Originally published as JCO Early Release 10.1200/JCO.2005.02.191 on November 30 2004

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Downregulation of Smac/DIABLO Expression in Renal Cell Carcinoma and Its Prognostic Significance

From the Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan; Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, UCLA School of Medicine, University of California at Los Angeles, Los Angeles, CA

Address reprint requests to Yoichi Mizutani, MD, Department of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan; e-mail: ymizutan{at}koto.kpu-m.ac.jp

	ABSTRACT

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PURPOSE: Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) was recently identified as a protein that is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing inhibitor of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune- and drug-induced apoptosis. However, little is known about the clinical significance of Smac/DIABLO in various cancers, including renal cell carcinoma (RCC). This study examined Smac/DIABLO expression in 78 healthy kidneys and 78 RCCs.

MATERIALS AND METHODS: The level of Smac/DIABLO expression was quantified by Western blot analysis using nonfixed fresh frozen tissues.

RESULTS: The expression of Smac/DIABLO was lower in RCC compared with the autologous normal kidney. Sixty-four (82%) of 78 of RCC expressed Smac/DIABLO, and 18% were negative, whereas 100% of normal kidney tissues were positive. In stage I/II RCC, 96% expressed Smac/DIABLO, whereas only 50% expressed Smac/DIABLO in stage III/IV. Smac/DIABLO expression inversely correlated with the grade of RCC. Patients with RCC expressing Smac/DIABLO had a longer postoperative disease-specific survival than those without Smac/DIABLO expression in the 5-year follow-up. Transfection with Smac/DIABLO cDNA enhanced tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) –mediated and cisplatin-mediated cytotoxicity in RCC.

CONCLUSION: The present study demonstrates for the first time that Smac/DIABLO expression was downregulated in RCC and that no Smac/DIABLO expression in RCC predicted a worse prognosis. In addition, transfection with Smac/DIABLO sensitized RCC to TRAIL/cisplatin-induced apoptosis. These results suggest that Smac/DIABLO expression in RCC may be used as a prognostic parameter, and that enhancement of Smac/DIABLO expression in RCC may potentiate immunotherapy and chemotherapy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Renal cell carcinoma (RCC) accounts for approximately 2% of all cancer cases worldwide.¹ Metastatic disease is often present at the time of diagnosis of RCC, and its poor response to chemotherapy and radiotherapy determines its poor prognosis. Cell death by apoptosis occurs when the intracellular apoptotic pathway is activated.² Signals inducing apoptosis can be very diverse and encompass the direct stimulation of death receptors or cellular stress induced by chemicals and irradiation. The ability to evade apoptosis may enhance the cells' propensity to malignancy. The classical apoptotic pathway consists of activation of the caspase family cascade. The effector caspase 3 serves to cleave the cellular protein substrates and brings about the apoptotic phenotype. Caspase 3 can be activated by either caspase 8 or by a signaling complex referred to as the apoptosome, consisting of cytochrome c, Apaf-1, and caspase 9. Cytochrome c allows the oligomerization of Apaf-1, thereby activating caspase 9 in the process. X-linked inhibitor of apoptosis protein (XIAP) has the potential to inhibit active caspase 3 and slows down the process at this step.³ Apoptogenic factors that are normally sequestered in the mitochondria are released into the cytosol during the mitochondria-dependent pathway for apoptosis. These factors include second mitochondria–derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO), endonuclease G, cytochrome c, and Omni/HtrA2.⁴ The release of cytochrome c into the cytoplasm is not always sufficient to initiate the caspase cascade. Endogenous inhibitors of apoptosis proteins (IAPs) including XIAP are present and thus prevent the activation of pro-caspases. Therefore, the inhibition of the activation of pro-caspases interferes with the activation of mature caspases. Murine Smac and its human ortholog DIABLO are 29-kD mitochondria precursor proteins proteolytically cleaved in the mitochondria to a 23-kD mature form and released into the cytosol after an apoptotic stimulus.^5,6 Smac/DIABLO acts as a dimer and contributes to caspase activation by sequestering IAPs.⁷

Recent studies have reported that overexpression of Smac/DIABLO can induce apoptosis and/or sensitize the resistant cancer cells to death receptor- or cytotoxic drug-induced apoptosis.^8,9 These findings suggest that Smac/DIABLO plays an important role in the regulation of apoptotic responses in cancer cells to both immune- and drug-mediated therapies. We hypothesized that the levels of Smac/DIABLO expression may decrease as a function of tumor cell progression. The expression of Smac/DIABLO in cancers has not been examined, with the exception of a recent study.¹⁰ There have been no reports on the expression of Smac/DIABLO in RCC and its potential clinical significance. This study was designed to test our hypothesis and investigate the level of Smac/DIABLO expression in RCCs compared with its expression in autologous normal kidneys and determine its prognostic significance.

