Clinical Cancer Research Joint Metastasis Research Society-AACR Conference on Metastasis Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
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Clinical Cancer Research Vol. 12, 3470-3477, June 1, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Anti-CD26 Monoclonal Antibody–Mediated G1-S Arrest of Human Renal Clear Cell Carcinoma Caki-2 Is Associated with Retinoblastoma Substrate Dephosphorylation, Cyclin-Dependent Kinase 2 Reduction, p27kip1 Enhancement, and Disruption of Binding to the Extracellular Matrix

Teruo Inamoto1,3, Tadanori Yamochi1, Kei Ohnuma1, Satoshi Iwata1, Shinichiro Kina1, Sakiko Inamoto1,3, Masaaki Tachibana2, Yoji Katsuoka3, Nam H. Dang4 and Chikao Morimoto1,4

Authors' Affiliations: 1 Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo; 2 Department of Urology, Tokyo Medical College, Tokyo, Japan; 3 Department of Urology, Osaka Medical College, Osaka, Japan; and 4 Department of Hematologic Malignancies, Nevada Cancer Institute, Las Vegas, Nevada

Requests for reprints: Chikao Morimoto, Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5549; Fax: 81-3-5449-5548; E-mail: morimoto{at}ims.u-tokyo.ac.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: CD26 is a 110-kDa cell surface glycoprotein with a role in tumor development through its association with key intracellular proteins. In this report, we show that binding of soluble anti-CD26 monoclonal antibody (mAb) inhibits the growth of the human renal carcinoma cells in both in vitro and in vivo experiments.

Experimental Design: Growth inhibition by anti-CD26 mAb was assessed using proliferation assay and cell cycle analysis. Anti-CD26 mAb, chemical inhibitors, dominant-negative, or constitutively active forms of specific signaling molecules were used to evaluate CD26-associated pathways. The in vivo growth-inhibitory effect of anti-CD26 mAb was also assessed in a human renal carcinoma mouse xenograft model.

Results: In vitro experiments show that anti-CD26 mAb induces G1-S cell cycle arrest associated with enhanced p27kip1 expression, down-regulation of cyclin-dependent kinase 2, and dephosphorylation of retinoblastoma substrate. Moreover, our data show that enhanced p27kip1 expression is dependent on the attenuation of Akt activity. Anti-CD26 mAb also internalizes cell surface CD26, leading to decreased binding to collagen and fibronectin. Experiments with a mouse xenograft model involving human renal carcinoma cells show that anti-CD26 mAb treatment drastically inhibits tumor growth in tumor-bearing mice, resulting in enhanced survival.

Conclusions: Taken together, our data strongly suggest that anti-CD26 mAb treatment may have potential clinical use for CD26-positive renal cell carcinomas.

CD26 is a 110-kDa surface glycoprotein with dipeptidyl peptidase IV activity able to cleave selected biological factors to alter their functions (1). CD26/dipeptidyl peptidase IV is involved in T lymphocyte signal transduction processes (2, 3) and regulates topoisomerase II{alpha} level in hematologic malignancies, affecting sensitivity to doxorubicin and etoposide (4). Moreover, our previous work showed that anti-CD26 monoclonal antibody (mAb) inhibits growth of CD26-positive T-cell malignancies (5, 6). Expressed on various tissues, including epithelial cells of liver, intestine, and kidney (1, 7), CD26 is involved in the development of certain human cancers, with lung adenocarcinomas, differentiated thyroid carcinomas, and metastatic prostate cancer being CD26 high and other histologic types of lung carcinomas, benign thyroid diseases, and primary prostate cancer being CD26 low (7–10). Notably, the role of CD26 in cancer biology depends on tumor types, as it is associated with high level of tumor aggressiveness in some and lower level in others (7–10). In a previous report, CD26 expression was detected on renal cell carcinoma (RCC) with unclear clinical significance (11). Extending these previous findings, we now define CD26 expression on normal and malignant tubular epithelial kidney tissues and establish that CD26 is an appropriate target for RCC treatment.

