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[Cancer Research 65, 3700-3706, May 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Estrogen Receptor–Binding Fragment–Associated Antigen 9 Is a Tumor-Promoting and Prognostic Factor for Renal Cell Carcinoma

Tetsuo Ogushi1, Satoru Takahashi1, Takumi Takeuchi1, Tomohiko Urano2, Kuniko Horie-Inoue3, Jinpei Kumagai1, Tadaichi Kitamura1, Yasuyoshi Ouchi2, Masami Muramatsu3 and Satoshi Inoue2,3

Departments of 1 Urology and 2 Geriatric Medicine, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan and 3 Research Center for Genomic Medicine, Saitama Medical School, Yamane, Hidaka-shi, Saitama, Japan

Requests for reprints: Satoshi Inoue, Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-3-5800-8652; Fax: 81-3-5800-6530; E-mail: INOUE-GER{at}h.u-tokyo.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The estrogen receptor–binding fragment–associated antigen 9 (EBAG9) has been identified as a primary estrogen-responsive gene in human breast cancer MCF7 cells. A high expression of EBAG9 has been observed in invasive breast cancer and advanced prostate cancer, suggesting a tumor-promoting role of the protein in malignancies. Here we show that intratumoral (i.t.) administration of small interfering RNA against EBAG9 exerted overt regression of tumors following s.c. implantation of murine renal cell carcinoma (RCC) Renca cells. Overexpression of EBAG9 did not promote the proliferation of culture Renca cells; however, the inoculated Renca cells harboring EBAG9 (Renca-EBAG9) in BALB/c mice grew faster and developed larger tumors compared with Renca cells expressing vector alone (Renca-vector). After renal subcapsular implantation, Renca-EBAG9 tumors significantly enlarged compared with Renca-vector tumors in BALB/c mice, whereas both Renca-EBAG9 and Renca-vector tumors were developed with similar volumes in BALB/c nude mice. No apparent difference was observed in specific cytotoxic T-cell responses against Renca-EBAG9 and Renca-vector cells; nonetheless, the number of infiltrating CD8+ T lymphocytes was decreased in Renca-EBAG9 subcapsular tumors. Furthermore, immunohistochemical study of EBAG9 in 78 human RCC specimens showed that intense and diffuse cytoplasmic immunostaining was observed in 87% of the cases and positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients. Multivariate analysis revealed that high EBAG9 expression was an independent prognostic predictor for disease-specific survival (P = 0.0485). Our results suggest that EBAG9 is a crucial regulator of tumor progression and a potential prognostic marker for RCC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor–binding fragment associated gene 9 (EBAG9) is an estrogen-responsive gene that we previously identified in MCF-7 human breast carcinoma cell line using a CpG-genomic binding site cloning method (1). EBAG9 protein, whose molecular size is 32 kDa by Western blot analysis, is expressed in estrogen target organs as well as several other tissues such as brain, liver, and kidney (2). The protein expression of EBAG9 is estrogen inducible, as it has been shown in ovariectomized mice treated with 17ß-estradiol administration (2). The physiologic function of EBAG9 has not been well defined, yet the molecule may be implicated in cancer pathophysiology, with several lines of evidence of the protein expression in malignancies, including breast (3), ovarian (4), prostate (5), and hepatocellular carcinomas (6). In prostate cancer (5), EBAG9 expression significantly correlated with advanced pathologic stages and high Gleason score (P = 0.0305 and P < 0.0001, respectively), suggesting the abundance of EBAG9 may relate to the progression of malignant tumors.

