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Clinical Cancer Research Vol. 9, 1021-1027, March 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Increased Activation of CCAAT/Enhancer Binding Protein-ß Correlates with the Invasiveness of Renal Cell Carcinoma1

Mototsugu Oya2, Akio Horiguchi, Ryuichi Mizuno, Ken Marumo and Masaru Murai

Department of Urology, Keio University School of Medicine, Tokyo 160-8582, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Positive inflammatory reactions in an aggressive phenotype are typical features of renal cell carcinoma (RCC). Although a high blood level of inflammatory cytokines, such as interleukin-6, interleukin-8, and tumor necrosis factor-{alpha}, has been observed in these patients, the mechanisms underlying this clinical phenomenon remain to be elucidated. CCAAT/enhancer binding protein (C/EBP) family are transcription factors which play a role in cell differentiation and inflammatory reactions. Among these, C/EBP-ß induces a variety of cytokines and thus may play a role in the pathogenesis of RCC. We studied the activation of C/EBP-ß determined by electrophoretic mobility shift assay in nine RCC cell lines and 44 tissue samples. Six cell lines showed an activation of C/EBP-ß, whereas three cell lines did not, and two of these three had no expression at all of C/EBP-ß protein. Of 44 tissue samples, 12 (27.3%) showed a >200% increase in the activity compared with the corresponding normal kidney tissues. Locally advanced cases had a significantly higher rate of increased C/EBP-ß activity (5 of 8 = 62.5% in advanced cases versus 7 of 36 = 19.4% in localized cases). Especially, all four cases with renal vein invasion had an increased C/EBP-ß activity. These data suggest that the increased activation of C/EBP-ß may contribute to promote tumor invasiveness and render a malignant phenotype of RCC, although it needs to be validated in a larger series.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent advances and the widespread use of such imaging modalities as ultrasonography and computed tomography have now made it possible to detect RCC3 asymptomatically at an early stage. These carcinomas can be successfully treated by a surgical resection. However, some patients demonstrate locally advanced RCC and/or distant metastases. These patients have a poor prognosis, and the therapeutic options for such patients are limited because chemotherapy and radiation therapy are ineffective. Cytokine therapy, such as IL-2 and IFN-{alpha}, is the current treatment modality; however, the response rate is 15–20% (1) . These tumors are occasionally associated with inflammatory reactions (2, 3, 4) . Cancer cells produce inflammatory cytokines, which work in an autocrine or paracrine manner, as well as circulate in the body and yield inflammatory reactions (5 , 6) . IL-6 has been thought to be a major causative cytokine because >=50% of patients with metastatic RCC have an increased level of IL-6 (2 , 7) . IL-8, an angiogenic cytokine secreted frequently from RCC, has been thought to be a causative cytokine of hypervascularity in RCC (8) . TNF-{alpha} is also secreted frequently from RCC and induces the expression of MMPs, which are known to work as key molecules related to cancer cell invasion (9, 10, 11) .

The C/EBP family of transcription factors belongs, together with activator protein-1 and activating transcription factor/cAMP response element-binding protein, to a class of DNA-binding proteins named basic leucin zipper proteins (12) . These proteins are characterized by their leucine zipper structure and the adjacent DNA-binding basic region, which are both located in the COOH-terminal half of the structure. The leucine zipper structure allows the C/EBP to dimerize as both homo and heterodimers. The DNA-binding regions of the dimerized proteins recognize a palindromic CCAAT motif in the promoter of the target genes. The originally characterized C/EBP is now named C/EBP-{alpha} (13) . C/EBP-{alpha} is expressed primarily in the liver, fat, and intestine (14) . In those tissues, the expression is restricted to terminally differentiated cells; therefore, its role in establishing and maintaining the state of terminal cell differentiation has been suggested. In contrast, C/EBP-ß is ubiquitously expressed (15) and is induced drastically by such inflammatory cytokines as IL-1, IL-6, and TNF-{alpha} (16) . C/EBP-ß was originally identified and named nuclear factor-IL6 as a DNA-binding protein responsible for IL-1-stimulated IL-6 induction (17) . The direct cloning of nuclear factor-IL6 revealed its homology with C/EBP. C/EBP-ß not only induces an IL-6 expression, but its activity is also induced by IL-6 and therefore may play a substantial role in IL-6-mediated autocrine growth in RCC. Furthermore, accumulating reports have demonstrated that C/EBP-ß induces IL-8 and TNF-{alpha} expression (18, 19, 20, 21) . In consideration of the inflammatory reactions observed frequently in RCC, C/EBP-ß may play a role in the oncogenesis and/or tumor development of RCC.