CD26 structure consists of three regions, an extracellular region, a 22-residue hydrophobic transmembrane region, and a 6-amino acid cytoplasmic region. The extracellular region contains a membrane-proximal glycosylated domain, a cysteine-rich domain, and a 260-amino acid COOH-terminal domain containing dipeptidyl peptidase IV activity. We now show that the anti-CD26 mAb 14D10, which recognizes the cell membrane-proximal glycosylated region starting with a 20-amino acid flexible stalk region of human CD26, induces cell cycle arrest, concomitantly blocking the adhesion of RCC cells to the extracellular matrix (ECM). In addition, anti-CD26 mAb-mediated growth arrest of RCC cells results from dephosphorylation of retinoblastoma substrate, decreased level of cyclin-dependent kinase (CDK) 2, and up-regulation of the Akt-dependent CDK inhibitor p27kip1. Furthermore, studies using a mouse xenograft model show that anti-CD26 mAb treatment inhibits RCC tumor growth in vivo. Our work hence suggests a potential role for CD26-targeted therapy in the treatment of human RCC.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies. Anti-CD26 mAb (IgG1) 14D10 and anti-CD45 mAb (IgG1) 2H4 were developed in our laboratory as described previously (12, 13), with the latter being used as isotype-matched control mAb. Rabbit mAb to Thr308 or Ser473-phosphorylated forms of Akt and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 and mouse mAb to protein kinase B{alpha}/Akt, ERK1/2, CDK4, CDK6, and phosphothreonine were from Cell Signaling Technology, Inc. (Beverly, MA), and mouse mAb to p21cip1/waf1, CDK2, p53, cyclin D1, retinoblastoma substrate, and p27kip1 were from BD PharMingen (Lexington, KY). Oct-1, and {alpha}-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA). LY294002, wortmannin, and PD98059 were from Calbiochem (San Diego, CA). Nocodazole was from Sigma-Aldrich (St. Louis, MO). Nocodazole (500 ng/mL from 1 mg/mL stock solution in DMSO), LY294002 (30 µmol/L from 50 mmol/L stock solution in DMSO), and PD98059 (30 µmol/L from 10 mmol/L stock solution in DMSO) were added to the culture medium 30 minutes before treatment with each antibody.

Cell culture and transfection procedures. Caki-2 (human renal carcinoma), LNCap (androgen-dependent prostate carcinoma), and DU-145 cells (androgen-independent prostate carcinoma) were kind gifts from Dr. Haruhito Azuma (Osaka Medical College, Osaka, Japan). VMRC-RCW (human renal carcinoma), Caki-1 (human renal carcinoma), and ACHN (human renal carcinoma) were obtained from Cell Resource Center for Biomedical Research (Tohoku University, Sendai, Japan). All cells were grown in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 µg/mL; Life Technologies, Inc., Gaithersburg, MD) or G418 (500 µg/mL; Sigma-Aldrich). The plasmid vectors (Upstate Biotechnology, Lake Placid, NY) used in exploring signaling pathway were as follows: the Myc-tagged NH2-terminal myristylated active Akt1 cDNA (Myr-Akt), dominant-negative form of Akt cDNA (d.n.-Akt), and hemagglutinin-tagged constitutive active mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) 1 cDNA (upstream of 44/42MAPK; c.a. MEK1), with all three constructs being placed in a pUSEamp vector. The plasmids were transfected into various cells using Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN). In each experiment, neomycin phosphotransferase II (Upstate Biotechnology) was probed to evaluate transfection efficacy.

Cell cycle analyses. Cells (1 x 106 per well) were incubated in medium alone or in the presence of anti-CD26 or isotype-matched control mAbs at indicated concentrations in the presence or absence of nocodazole. DNA contents were analyzed using propidium iodide as described previously (5) and were measured using FACSCalibur (Becton Dickinson Co., San Jose, CA) with CellQuest software (Becton Dickinson) and ModFit program (Becton Dickinson). In all experiments, at least >1 x 104 cells were sorted after gating out the fixation artifacts and cell debris.