In the present study, we investigated whether EBAG9 expression is critical in tumor development of renal cell carcinoma (RCC). RCC that comprises the majority of kidney cancer is one of the 10 most common malignancies in industrialized countries (7). The prognosis of patients with advanced RCC is poor, as 5-year survival rate is <5% (8), and the treatment of metastatic RCC remains a difficult clinical challenge. Development of new and alternative modalities of diagnosis and therapy for RCC is a clinical requisite. We used murine syngeneic renal adenocarcinoma model of Renca cells in this study and investigated whether gene silencing or overexpression of EBAG9 influences Renca cell growth and/or in vivo tumorigenesis. Administration of small interfering RNA (siRNA) against EBAG9 regressed s.c. Renca tumors. The proliferation of culture Renca cells constitutively expressing EBAG9 was not basically different from control Renca cells, whereas EBAG9-expressing cells grew faster in BALB/c mice and developed larger tumors. The tumor-promoting effect of EBAG9 in Renca tumors may relate to the suppression of antitumor immunity, as i.t. CD8+ T lymphocytes were reduced in renal subcapsular Renca tumors. The tumorigenic relevance of EBAG9 in Renca models further extended to clinicopathologic significance of the molecule in human RCC. EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients and it was an independent prognostic predictor for disease-specific survival. Our findings show that EBAG9 is a tumor-promoting factor and a potential prognostic marker in RCC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Rabbit anti-EBAG9 polyclonal antibody was generated against a fusion protein of glutathione S-transferase and EBAG9 (2). Rabbit polyclonal antihuman CD3 antibody (DakoCytomation, Carpinteria, CA), rat antimouse CD4 (L3T4; clone RM 4-5), rat antimouse CD8a (Ly-2; 53-6.7) monoclonal antibodies (BD PharMingen, San Diego, CA), and anti–ß-actin monoclonal antibody (Sigma, St. Louis, MO) were commercially purchased. Human EBAG9 cDNA was cloned into a mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA).

Tumor cells. Renca is a spontaneously arising murine RCC and was prepared as previously described (9, 10). Tumor cells were maintained in RPMI 1640 containing 10% FCS and antibiotics.

Mice. BALB/c mice and BALB/c nu/nu mice (Nisseizai, Tokyo, Japan) that were syngeneic to Renca cells were kept under specific pathogen-free conditions and fed dry food and water. All mice used for experiments were male at the age of 5 weeks.

Patients and tissue preparation. We investigated 78 tissue samples of RCC obtained from patients (14 females and 64 males) who underwent radical or partial nephrectomy at Tokyo University Hospital between 1990 and 1995. Patient information was retrieved from the review of patient charts. Staging and grading of the tumors were done according to the 1997 International Union Against Cancer tumor-node-metastasis classification and WHO histopathologic typing, respectively (11). The mean age of this population was 54 years (26-76 years) and the mean follow-up period was 60 months (2-78 months). For 32 patients with advanced tumors (pT2 or greater), adjuvant therapy was done, including immune therapy (n = 30), radiation (n = 5), and surgery for metastatic diseases in lung, colon, and pancreas (n = 8). During the follow-up period, 55 patients (70.5%) survived without evidence of disease, eight cases (10.3%) presented with tumor recurrence, and 15 cases (19.2%) died of disease. None died of other diseases.

Western blot analysis. Cells were lysed in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 8.0), 200 mmol/L NaCl, 20 mmol/L NaF2, 2 mmol/L EGTA, 1 mmol/L DTT, 2 mmol/L sodium vanadate, 0.5% v/v NP40 supplemented with a protease inhibitor cocktail Complete (Boehringer Manheim GmbH, Mennheim, Germany)]. Proteins were resolved by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were probed with rabbit anti-EBAG9 antibody or anti–ß-actin monoclonal antibody.

Tumor regression by EBAG9 small interfering RNA. Small interfering RNA (siRNA) duplex that targets EBAG9 was generated by Dharmacon (Lafayette, CO). The target sequence of EBAG9 siRNA was 5'-AAGAAGAUGCAGCCUGGCAAG-3'. Scramble II Duplex (Dharmacon) was used as a nontargeting control siRNA that does not possess homology with known gene targets in mammalian cells. The GC content of Scramble II Duplex was 57.9%, which was identical to that of EBAG9 siRNA.

To investigate in vivo silencing effect of EBAG9 siRNA in Renca tumors, i.t. injection of siRNA duplexes was done twice every week. Briefly, Renca cells (1 x 104 cellis) were implanted in the flank of BALB/c mice. Tumor size was measured weekly with a micrometer in two dimensions, and tumor volume was estimated according to the formula: (smallest diameter)2 x (longest diameter). When the volumes of tumors reached 300 mm3, siRNA duplexes (10 µg) were injected directly into tumors twice every week, along with 4 µL of GeneSilencer (Gene Therapy System, San Diego, CA) dissolved in 0.1 mL of Opti-MEM (Life Technologies, Gaithersburg, MD). Mice were sacrificed 4 weeks after treatment.

Generation of Renca cells stably expressing EBAG9. Renca cells were transfected with an expression vector pcDNA3, including human EBAG9 cDNA or vector alone using LipofectAMINE (Life Technologies). G418-resistant cells were selected and several independent clones were isolated.