	MATERIALS AND METHODS

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Patients
Surgical specimens were obtained from 78 patients with RCC. These patients were selected randomly for this study. They included 57 male and 21 female patients, ranging in age from 19 to 83 years. Histologic diagnosis revealed that 70, seven, and one patient had clear-cell carcinoma, papillary RCC, and Bellini duct carcinoma, respectively. Their histologic classification and staging data, according to the TNM system of classification (International Union Against Cancer, 6th edition, 2002), were: T1 (n = 54), T2 (n = 8), T3 (n = 12), T4 (n = 4); N0 (n = 74), N1 (n = 1), N2 (n = 3); M0 (n = 67), M1 (n = 11); stage I (n = 48), stage II (n = 6), stage III (n = 8), stage IV (n = 16), and G1 (n = 8), G2 (n = 48), G3 (n = 22), respectively. Specimens of normal kidney were collected from the same 78 patients with RCC. The paired samples were histologically confirmed RCC and normal kidney. Tissue specimens were also obtained from two patients with oncocytoma. The specimens were stored frozen at –80°C until use for the assay of Smac/DIABLO expression.

This study was performed after approval by a local human investigations committee. Informed consent was obtained from each patient.

RCC Cell Lines
NC65, ACHN, and Caki-1 human RCC cell lines^11,12 were maintained in monolayers on plastic dishes in RPMI-1640-1640 medium (Gibco Biocult, Glasgow, Scotland) supplemented with 25 mmol/L HEPES (Gibco Biocult); 2 mmol/L L-glutamine (Gibco Biocult); 1% nonessential amino acid (Gibco Biocult); 100 units/mL penicillin (Gibco Biocult); 100 µg/mL streptomycin (Gibco Biocult); and 10% heat-inactivated fetal bovine serum (Gibco Biocult), hereafter referred to as complete medium.

Measurement of Smac/DIABLO Expression in RCC and Normal Kidney by Western Blot Analysis and Definition of Smac/DIABLO Expression
The expression of Smac/DIABLO was determined by Western blot analysis as previously described.¹³ We electrophoresed 20 µg of the sample proteins on 7.5% polyacrylamide gels in Tris-glycin buffer and transferred it to nitrocellulose membranes. The membrane was blocked for 30 minutes in blocking buffer (5% skim milk in 1% Tween-phosphate-buffered saline) and probed first with the anti–Smac/DIABLO antibody (Imgenex, San Diego, CA) for 1 hour. The membrane was washed and then incubated with peroxidase-conjugated goat antirabbit immunoglobulin G (IgG) and developed with the use of an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). The relative expression of Smac/DIABLO protein was determined with a chemiluminescence imaging system and quantified by image analysis (Gel Doc 2000; BIO-RAD, Osaka, Japan).

The NC65 cell line constitutively expressed Smac/DIABLO and was used as the internal standard to compare assays. All samples were analyzed at the same time. Repeated measurements yielded the same results. When Smac/DIABLO expression was not visually observed by the Western blot analysis, it was regarded as no or negative expression. In contrast, expression of Smac/DIABLO was regarded as positive expression, if a visual band was detected by Western blot analysis regardless of the variation of the levels of expression. Positive expression meant unambiguous visual detection of Smac/DIABLO protein band by chemiluminescence and did not refer to the level of Smac/DIABLO expression.

Transient Transfection of RCC Cells With Smac/DIABLO cDNA
Transient transfection of RCC cells with Smac/DIABLO cDNA was determined as described by us previously.⁹ The transfection of RCC cell lines was performed with the pcDNA3.1 vector containing full-length Smac/DIABLO or an empty vector using the polycationic liposome reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The transfection was done according to the manufacturer's instructions. Overexpression of Smac/DIABLO was observed by this transfection procedure.⁹

Reagents
Recombinant human tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) was purchased from Peprotech (Rocky Hill, NJ). Cisplatin was supplied by Nippon Kayaku Co Ltd (Tokyo, Japan).