2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium assay. Cells were synchronized using double-thymidine block method as described previously (14), then released, and subjected to incubation in 96-well plates in medium alone or in the presence of anti-CD26 (0.1, 1, or 10 µg/mL) or isotype-matched control mAbs (0.1, 1, or 10 µg/mL) for a total volume of 100 µL (5 x 103 cells per well). After 24 hours of incubation in 37°C, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (Seikagaku, Tokyo, Japan) was added to each well. After another 2 hours of incubation, water-soluble formazan dye on bioreduction in the presence of an electron carrier, 1-methoxy-5-methylphenazinium, was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA). All samples were tested in triplicate. Values reported represent the mean of triplicated wells, and SE was within 15%.

Immunocytochemistry and immunohistochemistry. For fluorescent microscopy experiments, cells were treated and stained according to the methods described previously (3). In brief, Caki-2 cells (5 x 104/mL) were grown on coverslips in six-well plates in the presence or absence of anti-CD26 mAb. The cells were fixed in 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 in PBS and stained with anti-p27kip1 mAb and FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). After mounting with a ProLong Antifade kit (Molecular Probes, Eugene, OR), slides were examined by Olympus IX70 confocal microscope with 40 objective lenses (Olympus, Tokyo, Japan) using laser excitation at 488 nm. For immunohistochemistry, 11 primary RCC surgical specimens from patients were evaluated. For each, 10% formalin-fixed, paraffin-embedded specimens containing both the carcinoma and its adjacent nonneoplastic tissue were prepared. Slides were deparaffinized and then heated in a microwave processor for antigen retrieval in 10 mmol/L citrate buffer (pH 6) for 10 minutes. After blocking in 3% (v/v) bovine serum albumin, slides were incubated at 4°C overnight with the primary antibody (anti-CD26 mAb), washed with PBS, incubated for 30 minutes with FITC-conjugated anti-mouse IgG, and then analyzed using confocal laser microscopy. To serve as a control for nonspecific staining, duplicate sections were stained with isotype-matched mAb instead of the primary antibody. Two different pathologists checked the validity of the obtained results. All human specimens were obtained from the Department of Urology, Tokyo Medical College (Tokyo, Japan), and informed consents were obtained from all patients according to the format of the Institutional Review Board.

SDS-PAGE and immunoblotting. Preparation of whole-cell lysates and cell fractionations were done as described elsewhere (15). For detection of phosphorylated proteins, cells were harvested in NP40 buffer [1% NP40, 0.5% sodium deoxycholate, 5 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 8), 0.15 mol/L NaCl] containing 1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L NaF, 1 mmol/L Na3VO4, 10 µg/mL aprotinin, and 10 µg/mL leupeptin. The protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA). Specific antigens were probed by the corresponding mAbs followed by horseradish peroxidase–conjugated secondary Ig (Amersham Pharmacia Biotech, Piscataway, NJ). Western blots were visualized by the enhanced chemiluminescence technique (NEN, Boston, MA).

In vivo model. In vivo studies were approved by the Institute Animal Care and Use Committee. Female-specific pathogen-free BALB/c nu–/– mice (ages 8 weeks) were purchased from Charles River (Yokohama, Japan). All mice were pretreated by i.p. route with 0.2 mL anti-asialo-GM1 polyclonal antisera (25%, v/v; Wako, Osaka, Japan) 1 day before tumor transplant to eliminate host natural killer cell activity. For a xenograft model of RCC, mice were anesthetized with diethyl ether and subjected to direct s.c. inoculation of Caki-2 cells (1 x 106 per mouse) in 100 µL Matrigel (BD Biosciences, San Jose, CA). The studies involving the observation of tumor volume and survival advantage included five mice per group. Mice bearing established tumors (~5 mm in size) received PBS alone, anti-CD26 mAb, or isotype-matched control mAb by intratumoral injection in 0.1-mL volume of sterile PBS at 10 µg per dose as described previously (6). The mice were injected every 4 days for 60 days. Tumor-bearing mice were then monitored for tumor development and progression. Tumor size was determined by caliper measurement of the largest (x) and smallest (y) perpendicular diameters every 4 days and calculated according to the formula V = {pi}/6 x xy2. Cumulative proportion survival was assessed by Kaplan-Meier. Necropsies of moribund mice were done for evaluation of tumor, and tumors were removed to be frozen. After homogenization by Dounce homogenizer, frozen tissues were lysed in lysis buffer [1% SDS, 4 mol/L urea, 1 mmol/L EDTA, 150 mmol/L NaCl, 50 mmol/L Tris (pH 8)]. Each 50 µg of lysates was subjected to SDS-PAGE and immunoblotting to examine protein levels of p27kip1, phosphorylated Akt, and ß-actin.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cell surface CD26 is highly expressed on human RCC. Previous work showed CD26 expression is enhanced on RCC (11). We first evaluated CD26 expression level on surgically resected human RCC tissues from Japanese patients. Eleven consecutive surgically resected RCC specimens from the primary sites were examined for surface CD26 expression. CD26 was highly expressed on RCC tissues compared with normal renal cells surrounding the RCC (Fig. 1A, a and b ) particularly in normal proximal tubules (Fig. 1A, c and d). All 11 RCC tissues and 6 normal renal tissues surrounding RCC were evaluated for their CD26 expression intensity, revealing high expression on human RCC tissues compared with normal renal tissues (Fig. 1B). The RCC cell lines Caki-2, VMRC-RCW, Caki-1, and ACHN all exhibited high surface CD26 levels, with DU-145 and LNCap showing much lower expression (Fig. 1C). These data indicated that all RCC cells tested were highly CD26 positive, whereas tumor cells arising from other origins, including prostate cancers, exhibited much lower level.