Reverse transcription-PCR. Total cellular RNA of Renca cells was extracted using ISOGEN reagent (Nippon Gene, Tokyo, Japan) and first-stand cDNA was generated from 5 µg of total cellular RNA using a reverse transcriptase Omniscript RT (Qiagen, Tokyo, Japan) and random hexamers. To validate the expression of exogenous human EBAG9, reverse transcription-PCR (RT-PCR) was done using specific primers for human EBAG9 (sense 5'-GCTACACAAGATCTGCCTTT-3' and antisense 5'-CTTCTTCATTAGCCGTTGTG-3'). The amplification was done for 35 cycles at 62°C for annealing, using AmpliGold Taq polymerase (Perkin-Elmer, Boston, MA).

In vivo tumor challenge. For s.c. implantation, transfected Renca cells (1 x 104 cells per mouse) suspended in 0.1 mL of complete medium were injected in the flank of BALB/c mice. Tumor volume was calculated weekly. In survival analyses, Renca-bearing mice were followed up for 14 weeks after implantation.

For renal subcapcular implantation, tumors cells (1 x 104 cells per mouse) suspended in 0.1 mL of complete medium were inoculated into the subcapsule of the left kidney of BALB/c wild-type and nude mice. Mice were sacrificed 25 days after implantation and tumors were excised.

Cell proliferation assay. Cells were seeded at a density of 1 to 3 x 105 cells per dish into 10-cm dishes and hemocytometer counting was done every 2 days. Doubling time during exponential growth was determined by a formula: [incubation time (h) x log102] / [log10(cell number at sampling period) – log10(plating cell number)] (12).

Proliferation assays were done using the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt (WST-8) reagent (Nacalai, Kyoto, Japan; ref. 13). The assay is based on the conversion of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-like tetrazolium salt WST-8 to a water-soluble formazan by metabolically active cells and provides a quantitative determination of viable cells. Cells were seeded in 96-well plates at an initial density of 625 to 5,000 cells per well. At 1 hour after inoculation, cells were transfected with either EBAG9 siRNA or Scramble II Duplex (100 ng per well) using GeneSilencer reagent (Gene Therapy Systems). Assays were done on days 0, 2, and 4. For cells cultured up to day 4, medium was once exchanged on day 2. Spectrophotometric absorbance at 450 nm (for formazan dye) was measured with absorbance at 620 nm for reference.

Cytotoxicity assay. Renca-EBAG9 or Renca-vector cells were used as target cells. Splenocytes of Renca-bearing BALB/c mice were stimulated for 5 days in vitro with irradiated Renca cells at a splenocyte/tumor cell ratio of 20:1 in the presence of 1,000 IU/mL interleukin-2 and used as effector CTLs. Target cells were incubated with effector CTLs at various E/T ratios in a final volume of 200 µL for 18 hours at 37°C. Lactate dehydrogenase release from cells with a damaged membrane was examined using CytoTox-ONE Reagent (Promega, Madison, WI) and fluorescence was measured with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Experiments were done in triplicate.

Immunohistochemistry. Immunohistochemical studies were done using the streptavidin-biotin amplification method with horseradish peroxidase detection. Paraffin sections of tumors were blocked in 0.3% H2O2 (30 minutes) and in 10% FCS (30 minutes), incubated overnight with specific antibodies against CD3, CD4, or CD8a for Renca tumors (1:20 dilution), or with purified rabbit anti-EBAG9 antibody for human RCC (1:40 dilution). Sections were incubated with biotinylated rabbit antirat immunoglobulin G or antirabbit EnVison+ reagent (DakoCytomation), developed by diaminobenzidine (Sigma), and counterstained with hematoxylin (Sigma). Negative controls were done for each slide, using nonimmune immunoglobulin G.

In Renca experiments, numbers of tumor-infiltrating lymphocytes (TILs) positive for CD3, CD4, or CD8 expression were microscopically examined in the high-power field of view at a magnification of 400x (14). BALB/c mouse spleen specimen was used as a positive control.

In RCC examination, immunoreactivity scores of EBAG9 expression were determined by two pathologists according to percentages of positive cells. Human breast cancer section (DakoCytomation) was used as a positive control. Positivity was 0% to 4% for immunoreactivity score of 0 (negative), 5% to 24% for a score of 1+, 25% to 49% for a score of 2+, and 50% to 100% for a score of 3+. Sections that had ≥25% positive cells but apparent lower intensity compared with positive controls were scored as immunoreactivity score of 1+. Immunoreactivity scores of 1+, 2+, and 3+ were defined as positive staining. If immunoreactivity scores were different between two pathologists, the average immunoreactivity score was adopted. If several types of histology were included in one section, immunoreactivity score of predominant histology was used.