Cytotoxicity Assay
Microculture tetrazolium dye assay was used to determine tumor cell lysis as previously described.^14,15 Briefly, 100 µL of target cell suspension (2 x 10⁴ cells) was added to each well of 96 well flat-bottom microtiter plates (Corning Glass Works, Corning, NY), and each plate was incubated for 24 hours at 37°C in a humidified 5% CO₂ atmosphere. After incubation, the supernatants were aspirated, and tumor cells were washed three times with RPMI-1640 medium, and 200 µL of drug solution or complete medium for control were distributed in the 96-well plates. Each plate was incubated for 24 hours at 37°C. Following incubation, 20 µL of microculture tetrazolium dye working solution (5 mg/mL; Sigma Chemical Co, St Louis, MO) was added to each culture well and the cultures were incubated for 4 hours at 37°C in a humidified 5% CO₂ atmosphere. The culture medium was removed from the wells and replaced with 100 µL of isopropanol (Sigma Chemical Co) supplemented with 0.05 normal HCl. The absorbance of each well was measured with a microculture plate reader (Immunoreader; Japan Intermed Co Ltd, Tokyo, Japan) at 540 nm. The percent cytotoxicity was calculated by the following formula:

Statistical Analysis
All determinations were made in triplicate. For statistical analysis, Student's t test and a ² test were used. Postoperative disease-specific survival was determined by the Kaplan-Meier method. The Cox-Mantel test was used to establish the statistical difference in survival between RCC patients with and without Smac/DIABLO expression. A P value .05 was considered significant.

	RESULTS

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Expression of Smac/DIABLO in RCC Cell Lines, RCC, and Normal Kidneys
The levels of Smac/DIABLO in cell lysates of RCC cell lines, RCC, and normal kidneys were determined by Western blot analysis as described in Materials and Methods. The NC65, ACHN, and Caki-1 RCC cell lines all expressed Smac/DIABLO albeit at different levels (Fig 1A). NC65 expressed the highest level of Smac/DIABLO and Caki-1 expressed the lowest level. The expression level of Smac/DIABLO in normal kidneys was higher than that in the NC65 line, and the level of Smac/DIABLO expression in most RCCs was lower, as represented in Figure 1B.

View larger version (27K): [in this window] [in a new window]   Fig 1. (A) Expression of second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) in renal cell carcinoma (RCC; T) cell lines. (B) Expression of Smac/DIABLO in RCC and the normal (N) kidney.

View larger version (39K): [in this window] [in a new window]   Fig 2. (A) Expression of second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) in renal cell carcinoma (RCC; T) and the normal (N) kidney. (B) Expression of Smac/DIABLO in RCC and the N kidney. (C) Expression of Smac/DIABLO in primary and metastatic RCC (PT and MT, respectively). Case 12, brain metastasis; case 13, bone metastasis; case 14, bone metastasis.

View larger version (17K): [in this window] [in a new window]   Fig 3. Expression of second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) in oncocytoma. N, normal kidney; T, oncocytoma.

Correlation Between Smac/DIABLO Expression and Postoperative Disease-Specific Survival in Patients With RCC
RCC patients undergoing radical nephrectomy were evaluated for the postoperative clinical course. Postoperative disease-specific survival was estimated by Kaplan-Meier analysis. Based on this analysis, patients with RCC were divided into two groups; namely, those with positive Smac/DIABLO expression and those with negative expression, as described in Materials and Methods. Patients with RCC with positive Smac/DIABLO expression had a longer disease-specific survival compared with those with negative expression in the 5-year follow-up (Fig 4). Moreover, it is noteworthy that only one patient with RCC with positive Smac/DIABLO expression died in this study, and the expression of Smac/DIABLO was very low in the primary tumor and negative in the metastatic tumor (representative case 14, Fig 2C). These findings suggest that the level of Smac/DIABLO expression in RCC may be a prognostic indicator, and that positive Smac/DIABLO expression in RCC may be a good prognostic sign.