Figure 1
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  		Fig. 1. Cell surface expression of CD26 in human RCC. A, immunohistochemical analysis of CD26 expression was done on surgically resected RCC specimens of primary sites. RCC stained with H&E (a) and anti-CD26-FITC (b). Normal renal structures adjacent to RCC tissues in (a) and (b) were stained with H&E (c) and anti-CD26-FITC (d). Representative of 11 consecutive specimens. Original magnification, x200. N, normal renal tissue; C, connective tissue; T, tumor. B, fluorescent intensities of CD26 were evaluated from 11 RCC tissue samples and 6 adjacent normal renal tissues. Fluorescent intensity was determined as follows: strong positive (T; Fig. 1A, b), positive (Fig. 1A, d), weak positive (C; Fig. 1A, b), or none. Identical experiments were repeated thrice with similar results. C, surface expression of CD26 on various cell lines was analyzed by flow cytometry. Red line, CD26 histograms were obtained by staining with mouse anti-CD26 mAb followed by staining with rabbit anti-mouse IgG-FITC conjugate. Black line, control histograms represent background fluorescence obtained by staining of same cell cultures with isotype-matched control mAb.

 
Anti-CD26 mAb induces cell cycle arrest at G1-S checkpoint and growth inhibition of RCC. Because we showed previously that anti-CD26 mAb treatment induced cell cycle arrest at G1-S in CD26-positive T-cell lymphoma and T-cell clones (5, 6), we first examined anti-CD26 mAb effect on cell cycle progression of CD26-positive RCC cell lines. Treatment of Caki-2 cells with anti-CD26 blocked cell cycle progression at G1-S (Fig. 2A ), an effect better visualized when cells were treated with nocodazole to induce M-phase cell cycle arrest (Fig. 2B). Caki-2 cells exhibited a significant increase in G0-G1 from 14 to 28 hours after initiation of anti-CD26 treatment (Fig. 2C), whereas anti-CD26 mAb-mediated G0-G1 arrest occurred in a dose-dependent manner, peaking at 10 µg/mL (Fig. 2D). Consistent with the observed anti-CD26 mAb-induced cell cycle arrest G1-S, growth of Caki-2, VMRC-RCW, Caki-1, ACHN, and DU-145 cells was inhibited in a dose-dependent manner when treated with anti-CD26 mAb for 24 hours (Fig. 3 ). However, anti-CD26 mAb had minimal growth-inhibitory effect on LNCap cells, indicating the specific antitumor effect of anti-CD26 mAb.