Statistical analyses. Comparisons between different groups of Renca samples were analyzed with nonparametrical Mann-Whitney U test. The associations between EBAG9 immunoreactivity and clinicopathologic characteristics were evaluated by Student's t test or Fisher's exact probability test. Disease-specific survival was computed by Kaplan-Meier method and the curves were compared by log-rank test. Multivariate analysis of prognostic factors was done using Cox proportional hazard regression model. Computations were done with the StatView 5.0J software (SAS Institute, Inc., Cary, NC). All Ps are two sided and evaluated as significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene silencing of EBAG9 suppressed in vivo tumor growth of Renca cells. To determine the role of EBAG9 in tumor growth of renal cancer cells, we investigated the effects of synthesized siRNA duplexes targeting EBAG9 on s.c. tumor models of Renca cells implanted in syngeneic BALB/c mice. Intratumoral injection of EBAG9 siRNA reduced the protein levels of endogenous EBAG9 compared with the levels of EBAG9 in parental Renca cells or in the Renca tumor treated with control scrambled siRNA duplexes (Fig. 1A). Under the treatment of scrambled siRNA, s.c. implanted Renca cells developed prominent tumors, whereas the injection of EBAG9 siRNA suppressed tumor growth of Renca cells (Fig. 1B and C). After 4-week treatments, the volume of tumors with EBAG9 siRNA treatment was significantly smaller than that with scrambled siRNA (3,854 ± 665 versus 6,315 ± 1,053 mm3, n = 5; P = 0.0472). We infer that tumor growth is modulated by EBAG9 expression, implicating EBAG9 as a tumor-promoting factor in renal carcinoma.



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  		Figure 1. Expression of EBAG9 siRNA suppresses tumor growth derived from murine renal cell carcinoma Renca cells in BALB/c mice. S.c. primary Renca tumors were established by midflank injections of 10,000 tumor cells and i.t. injections of either control scrambled siRNA or EBAG9 siRNA duplexes together with a transfection reagent GeneSilencer were done twice a week in five mice per group when the initial tumor volumes reached 300 mm3. Mice were sacrificed after 4 weeks of siRNA administration and tumors were homogenized for protein extraction. A, Western blot analysis of lysates from in vitro culture Renca cells and tumor samples expressing either control scrambled siRNA or EBAG9 siRNA. B, representative mice after 4 weeks of siRNA treatment. Top, mouse treated with control scrambled siRNA. Bottom, mouse treated with EBAG9 siRNA. C, tumor volume in EBAG9 siRNA–treated mice (n = 5) is reduced compared with control mice (n = 5). *, P < 0.05 at 4 weeks (EBAG9 siRNA versus scrambled siRNA).

 
Generation of Renca cells stably expressing EBAG9. To explore whether constitutive EBAG9 expression influences tumor growth, we generated Renca cells stably expressing human EBAG9. We selected two Renca-EBAG9 cell clones 3 and 4 that express human EBAG9 mRNA as confirmed by RT-PCR using human EBAG9–specific primers (Fig. 2A, top). The amounts of EBAG9 proteins in Renca-EBAG9 cells were ~2.0 fold increased compared with those in parental Renca cells and Renca-vector cell clones 1 and 2, which were transfected with pcDNA3 empty vector (Fig. 2A, bottom). In terms of cell growth rate, doubling time of culture Renca-EBAG9 cells was not significantly different from that of Renca-vector cells (Fig. 2B). Proliferation of Renca cells was further analyzed by a colorimetric MTT-like assay using a tetrazolium monosodium salt WST-8 that is converted to a water-soluble formazan by metabolically active cells (Fig. 2C). Neither EBAG9 overexpression nor RNA interference against EBAG9 did not significantly influence the growth of Renca cells. Moreover, EBAG9 overexpression did not influence the incorporation of bromodeoxyuridine in culture Renca cells (data not shown). The results suggest that stable expression of EBAG9 itself does not accelerate the proliferation of culture tumor cells.



