Original Paper
Urologia
Received: June 23, 2012 Accepted after revision: November 6, 2012 Published online: December 22, 2012
Urol Int 2013;90:233–239 DOI: 10.1159/000345608
Internationalis
Downregulation of Fumarate Hydratase Is Related to Tumorigenesis in Sporadic Renal Cell Cancer Yun-Sok Ha a, f Yoshitomo Chihara e Hyung-Yoon Yoon a, b Yong-June Kim a, b Tae-Hwan Kim c Seung Hyo Woo d Seok-Joong Yun a, b Isaac Yi Kim f Yoshihiko Hirao e Wun-Jae Kim a, b a Department of Urology, College of Medicine, Chungbuk National University and b BK21 Chungbuk Biomedical Science Center, School of Medicine, Chungbuk National University, Cheongju, c Department of Urology, College of Medicine, Kyungbuk National University, Daegu, and d Department of Urology, Eulji University Hospital, Eulji University College of Medicine, Daejeon, South Korea; e Department of Urology, Nara Medical University, Kashihara, Japan; f Section of Urologic Oncology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, N.J., USA
Key Words Fumarate hydratase ⴢ Renal cell cancer ⴢ mRNA ⴢ Tumorigenesis
Abstract Objective: Although germline mutations of fumarate hydratase (FH) are a useful molecular marker of hereditary leiomyomatosis and renal cell cancer (RCC) syndrome, their clinical significance in sporadic RCC has not been studied in detail. The aim of the present study was to investigate possible correlations between the expression of FH and the clinical implications of sporadic RCC. Materials and Methods: FH mRNA levels were evaluated in 140 tumor specimens from patients with primary RCC and in 62 specimens of corresponding normal-appearing kidney tissue using real-time quantitative polymerase chain reaction. Immunohistochemical staining was performed on 6 normal surrounding tissues and 71 RCC tissues. Results: FH mRNA levels were significantly lower in tumor tissues than in matched normal-appearing kidney tissues (p = 0.031). In all normal tissues, FH staining intensity was strong. However, the expression of FH showed
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no significant correlation with the pathological and clinical characteristics of patients with sporadic RCC. Conclusions: Our results showed that FH mRNA expression decreased significantly in correlation with the transition from normal renal parenchyma to RCC. FH may be an indicator or tumorigenesis in sporadic RCC and could be a potential target for therapies against RCC in the future. Copyright © 2012 S. Karger AG, Basel
Introduction
Renal neoplasm has been the subject of numerous cytogenetic and molecular genetic studies [1, 2]. Renal cell cancer (RCC) is the most common type of kidney cancer and accounts for approximately 90% of all renal malignancies [3, 4]. RCC refers to cortically-derived tumors of the renal parenchyma and encompasses a heterogeneous group of cancers [5]. In recent decades, great strides have been made in understanding the molecular mechanisms of RCC. As a result of these studies, the vascular endothelial growth factor (VEGF) and mammalian target of rapaWun-Jae Kim Department of Urology, College of Medicine Chungbuk National University, 62, Kaeshin-dong Heungduk-ku, Cheongju, Chungbuk 361-711 (South Korea) E-Mail wjkim @ chungbuk.ac.kr
mycin (mTOR) pathways were identified as fundamental to the biology of RCC [6]. This biologic insight provided a rationale for targeting these growth factor signaling pathways in RCC. However, the therapeutic targeting of the tumor cells themselves is highly desirable. Fumarate hydratase (FH) and succinate dehydrogenase are two integral enzyme components of the Krebs cycle, which in addition to their essential role in the tricarboxylic acid (TCA) cycle, can also act as tumor suppressors [7]. Patients with hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome are at risk for cutaneous and uterine leiomyomas and solitary papillary RCC. Mutations in the FH gene cause this autosomal dominant syndrome [8, 9]. It has been proposed that ‘pseudohypoxic’ stabilization of hypoxia-induced factor1␣ (HIF-1␣) by fumarate accumulation contributes to tumorigenesis in HLRCC [10, 11]. Although only about 2% of individuals with RCC have a positive family history, the elucidation of the molecular pathology of familial RCC syndromes has provided important insights into the pathogenesis of sporadic forms of RCC, such as the role of mutations in the von Hippel-Lindau tumor suppressor gene [12, 13]. Only a few studies have investigated whether FH mutations are associated with sporadic RCC [14, 15]. In all these studies combined, somatic FH mutations were not detected in a total of 60 conventional RCCs or in 38 non-conventional cell RCCs. Thus, there is little evidence that somatic FH mutations are frequent in sporadic RCC. However, the expression levels of FH in sporadic RCC tissues have not been examined to date. The aim of the present study was to investigate the possible correlation between the expression of FH and RCC. To our knowledge, this is the first study that used sporadic RCC tissues and real-time quantitative polymerase chain reaction (qPCR) analysis to identify the correlation between FH gene expression and clinicopathological parameters in sporadic RCC.