An increased expression of C/EBP-ß in solid cancer is observed in breast cancer (22) , ovarian tumors (23) , and colorectal cancer (24) . In contrast, a diminished expression has also been described in squamous cell carcinoma (25) . The descriptions of these reports are limited to the quantitative evaluation of C/EBP-ß protein. Because C/EBP-ß is a transcription factor, evaluating the activity is thus considered to be more biologically significant than to evaluate the amount of the expressed protein levels. A constitutive activation of C/EBP-ß has been reported in head and neck squamous carcinoma cell lines, which produce proinflammatory cytokines (26) . Thus far, the activation of C/EBP-ß has not yet been evaluated using tumor specimens of solid cancer. We studied the activation of C/EBP-ß in 44 RCC tissue specimens of different grades and stages determined by EMSA. In this study, we investigate whether or not an increased activation of C/EBP-ß correlates with the pathologically determined tumor invasiveness of RCC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Culture.
All nine RCC cell lines were cultured in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) with 10% fetal bovine serum, streptomycin (100 µg/ml; Life Technologies, Inc.), and penicillin (100 µg/ml; Life Technologies, Inc.). KU-2 and KU19-20 are clear cell renal carcinoma cell lines established in our institute (27 , 28) . Caki-1, Caki-2, ACHN, 769P, 786-O, SW-839, and A498 were purchased from the American Tissue Culture Collection (Rockville, MD).

Patient Characteristics and Tissue Samples.
A total of 44 RCCs was surgically removed in the Department of Urology, Keio University School of Medicine. Tumor tissue specimens as well as normal kidney tissue specimens were taken and immediately cast into liquid nitrogen. All samples were stored in a deep freezer at -80°C. The patient data and tumor characteristics are shown on Table 1Citation . Tumor grading and staging were done according to the Tumor-Node-Metastasis classification (29) . pT1 and pT2 are localized tumors, whereas pT3 is invasive disease into the perinephric tissue (pT3a) or renal veins (pT3b). pT1 is defined as a tumor measuring <=7 cm in size and subdivided into either pT1a (<=4 cm) or pT1b (>4 cm). Patients 4, 35, 36, and 42 and 10 and 42 had metastatic disease in the lung and bone, respectively. The materials included 40 clear cell renal carcinomas and 4 papillary cell renal carcinomas.


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  		Table 1 C/EBP-ß activation in RCCa

 
Extraction of Proteins.
Stored samples were crushed in liquid nitrogen, homogenized using a homogenizer in cell lysis buffer [20 mM HEPES (pH 7.9), 0.2% NP-40, 10% glycerol, 400 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 1 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride (all Sigma, St. Louis, MO), and a protease inhibitor mix (complete protease inhibitor cocktail set; Boehringer Mannheim, Indianapolis, IN)], and then incubated on ice for 15 min. The supernatants were taken and stored at -80°.

EMSA and Supershift Assay.
Twenty micrograms of the supernatants were incubated with poly dIdC (3 µg; Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) in a binding buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM DTT, and 4 vol % glycerol] with double-stranded oligonucleotides for the consensus binding sites of C/EBP (5'-TGCAGATTGCGCAATCTGCA-3'; Santa Cruz Biotechnology, Santa Cruz, CA) labeled with [{gamma}-32P]dATP by T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA) for 20 min at room temperature. For supershift assay, 2 µg of antibody for C/EBP-{alpha}, ß, {gamma}, {delta}, and {epsilon} (Santa Cruz Biotechnology) were added to the samples and incubated for 30 min. The samples were separated on nondenaturing 4% polyacrylamid gel in 0.5 x Tris-borate EDTA. The gels were then dried and autoradiographed. The signals were quantitated using a densitometer (Image 1.52 program; NIH, Bethesda, MD). The values (tumor tissue/normal tissue) are shown on Table 1Citation . Using densitometric analyses, we defined increased activation as a ratio (T/N) of >2.

IL-6, IL-8, and TNF-{alpha} Determination by ELISA.
The cells were cultured in triplicate in serum-free medium for 24 h. Cell-free culture supernatants were analyzed for IL-6, IL-8, and TNF-{alpha} protein content by ELISA using commercial kits according to the manufacturer’s instructions (R&D System, Minneapolis, MN).