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  		Figure 2. Overexpression of EBAG9 in Renca cells does not accelerate culture cell growth. A, RT-PCR analysis and Western blot analysis of Renca cells stably expressing human EBAG9 (Renca-EBAG9) or empty vector (Renca-vector). Top, human EBAG9 mRNA is expressed in clones 3 and 4 of Renca-EBAG9 cells. Empty pcDNA3 vector was used as a negative control and pcDNA3 including EBAG9 cDNA as a positive control. Bottom, EBAG9 protein is overexpressed in Renca-EBAG9 clones compared with Renca-vector clones or parental Renca cells. B, doubling time of culture Renca cells. The numbers of cells in the exponential growth were counted every 2 days and doubling time was calculated according to a formula as described in Materials and Methods (n = 5 for each). C, proliferative assay using WST-8 tetrazolium salt. Cells seeded into 96-well plates were transfected with control scrambled siRNA or EBAG9 siRNA duplexes (100 ng per well) and cell proliferation was evaluated on days 0, 2, and 4 (n = 3 for each). Absorbance at 450 nm (for formazan dye) was measured with absorbance at 620 nm for reference.

 
EBAG9 promotes in vivo tumor growth of Renca cells. In spite of little difference of propagation abilities between Renca-EBAG9 cells and Renca-vector cells in culture, Renca-EBAG9 cells s.c. implanted into BALB/c mice developed >4-fold larger tumors compared with Renca-vector cells at 4 weeks after inoculation (Fig. 3A and B). Mean tumor volumes at 4 weeks were 1,712 ± 506 mm3 for Renca-EBAG9 cell clones 3 and 4 versus 366 ± 110 mm3 for Renca-vector cell clones 1 and 2 (P = 0.0055; Fig. 3B).



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  		Figure 3. Renca cells stably expressing EBAG9 develop large tumors in BALB/c mice. A, representative mice 4 weeks after the inoculation of tumor cells. B, volumes of tumors derived from Renca-EBAG9 cells are significantly larger compared with Renca-vector cells in BALB/c mice. S.c. primary tumors were established by midflank injections of 10,000 tumor cells. **, P < 0.01 at 4 weeks (Renca-EBAG9 versus Renca-vector). Renca-vector 1, n = 16; Renca-vector 2, n = 6; Renca-EBAG9 3, n = 6; Renca-EBAG9 4, n = 8. C, poorer prognosis of mice inoculated with Renca-EBAG9 cells compared with mice with Renca-vector cells. Disease-specific survival on day 100: P = 0.0412 (Renca-EBAG9 versus Renca-vector). Renca-vector 1, n = 9; Renca-vector 2, n = 8; Renca-EBAG9 3, n = 8; Renca-EBAG9 4, n = 10.

 
In terms of prognosis of mice harboring Renca tumors, 23.5% of mice with Renca-vector cells (n = 17) survived on day 100 after tumor challenge whereas only 5.6% of mice with Renca-EBAG9 cells (n = 18) survived at the same period (Fig. 3C; P = 0.0412 by log-rank test). Systemic metastases, including tumor dissemination into peritoneum and distant metastases of lung and liver, were reasons for death in all deceased cases.

EBAG9 suppresses host immune surveillance. To determine whether aberrant EBAG9 expression in Renca cells affects the local immune responses in tumors, we implanted Renca-EBAG9 cells or Renca-vector cells under the renal capsule of BALB/c mice and immunodeficient BALB/c nude mice. Both Renca cell lines formed macroscopic tumors in all of the cancer-bearing hosts by day 25 (Fig. 4A). In conventional BALB/c mice, Renca-EBAG9 tumors grew significantly larger compared with Renca-vector tumors (Fig. 4A and B). Mean volumes of tumors on day 25 in BALB/c mice were 856 ± 162 mm3 (n = 19) for Renca-EBAG9 clones 3 and 4 versus 149 ± 59 mm3 (n = 18) for Renca-vector clones 1 and 2 (Fig. 4B; P < 0.0001). In immunodeficient BALB/c nude mice, both Renca-vector cells and Renca-EBAG9 cells developed extensive tumors compared with tumors in BALB/c mice and there was no significant difference in tumor volumes between Renca-vector cells and Renca-EBAG9 cells (Fig. 4A and B). Mean volumes of tumors on day 25 in BALB/c nude mice were 2,215 ± 227 mm3 (n = 18) for Renca-EBAG9 clones 3 and 4 versus 1,802 ± 240 mm3 (n = 23) for Renca-vector clones 1 and 2 (Fig 4B; P = 0.118). These results may suggest that aberrant EBAG9 expression in Renca cells hampers a local primary immune response that retards the growth of tumors rather than potentiates the intrinsic tumorigenicity of the tumor cells.