View larger version (12K): [in this window] [in a new window]   Fig 4. Relationship between second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) expression and postoperative disease-specific survival in patients with renal cell carcinoma. Solid line, 64 patients with positive Smac/DIABLO expression; dashed line, 14 patients with negative Smac/DIABLO expression.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
For the first time, we present evidence that Smac/DIABLO expression was downregulated in RCC compared with autologous normal kidneys, and that the level of Smac/DIABLO expression inversely correlated with both the progression of the stage and the increase of the grade of RCC. Furthermore, this study shows that RCC patients with positive Smac/DIABLO expression had a longer disease-specific survival as compared with those with negative expression in the 5-year follow-up. Although the data reported here correspond to a small number of patients during a short-term follow-up, these findings suggest that Smac/DIABLO in RCC may play an important role in regulating apoptosis and may be of prognostic value in RCC.

Patients with RCC respond very poorly to chemotherapy and radiotherapy.¹⁶ RCC cell lines have been described to be resistant to apoptosis-inducing stimuli. A set of cell lines derived from human RCC almost completely lacked the expression of caspase 3 and further expressed other caspases at low levels.¹⁷ Such alteration might contribute to RCC development. A recent study by Gerhard et al¹⁸ examined the functional competence of the apoptosome in RCC cell lines and RCC fresh tissues. They found that the apoptosome is structurally and functionally intact in both RCC cell lines and primary RCC by the criteria of adding exogenous cytochrome c. These findings suggested that the apoptosome may not be directly involved in resistance. Their study, however, did not examine the activation of the apoptosome and apoptosis by intrinsic cytochrome c and the role of Smac/DIABLO in the activation. The interaction of the apoptosome with low expression of cytochrome c or Smac/DIABLO may not be sufficient to trigger the apoptosome. The present study shows that low expression of Smac/DIABLO with a possibly intact apoptosome may be associated with resistance and suggests the therapeutic effect of overexpressing Smac/DIABLO in the reversal of resistance.

The present study has shown that the expression of Smac/DIABLO in RCC was significantly lower than that in the normal kidney, and approximately 20% RCC lacked Smac/DIABLO expression, though all normal kidney specimens expressed Smac/DIABLO. A recent study by Yoo et al¹⁰ has reported analysis of archival tissues of carcinoma and sarcoma by immunohistochemical analysis for the expression of Smac/DIABLO. Smac/DIABLO expression was observed in 62% of carcinomas and 22% of sarcomas. The level of Smac/DIABLO expression varied depending on the individual tumor. For instance, two of 10 prostate carcinomas were positive for Smac/DIABLO, whereas the remaining eight were negative. Normal tissues adjacent to the cancer showed various degrees of Smac/DIABLO expression. However, in this report, there were no data on the expression of Smac/DIABLO in RCC. Our studies and those of Yoo et al,¹⁰ demonstrating that the expression level of Smac/DIABLO in carcinoma and sarcoma is significantly different from the corresponding noncancerous tissues, suggest strongly that Smac/DIABLO might play a role in the development of cancer.

This study is the first to demonstrate that Smac/DIABLO expression in RCC predicts the clinical outcome. The precise reasons responsible for this relationship remain unclear at present. Since Smac/DIABLO is a proapoptotic regulatory molecule, it is reasonable to assume that despite treatments, clones of cells that do not express Smac/DIABLO will not undergo apoptosis and will be selected to grow more easily and rapidly than clones that overexpress Smac/DIABLO. In addition, the current study has shown that Smac/DIABLO was less expressed in the metastatic RCC than in the primary RCC. These findings suggest that Smac/DIABLO agonists may provide a therapeutic means of preventing metastasis and growth of RCC.

Cytotoxic chemotherapy, an integral part of the therapeutic approach for many solid tumors, has shown little or no antitumor activity against RCC and has played no role in either an adjuvant or a neoadjuvant support therapy.¹⁶ Immunotherapy including interleukin-2 and interferon alfa is relatively effective against metastatic RCC, and the overall response rate of immunotherapy and/or chemotherapy has gradually improved. However, the response rate is approximately 20%, and metastasis and recurrence still remain major problems in the therapy for RCC.¹⁹ Therefore, new therapeutic approaches are required. The down-regulation of Smac/DIABLO expression in RCC compared with the normal kidney identifies Smac/DIABLO as a molecular therapeutic target. Our observation that overexpression of Smac/DIABLO in RCC by transfection resulted in high sensitivity to TRAIL/cisplatin–mediated killing, may be clinically relevant in the management of patients with RCC. The endogenous low level of Smac/DIABLO in RCC may not be adequate to neutralize the antiapoptotic mechanism regulated by IAPs. Thus, immunotherapy/chemotherapy in combination with Smac/DIABLO agonists may be a promising strategy against RCC. Furthermore, enhancement of Smac/DIABLO expression by gene therapy may also provide a novel therapeutic means of overcoming the resistance of RCC to immunotherapy/chemotherapy.