Immunoblotting.
The extracted protein (20 µg) with sample buffer containing 2-mercaptoethanol was separated on 12% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was incubated with 5% skim milk in Tris-buffered saline overnight. The primary antibodies for C/EBP-{alpha}, ß, {gamma}, {delta}, and {epsilon} were reacted (Santa Cruz Biotechnology). Anti-ß-actin mouse monoclonal antibody (Sigma) was used as an internal control. Immunodetection was performed using an enhanced alkaline phosphatase detection system with avidin-biotin complex (Bio-Rad Laboratories).

Immunohistochemistry.
Five-micron sections from formalin-fixed, paraffin-embedded tissue specimens were deparaffinized in xylene and dehydrated in graded ethanol, followed by PBS. Antigen was retrieved by heating at 121°C for 10 min in 10 mM sodium citrate (pH 6.0) and then was incubated with 0.3% H2O2 to quench the endogenous peroxidase activity. The slides were blocked in 10% goat serum and incubated with mouse monoclonal anti-C/EBP-ß antibody (1:50; Santa Cruz Biotechnology) for 24 h at 4°C. After washing, the slides were incubated with Simple stain max p.o. (Nichirei, Tokyo, Japan) for 30 min and 3,3'-diaminobenzidine, counterstained by hematoxylin.

Statistics.
The differences in the C/EBP-ß activation between the subgroups of RCC were tested for significance using Fisher’s exact probability test. A P < 0.05 was considered to indicate significance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of C/EBP-ß in RCC Cell Lines.
Nine cell lines were used to investigate the activation of C/EBP family proteins. The consensus oligonuleotides of C/EBP was used to evaluate the binding activity determined by EMSA. To specifize the activity, a supershift assay was done adding the respective antibodies of the five isoforms. Of nine cell lines, six showed the activation. Caki-1 and KU19-20 cells had a high activity, whereas ACHN, 769P, 786-O, and SW-839 showed a weak activity (Fig. 1A)Citation . These bands in EMSA were supershifted by the C/EBP-ß antibody but not by the other antibodies (Fig. 1B)Citation . These data showed that the activation of C/EBP depends solely on the activation of C/EBP-ß. We performed immunoblotting of the lysates of the nine cell lines which revealed no expression of C/EBP-{alpha}, {gamma}, {delta}, and {epsilon} (data not shown). In these RCC cell lines, C/EBP-ß is the only expressed C/EBP family proteins in RCC cell lines. Of three cell lines without any activation of C/EBP, KU2 showed the expression of C/EBP-ß proteins detected by immunoblotting, whereas Caki-2 and A498 showed no expression (Fig. 1C)Citation . Although almost the same amount of C/EBP-ß protein is expressed in 769P and SW-839 cells as that of Caki-1 and KU19–20 cells, the activation of C/EBP-ß is much lower in 769P and SW-839 cells. Therefore, the activation did not entirely depend on the expressed amount of C/EBP-ß proteins in the RCC cell lines.



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  		Fig. 1. A, activation of C/EBP determined by EMSA. Activation is observed in six cell lines: (a) Caki-1 and (b) KU19-20 showed a high activation; (c) ACHN, (d) 769P, (e) 786-O, and (f) SW-839 showed a weak activation. In B, supershift bands were only observed by adding C/EBP-ß antibody. C, immunoblots with C/EBP-ß antibody. Caki-2 and A498 had no expression. KU2 had an expression of C/EBP-ß protein, although no C/EBP-ß activation was observed. ß-actin was used as an internal control.

 
Cytokine Secretion of RCC Cell Lines.
IL-6, IL-8, and TNF-{alpha} secretion was determined by ELISA (Fig. 2)Citation . TNF-{alpha} production was detected only in Caki-1 and 786-O cells at a very low level (1.64 ± 0.4 and 0.78 ± 0.2, respectively). On the whole, the C/EBP-ß activity determined by EMSA correlated with the production of IL-6 or IL-8, e.g., Caki-1 and KU19-20 with a high C/EBP-ß activity produced a high level of IL-6 or IL-8, whereas KU2 and Caki-2 without any C/EBP-ß activity produced scarcely any IL-6 or IL-8. Exceptionally, A498 produced IL-6 and IL-8, although no C/EBP-ß activity was detected. Other transcription factors, such as activator protein-1 or Oct-1, may play a role in the production of such cytokines in A498 cells.