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  		Figure 4. EBAG9 overexpression promotes renal subcapsular tumor growth by Renca cells in wild-type BALB/c mice. A, representative tumors 25 days after the inoculation of tumor cells (10,000 cells). B, volumes of Renca-EBAG9 tumors are larger than Renca-vector tumors in BALB/c mice, whereas no significant difference of tumor volumes between Renca-vector and Renca-EBAG9 in BALB/c nude mice. **, P < 0.0001 on day 25 (Renca-EBAG9 versus Renca-vector). Renca-vector 1, n = 12; Renca-vector 2, n = 11; Renca-EBAG9 3, n = 9; Renca-EBAG9 4, n = 9. C, lysis of Renca-EBAG9 and Renca-vector cells by tumor-specific CTLs. Splenocytes from Renca-bearing mice were cultured with Renca cells at a ratio of 20:1 pulsed with interleukin-2 (1,000 units/mL) for 5 days. Lactate dehydrogenase release from cells with a damaged membrane was examined using CytoTox-ONE Reagent and fluorescence was measured with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. D, numbers of tumor infiltrating-lymphocytes positive for CD3, CD4, or CD8 immunostaining were microscopically examined in the high-power field of view at a magnification of 400x. BALB/c mouse spleen specimen was used as a positive control. *, P < 0.05 (Renca-EBAG9 versus Renca-vector).

 
To investigate whether the progression of Renca-EBAG9 tumors depends on a reduced sensitivity of the cells to tumor-specific CTLs, we did cytotoxicity assays. Effector CTLs were derived from splenocytes of Renca-bearing BALB/c mice, after a 5-day restimulation with Renca cells in the presence of interleukin-2. (Fig. 4C). Renca-EBAG9 cells and Renca-vector cells were equally lysed by tumor-specific CTLs, suggesting that EBAG9 expression itself does not affect the sensitivity of Renca cells to CTL lysis.

To assess whether EBAG9 modulates the subtype-specific reactivity of T lymphocytes against tumors, we examined the numbers of TILs in renal subcapsular Renca tumors developed in BALB/c mice (Fig. 4D). No significant differences in numbers of CD3+ and CD4+ T cells were observed between Renca-vector and Renca-EBAG9 tumors, whereas the number of CD8+ T cells in Renca-EBAG9 tumors was significantly decreased compared with that in Renca-vector tumors (P < 0.05).

Expression of EBAG9 protein in human renal cell carcinoma tumors. The finding that EBAG9 modulated the growth of Renca tumors led us to the notion whether the molecule contributes to the progression of RCC in human tissues. EBAG9 expression was evaluated immunohistochemically in 78 RCC whole tissue specimens including normal lesions. In noncarcinomatous lesions, a weak and scattered immunostaining of EBAG9 was observed in the cytoplasm of the mesangial cells (Fig. 5A) as well as on the luminal surface of the renal tubular cells (data not shown). The levels of EBAG9 expression in normal renal tissues corresponded to immunoreactivity score of 0. In RCC tumors, 10 of 78 cases (13%) had negative immunoreactivity of EBAG9, whereas 68 of cases (87%) showed EBAG9 positivity. With regard to EBAG9-positive RCC tumors, the cancer cells generally retain intense and diffuse staining patterns in the cytoplasm or on the membrane (Fig. 5B, C, and D). The levels of EBAG9 positivity were immunoreactivity score of 1+ for 18 RCC tumors (23%), 2+ for 31 tumors (40%), and 3+ for 19 tumors (24%). With respect to RCC histology, clear cell tumors displayed an intense membrane staining as well as a diffuse cytoplasmic staining of EBAG9 (Fig. 5B; immunoreactivity score, 2+). Sarcomatoid tumors showed an intense and frequent cytoplasmic immunoreactivity (Fig. 5C; immunoreactivity score, 3+). Lung metastatic tumors showed the highest EBAG9 staining, predominantly in the cytoplasm (Fig. 5D; immunoreactivity score, 3+).