Figure 2
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  		Fig. 2. Anti-CD26 mAb-mediated cell cycle arrest at G1-S checkpoint in RCC. A, Caki-2 cells were treated with medium alone (med), anti-CD26 mAb (CD26mAb), or isotype-matched control mAb (iso) for 21 hours, and cell cycle analysis was done by ModFit program. Histograms were made by CellQuest software. Data are representative of three independent experiments. B, nocodazole was added to Caki-2 cells 30 minutes before administration of medium, anti-CD26 mAb, or isotype-matched control mAb. Data are representative of three independent experiments. C, Caki-2 cells were treated with isotype-matched control mAb or anti-CD26 mAb. After the indicated time period, cell cycle analysis was done. Points, mean of triplicated tests; bars, SE. D, Caki-2 cells were treated with isotype-matched control mAb or anti-CD26 mAb at a concentration of 0.1, 1, and 10 µg/mL. Caki-2 cells were collected for cell cycle analysis at 21 hours after antibody administration.

 

Figure 3
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  		Fig. 3. Inhibitory effect of anti-CD26 mAb on RCC proliferation. Synchronized 5 x 103 cells per well of Caki-2, VMRC-RCW, Caki-1, ACHN, DU-145, and LNCap were incubated in 96-well plates in the presence of either anti-CD26 or isotype-matched control mAbs. After 24 hours of antibody treatment, water-soluble formazan dye on bioreduction in the presence of an electron carrier, 1-methoxy-5-methylphenazinium, was measured at 450 nm using a microplate reader as described in Materials and Methods, and growth-inhibitory ratio was calculated as % reduction of A450 nm.

 
Enhancement of p27kip1 expression, reduction of CDK2, and dephosphorylation of retinoblastoma substrate are associated with anti-CD26 mAb-mediated G1-S cell cycle arrest through phosphatidylinositol 3-kinase/Akt and ras/raf/MEK/ERK(MAPK) pathways. Because the cell cycle is strictly regulated by regulators (16), we next evaluated the levels of cell cycle regulators in Caki-2 cells following anti-CD26 mAb treatment. Enhanced expression of p27kip1, reduction of CDK2, and dephosphorylation of retinoblastoma substrate were observed, with no detectable changes in p21cip/waf1, p53, cyclin D1, CDK4, and CDK6 (Fig. 4A ). Markedly enhanced p27kip1 level was detected in the nuclei of anti-CD26-treated cells (Fig. 4B), corroborated by results of cell fractionation study (Fig. 4C). Moreover, immunoprecipitation analysis with anti-p27kip1 mAb revealed that anti-CD26 mAb treatment reduced the amount of phosphothreonine residue of p27kip1, suggesting that accumulation of p27kip1 is nuclei specific (Fig. 4D; ref. 17). These results strongly suggested that anti-CD26-mediated up-regulation of p27kip1 occurred mainly in the nucleus, being potentially responsible for cell cycle arrest in RCC.


Figure 4
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  		Fig. 4. Anti-CD26 mAb-mediated induction of p27kip1 expression. A, Caki-2 cells were treated with medium alone, anti-CD26 mAb, or isotype-matched control mAb. At 21 hours after antibody administration, cells were harvested, lysed, subjected to SDS-PAGE, and probed by specific antibody to p27kip1, p21cip1/waf1, p53, CDK2, CDK4, CDK6, cyclin D1, and phosphorylated retinoblastoma substrate (Rb). B, Caki-2 cells (5 x 104/mL) were grown on coverslips in six-well plates in medium alone (a), anti-CD26 mAb (b), or isotype-matched control mAb (c). After 21 hours of incubation, cells were subjected to immunocytochemistry as described in Materials and Methods and stained with mouse anti-p27kip1 mAb followed by FITC-conjugated anti-mouse IgG. Bar, 50 µm. C, after 21 hours of treatment with medium alone, anti-CD26 mAb, or isotype-matched control mAb, Caki-2 cells were harvested for cell fractionation. Each sample was probed with antibodies against p27kip1. To determine fractionation purity, Oct-1 and {alpha}-tubulin were probed with specific antibodies for cytoplasmic and nuclear fractions, respectively. D, for immunoprecipitation, whole-cell lysates were precipitated with anti-p27kip1 mAb, subjected to SDS-PAGE, and probed by specific antibody to phosphothreonine.