Materials and Methods Patients and Tissue Specimens Between April 1996 and December 2010, tissue samples from 140 patients with primary RCC who underwent radical or partial nephrectomy were collected. Matched normal-appearing kidney tissues were obtained from 62 patients. The pathology samples were re-examined by a pathologist to confirm the presence of tumor or normal tissue. All patients gave written informed consent, and the study was approved by the institutional ethics committee. All tumors were macrodissected within 15 min of surgical resection, fresh frozen in liquid nitrogen, and stored at –80 ° C until use. Each cancer specimen was confirmed as representative tumor tis
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sue by analysis of adjacent tissue in fresh frozen sections from nephrectomy specimens. Staging was based on pathological findings according to the American Joint Committee on Cancer classification [16], and histological differentiation was graded according to Fuhrman’s nuclear grading system [17]. All patients were evaluated postoperatively every 3 months for the first 2 years, every 6 months for the next 2 years, and yearly thereafter. The definitions of disease progression included local recurrence, lymph node metastasis, and distant metastasis by CT scan and bone scan. RNA Extraction and Construction of cDNA RNA was isolated from tissue using 1 ml of TRIzol (Invitrogen, Carlsbad, Calif., USA) and homogenization in a 5-ml glass tube. The homogenate was transferred to a 1.5-ml tube and then mixed with 200 l of chloroform. After incubation for 5 min at 4 ° C, the homogenate was centrifuged for 13 min at 13,000 rpm at 4 ° C. The upper aqueous phase was transferred to a clean tube and then 500 l of isopropanol was added. The mixture was incubated for 60 min at 4 ° C and centrifuged for 8 min at 13,000 rpm at 4 ° C. The upper aqueous phase was discarded and the pellet was mixed with 500 l of 75% ethanol followed by centrifugation for 5 min at 13,000 rpm at 4 ° C. After discarding the upper aqueous layer, the pellet was dried at room temperature, dissolved in diethylpyrocarbonate-treated distilled water, and stored at –80 ° C. The quality and integrity of the RNA were confirmed by agarose gel electrophoresis and ethidium bromide staining, followed by visual inspection under ultraviolet light. cDNA was prepared from 1 g of total RNA using a First-Strand cDNA Synthesis Kit (Amersham Biosciences Europe GmbH, Freiburg, Germany) according to the manufacturer’s protocol.
Real-Time qPCR Real-time PCR amplification was performed using a Rotor Gene 6000 instrument (Corbett Research, Mortlake, N.S.W., Australia) to quantify the expression of FH. Real-time PCR assays were carried out in microreaction tubes (Corbett Research) using SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan). The primers used for amplification of FH (130 base pairs) were sense (5ⴕ-GTA TTA TGG CGC CCA GAC C-3ⴕ) and anti-sense (5ⴕ-ATC CTG GTT TAC TTC AGC GG-3ⴕ). The PCR reaction was performed in a final volume of 10 l consisting of 5 l of 2! SYBR Premix Ex Taq buffer, 0.5 l each of 5ⴕ- and 3ⴕ-primer (10 pmol/l), and 1 l of the sample cDNA. The product was purified with a QIAquick Extraction Kit (Qiagen, Hilden, Germany), quantified with a spectrophotometer (MBA 2000; PerkinElmer, Fremont, Calif., USA), and then sequenced with an automated laser fluorescence sequencer (ABI Prism 3100 Genetic Analyzer; Applied Biosystems, Foster City, Wisc., USA). Tenfold serial dilutions of a known concentration of the product (from 100 to 0.1 pg/l) were used to establish the standard curve for real-time PCR. The real-time PCR conditions were as follows: 1 cycle of 20 s at 96 ° C, followed by 40 cycles of 2 s at 96 ° C for denaturation, 15 s at 56 ° C for annealing, and 15 s at 72 ° C for extension. The melting program was performed at 72–95 ° C with a heating rate of 1 ° C per 45 s. Spectral data were captured and analyzed using Rotor-Gene Real-Time Analysis Software 6.0 Build 14 (Corbett Research). All samples were run in triplicate. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed as an endogenous RNA reference gene and gene expression was normalized to the expression of GAPDH.