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  		Figure 5. EBAG9 immunostaining in human kidney and renal cell carcinoma specimens. A, normal kidney (immunoreactivity score, 0). EBAG9 is weakly expressed in the mesangial cells (arrowheads). B, clear cell carcinoma (immunoreactivity score, 1+). EBAG9 is immunostained predominantly on the cell membrane in cancerous regions. C, spindle cell carcinoma (immunoreactivity score, 3+). Intense immunostaining of EBAG9 is observed in the cytoplasm of sarcomatoid cancerous regions. D, lung metastatic tumors (immunoreactivity score, 3+). Intense immunoreactivity of EBAG9 is observed in metastatic tumors with high immunoreactivity scores. Bars, 50 µm.

 
A significant association between EBAG9 immunoreactivity and clinicopathologic variables was observed in RCC patients (Supplementary Table 1). EBAG9 positivity (immunoreactivity score, >1+) was significantly correlated with advanced pathologic tumor stages, positivity of vascular infiltration, and nonclear cell histology (P = 0.0017, P = 0.0109, and P = 0.0126, respectively).

In Kaplan-Meier analysis of the RCC patients, those in which the tumor had high EBAG9 immunoreactivity (immunoreactivity score, 3+) showed a shorter disease-specific survival (Fig. 6) compared with patients showing low or negative EBAG9 immunoreactivity (immunoreactivity score, 0-2+). The 5-year disease-specific survival in cases with EBAG9 immunoreactivity score of 3+ was 55%, whereas 91.2% of patients with low or negative EBAG9 immunoreactivity were alive during the same period.



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  		Figure 6. Association of immunocytochemical staining for EBAG9 with disease-specific survival of 78 RCC patients. Five-year disease-specific survival of the patients with high EBAG9 immunoreactivity (immunoreactivity score, 3+; n = 19) was significantly worse than the patients with immunoreactivity scores of 0-2+ (n = 59; 55% versus 91%, P = 0.0007, by log-rank test).

 
In univariate Cox proportional hazards analysis for a 5-year disease-specific survival, established prognostic factors including infiltration, pathologic stage, and metastatic status are the most significant univariate variables of survival (Supplementary Table 2; P < 0.0001 for all). Lower EBAG9 immunoreactivity as well as negativity of lymph node status or vascular infiltration are also involved in significant univariate survival predictors (P = 0.0007, 0.0002, and 0.0003, respectively). In multivariate Cox proportional hazards analysis, negative metastatic status is the most significant predictor of survival (Supplementary Table 3; P < 0.001; relative risk, 42.53) Notably, high EBAG9 immunoreactivity is associated with disease-specific death in multivariate analysis (P = 0.0485; relative risk, 5.09). These results indicate that high immunoreactivity of EBAG9 is a potential poor prognostic variable in RCC patients.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows the first evidence regarding a tumor-promoting role of EBAG9 in vivo. We showed that Renca tumors overexpressing EBAG9 had a much aggressive phenotype with poorer prognosis compared with Renca tumors expressing empty vector, although the effects of EBAG9 on culture cell proliferation was relatively minimal. EBAG9 immunoreactivity was detected in most of human RCC samples and high amounts of EBAG9 protein may associate with poor prognosis of the patients. Our findings suggest that EBAG9 is a tumor-promoting factor in RCC yet does not function as an essential oncogene by itself. The present results lead us to the notion that EBAG9 potentiates tumor growth by altering tumor microenvironment.

Decreased local immune responses may be one of the critical mechanisms that change tumor microenvironment. In antitumor immunity, T lymphocyte–mediated immune surveillance is thought a principal host defense mechanism (15). Although tumors such as RCC are immunogenic and could be targeted by tumor-specific CTL or natural killer cells, antitumor immune reactions are not completely effective to reject tumor cells so that tumors continue to grow progressively (16). In our cytotoxicity assay, there was no significant difference of CTL lysis between Renca-EBAG9 and Renca-vector cells, suggesting that overexpression of EBAG9 may not particularly alter the presentation of tumor-associated antigens or the levels of MHC class I molecule expression. In TIL assay, however, CD8+ T cells seemed specifically reduced by aberrant EBAG9 expression. We suspect that generation of immunosuppressive factors or apoptosis activation may result in the reduction of CD8+ TIL, leading to hamper antitumor immunity.

