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Keywords:

  • tumor-associated antigen;
  • 90K/Mac-2 binding protein;
  • immunogenicity;
  • lung carcinoma;
  • tumor marker;
  • immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

The authors attempted to obtain shared proteins among lung carcinoma cells by column chromatographies. A glycoprotein with approximately 500 kDa isolated from QG56 cells showed an identical amino acid sequence to 90K/Mac-2 binding protein (M2BP). This protein has been reported to be highly expressed and to modulate the expression of surface molecules involved in immune responses on cultured cancer cells. Therefore, it would be beneficial for M2BP to be targeted in cancer immunotherapy.

METHODS

The authors analyzed the expression of M2BP in lung carcinoma cells and M2BP's immunogenicity as a tumor antigen. Eight cultured lung carcinoma cell lines and 28 tumor tissues from patients with lung carcinoma were examined for the expression of M2BP mRNA and protein. Sera from cancer patients (n = 23) and healthy donors (n = 19) were studied for their reactivity to M2BP peptides by enzyme–linked immunosorbent assay.

RESULTS

Seven of the 8 (87.5%) lung carcinoma cell lines and 17 of the 28 (60.7%) tumor tissues expressed high levels of M2BP mRNA. Most of the M2BP mRNA-positive cancer cell lines and tumors also showed M2BP protein expression. The serum levels of antibodies to M2BP were elevated in 30.4% of the patients. In addition, M2BP-specific immunoglobulin G was observed in all patients with anti-M2BP antibodies.

CONCLUSIONS

M2BP is highly expressed in lung carcinoma cells and is sufficiently immunogenic to elicit specific immunity to this molecule in patients with lung carcinoma. M2BP is expected to be useful as a tumor marker and a target antigen in cancer immunotherapy. Cancer 2002;95:1954–62. © 2002 American Cancer Society.

DOI 10.1002/cncr.10899

For the last decade, efforts to characterize molecules that are specifically recognized on human tumor cells by the immune system have led to the identification of a large number of tumor associated-antigens (TAAs) such as the MAGE and BAGE families, gp100, SART-1, tyrosinase, MUC-1, and HER-2/neu.1–9 Some TAAs had been established as targets for cancer immunotherapy in several clinical trials.10, 11 Conversely, in lung carcinoma, no available cancer immunotherapy has been established. It is necessary to define novel TAAs which are highly expressed in cancer cells and can evoke a strong antitumor immune response in patients with lung carcinoma. Recently, we reported the expression of MUC-4 mucin in lung cancer.12 In the process of extracting MUC-4 protein from QG56 cells, a lung carcinoma cell line, a glycoprotein whose molecular mass size was > 500 kDa was co-extracted with MUC-4 by gel-filtration and anion exchange chromatographies. This protein was also obtained from other lung carcinoma cell lines studied. The N-terminal amino acid sequence of this protein was identical with that of 90K/Mac-2 binding protein (M2BP).

M2BP has been identified as an ∼90-70 kDa secreted glycoprotein that binds to the human macrophage-associated lectin, Mac-2, and has been known as a member of the macrophage scavenger receptor superfamily that contains the cystein-rich domain.13–16 M2BP has been reported to up-regulate the expression of MHC and cell-adhesion molecules on cultured tumor cells17 and was found at high levels in sera from patients with various types of cancers, such as breast, colon, stomach, ovary, and lung.13, 16, 18–22 Serum levels of M2BP were also elevated in patients with viral infections, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection.21–23 M2BP has also been shown to be a potent immune stimulator with positive effects on the generation of natural killer/lymphocyte-activated killer (NK/LAK) cells from peripheral blood mononuclear cells.16, 24 Therefore, this protein is thought to play a crucial role in cellular immune response in hosts. However, the details of its physiologic function remain unclear, and the possibility of utilizing M2BP as a target antigen in immunotherapy for cancer is not known.