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  		Fig. 2. The level of IL-6 and IL-8 secretion was determined by ELISA. The results represent the mean ± SD of cell-free culture supernatants per milliliter.

 
Activation of C/EBP-ß in RCC.
Forty-four tumor specimens and the corresponding normal kidney specimens were lysed in the cell lysis buffer described in "Materials and Methods." EMSA was performed, and the intensity of the bands was quantitated (Table 1)Citation . An example of EMSA is shown on Fig. 3ACitation . We defined an increased activity of C/EBP-ß if the ratio of intensity (tumor:normal tissue) was >2. Of the 44 tissue specimens, 12 cases (27.3%) showed a >200% increased activity compared with the corresponding normal kidney tissues. Correlations were analyzed according to the pathological findings. The pT category correlated with the increased activity of C/EBP-ß, namely, locally advanced cases had a significantly higher rate of enhanced C/EBP-ß activity [62.5% (5 of 8) in advanced cases versus 19.4% (7 of 36) in localized cases; Table 2Citation ]. There are two types of invasion of RCC, namely, perinephric adipose tissue invasion (categorized as pT3a) and renal vein invasion (categorized as pT3b). Especially, all four cases with renal vein invasion (pT3b) had an enhanced C/EBP-ß activity. No correlation was observed regarding pN, pM, or the tumor grade.



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  		Fig. 3. A, an example of EMSA in consecutive RCC specimens and the corresponding normal tissues. Patients 37, 38, and 39 had an enhanced C/EBP-ß activation. B, the immunoblotting by the C/EBP-ß antibody in the specimens shown in A (36–39). Increased activation of C/EBP-ß in the tumor tissues in Pt 37–39 correlates with the increased amount of C/EBP-ß protein. In Pt 36, without any increased activation, C/EBP-ß protein expression was not increased. ß-actin was used as an internal control.

 

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  		Table 2 Correlation between the C/EBP-ß activation and the pathological findingsa

 
Activation of C/EBP-ß Correlates with the Expressed Amount of C/EBP-ß Protein in Most Cases of RCC.
The ratio of expressed protein (tumor/normal tissue) as determined by a densitometer is shown in Table 1Citation , and an example of immunoblotting is shown in Fig. 3BCitation . Increased activation of C/EBP-ß in the tumor tissues in Pt 37–39 correlates with the increased amount of C/EBP-ß protein. Two specimens (8 and 19) showed a >200% increase in the expressed amount of C/EBP-ß protein compared with the corresponding normal tissues; however, no enhanced activation was observed (Table 1)Citation . As already shown, the activation of C/EBP-ß in RCC cell lines did not entirely depend on the expressed amount of protein. This phenomenon is also observed in some RCC tissues. A subtype of RCC was thus suggested in which C/EBP-ß is not activated despite a considerable amount of C/EBP-ß protein expression.

Increased C/EBP-ß Activation in RCC Correlates with the Immunohistochemical Nuclear Staining of C/EBP-ß.
Activated transcription factors translocate into the nucleus. Therefore, to confirm the activation of C/EBP-ß in RCC, an immunohistochemical method was used to detect the localization, i.e., nuclear staining by C/EBP-ß antibody. We analyzed 20 specimens (25–44), and the correlation was investigated between the C/EBP-ß activity determined by EMSA and nuclear staining. In normal kidney tissues, weak nuclear staining was observed in renal tubular epithelial cells (Fig. 4A)Citation . In a specimen from patient 37 which had an increased C/EBP-ß activity determined by EMSA, the nucleus of most cells was positively stained by C/EBP-ß antibody (Fig. 4B)Citation . In contrast, in a specimen from patient 39, which had an increased C/EBP-ß activity but weaker than that patient 37, there were fewer positively stained cells (Fig. 4C)Citation . In patient 31, without any enhanced activation, positively stained cells were scarcely observed (Fig. 4D)Citation . These correlations were observed in the other specimens (data not shown). In summary, the C/EBP-ß activity correlated with positive nuclear staining by C/EBP-ß antibody.