 
Because anti-CD26 mAb induced nuclear accumulation of p27kip1 protein, concomitantly reducing the phosphorylated form of p27kip1 (Fig. 4B-D), we hypothesized that the phosphatidylinositol 3-kinase/Akt pathway is involved in this process (17, 18). Elucidating the particular signaling pathways involved in anti-CD26 mAb-mediated up-regulation of p27kip1, we found that activated Akt was significantly abrogated in Caki-2 cells as early as 7 hours and as late as 21 hours after antibody treatment (Fig. 5A, b, lanes 16-21 ). Attenuation of 44/42MAPK was also observed as late as 21 hours following antibody treatment of Caki-2 cells (Fig. 5A, d, lanes 19-21).


Figure 5
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  		Figure 5. Anti-CD26 mAb-mediated enhancement of p27kip1 in RCC via attenuation of phosphorylated Akt rather than phosphorylated 44/42MAPK. A, Caki-2 cells were treated with medium only, anti-CD26 mAb, or isotype-matched control mAb. Caki-2 cells were immediately collected for preparation of whole-cell lysates at 0, 5, 10, and 30 minutes and 1, 7, 21, and 42 hours after administration of indicated panel of antibodies. B, for whole-cell lysate preparation, Caki-2 cells were preincubated in the presence or absence of PD98059 and LY294002 30 minutes before treatment with medium alone, isotype-matched control mAb, or anti-CD26 mAb at 10 µg/mL. After 21 hours of antibody treatment, cells were harvested and subjected to SDS-PAGE and immunoblotting for p27kip1 (a), phosphorylated Akt (b), protein kinase B{alpha}/Akt (c), phosphorylated 44/42MAPK (d), 44/42MAPK (e), and ß-actin (f). C, SDS-PAGE and immunoblotting of whole-cell lysates of Caki-2 cells that express a His/Myc-tagged dominant-negative Akt (Caki-2-d.n.-Akt), His/Myc-tagged myristylated constitutively active Akt (Caki-2-myr-Akt), and control vector (Caki-2-pUSEamp). Transient transfectants of Caki-2-pUSEamp, Caki-2-d.n.-Akt, and Caki-2-myr-Akt were made, and after 48 hours, each of the three transfectants was treated by medium alone, 10 µg/mL anti-CD26 mAb, or 10 µg/mL isotype-matched control mAb. Twenty-one hours after antibody treatment, cells were collected, lysed, subjected to SDS-PAGE and immunoblotting, and probed with specific antibody to p27kip1 (a), phosphorylated Akt (b), protein kinase B{alpha}/Akt (c), Myc (d), neomycin phosphotransferase II (e), and ß-actin (f). D, in vitro proliferation assay of Caki-2-parental, Caki-2 pretreated by PD98059, Caki-2 pretreated by LY294002, Caki-2-pUSEamp, Caki-2-d.n.-Akt, and Caki-2-myr-Akt. Caki-2 cells were preincubated in the presence or absence of PD98059 (Caki-2 PD) and LY294002 (Caki-2 LY) for 30 minutes before treatment with isotype-matched control or anti-CD26 mAbs. Transient transfectants of Caki-2-pUSEamp, Caki-2-d.n.-Akt, and Caki-2-myr-Akt were established 48 hours before treatment with isotype-matched control mAb or anti-CD26 mAb. Anti-CD26 and isotype-matched control mAbs were administered at 10 µg/mL to each cell type, and then cells were subjected to in vitro cell proliferation assay. Independent tests were examined in triplicates. Columns, mean; bars, SE.

 
To further characterize the specific pathway with a key role in the anti-CD26 mAb-induced enhancement of p27kip1 expression, specific inhibitors against each pathway as well as genetically introduced His/Myc-tagged dominant-negative form of Akt (Caki-2-d.n.-Akt) and constitutively active His/Myc-tagged myristylated Akt (Caki-2-myr-Akt) were used (Fig. 5B-D). Caki-2 pretreated with LY294002 (Fig. 5B, e, lanes 3, 6, and 9) and Caki-2-d.n.-Akt (Fig. 5C, a, lanes 7-9) up-regulated endogenous p27kip1 protein level, more importantly resulting in additive up-regulation of p27kip1 when combined with anti-CD26 mAb. In contrast, Caki-2-myr-Akt exhibited only sparse basal level of p27kip1, completely abolishing anti-CD26 mAb-induced up-regulation of p27kip1 (Fig. 5C, a, lanes 10-12). Meanwhile, PD98059, a specific inhibitor of MEK1, did not cause any detectable change in p27kip1 (Fig. 5B, e, lanes 2, 5, and 8). These findings strongly suggested that the phosphatidylinositol 3-kinase/Akt pathway is more potent than the ras/raf/MEK/ERK pathway in mediating anti-CD26 mAb-induced p27kip1 accumulation.