IAPs such as XIAP are highly expressed in various cancers and are associated with poor prognosis and resistance to apoptosis.^20,21 Preliminary experiments demonstrated that the expression of XIAP in RCC was higher than that in the normal kidney. Since XIAP blocks apoptosis at the effector phase, strategies targeting XIAP may be especially effective at overcoming resistance to apoptosis. Smac/DIABLO is able to bind to IAP family members, and XIAP is a predominant Smac/DIABLO binding protein. Smac/DIABLO binds to XIAP, displaces XIAP from caspase-9, promotes cleavage of effector caspases, and induces apoptosis.^22,23 Therefore, the measurement of XIAP expression as well as Smac/DIABLO expression may be necessary for the accurate evaluation of the efficacy of therapy with Smac/DIABLO.

Drugs that can antagonize IAPs may have benefits, particularly when combined with chemotherapeutic drugs or TRAIL. For instance, Arnt et al²⁴ found that the first four amino acids of Smac/DIABLO increased apoptosis in cell lines treated with paclitaxel, etoposide, camptothecin, and doxorubicine.

Cancer therapy using TRAIL or anti-DR4/5 monoclonal antibody is currently being investigated in clinical trials due to their low toxicity to normal tissues.^25,26 However, not all tumors respond to TRAIL, and resistance to TRAIL has been shown to be overcome by drugs,^27,28 by overexpression of Smac/DIABLO, or by Smac/DIABLO peptides.^8,9,24 Thus, analysis of the expression of Smac/DIABLO in cancer may be helpful for determining therapeutic modalities such as TRAIL therapy.

The findings of this study showed that patients with RCC with positive Smac/DIABLO expression had a longer disease-specific survival than those with negative expression. Fundamentally, patients without metastasis or recurrence received no postoperative treatments. The first- and the second-line treatments for metastasis or recurrence were intramuscular interferon alfa monotherapy and combination therapy with intramuscular interferon alfa and intravenous interleukin-2, respectively. The third-line treatment or surgery was dependent on each patient. Therefore, differing therapies may in part account for the different survival curves.

The dramatic postoperative disease-specific survival advantage for Smac/DIABLO-positive RCCs is the central issue in this study. In stage III/IV RCC patients (n = 24), 10 patients (83%) with negative Smac/DIABLO expression (n = 12) died of RCC. In contrast, only one patient (8%) with positive expression (n = 12) died. However, the numbers do not permit multivariate analysis. Therefore, it is not clarified whether Smac/DIABLO expression adds prognostic importance to tumor stage or not. The potential biologic significance of Smac/DIABLO expression as a component of a more generalized problem of apoptotic resistance warrants further investigation and suggests future clinical study.

In conclusion, the current study demonstrated that Smac/DIABLO expression was downregulated in RCC, and that negative Smac/DIABLO expression was a poor prognostic sign. Furthermore, elevated Smac/DIABLO expression by transfection rendered resistant RCC cells sensitive to TRAIL/cisplatin-mediated cytotoxicity. These findings suggest that the assessment of Smac/DIABLO expression may be useful in the management of RCC. Since Smac/DIABLO expression could be used as a prognostic parameter in patients with RCC, the accurate prediction of prognosis may help select patients for more intensive surgical or immunochemotherapeutic approaches in combination with Smac/DIABLO agonists. However, further studies are needed to determine the regulatory effects of Smac/DIABLO expression in RCC.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The authors indicated no potential conflicts of interest.

	Acknowledgment

 
We acknowledge the assistance of Ms Yukako Morioka and Ms Kate Dinh in the preparation of this manuscript.

	NOTES

 
Supported in part by grants in aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 15390496), and from the US Department of Defense (DAMD17-02-1-0023; B.B.).

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
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Submitted February 26, 2004; accepted July 20, 2004.

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