In the current study, we report mRNA and protein expressions of M2BP in cultured lung carcinoma cells and tumor tissues from patients with primary lung carcinoma and M2BP immunogenicity to assess the possibility of using this protein as a tumor marker and as a target antigen in cancer immunotherapy.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Cell Lines

Eight lung carcinoma cell lines were used in the current study. QG56 cells (squamous cell carcinoma) were provided by Tatsuhiko Narita, M.D., Ph.D. (Laboratory of Experimental Pathology, Aichi Cancer Center Research Institute, Nagoya, Japan). EBC-1 and LK-2 (adenocarcinoma) and SBC-2 cells (small cell carcinoma) were obtained from the Japanese Collection of Research Bioresource (Tokyo, Japan). IGH-KS and YMN-KSH (adenocarcinoma) and MSK-KS and SKB-KSH (squamous cell carcinoma) were established as reported previously25, 26 from primary tumors resected at the time of operation from patients with lung carcinoma by grafting the tumor tissues subcutaneously into female severe combined immunodeficiency mice that were purchased from Charles River Inc. (Hino, Japan). For control samples, we used seven human breast carcinoma cell lines (SK-BR-3, MCF-7, ZR75-1, MDA-MB-231, NZK-K1, BT-20, and T-47D), five human hematopoietic neoplastic cell lines (Jurkat, T2, Daudi, 221A2.1, and K562), and two murine neoplastic cell lines (P815 and Colon 26). NZK-K1 cells were established from the primary tumor of a 45-year old patient with breast cancer.25 ZR75-1, MDA-MB-231, BT-20, T-47D, and T2 cells were purchased from American Type Culture Collection (Rockville, MD), and MCF-7, SK-BR-3, Daudi, K562, Jurkat and P815 cells were purchased from Dainippon Seiyaku Co. Ltd., (Osaka, Japan). 221A2.1 cells were given by Dr. Ichiro Kawashima (TaKaRa, Otsu, Japan), and Colon 26 cells were given by Dr. Eiichi Hara (Saitama Cancer Center, Kita-adachi, Japan). These cells were maintained in Dulbecco's modified Eagle medium (Nakarai Tesque, Kyoto, Japan) containing 10% heat inactivated fetal calf serum, 2 ML-glutamine, 100 U/mL penicillin G, 0.1 mg/mL streptomycin, and 0.2 mg/mL amphotericin B (all from GIBCO BRL, Tokyo, Japan) at 37 °C in a humidified atmosphere at 5% CO2.

Tissue Samples and Sera

Primary lung carcinoma tissues and normal lung tissues adjacent to the tumors were resected from 28 patients (17 males, 11 females) at the time of operation. Histologic subtypes of the cancer tissues were as follows: 13 adenocarcinomas, 11 squamous cell carcinomas, 2 small cell carcinomas, 1 large cell carcinoma, and 1 adenosquamous cell carcinoma.

Blood samples were obtained from 23 patients with lung carcinoma and 19 healthy donors. Samples were allowed to clot, and the sera were stored at −20 °C until use. We obtained the approval of the university ethics committee and informed consent from each patient and volunteer.

Preparation of Rabbit Antisera Directed Against M2BP Peptides

From the three dimensional structure of M2BP deduced from its amino acid sequence,15 we prepared three oligopeptides which were derived from hydrophilic regions in the β-turns: M2BP104–117; KSLGWLKSNCRHER, M2BP185–197; LWKEPGSNVTMSV, and M2BP441–460; KYSSDYFQAPSDYRYYPYQS, which were synthesized by the F moc method using NovaSyn Gem (Nova Bilchem, Darmstadt, Germany) in the Central Research Center, Shiga University of Medical Science. These peptides were proven to be over 95% pure by reverse-phase high performance liquid chromatography. Female New Zealand rabbits (Charles River Inc.) were immunized with 200 μg keyhole limpet hemocyanin-conjugated M2BP peptides emulsified in 500 μL of complete Freund adjuvant (Nakalai Tesque). Three weeks after the immunization, the rabbits were boosted twice with the same peptides emulsified in incomplete Freund adjuvant (Nakalai Tesque) at an interval of three weeks. Two weeks after the last immunization, the rabbits were bled, and the antibodies were purified by affinity column chromatography (rec-Protein A Sepharose 4B, Zymed, San Francisco, CA).