To confirm the above results, Caki-2 pretreated with LY294002 and PD98059, Caki-2-d.n.-Akt, and Caki-2-myr-Akt were evaluated for cell proliferation activity. Caki-2 pretreated with LY294002 and Caki-2-d.n.-Akt displayed drastically increased anti-CD26 mAb-mediated growth inhibition, whereas Caki-2-myr-Akt completely abolished this effect (Fig. 5D). On the other hand, pretreatment of Caki-2 with PD98059 did not enhance anti-CD26 mAb-mediated growth inhibition. Taken together, the above results showed that the phosphatidylinositol 3-kinase/Akt pathway was more potent than the ras/raf/MEK/ERK pathway in regulating anti-CD26 mAb-mediated growth arrest and that Akt activity has an effect on anti-CD26 mAb-induced expression of p27kip1 and growth arrest.

Anti-CD26 mAb regulates cell adhesion to ECM through internalization of cell surface CD26 and reduces in vivo tumorigenicity of Caki-2 associated with prolonged survival. Because CD26 plays a role in cell adhesion to the ECM proteins (19, 20), we examined the effect of anti-CD26 mAb on cellular interaction with the ECM. Anti-CD26 antibody inhibited Caki-2 binding to fibronectin and type I collagen (Fig. 6A ) associated with antibody-mediated CD26 internalization (Fig. 6B). These findings thus suggested that contact inhibition may play a contributing role to the observed anti-CD26-mediated up-regulation of p27kip1 (21, 22).


Figure 6
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  		Fig. 6. Antitumor effect of anti-CD26 mAb in mouse xenograft model of Caki-2. A, effect of anti-CD26 mAb on cell adhesion to ECM. Caki-2 cells treated with medium only, anti-CD26 mAb, or isotype-matched control mAb were plated onto 60-mm dishes (3 x 106 per dish) coated with collagen I (CL), fibronectin (FN), or laminin (LN) and cultured for 21 hours. The adhesive ability of cancer cells was expressed as the mean number of cells that had attached to the bottom surface of the dish. Columns, mean number of cells per field of view; bars, SE. Values for invasion were determined by calculating the average number of adhesive cells per mm2 over three fields per assay and expressed as an average of triplicate determinations. Adhesive cells (%): adhesive cells / adhesive cells + nonadhesive cells. B, Caki-2 cells were treated with anti-CD26 mAb on ice, or isotype-matched control mAb, followed by washing in ice-cold PBS twice and subsequently incubated at 37°C for 12 hours. Cell were collected and stained with FITC-conjugated anti-mouse IgG. Expression status of cell surface CD26 was analyzed by flow cytometry. To detect total CD26 level, including the internalized CD26 fraction, cell membrane permeabilization method was used (26). Filled histogram, positive control, which was incubated 30 minutes with anti-CD26 mAb. Open histogram, status of CD26 after treatment. C, Caki-2 cells (1 x 106) were inoculated s.c. into the left flank of mice. CD26 expression of Caki-2 cells after tumor implantation into the mouse was similar to its level before tumor implantation. Mice were treated with intratumoral injection of PBS only (medium; n = 5), anti-CD26 mAb (n = 5), or isotype-matched control mAb (n = 5) on the day when the tumor mass became visible (5 mm in size). Tumor size and cumulative survival were monitored. D, resected specimens were immediately frozen for whole-cell lysate preparation and lysed by lysis buffer as described in Materials and Methods. Protein (50 µg) was applied for SDS-PAGE and immunoblotting for p27kip1, phosphorylated Akt, and ß-actin. R', RR, RL, RL', and LL, names of mice in each treatment group.