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  		Fig. 4. Immunohistochemical analysis using C/EBP-ß antibody. In A, normal kidney tissue showed weak staining in renal tubular epithelial cells. In B, cancer cells (clear cell type) are mostly positive for nuclear staining (Patient 37). In C, in the specimen with weaker C/EBP-ß activity than specimen 37, fewer positively stained cells are observed (Patient 39). In D, in a specimen without any enhanced activation, positively stained cells are scarcely observed (Patient 31). Scale bar, 50 µm.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cytokine secretion in RCC has been suggested to be related with the clinical features of RCC, such as positive inflammatory reactions, hypervascularity, and resistance to immunotherapy (2 , 7 , 30 , 31) . IL-6, IL-8, and TNF-{alpha} are the most frequently detected cytokines in RCC (8 , 9) . Notably, these cytokines are all inducible by the transcription factor C/EBP-ß. Furthermore, the expression of C/EBP-ß can be stimulated by these inflammatory cytokines (16) . Therefore, C/EBP-ß may play a role in the putative autocrine growth stimulation by IL-6 creating a persistent and vicious cycle of proliferation. IL-8 and TNF-{alpha} have been suggested to have some roles in cancer cell invasion by the induction of MMPs (8, 9, 10, 11) .

We first evaluated the activation of C/EBP family by EMSA using consensus oligonucleotides common to all isoforms. Supershift assay showed the activation depends solely on the ß isoform. Other isoforms were not expressed in all RCC cell lines determined by immunoblotting. These data suggest that C/EBP-ß is the sole member of the C/EBP family expressed in RCC. On the present study, the activation of C/EBP-ß did not necessarily correlate with the expressed amount of C/EBP-ß proteins. This phenomenon was observed both in the RCC cell lines and tissues. This suggests that a constitutive activation is observed in selected cells, in which the upstream activation of the signal pathways, such as mitogen-activated protein kinases, are constitutively activated by external stimuli, such as IL-6 (32) , and transcriptional machinery is constantly at work. In other cells without any activation of C/EBP-ß despite the fact that a considerable amount of C/EBP-ß protein is expressed, upstream signal cascade may not be activated by the external stimuli, and/or upstream signal molecules may not be functional.

In our study, a high percentage of increased C/EBP-ß activity was observed in RCC that was not confined to the kidney. Therefore, the increased activity of C/EBP-ß may be related to the invasiveness of RCC, although additional studies in a larger series need to be validated. Sundfelt et al. (23) demonstrated that an increased C/EBP-ß expression was observed in all proliferative and dedifferentiated malignant ovarian tumor tissues. This suggests that C/EBP-ß plays a role in the early oncogenesis of ovarian tumors. In contrast, the stage-specific increased expression of C/EBP-ß was observed in colorectal cancer, in which low-stage tumors had the increased expression (24) . A correlation with the local invasiveness of cancer shown in our study has not yet been described in other solid cancers. Increased activation of C/EBP-ß may be a late event in RCC oncogenesis.

On the basis of our data, C/EBP-ß may play a potential role in the invasion of RCC. Recent advances in molecular biology have led to the recognition of various mechanisms of cancer invasion. An increased degradation of collagen in the extracellular matrix mediated by MMPs has been suggested to play an essential role in cancer invasion (33) . Notably, C/EBP-ß induces MMP-1. Furthermore, MMP-2 promoter contains cis-acting C/EBP-ß regulator elements (34) . C/EBP-ß can work as a key transcription factor by inducing such MMPs. Another mechanism is conceivable through the activation of cytokines, including IL-6, IL-8, and TNF-{alpha}. TNF-{alpha} induces MMPs, such as membrane type 1-MMP, MMP-2, and MMP-9 (10 , 35) . IL-8 can also up-regulate MMP-2 and MMP-9 (36 , 37) . IL-6 has been shown to enhance the invasiveness of cancer cells, although the underlying mechanism remains unknown (38 , 39) .


   FOOTNOTES
 
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.

1 Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 To whom requests for reprints should be addressed, at Department of Urology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3825; Fax: 81-3-3225-1985; E-mail: moto-oya{at}sc.itc.keio.ac.jp Back

3 The abbreviations used are: RCC, renal cell carcinoma; IL, interleukin; TNF, tumor necrosis factor; MMP, matrix metalloprotease; C/EBP, CCAAT/enhancer binding protein; EMSA, electrophoretic mobility shift assay. Back

Received 2/26/02; revised 10/28/02; accepted 11/ 5/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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