Measurement of M2BP Concentrations in Lysate from Cancer Cells

Cells (2 × 107) were suspended in lysis buffer (50 mM Tris-HCl, 150 mM Na Cl, 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM phenylmethane sulfonyl fluoride (PMSF), 1% Triton-X100; Nakalai Tesque), left on ice for 30 minutes, and then centrifuged at 15,000 × g for 15 minutes. The supernatant was collected and measured for the protein concentration. The concentration of M2BP contained in the cell lysate from lung carcinoma cell lines was measured using an M2BP enzyme–linked immunosorbent assay kit (Bender Medsystems, Vienna, Austria) according to the manufacturer's instructions.

Immunohistochemical Study

Tissue samples were fixed in 10% formalin, embedded in paraffin, sliced into 20 μm thickness, and mounted on glass slides. The sections were treated with 2% trypsin and then incubated in 5% normal goat serum to block nonspecific bindings for 30 minutes. After washes in phosphate–buffered saline (PBS), the sections were incubated with rabbit antisera diluted at 1:5000 overnight at room temperature. Then, the sections were washed in PBS three times and incubated with biotinylated anti-rabbit immunogloblins (1:2000) followed by incubation in horse radish peroxidase (HRP)-conjugated avidin solution at room temperature for 90 minutes (Vector Laboratories, Inc., Burlingame, CA). They were colored by incubation with 0.4 mg/mL 3.3′-diaminobenzidine for 10 minutes at room temperature. They were washed in tap water, counter-stained with 0.1% hematoxylin solution, and mounted in 50% glycerol.

Northern Blot Analysis

The 2.2-kb full length cDNA of M2BP was prepared for use in hybridizing M2BP mRNA by reverse transcriptase polymerase chain reaction (PCR) from cDNA obtained from QG56 cells. Total RNA extracted from QG56 cells was reverse-transcribed with avian myeloblastosis virus (AMV) transcriptase (GIBCO BRL), yielding cDNA. The template DNA was transferred to a tube containing 2.5 U Taq polymerase (GIBCO BRL) and oligonucleotide primers (sense: 5′ GCC TGA CCA CGC TCC ATA 3′; antisense: 5′ GAC CAG TGA CAA GGG CAG 3′) and subjected to three-step thermal cycles (94 °C, 1 minute; 60 °C, 1 minute; 72 °C, 2 minutes; 35 cycles), to amplify the M2BP cDNA. The resulting PCR product was purified using a GENECLEAN II kit (BIO 101 Inc., Vista, CA).

Two hundred μg tumor samples or 107 cancer cells were minced, placed in tubes containing 1 mL ISOGEN solution (Nippongene Co. Ltd., Tokyo, Japan), and homogenized using a Teflon homogenizer (Iwaki, Tokyo, Japan). After adding 200 μL of chloroform, the homogenate was vortexed and centrifuged at 15,000 rpm at 4 °C for 15 minutes. The aqueous phase containing nucleic acids was collected, precipitated with isopropanol, and dissolved in Tris/EDTA solution. In order to eliminate contaminating genomic DNA, the precipitates were incubated with 500 U/mL DNAse I (Pharmacia Biotech, Tokyo, Japan) for 60 minutes at 37 °C. After phenol-chloroform extraction, the concentration of the total RNA was measured and stored at −70 °C until use. RNA (15 μg) was fractionated by electrophoresis through 0.9% agarose-formaldehyde gel, blotted onto a nitrocellulose membrane, and hybridized in a formamide-based hybridization solution at 42 °C with 32P-(dCTP) labeled cDNA probe. The blots were washed twice at room temperature in 2 × sodium chloride, sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) for 20 minutes and twice at 55 °C in 0.1 × SSC, 0.1% SDS for 10 minutes and exposed at −80 °C to X-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan). Intactness and quantity of the analyzed RNA samples were monitored by ethidium-bromide staining for 18S and 28S ribosomal RNA.

