be differentially expressed during developmental processes (45, 46). Furthermore, UCHL1-deficient mice exhibit pathologic changes, such as decreased spermatogonial stem cell proliferation (47), whereas overexpression of UCHL1 arrests spermatogenesis in transgenic mice (48). This led us to postulate that UCHL1 is involved in cell proliferation. Overexpression of UCHL1 in MZ1940RC cells increased the growth and migration rates (Fig. 5). Similar data were obtained from EBV-infected cells, where UCHL1 may play a role in the transition from slow to a rapid proliferating cell or may be a consequence of this adaptation (42). The mechanisms by which UCHL1 may contribute to the growth phenotype of certain cell types remains as yet unknown. Such analyses can be done using UCHL1-specific inhibitors or small interfering RNA approaches (9). These experiments might lead to the characterization of factors inhibiting UCHL1 function, which could be used for the design of novel therapeutic strategies for the treatment of RCC patients. However, yeast two-hybrid assays showed that UCHL1 interacts with JAB1, a c-jun activating domain binding to the cyclin-dependent kinase inhibitor p27(kip1) (49), suggesting that UCHL1 contributes to the degradation of p27(kip1) degradation via its interaction and nuclear translocation with JAB1.

There exists several UCHL1 variants, which are associated with Parkinson disease (50) and may play distinct roles in proteasomal degradation. In this context, the UCHL1-specific activity most consequential for RCC tumorigenesis is not yet known. Further biochemical and functional analysis of UCHL1 and other family members will help to elucidate the role of UCHL1 in the complex mechanisms involved in tumor genesis, in particular the development of metastasis.

In summary, UCHL1 identified by proteome-based analyses is a promising diagnostic, prognostic, and therapeutic marker for RCC, which is associated with tumor progression and the VHL status. The study further points out the usefulness of these technologies for the identification of other diagnostic markers relevant for this malignancy.


   Acknowledgments
 
We thank Liis Pellenen and Claudia Stoerr for excellent secretarial help.