The alteration in cell surface glycosylation could be implicated in the modulation of tumor microenvironments (17, 18). It has been recently shown that tumor-associated ganglioside expression in human RCC cells suppresses nuclear factor-{kappa}B activation in T cells and mediates T-cell apoptosis (19, 20). RCC display increased levels of gangliosides including GM2, GM1, and GD1a (21) as well as several disialogangliosides (22), which may inhibit the function of antigen-presenting cells (23) or modulate tumor vascularization (24). It has been recently shown that tumor-associated O-linked glycan antigens Tn and TF were expressed in transfected cells expressing RCAS1 (receptor-binding cancer antigen expressed on SiSo cells; ref. 25), whose cDNA has been found to be a homologue of EBAG9 (26).

Another possible explanation is that EBAG9 may stimulate angiogenesis by up-regulating growth factors or cytokines. There are literatures that suggest that vascular endothelial growth factor (VEGF) could be involved in RCC tumor progression. Mutations of the von Hippel-Lindau tumor suppressor gene, which are often observed in hereditary RCC and sporadic clear cell RCC, result in overproduction of VEGF through a mechanism involving hypoxia-inducible factor {alpha} (27, 28). It has been recently shown that VEGF interferes with the development of T cells at pathologically relevant concentrations in vivo (29); thus, the growth factor may contribute to tumor-associated immune deficiencies.

It has been generally accepted that tumor cells may escape from immune surveillance by expressing the EBAG9 homologue RCAS1, which targets RCAS1 receptor–expressing immune cells and induces apoptosis (26). Nakashima et al. identified the RCAS1 cDNA through expression cloning using the 22-1-1 monoclonal antibody that they originally generated (30). Engelsberg et al. recently showed, however, that the 22-1-1 epitope was distinct from the products encoded by RCAS1 cDNA, because the RCAS1 protein was not recognized by the 22-1-1 antibody, whereas the 22-1-1 antibody recognized the tumor-associated O-linked glycan antigens (25). They showed that their raised polyclonal antibody recognized a ~35-kDa protein, consistent with the immunoblotting results using our polyclonal antibody. On the contrary, the putative RCAS1 protein recognized by the 22-1-1 antibody was identified as an ~80-kDa membrane molecule expressed on human uterine cancer cells (26, 30). Although there are a number of publications concerning RCAS1 in cancers from the point of view as the 22-1-1 antigen, we consider that a pathophysiologic role of EBAG9 in tumor immunology needs to be properly evaluated. The present article may provide new insights into an EBAG9-mediated in vivo function in cancer progression.

We have previously reported that the immunoreactivity of EBAG9 was mainly observed in the cytoplasm of normal epithelial cells with a granular staining pattern, or particularly in perinuclear regions (3, 5). In carcinoma tissues, an intense staining of the cell surface could be also observed such as in prostate cancer or hepatocellular carcinoma. The expression of RCAS1 immunoreactivity recognized by antibodies against recombinant RCAS1 was localized to perinuclear structures, suggesting that the protein is predominantly distributed in the Golgi system (25). Given that EBAG9 is a Golgi-predominant protein that could be trafficking from the perinuclear regions to the cell surface membrane, it is likely that EBAG9 immunoreactivity could be observed in both cytoplasm and cell surface of cancerous tissues with the abundant expression of EBAG9. Notably, EBAG9 immunoreactivity in RCC with advanced stages such as sarcomatoid or metastatic tumors was cytoplasmic predominant (Fig. 5C and D). Further studies using confocal or electron microscopic examination may elucidate the dynamic distribution of EBAG9.

As we showed that there are several types of cancer that intensely express EBAG9 and the expression levels of EBAG9 may relate to advanced tumor grades (3–6), it is likely that the tumor-promoting effect of EBAG9 is a general event in malignancies regardless of their estrogen dependency. We also observed the lack of association between sex and EBAG9 expression in human RCC in our clinicopathologic study (Supplementary Table 1). Thus, EBAG9 could be a therapeutic target for various tumors constitutively expressing the molecule.

In summary, we show that EBAG9 is a tumor-promoting factor in both murine Renca RCC and human RCC. We propose that EBAG9 immunoreactivity is a new potential biomarker for prognosis of RCC and a treatment modality targeting EBAG9 will provide a novel therapeutic option for advanced RCC.


   Acknowledgments
 
Grant support: Ministry of Health, Labor and Welfare Japan, the Ministry of Education, Culture, Sports, Science and Technology Japan.

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.

We thank T. Suzuki for her technical assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org).

Received 9/28/04. Revised 2/15/05. Accepted 2/25/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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