Purification of M2BP from Lung Carcinoma Cell Lines

Ten liters of serum-free, 48-hours conditioned Dulbecco's modified Eagle's medium were collected from lung carcinoma cells. Proteins were precipitated from the medium by adding solid ammonium sulfate to 70% saturation (8 hours at 4 °C). The precipitate was dissolved in a minimal volume of PBS, dialyzed against overnight 25 mM Tris (pH 8.5), and then applied onto Hi-Load Superdex 200 pg column (2.6 × 60 cm; Amersham Biosciences, Buckinghamshire, UK) at a flow rate of 0.3 mL/minute. The first peak fraction was collected and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). For amino acid sequence analysis, the peak fraction was applied into Resource Q column (Amersham Biosciences) pre-equilibrated with 20 mM phosphate buffer (pH 7.5). The column was washed and the absorbed proteins were eluted by a stepwise NaCl gradient (0–1.0 M) at a flow rate of 0.3 mL/minute. Peak fractions, which were eluted with 0.2, 0.3, and 0.4 M NaCl, were pooled, dialyzed against overnight PBS, and concentrated with Amicon YM10 membrane (Millipore, Ltd., Bedford, USA). The concentrated proteins were subjected to SDS-PAGE, transferred onto the polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA), and sequenced by automated Edman degradation using a protein sequencer (Applied Biosystems Model 473, Foster City, CA).

Enzyme-linked Immunosorbent Assay

Ninety-six cell microtiter plates (Nalgen Nunc, Rochester, NY) coated with 10 μg/mL M2BP peptides were incubated with sera diluted at 1:50 to 1:400 for 60 minutes at 37 °C, following incubation in PBS containing 2% fetal calf serum for 60 minutes at 37 °C. After washes with PBS containing 0.02% Tween 20, the plates were incubated with HRP-conjugated anti-human immunoglobulin antibody (Organon Teknica Corp., West Chester, PA) diluted at 1:1000 for 45 minutes at 37 °C, washed three times, and colored by adding 50 μL of 0.05 M o-phenylenediamine (GIBCO BRL). The reaction was stopped with 50 μL of 4N HCl, and the absorbance at 492 nm was measured on a microplate reader (Titertek™ Mutiskan MCC/340 Mk II, Labsystems, Tokyo, Japan). To determine isotypes of anti-M2BP immunoglobulins, HRP-conjugated anti-human immunoglobulin (Ig) M and IgG antibodies (DAKO, Kyoto, Japan) were used as secondary antibodies.

Statistical Analyses

The differences between groups with and without M2BP mRNA overexpression were evaluated using the Fisher chi-square test. We defined the normal upper cut-off value of anti-M2BP antibody in sera as mean + 2 standard deviation of the absorbance in 19 healthy donors. The Mann-Whitney U test was used to compare the two proportions.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Amino Acid Sequence of the Protein of > 500 kDa Extracted from Lung Carcinoma Cells

Proteins purified from the supernatant of QG56 cells by Superdex 200 and Resource Q chromatographies as described in materials and methods were subjected to SDS-PAGE and stained with periodic acid-Schiff (PAS). A single glycoprotein with size > 500 kDa was obtained (Fig. 1). The sequence of the N-terminal amino acids of this protein was as follows: VNDGDMRLADGGATNQGRVEIFYRGQWGTV(C)DNLWDLTDA(S)VV(C)RA(L)GFE(N)ATQALGRAA (residues in parentheses are uncertain). It was identical to the sequence of M2BP17–78 reported by Koths et al.15

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Figure 1. Preparation of Mac–2 binding protein from the culture supernatant of lung carcinoma cells. Proteins extracted from the supernatant of QG56 cells by gel-filtration and anion exchange chromatographies were subjected to sodium dodecyl sulfate poly acrylamide gel electrophoresis and stained with periodic acid-Schiff (left lane). A molecular weight marker (200 kDa) was applied in the right lane.

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M2BP mRNA Expression in Tumor Cell Lines and Lung Carcinoma Tissues

Cultured neoplastic cell lines including eight human lung carcinoma, seven human breast carcinoma, five human hematopoietic neoplastic, and two murine neoplastic cell lines were examined for their M2BP mRNA expression by Northern hybridization. Seven of eight lung carcinoma cell lines (87.5%) and five of the seven breast carcinoma cell lines (71.4%) exhibited high levels of M2BP mRNA expression (Fig. 2). Conversely, only one of the five hematopoietic cell lines and none of the two murine cell lines were positive for M2BP mRNA expression.