   Footnotes
 
Grant support: Bundesministerium für Bildung und Forschung (Bonn) project grant 031U101H (B. Seliger), the Mildred Scheel Foundation (Bonn) grant 106241 (B. Seliger), and the Wilhelm Roux Program (Halle) grant 13/21.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/ 4/06; revised 7/18/06; accepted 9/12/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Eytan E, Armon T, Heller H, Beck S, Hershko A. Ubiquitin C-terminal hydrolase activity associated with the 26 S protease complex. J Biol Chem 1993;268:4668–74.[Abstract/Free Full Text]
  2. Ciechanover A, Schwartz AL. The ubiquitin-proteasome pathway: the complexity and myriad functions of proteins death. Proc Natl Acad Sci U S A 1998;95:2727–30.[Free Full Text]
  3. Tobias JW, Varshavsky A. Cloning and functional analysis of the ubiquitin-specific protease gene Ubp1 of Saccharomyces cerevisiae. J Biol Chem 1991;266:12021–8.[Abstract/Free Full Text]
  4. Pickart CM, Rose IA. Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides. J Biol Chem 1985;260:7903–10.[Abstract/Free Full Text]
  5. Larsen CN, Krantz BA, Wilkinson KD. Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases. Biochemistry 1998;37:3358–68.[CrossRef][Medline]
  6. Wilkinson KD, Lee KM, Deshpande S, Duerksen-Hughes P, Boss JM, Pohl J. The neuron-specific protein PGP 9.5 is a ubiquitin carboxy-terminal hydrolase. Science 1989;246:670–3.[Abstract/Free Full Text]
  7. Chung CH, Baek SH. De-ubiquitinating enzymes: their diversity and emerging roles. Biochem Biophys Res Commun 1999;266:633–40.[CrossRef][Medline]
  8. Wilkinson KD. Ubiquitination and deubiquitination: targeting of proteins for degradation of the proteasome. Semin Cell Dev Biol 2000;11:141–8.[CrossRef][Medline]
  9. Liu Y, Fallon L, Lashuel HA, Liu Z, Lansbury PT, Jr. The UCHL1 gene encodes two opposing enzymatic activities that affect {alpha}-synuclein degradation and Parkinson's disease susceptibility. Cell 2002;111:209–18.[CrossRef][Medline]
  10. Hibi K, Liu Q, Beaudry GA, et al. Serial analysis of gene expression in non-small lung cancer. Cancer Res 1998;58:5690–4.[Abstract/Free Full Text]
  11. Hibi K, Westra WH, Borges M, Goodman S, Sidransky D, Jen J. PGP9.5 as a candidate tumor marker for non-small-cell lung cancer. Am J Pathol 1999;155:711–5.[Abstract/Free Full Text]
  12. Takano T, Miyauchi A, Matsuzuka F, et al. PGP9.5 mRNA could contribute to the molecular-based diagnosis of medullary thyroid carcinoma. Eur J Cancer 2004;40:614–8.[CrossRef][Medline]
  13. Sato N, Fukushima N, Maitra A, et al. Discovery of novel targets for aberrant methylation in pancreatic carcinoma using high-throughput microarrays. Cancer Res 2003;63:3735–42.[Abstract/Free Full Text]
  14. Yamazaki T, Hibi K, Takase T, et al. PGP9.5 as a marker for invasive colorectal cancer. Clin Cancer Res 2002;8:192–5.[Abstract/Free Full Text]
  15. Tezel E, Hibi K, Nagasaka T, Nakao A. PGP9.5 as a prognostic factor in pancreatic cancer. Clin Cancer Res 2000;6:4764–7.[Abstract/Free Full Text]
  16. Al-Katib AM, Mohammad RM, Maki A, Smith MR. Induced expression of a ubiquitin COOH-terminal hydrolase in acute lymphoblastic leukaemia. Cell Growth Differ 1995;6:211–7.[Abstract]
  17. Brichory F, Beer D, LeNaour F, Giordano T, Hanash S. Proteomics-based identification of protein gene product 9.5 as a tumor antigen that induces a humoral immune response in lung cancer. Cancer Res 2001;61:7908–12.[Abstract/Free Full Text]
  18. Takase T, Hibi K, Yamazaki T, et al. PGP9.5 overexpression in esophageal squamous cell carcinoma. Hepatogastroenterology 2003;50:1278–80.[Medline]
  19. Otsuki T, Yata K, Takata-Tomokuni A, et al. Expression of protein gene product 9.5 (PGP9.5)/ubiquitin-C-terminal hydrolase 1 (UCHL-1) in human myeloma cells. Br J Haematol 2004;127:292–8.[CrossRef][Medline]
  20. Gavioli R, Frisan T, Vertuani S, Bornkamm GW, Masucci MG. c-myc overexpression activates alternative pathways for intracellular proteolysis in lymphoma cells. Nat Cell Biol 2001;3:283–8.[CrossRef][Medline]
  21. Bittencourt Rosas SL, Caballero OL, Dong SM, da Costa Carvalho Mda G, Sidransky D, Jen J. Methylation status in the promoter region of the human PGP9.5 gene in cancer and normal tissues. Cancer Lett 2001;170:73–9.[CrossRef][Medline]
  22. Mandelker DL, Yamashita K, Tokumaru Y, et al. PGP9.5 promoter methylation is an independent prognostic factor for esophageal squamous cell carcinoma. Cancer Res 2005;65:4963–8.[Abstract/Free Full Text]
  23. Kim WY, Kaelin WG. Role of VHL gene mutation in human cancer. J Clin Oncol 2004;22:4991–5004.[Abstract/Free Full Text]
  24. Herman JG, Latif F, Weng Y, et al. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A 1994;91:9700–4.[Abstract/Free Full Text]
  25. Iwai K, Yamanaka K, Kamura T, et al. Identification of the von Hippel-Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex. Proc Natl Acad Sci U S A 1999;96:12436–41.[Abstract/Free Full Text]