 
We also investigated the effect of anti-CD26 mAb treatment on Caki-2 growth in a xenograft mouse tumor model. Mice treated with anti-CD26 mAb had a lower rate of tumor development than controls, leading to enhanced survival (Fig. 6C). To define the molecular events occurring in inoculated tumors, tumor mass was removed at day 60 after initial treatment for postmortem biochemical analyses of tissue lysates. Our results showed enhanced expression of p27kip1 protein with concomitant inactivation of Akt in the tumor mass (Fig. 6D), consistent with our in vitro data (Figs. 4 and 5). Taken together, these results indicated that CD26 may be an appropriate molecular target for RCC therapy.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show the antitumor effect of anti-CD26 mAb in an in vitro and in vivo model. Importantly, our study suggests the potential role of CD26 as a molecular target in human RCC, a highly malignant disease that is resistant to standard treatment with chemotherapy or biologics (23). Although highly expressed in RCC, particularly the clear cell type (11), CD26 role in human RCC is poorly understood. Our present results indicate that anti-CD26 mAb induces RCC G1-S cell cycle and growth inhibition, concomitantly blocking cell adhesion. Our data also suggest that targeting CD26 is a potentially effective therapeutic strategy for selected neoplasms, including human RCC. Of interest is that tumors can result from cell cycle dysregulation, with various cancers expressing low levels of CDK inhibitors, including p27kip1 (16).

Although recent work showed that activated Akt regulates both p27kip1 subcellular localization and degradation (17, 24), we show that combining anti-CD26 mAb with LY294002 or a dominant-negative form of Akt has an additive effect on p27kip1 accumulation. In contrast, constitutively active Akt abolishes not only basal p27kip1 protein level but also anti-CD26 mAb-induced p27kip1 overexpression, with resultant amelioration of growth inhibition by anti-CD26 mAb. These observed results strongly suggest that Akt negatively regulates total p27kip1 protein level, and perturbation of CD26 by its specific antibody engages the same pathway in RCC, leading to an increase in G0-G1 phase. Meanwhile, the mechanisms involved in anti-CD26 mAb-induced inactivation of Akt resulting in p27kip1 accumulation in RCC remain to be elucidated. One possible explanation may involve the potential interaction between CD26 and the docking sites for Akt. Binding of anti-CD26 antibody causes CD26 internalization, leading to signal transduction. Because Akt is a lipid-binding protein kinase, which becomes activated as a result of recruitment to docking sites consisting of phosphatidylinositol phosphate in the plasma membrane (25), antibody-induced CD26 internalization may affect Akt docking and activation in RCC. Moreover, because anti-CD26 mAb blocks ECM binding and because p27kip1 is up-regulated during contact inhibition (21), anti-CD26 mAb may induce cellular mechanisms associated with contact inhibition in selected CD26-positive adhesive tumors.

Our present study shows that CD26 is highly expressed in human RCC and that anti-CD26 mAb binding engages key signaling pathways, resulting in G1-S arrest. Our subsequent in vivo experiments further indicate that CD26 is an appropriate molecular target for RCC therapy by showing that anti-CD26 mAb treatment leads to loss of tumorigenicity. We postulate that the potent antitumor effect of anti-CD26 mAb observed in our study may be used in the future as novel therapeutic approaches against various CD26-positive malignancies, including RCC.


   Acknowledgments
 
We thank Y. Urasaki and Y. Itoh for their technical assistance.


   Footnotes
 
Grant support: Grant-in-Aid of Ministry of Education, Science, Sports, and Culture (K. Ohnuma and C. Morimoto) and Ministry of Health, Labor, and Welfare, Japan (C. Morimoto); Osaka Kidney Foundation grant OKF05-0002 (T. Inamoto); and The Yasuda Medical Foundation.

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

Received 2/16/06; revised 3/22/06; accepted 3/29/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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T. Inamoto, T. Yamada, K. Ohnuma, S. Kina, N. Takahashi, T. Yamochi, S. Inamoto, Y. Katsuoka, O. Hosono, H. Tanaka, et al.
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