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Figure 2. Northern blot analysis of Mac–2 binding protein (M2BP) mRNA expression in a variety of neoplastic cell lines. Eight human lung carcinoma cell lines, seven human breast carcinoma cell lines, five human hematopoietic neoplastic cell lines, and two murine cancer cell lines were examined for M2BP mRNA expression. Twenty μg of RNA extracted from the cells were electrophoresed, transferred onto the nitrocellurose membrane, and hybridized with M2BP cDNA probe. The lower panel shows 28 S ribosome RNA stained with ethidium bromide in each lane.

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Tumor tissues resected from 28 patients with lung carcinoma were examined for their M2BP mRNA expression. Seventeen (60.7%) exhibited high levels of M2BP mRNA expression in tumors. Of the 17 M2BP-positive cases, 14 cases showed the overexpressed M2BP mRNA in tumor tissues but no expression in the adjacent normal lung tissues; the remaining 3 cases showed the overexpressed M2BP mRNA in both tumor tissues and normal lung tissues, as shown in Case 3 (Fig. 3).

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Figure 3. Northern blot analysis of Mac–2 binding protein (M2BP) mRNA expression in tumor tissues from patients with lung carcinoma. Twenty μg of RMA extracted from the tumor tissues or adjacent normal lung tissues were electrophoresed, transferred onto the nitrocellurose membrane, and hybridized with M2BP cDNA probe. Expression levels of M2BP mRNA in the tumor tissues (T) and the adjacent normal lung tissues (N) are shown for each case. The lower panel shows 28 S ribosome RNA stained with ethidium bromide in each lane.

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Expression of M2BP in Lung Carcinoma Cell Lines and Cancer Tissues

To examine the expression of M2BP in lung carcinoma cells, the cell lysate was examined as described in materials and methods. Seven of the eight lung carcinoma cell lines studied contained M2BP, ranging from 51.7 to 550 ng/mg protein (Fig. 4). Only one cell line, SKB-KSH, was deficient in M2BP expression. This finding was compatible with the result of Northern hybridization with the lung carcinoma cell lines (Fig. 1). The tumor samples were studied for M2BP expression by immunohistochemical study using prepared anti-M2BP antibody. In the lung carcinoma tissues, many tumor cells were stained strongly and diffusely in their cytoplasm (Fig. 5C,D). Some bronchial epithelial cells were weakly stained in their apical portions (Fig. 5A). Tissues in which more than 10% of the 300 tumor cells counted were positively stained were judged positive. Eleven of the 27 tissues (40.7%) were positive for M2BP expression.

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Figure 4. Measurement of the concentration of M2BP in lung carcinoma cell lines. Cell lysate prepared from eight lung carcinoma cell lines was examined for concentration of Mac–2 binding protein (M2BP) using an M2BP enzyme–linked immunosorbent assay kit. K562 cells deficient in M2BP mRNA expression were used as a negative control.

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Figure 5. Immunohistochemical study of Mac–2 binding protein (M2BP) expression in lung carcinoma tissues and normal lung tissues. Tissue samples from the tumor tissues or normal lung tissues were examined for M2BP expression using rabbit antisera reactive with M2BP peptides. A) Normal lung tissue incubated with rabbit antisera. B) Squamous cell carcinoma tissue incubated with normal rabbit sera. C) Squamous cell carcinoma tissue incubated with rabbit antisera. D) Adenosquamous cell carcinoma tissue incubated with rabbit antisera.

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Relationship Between Clinicopathologic Characteristics in Patients with Lung Carcinoma and Mac-2 BP Expression in Their Tumors

Table 1 shows the relationship between M2BP expression in the tumors and their histologic types in lung carcinoma patients. Positive cases for M2BP mRNA and protein expression were 46.7% and 33.3% in adenocarcinomas and 77.8% and 44.4% in squamous cell carcinomas, respectively. There were no statistically significant differences among these histologic types. In other histologic types (adenosquamous, small cell, and large cell carcinomas), there were insufficient numbers of each subtype to evaluate the histologic preference. Although patients in advanced stages tended to be positive for M2BP in their tumors compared to patients in early stages (advanced vs. early stages: 60.0% and 27.8%), that tendency not statistically significant. Furthermore, there was no significant difference in postoperative survival between cancer patients with and without overexpressed M2BP (data not shown).