  26. Krek W. VHL takes HIF's breath away. Nat Cell Biol 2000;2:423–7.[CrossRef][Medline]
  27. Semenza GL. HIF-1 and mechanisms of hypoxia sensing. Curr Opin Cell Biol 2001;13:167–71.[CrossRef][Medline]
  28. Goulx I, Lee S. Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein. Mol Cell Biol 2002;22:5319–36.[Abstract/Free Full Text]
  29. Motzer RJ, Russo P. Systemic therapy for renal cell carcinoma. J Urol 2000;163:408–17.[CrossRef][Medline]
  30. Seliger B, Kellner R. Design of proteome-based studies in combination with serology for the identification of biomarkers and novel targets. Proteomics 2002;2:1641–51.[CrossRef][Medline]
  31. Seliger B, Lichtenfels R, Kellner R. Detection of renal cell carcinoma-associated markers via proteome- and other ‘ome’-based analyses. Brief Funct Genomic Proteomics 2003;2:194–212.
  32. Craven RA, Hanrahan S, Totty N, et al. 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. Proteomics 2006;6:3880–93.[CrossRef][Medline]
  33. Lichtenfels R, Kellner R, Bukur J, Brenner W, Ackermann A, Seliger B. Heat shock protein expression and anti-heat shock protein reactivity in renal cell carcinoma. Proteomics 2002;2:561–70.[CrossRef][Medline]
  34. Kellner R, Lichtenfels R, Atkins D, et al. Targeting of tumor associated antigens in renal cell carcinoma using proteome-based analysis and their clinical significance. Proteomics 2002;2:1743–51.[CrossRef][Medline]
  35. Lichtenfels R, Kellner R, Atkins D, et al. Identification of metabolic enzymes in renal cell carcinoma utilizing PROTEOMEX analyses. Biochim Biophys Acta 2003;1646:21–31.[Medline]
  36. Seliger B, Menig M, Lichtenfels R, et al. Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy. Proteomics 2003;3:979–90.[CrossRef][Medline]
  37. Seliger B, Lichtenfels R, Atkins D, et al. Identification of fatty acid binding proteins as markers associated with the initiation and/or progression of renal cell carcinoma. Proteomics 2005;5:2631–40.[CrossRef][Medline]
  38. Thoenes W, Storkel S, Rumpelt HJ. Histopathology and classification of renal cell tumors (adenomas, oncocytomas, and carcinomas). The basic cytological and histopathological elements and their use for diagnostics. Pathol Res Pract 1986;181:125–43.[Medline]
  39. Jung D, Hilmes C, Knuth A, Jaeger E, Huber C, Seliger B. Gene transfer of the Co-stimulatory molecules B7-1 and B7-2 enhances the immunogenicity of human renal cell carcinoma to a different extent. Scand J Immunol 1999;50:242–9.[CrossRef][Medline]
  40. Cohen HT, McGovern FJ. Renal-cell carcinoma. N Engl J Med 2005;353:2477–90.[Free Full Text]
  41. Spataro V, Norbury C, Harris AL. The ubiquitin-proteasome pathway in cancer. Br J Cancer 1998;77:448–55.[Medline]
  42. Ovaa H, Kessler BM, Rolén U, Galardy PJ, Ploegh HL, Masucci MG. Acitivity-based ubiquitin-specific protease (USP) profiling of virus-infected and malignant human cells. Biotechnol 2004;101:2253–8.
  43. D'Andrea V, Malinovsky L, Berni A, et al. The immunolocalization of PGP9.5 in normal human kidney and renal cell carcinoma. G Chir 1997;18:521–4.[Medline]
  44. Hoek K, Rimm DL, Williams KR, et al. Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas. Cancer Res 2004;64:5270–82.[Abstract/Free Full Text]
  45. Satoh Jl, Kuroda Y. Ubiquitin C-terminal hydrolase-U (PGP9.5) expression in human neural cell lines following induction of neuronal differentiation and exposure to cytokines, neurotrophic factors, or heat stress. Neuropathol Appl Neurobiol 2001;27:95–104.[CrossRef][Medline]
  46. Kwon J, Wang YL, Setsuie R, et al. Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice. Biol Reprod 2004;71:515–21.[Abstract/Free Full Text]
  47. Kwon J, Mochida K, Wang YL, et al. Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis. Biol Reprod 2005;73:29–35.[Abstract/Free Full Text]
  48. Wang YL, Liu W, Sun YJ, et al. Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermato-genesis in transgenic mice. Mol Reprod Dev 2006;73:40–9.[CrossRef][Medline]
  49. Caballero OL, Resto V, Patturajan M, et al. Interaction and colocalization of PGP9.5 with JAB1 and p27(Kip1). Oncogene 2002;21:3003–10.[CrossRef][Medline]
  50. Chung KK, Dawson VL, Dawson TM. New insights into Parkinson's disease. J Neurol 2003;250 Suppl 3:15–24.



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
V. M. Howell, A. Gill, A. Clarkson, A. E. Nelson, R. Dunne, L. W. Delbridge, B. G. Robinson, B. T. Teh, O. Gimm, and D. J. Marsh
Accuracy of Combined Protein Gene Product 9.5 and Parafibromin Markers for Immunohistochemical Diagnosis of Parathyroid Carcinoma
J. Clin. Endocrinol. Metab., February 1, 2009; 94(2): 434 - 441.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seliger, B.
Right arrow Articles by Lichtenfels, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seliger, B.
Right arrow Articles by Lichtenfels, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online