Ha /Chihara /Yoon /Kim /Kim /Woo /Yun / Kim /Hirao /Kim
Immunohistochemical Staining Six normal surrounding tissues and 71 RCC tissues were fixed in 10% neutral-buffered formalin and then embedded in paraffin. Tissue sections were cut and placed on Superfrost Plus microscope slides (Fisher Scientific). Using the Benchmark XT automated immunohistochemistry stainer (Ventana Medical Systems, Inc., Tucson, Ariz., USA), slides were stained as follows. Detection was done using the Ventana Ultraview DAB Kit (Ventana Medical Systems). Sections were deparaffinized using EZ Prep solution. CC1 standard (pH 8.4 buffer contained Tris/borate/ EDTA) was used for antigen retrieval. DAB inhibitor (3% H2O2, endogenous peroxidase) was blocked for 4 min at 37 ° C temperature. Sections were incubated with an anti-FH primary antibody (Abcam, Inc., San Diego, Calif., USA) for 40 min at 37 ° C, and a secondary antibody of Universal HRP Multimer for 8 min at 37 ° C. Slides and then DAB+ H2O2 substrate followed by hematoxylin and bluing reagent were counterstained for 8 min at 37 ° C. Reaction buffer (pH 7.6 Tris buffer) was used as washing solution. Staining intensity was evaluated. FH localized primarily to the cytoplasm. Staining intensity was classified as follows: none, weak, moderate and strong. Each specimen was examined and scored separately by three investigators, and discrepancies were discussed until agreement was reached.
Statistics To normalize the highly skewed distribution of FH mRNA expression, the data were examined as the natural log and subsequently back transformed for the model results [18]. Results were expressed as a geometric mean (anti-log 95% confidence interval) [18]. The paired t test was used to calculate the significance of differences between data from tumor tissues and matched normal tissues. To compare gene expression levels among the groups, the two-sample t test or ANOVA were performed. Statistical analysis was performed using SPSS 12.0 software (SPSS, Inc., Chicago, Ill., USA), and a p value !0.05 was considered statistically significant.
Results
Baseline Characteristics The baseline characteristics of the enrolled patients are listed in table 1. The mean age of the subjects was 57.9 8 12.6 years (range 21–83). The mean postsurgical follow-up period was 55.9 8 43.2 months (range 1.0–156.8). Males and females accounted for 104 patients (74.3%) and 36 patients (25.7%), respectively. 84 patients (60.0%) received an open radical (n = 80) or partial (n = 4) nephrectomy. Laparoscopic surgery was performed in 56 patients (40.0%), including 53 patients treated with radical nephrectomy and 3 with partial nephrectomy. Expression of FH in Tumor and Surrounding Matched Normal-Appearing Tissues The FH mRNA expression in 62 RCC specimens was compared to that in matched normal-appearing tissues. FH mRNA expression was significantly lower in tumor Downregulation of Fumarate Hydratase in RCC
Table 1. Demographic characteristics and histopathological data
Variables
Incidence (%) or value
Median age, years (range) Median follow-up periods, months (range) Gender male female Histologic subtype conventional papillary chromophobe unclassified Pathologic T stage pT1a pT1b pT2 pT3 pT4 Nuclear grade 1 2 3 4 Progression no yes
58 (21–83) 43.6 (1.0–156.8) 104 (74.3) 36 (25.7) 118 (84.3) 16 (11.4) 5 (3.6) 1 (0.7) 60 (42.9) 34 (24.3) 21 (15.0) 20 (14.3) 5 (3.6) 26 (18.6) 58 (41.4) 43 (30.7) 13 (9.3) 116 (82.9) 24 (17.1)
tissue (11.92 ! 103 copies/l, 8.12–17.48) than in normal tissue (24.23 ! 103 copies/l, 15.46–37.97) (p = 0.031). Figure 1 showed these results expressed by natural log scale. Correlation of FH Expression with Clinical Parameters in RCC Table 2 summarizes the correlation between FH expression and clinicopathological parameters in patients with RCC. Tumor size correlated marginally with FH mRNA levels. Pathological stage T2 tumors showed an aberrantly high FH mRNA expression compared to the other stages. Other pathological parameters, including node status, metastasis, lymphovascular invasion and histologic subtype, were not associated with the expression of FH (fig. 2). Relationship between Protein Expression Levels and Pathological Features In all normal surrounding tissues, FH staining intensity was strong (fig. 3a, b). Among 71 RCC tissues, various intensities of FH cytoplasmic staining were observed (fig. 3c–f). There were no relations between clinicopathological characteristics and FH protein expression levels. Urol Int 2013;90:233–239
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Table 2. FH mRNA expression stratified by clinicopathological
Parameters Age Gender Tumor size Pathologic T stage
Nuclear grade
Progression a