Table 1. Relationship Between Clinicopathologic Characteristics of Patients with Lung Carcinoma and Mac–2 Binding Protein Expression in Their Lung Tumors
CharacteristicnmRNA (%)Protein (%)
All2817 (60.7)11 (39.3)
Age   
 ≤ 65 years128 (66.7)5 (41.7)
 > 65 years169 (56.3)6 (37.5)
Gender   
 Male1811 (61.1)6 (33.3)
 Female106 (60.0)5 (50.0)
Histology   
 Adenocarcinoma157 (46.7)5 (33.3)
 Squamous cell97 (77.8)4 (44.4)
 Small cell22 (100)1 (50.0)
 Large cell20 (0)0 (0)
 Adenosquamous cell11 (100)1 (100)
Clinical stage   
 1 + 21811 (61.1)5 (27.8)
 3 + 4106 (60.0)6 (60.0)

Anti-M2BP Antibody in Sera from Patients with Lung Carcinoma

To examine the immunogenicity of M2BP, we analyzed reactivity of sera from 23 lung carcinoma patients and 19 healthy donors with M2BP by ELISA. Serially diluted sera from the patients (1:25 to 1:200) were incubated in 96 well plates coated with a mixture of three prepared M2BP peptides, followed by incubation with HRP-anti-human Ig antibody. The absorbance at a dilution of 1:50 was compared between the cancer patients and the healthy donors. The mean absorbances of the patients and healthy donors were 0.173 ± 0.118 and 0.087 ± 0.048, respectively (P = 0.047; Fig. 6A). When we defined the mean + 2 standard deviation of the absorbance in sera from healthy donors as the normal upper cut-off value (0.296), 7 of the 23 patients' sera (30.4%) were positive for anti-M2BP antibodies. To determine subclasses of immunogloblins reactive with M2BP, we used anti-human IgM and IgG antibodies as secondary antibodies in ELISA. All of the patients with anti-M2BP antibody possessed high titers of IgG specific for M2BP (Fig. 6B).

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Figure 6. Reactivity of sera from patients with lung cancer with Mac–2 binding protein (M2BP) peptides. The sera from 29 patients with lung carcinoma and 19 normal healthy donors were tested for their reactivity with M2BP peptides by enzyme–linked immunosorbent assay (ELISA). A) The absorbance (492 nm) at a dilution of 1:50 for each case was plotted and compared between the cancer patients and normal volunteers. B) To determine isotypes of immunoglobulins (Ig) specific for M2BP in sera, anti-human IgM or IgG monoclonal antibody was used in ELISA.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A number of tumor antigens have been identified, and some of them are used to target immunotherapy for cancer.10, 11 However, the outcome of the clinical therapeutics targeting these TAAs is not satisfactory. In lung carcinoma, no available TAAs to target have been identified. A tumor-associated antigen, M2BP, has been initially isolated from the culture supernatant of a breast carcinoma cell line.13 This protein has been reported as an ∼90-70 kDa secreted glycoprotein, which could be a ligand of galectin-3 (formerly known as Mac-2).15 cDNA cloning revealed that it contains a number of cysteines and N-glycosylation sites and that its N-terminal domain shares a similar sequence with an extracellular domain of the macrophage scavenger receptor.15, 16 It was also found that M2BP serves as a ligand for galectin-1 and mediates the cell-aggregating activity by this lectin.27 These characteristics allow us to assume that M2BP is responsible for metastatic activity of cancer cells.28 On the other hand, M2BP is thought to play a part in cellular immune responses in hosts. M2BP can up-regulate the expression of MHC Class I molecules in breast carcinoma cell lines17 and activate NK and LAK cells in vitro. Also, this protein can induce interleukin-2 secretion in mitogen-stimulated peripheral blood monocytes16, 24 and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on tumor endothelial cells.29 Some clinical data show that M2BP is found at high levels in the sera of patients with various types of cancers or viral infections, including HIV and HCV infection,13, 16, 18–22 and that serum levels of M2BP predict disease progression or responsiveness to therapy in patients with HCV or HIV infection.23, 30–33 These findings suggest that M2BP may modulate the immune response to tumors in cancer patients.

In the current study, we attempted to isolate shared proteins among lung carcinoma cells by two column chromatography. A glycoprotein of over 500 kDa was isolated; this glycoprotein showed an identical amino acid sequence to M2BP (Fig. 1). Since M2BP is thought from cDNA cloning to be an ∼90-70 kDa of protein, the protein of over 500 kDa extracted from the lung carcinoma cell lines was thought to be either highly glycosylated or polymerized via its disulfide bond. Northern hybridization analysis showed that M2BP mRNA was overexpressed in 86.5% of the lung carcinoma cell lines and in 68% of the lung carcinoma tissues studied (Figs. 2,3). In contrast, normal lung tissues showed no expression of M2BP mRNA, with the exception of three cases showing M2BP mRNA in both tumors and lung tissues. In these cases, we cannot eliminate the possibility that the adjacent lung tissues studied were involved with cancer or related inflammation. Although not all of the cases with M2BP mRNA expression exhibited its protein expression as shown in immunohistochemical study with lung carcinoma tissues (Table 1), all of the M2BP mRNA-positive lung carcinoma cell lines expressed its protein (Figs. 2,4). To detect M2BP expressed in lung carcinoma cell lines, anti-M2BP monoclonal antibody, SP2 reactive with natural structures of the Mac2 mucin-like binding domain, was used. However, anti-M2BP antibody used in immunohistochemical staining was prepared by immunizing rabbits with synthetic oligopeptides derived from M2BP core. This polyclonal antibody is expected to react with the core peptide or poorly glycosylated M2BP, not with highly glycosylated M2BP. These findings suggest that cancer cells in some of the lung tumors are highly glycosylated, masking M2BP core protein, and that cultured lung carcinoma cells may have lost carbohydrate chains on the core protein while maintained in an artificial condition. The fact that the same size M2BP mRNA (2.2 kb) was detected in all of the positive cases by Northern hybridization may eliminate the possibility that transcriptional events such as alternative splicing or deleted transcripts abrogate the translation of M2BP. Also, it is probable that the binding affinity of antibody against M2BP is different under various chemical and physical conditions. Many proteases and detergents reduce the immunoreactivity to natural structures of M2BP, but some exoglycosidases such as neuraminidase and β-galactosidase can increase the affinity of the anti-M2BP antibody, SP-2, to its epitope of this protein.14

There have been some reports showing no relationship between serum levels of M2BP in cancer patients and clinical staging or prognosis.34, 35 In the current study, protein expressions of M2BP were correlated with clinical staging in patients with lung carcinoma (Table 1: Stage I and II, 27.8%; Stage III and IV, 60%). However, that was not statistically significant because the total numbers of patients studied were not sufficient for this analysis.

Antitumor immune responses may result in eliciting autoantibodies directed against various TAAs. Previous reports showed that several autoantigens trigger the generation of autoantibodies among patients with malignant disease.12, 36–43 For example, anti-p53 antibodies were detected in 3–65% of sera of patients with cancer,38, 39 and anti-HER2/neu antibodies were detected in 11% of patients with breast carcinoma.40 Also, antibodies reactive with cancer/testis antigen, such as MAGE, GAGE, BAGE, SSX, and NY-ESO-1, were found in 4.2–12.5% of sera of patients with melanoma and carcinoma of the lung, breast, and ovary.41, 42 Antibodies against mucins were elicited in 8.3% of patients with various types of cancer.37 We have previously reported that high levels of anti-MUC4 antibodies (either IgM or IgG) are found in 29% of patients with lung carcinoma.12 In comparison with the data described above, M2BP is among the most immunogenic antigens in lung carcinoma. Surprisingly, all of the immunoglobulins specific for M2BP detected in patients with lung carcinoma were found to be IgG (Fig. 6). In contrast, antibodies against TAAs described above were dominantly IgM. Class switching from IgM to IgG is required for cytokines produced by helper T lymphocytes after recognition of antigens by the T lymphocytes. This suggests that M2BP has antigenic determinants that can elicit humoral immunity to this molecule as well as prime the helper function in hosts. Thus, since M2BP abundantly expressed in lung carcinoma has both immunoregulatory functions and strong immunogenicity in itself, targeting this unique molecule is expected to be useful in immunotherapy for lung carcinoma.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Mr. Giyouzan Yamazaki and Mr. Noboru Urushiyama (Central Research Center, Shinga University of Medical Science) and the staff of the Division of Surgical Pathology, Shiga University of Medical Science, Otsu, Japan for their technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES